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Med-MDPI/cardiogenetics/cardiogenetics-10-02-00005.txt

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+Cardiogenetics was launched in 2011 and it has been published over the past nine years by PAGEPress Publications [1]. Dr. Giuseppe Limongelli has served as its Editor-in-Chief [2] since its inception and, together with Dr. Lia Crotti, who became co-Editor-in-Chief in 2019, will remain active in this role.We are delighted to take over the publication of Cardiogenetics from PAGEPress and perpetuate the legacy of this journal, and ensure that we serve well the genetics and the cardiology communities. Cardiogenetics complements very well the MDPI portfolio of medical and life sciences journals [3,4], especially Journal of Cardiovascular Development and Disease [5], Hearts [6], and Genes [7] and strengthen the trans-disciplinary bridge between basic sciences and applied sciences across MDPI journals.We will publish only one quarterly issue in 2020, and regularly publish four quarterly issues from 2021. Enjoy publishing your work in Cardiogenetics [8]!Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/cardiogenetics/cardiogenetics-10-02-00006.txt

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+Anomalous Left Coronary Artery from the Pulmonary Artery (ALCAPA) is a rare coronary artery anomaly which accounts for 0.25–0.5% of all congenital cardiac diseases, where most die within the first year of life. We present a case report of a 50-year-old lady who presented to hospital with persistent palpitations. Her admission electrocardiogram found her to be in Atrial Fibrillation (AF). She was rate-controlled and subsequently discharged. Despite that, she represented with further episodes of AF and was referred for an outpatient transthoracic echocardiogram. This revealed a dilated right coronary artery, retrograde flow in the left coronary artery and collateral flow in the myocardium. To investigate, the patient had undergone further imaging which confirmed the diagnosis. As such, she was later shortlisted for surgical intervention. Conclusively, our case exemplifies the role of multimodal imaging to identify the features of ALCAPA and may be useful for the purposes of surgical intervention.Anomalous Left Coronary Artery from the Pulmonary Artery (ALCAPA), also referred to as Bland–White–Garland syndrome, is a rare coronary artery anomaly which accounts for 0.25–0.5% of all congenital cardiac diseases and has a reported incidence of 1 in 300,000 live births [1]. Most cases of ALCAPA die in infancy, whereas 10% survive to adulthood. The adult type of ALCAPA presents with the heightened risk of myocardial ischemia, heart failure and arrhythmia [2], the presentation of which is highlighted in this rare case involving a 50-year-old lady.A 50-year-old Caucasian lady, with a background of migraines, presented to our hospital in October 2014 with persistent palpitations. She reported that her episodes, along with shortness of breath and chest tightness had increased in frequency and severity over the past year. She did not report any chest pain, orthopnoea, paroxysmal nocturnal dyspnoea, presyncope or syncope. There is no known family history of ischaemic cardiac disease but all her siblings suffered from arrhythmias. Other than being an ex-smoker, she did not suffer from diabetes mellitus, hypertension or hypercholesterolaemia and worked as a horse-riding coach. Her admission vital signs were as follows: respiratory rate 18/min, oxygen saturations of 95% (room air), temperature 36.6 degrees Celsius, blood pressure of 120/63 and pulse 122 (irregular).Her admission electrocardiogram showed Atrial Fibrillation (AF) with left axis deviation and partial left bundle branch block (Figure 1). Her admission chest X-ray was normal (Figure 2) and a troponin-T (ng/L) level was also normal (<13.0). She was subsequently reverted to sinus rhythm and an eventual diagnosis of paroxysmal AF was made, managed by beta-blocker and warfarin. The warfarin was subsequently changed to a direct oral anticoagulant. She remained stable until two further episodes of AF in January 2018 and March 2019, chemically cardioverted with flecainide in accident and emergency. She was then referred to cardiology.An outpatient transthoracic echocardiogram revealed moderate left ventricular dilatation (and mild dilatation of the right ventricle) with preserved systolic function, severe left atrial dilatation and moderate mitral regurgitation. Furthermore, an anomalous origin of the left coronary artery was also suspected because of the retrograde flow noted in the left anterior descending and left circumflex coronary arteries. A dilated right coronary artery and extensive collateral flow across the myocardium was also visualised (Figure 3).For further assessment, she underwent cardiac Magnetic Resonance Imaging (MRI) to investigate a possible cardiomyopathy. This showed dilatation of the left ventricle and an ejection fraction of 51%. Late gadolinium enhancement sequences also found a previous myocardial infarction in the LAD territory. In view of the cardiac MRI findings, she had a diagnostic coronary angiogram. This showed a large aneurysmal right coronary artery of normal origin but the left coronary artery could not be cannulated, suggesting an aberrant origin (Figure 4).In order to delineate the origin of the left coronary artery, a Computer Tomography Coronary Angiogram (CTCA) was later performed and illustrated that both arteries were diffusely aneurysmal. Whilst the right coronary artery originated from the right coronary sinus, the left coronary artery anomalously originated from the left anterior pulmonary sinus of the main pulmonary artery (Figure 5). Furthermore, there was a right dominant coronary artery system with multiple collateral vessels (also visualised on the coronary angiogram) and no evidence of coronary artery stenosis. This suggested a diagnosis of ALCAPA.She was referred for surgical intervention following a joint cardiology and cardiothoracic surgery team discussion.This case report describes a 50-year-old lady who presented with AF and was incidentally found to have myocardial ischaemia and early stages of heart failure, all of which a likely consequence of ALCAPA syndrome.This extremely rare congenital anomaly was first clinically described by Bland, White and Garland in 1933 [3]. As previously discussed, there are two variants of ALCAPA syndrome: infant and adult type. Prior to birth, the ductus arteriosus equalises the systemic and pulmonary arterial circulations, ensuring antegrade blood flow. After birth, however, the ductus arteriosus is physiologically occluded and, because of the decreased pulmonary pressure, the flow in the left coronary artery also decreases—so much that it reverses [4]. This is known as the steal phenomenon, whereby blood is carried away from the myocardium and towards the pulmonary artery. In the adult type of ALCAPA, inter-coronary collaterals form to compensate for the lack of blood supply [5]. The extent of compensation through these collaterals determines the extent of myocardial ischemia and mitral insufficiency [4,5]. In our case, extensive inter-coronary collaterals were found via CTCA and diagnostic coronary angiography, possibly explaining why few complications were found. It might also justify why 15% of patients who do not undergo surgical intervention survive childhood [6].Despite that, this might be an underestimation and the true incidence of this rare congenital anomaly is thought to be higher with the increasing use of multimodal imaging. CTCA is considered the radiographic modality of choice for diagnosing ALCAPA [7]. With the help of three-dimensional reconstruction, CTCA can narrate the course and location of arteries which assists with planning for coronary reimplantation [8]. In addition, a dilated right coronary artery and inter-coronary collaterals may be visualised better by CTCA compared to cardiac MRI. In contrast, cardiac MRI may provide better functional evaluation and can elicit the steal phenomenon [9,10]. Myocardial infarcts (via late gadolinium enhancement sequences), mitral valve insufficiency and left ventricular dilatation are better detected by cardiac MRI—additional features of ALCAPA [9]. Furthermore, diagnostic coronary angiography can provide more accurate quantifications than CTCA and possesses the ability for possible intervention (but is associated with low procedural complications) [11].Transthoracic echocardiography tends to be the first imaging modality in most clinical scenarios, and, as evident in our case, can be used to suspect ALCAPA. Some of the reported echocardiographic features of ALCAPA include [12], mitral regurgitation, retrograde flow in the left coronary artery, dilated right coronary artery, collateral flow in the myocardium and an aberrant origin of the left main stem (Figure 3). This justifies the role of transthoracic echocardiography, a readily available and inexpensive tool that may uncover features of ALCAPA early on.Here, we present a case of ALCAPA syndrome of the adult variant, incidentally found in a middle-aged lady. Features of ALCAPA were first discovered using transthoracic echocardiography, a relatively inexpensive, non-invasive and readily available diagnostic imaging modality. We later confirmed our diagnosis using other imaging modalities, whilst excluding other potential differentials. Our case report supports the role of multimodal imaging where transthoracic echocardiography may suspect ALCAPA before the use of CTCA, cardiac MRI and diagnostic coronary angiography which may together plan for coronary reimplantation or other forms of surgical interventions.M.S.: Leading the case report, writing the manuscript/paper (introduction, case presentation, follow up, discussion and conclusion) and constructing a team to complete the report. They were also responsible for composing figures for the report and gaining signed consent. A.T.G.: Assisted with selecting and interpreting echocardiogram images and provided captions. They also captioned the selected videos and helped review the written manuscript. M.E.-H.: Offered advice and jointly-assisted with retrieval of angiogram still image. A.K.S.: Selecting and interpreting cardiac MRI images. They also selected videos for the case report and reviewed the written manuscript. R.I.: Played an advisory role and reviewed the written manuscript. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The authors declare that there is no conflict of interest regarding the publication of this case report.12-lead electrocardiogram taken on initial admission. It shows atrial fibrillation with a rapid ventricular rate of 122, with evidence of left axis deviation and partial left bundle branch block.PA chest X-ray taken on initial admission. Lungs were found to be clear with no focal abnormality identified. The heart and mediastinum were reported as normal.Transthoracic echocardiogram. (A): Modified parasternal long axis view showing a dilated right coronary artery to increase blood supply to the myocardium. (B): Modified apical 2-chamber view focused on the inferior wall showing prominent retrograde coronary flow from the septal perforator coronary arteries. (C): Modified apical 4-chamber view focused on the right ventricle lateral wall showing prominent retrograde coronary flow from the septal perforator coronary arteries extending towards the apex, also known as the “Christmas tree” appearance. (D): Parasternal short axis view at the level of the papillary muscles showing prominent retrograde coronary flow from the septal perforator coronary arteries.Angiographic image of the coronary circulation and collaterals. Right coronary artery appears dilated and tortuous with collaterals following across to the left side of the heart.Computer tomography coronary angiogram (CTCA) with contrast. Heart rate control and coronary vasodilatation was achieved using an oral beta-blocker and sublingual glyceryl trinitrate spray, respectively. The right coronary artery (RCA) appears diffusely dilated and has a normal origin from the right coronary sinus. The left coronary artery (LCA) has an anomalous origin from the main pulmonary artery (MPA). There is a right dominant coronary artery system, where multiple collaterals arise from the RCA, spanning towards the LCA. The left main stem (LMS) is also dilated and divides into the left anterior descending artery (LAD) and the left circumflex artery (LCx). Both RCA and LCA, with the collaterals, appear tortuous. There is no evidence of coronary artery calcification. In summary, CTCA with contrast confirms that there is an anomalous origin of the left coronary artery from the main pulmonary artery (ALCAPA). (A,B) Volume rendering demonstrating anomalous origin of the LCA from the MPA and marked dilatation and tortuosity of the coronary arteries. Collaterals between RCA and LCA are also seen. (C) Axial view demonstrating the marked tortuosity of the RCA. (D) Axial view of the origin of LCA from MPA (*). (E) Multiplanar reconstruction demonstrating a dilated and tortuous RCA.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/cardiogenetics/cardiogenetics-10-02-00007.txt

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+Objectives: Fetuin-A is a circulating calcification inhibitor that prevents coronary artery calcification (CAC) by increasing calcium phosphate solubility and inhibiting VSMC differentiation and apoptosis. In this study, we investigated the correlation between rs4918 and CAC in patients with coronary artery disease (CAD). Methods: Forty-two healthy individuals and eighty-one CAD patients were recruited in the present study. The CAC score (CACS) was measured by CT angiography and the genotype analysis of rs4918 single-nucleotide polymorphism SNP was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Results: The CACS was significantly higher in CAD patients compared to healthy individuals (p < 0.001); however, there was no significant difference between the mean CACS in the presence and absence of rs4918 (p = 0.792). The mean calcium score of the left main coronary artery (LMCA) was significantly lower in carriers of the rs4918 allele (p = 0.036). The frequency of rs4918 SNP was almost similar in the control group and CAD patients (p = 0.846). Conclusions: in patients with CAD, we found no significant association between rs4918 SNP and CACS, indicating that carriers of this allele are not at increased risk of developing cardiovascular diseases compared with those without.Coronary artery disease (CAD) is one of the major causes of mortality worldwide, and atherosclerosis is the primary etiology [1]. Coronary artery calcification (CAC) is the characteristic feature of atheroma plaque, and its extent is increased with the progression of the lesion. Calcified plaques are associated with an almost 1.7-fold higher incidence of mortality, independent of other cardiovascular risk factors [2]. Although traditional Framingham risk factors, including age, sex, hyperlipidemia, hypertension, high body mass index, diabetes, and cigarette smoking, influence the severity of atherosclerosis and the extent of CAC, these environmental factors account for only 40% of the inter-individual variety, and more than 40% of the variations in the extent of CAC are due to genetic factors [3].It is believed that the transdifferentiation of VSMCs to the osteoblastic lineage and the loss of balance between inhibitors and inducers of calcification are the prominent etiologies of ectopic calcification. In this context, fetuin-A has been identified as an important inhibitor of ectopic calcification.Fetuin-A, also called alpha2-Heremans Schmid glycoprotein (AHSG), is a 62 kDa glycoprotein and a member of the cystatin superfamily which is synthesized and secreted from the liver and is present abundantly in the circulation and extracellular space [4]. It is a potent inhibitor of calcification; nearly 50% of blood capacity to inhibit ectopic calcification is attributed to fetuin-A activity [5]. It inhibits vascular calcification by binding to hydroxyapatite crystals and forming the fetuin mineral complex (FMC) that enhances the calcium phosphate blood solubility [6]. Furthermore, it has anti-inflammatory activity by preventing the production and release of tumor necrosis factor (TNF) α from activated macrophages, which have a central role in the pathophysiology of CAD [7].Several in vitro and in vivo studies have demonstrated its role in the pathogenesis of CAD. Fetuin-A-deficient mice on a calcium- and vitamin D-rich diet developed ectopic calcification, which clearly confirmed its role as a calcification inhibitor [8].It was shown that fetuin-A is directly associated with the intimal–medial thickness (IMT), a hallmark of subclinical atherosclerosis, which points to its atherogenic property [9]. In dialysis and end-stage renal disease (ESRD) patients, the reduction in this protein has been reported to correlate with the acceleration of vascular calcification and all-cause and cardiovascular death [10,11]. Genetic studies have demonstrated the impact of the AHSG gene variations on its serum level, CVD complications, and morality [4,11]. The role of AHSG polymorphisms and haplotypes in the progression of the calcified plaque was demonstrated in a group of European American diabetic patients with subclinical atherosclerosis and without advanced renal dysfunction [12]. Higher mortality rate, vascular calcification, and lower fetuin-A concentrations have been reported in ESRD patients carrying rs4918 single-nucleotide polymorphism (SNP) [11]. Additionally, this allele has been shown to be associated with arterial stiffness in patients with normal kidney function [13]. These data suggest that genetic variations in the AHSG gene may influence the extent of CAC.To our knowledge, no clinical study has evaluated the relationship between the rs4918 allele and CAC in patients with CAD. In the present study, the correlation between the rs4918 polymorphism and coronary artery calcification was evaluated, and its frequency in CAD participants and healthy individuals was studied.Eighty-one patients diagnosed with ischemic heart disease who fulfilled the inclusion and exclusion criteria entered the study. The inclusion criteria included age above 35 years and the presence of coronary artery disease, and the exclusion criteria involved disturbed calcium and phosphorus homeostasis, acute or chronic kidney disease (CKD), malignancies, bone disorders, primary and secondary hyperparathyroidism, and active infectious diseases.The calcium score was measured in four main coronary arteries, including the left main coronary artery (LMCA), right coronary artery (RCA), circumflex artery (CX), and left anterior descending artery (LAD) using CT angiography.The control group consisted of forty-two subjects free of any cardiovascular diseases (including myocardial infarction, angina, stroke, transient ischemic attack, heart failure, having current atrial fibrillation, taking nitroglycerin, or undergoing angioplasty, coronary artery bypass graft, valve replacement, pacemaker or defibrillator implantation, or any surgery on the heart or arteries); any chronic diseases; and CVD risk factors including age (above 50 years for women and 45 years for men), sex, history of death due to CVD in the first-degree family, smoking, diabetes mellitus, and hypertension. Additionally, all their biochemical measurements had to be in the normal range.Clinical examinations, biochemical assays, calcium score measurements, and blood sampling were conducted in the cardiology department of the Razavi hospital in Mashhad, Iran, from 2014 to 2018. Ethical approval was obtained from the university medical ethics committee. All the participants signed written informed consent.Demographic and clinical data including age, sex, history of previous and current diseases and medications, weight, height, smoking status, and family history of CVD were collected in a questionnaire. Biochemical parameters of lipid profile (total cholesterol, triglycerides, high- and low-density lipoprotein), and fasting blood sugar (FBS) were determined for all the participants by standard laboratory protocols.Genomic DNA was extracted from whole blood samples obtained from study participants using the FavorPerp blood genomic DNA extraction mini kit (Favorprep, South Korea) according to the manufacturer’s protocol. DNA concentration and purity was determined using Nanodrop spectrophotometer (Thermofisher Scientific, Waltham, MA, USA) and DNA extracts were stored at −20 °C.The amplification of the targeted gene (AHSG) and analysis of the polymorphism was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The 309 base pairs fragment of AHSG gene was amplified using forward primer 5′-TGTGAGGAAATTGGGTGCCA-3′ and reverse primer 3′-GACCACACCCATGAAGGTGT-5′ (Bioneer, Daejeon, South Korea).The PCR reaction mixture contained 2 ng of DNA, 0.6 μM of each forward and reverse primer, and 1× Taq DNA Polymerase 2× Master Mix Red (Ampliqon PCR enzymes and reagents, city, Denmark). The cycling program for sequence amplification was the denaturation of DNA strands at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 25 s, and extension at 72 °C for 20 s, with a final extension at 72 °C for 5 min, using the MyCycler thermocycler (Bio-Rad, Hercules, CA, USA).To identify the rs4918 single-nucleotide polymorphism (SNP), the amplified gene (PCR product) was treated with the restriction enzyme Sac I (SstI) (Jena Bioscience, Hannover, Germany) for 1 h at 37 °C. The digested products were electrophoresed through loading on a 1.5% (w/v) agarose gel stained with gel red (Biotium, Fremont, CA, USA), using TBE (1.1 mM Tris base, 900 mM boric acid, 25 mM EDTA) as a running buffer, and visualized under UV light transillumination (Uvitec, Cambridge, UK).The comparison of the coronary artery calcium scores between different groups was performed by an independent samples t-test. The difference in SNP frequency between different groups was determined by chi-squared statistics. p values of 0.05 or less were considered statistically significant for all tests. All the statistical analyses were performed using SPSS version 16.Details of the study participants’ characteristics including age; family history of CVD; sex; concomitant conventional CVD risk factors including hypertension, type two diabetes mellitus, and dyslipidemia; biochemical profiles; calcium score; and genotype frequency are listed in Table 1.The coronary artery calcium score was significantly higher in CAD patients compared to healthy individuals (p < 0.001); however, no significant difference was observed in the CACS in the presence and absence of rs4918 SNP (p = 0.792).The calcium score of four main coronary arteries was measured; the highest CACS was observed in the LAD artery, and the LMCA had the lowest CACS (Table 2).The calcium score of the LMCA was significantly lower in carriers of the rs4918 allele (p = 0.036) compared with the CACS of LMCA in individuals without this allele; on the other hand, an insignificantly higher CACS was observed in the LAD artery in the presence of this SNP compared with the CACS in its absence (p = 0.120) (Figure 1).The frequency of rs4918 SNP was similar in the control group and CAD patients (p = 0.846). The frequency of the rs4918 SNP in different calcium score groups was assessed and is presented in Table 3. Individuals were divided into three groups according to their coronary artery calcium score. Individuals with a CACS between 0 and 100 were assigned to group 1, with one between 101 and 400 were assigned to group 2, and with a CACS above 400 were assigned to group 3. There was no significant difference in the frequency of this SNP in these groups (p > 0.05).Coronary artery calcification has been considered as a non-traditional risk factor of cardiovascular disorders and events.In the present study, the coronary calcium score was measured by the noninvasive and quantifiable CT angiography technique, and the correlation between the obtained coronary calcium score and the AHSG genotype was assessed. According to the results, the calcium score was significantly higher in CAD patients compared to healthy individuals, and no difference was observed between the mean calcium scores in the presence and absence of the rs4918 polymorphism.Individuals in the control group were free of apparent coronary vasculopathy and CVD risk factors, on the other hand, while participants in the patients’ group were diagnosed with coronary artery disease and had undergone either percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) intervention or had myocardial infarction (MI). Higher CACS in this group is a representative of the association of calcification with atheroma plaque. Moreover, patients had at least one CVD risk factor, including hypertension, diabetes, dyslipidemia, cigarette smoking, and positive family history, which triggered the formation and progression of a plaque and subsequent calcified lesion by damaging the endothelial layer.In this project, the role of rs4918 polymorphism in the inter-individual variability of atherosclerotic calcification was studied; therefore the correlation between the rs4918 polymorphism and the coronary artery calcium score in CAD patients and healthy individuals was assessed.Our results indicate that the rs4918 allele is distributed equally between the healthy individuals and group of CAD patients, which indicates that carriers of this allele are not at risk of developing cardiovascular diseases. Cozzolino et al. reported a similar frequency of this allele among Italian hemodialysis patients and a healthy population [14]. Moreover, we found no correlation between this SNP and coronary artery calcified plaque. This is in agreement with the results of the Lehtinen group, who analyzed 11 polymorphisms in the AHSG gene and found no association between the rs4918 allele and coronary artery calcified plaque, diabetes mellitus, BMI, and fetuin-A serum levels in a group of American diabetic patients [12]. In another study, Bellia et al. reported the association of fetuin-A levels with serum calcium and rs4918 polymorphism but not with coronary artery calcification in individuals with a low risk for developing CAD and without apparent coronary vasculopathy [15].Although in animal studies AHSG-deficient mice developed widespread ectopic calcification of soft tissue and vasculature, which clearly confirmed its role as a calcification inhibitor [8], clinical findings regarding the impact of AHSG genotypes on CAC are still a matter of debate, and there are inconsistencies in the literature concerning the role of fetuin and its genotypes in the pathogenesis of CVD and CAC.Fetuin-A has been studied widely in end-stage renal disease and dialysis patients, where CVD and vascular calcification are common complications and the main cause of death in these patients. Low plasma levels of fetuin-A have been reported in these patients. Furthermore, carriers of the rs4918 polymorphism had lower fetuin-A levels and were more liable to develop vascular calcification, particularly in the presence of chronic inflammation [11]. In another study, Verdujin et al. showed that, although serine allele carriers were predisposed to lower serum fetuin-A concentrations, which were associated with increased mortality risk, the Thr256Ser polymorphism had a minor effect on mortality [16].In the general population and non-dialyzed patients, however, conflicting data are available regarding the genetic contribution of AHSG in CVD and CAC. Fisher et al. reported that AHSG polymorphisms were positively correlated with fetuin-A levels and the elevated concentration increased the risk of MI. In addition, the rs4917 allele was particularly associated with the incidence of MI [4]. In contrast, Roos et al. observed that fetuin-A levels were inversely associated with arterial stiffness, but the SerSer genotype of rs4918 SNP was significantly associated with arterial stiffness in male patients with normal renal function [13]. On the other hand, others reported no significant association between the AHSG polymorphisms and its levels and coronary artery calcification [12,17].Analyzing the calcium score of cardiac arteries of CAD patients, the CACS of LMCA was significantly lower in carriers of the rs4918 allele compared to individuals without this allele. In contrast, an insignificant higher CACS was observed in the presence of this SNP in LAD artery compared with the mean CACS in its absence. To explain these findings, differences in ethnicity and small sample size should be taken into account.The protein-coding rs4918 allele, also known as Thr256Ser, is situated in the coding region of the AHSG gene in exon 7 that encodes Thr256Ser amino acid substitution [18]. This protein-coding polymorphism determines the required amino acids for protein synthesis. It should be noted that it is the regulatory or promoter region of a DNA that determines the expression of mRNA and the quantity of translated protein, and the coding region determines the properties and structure of a protein and not its concentration.However, according to the obtained reports either the structure of the protein, concentration or both can be affected by this allele especially in ESRD and dialysis patients. The transcription of AHSG gene is mediated by C/EBP and NF-1 transcription factors [19] and pro-inflammatory cytokines cause loss of transcriptional domain of C/EBP in acute inflammation, which ultimately leads to reduced serum fetuin-A concentration [20]. Furthermore, the expression of the AHSG gene can be mediated by cytokine stimulation. In vitro studies showed that TNFα [21], IL-1, and IL-6 [22] down-regulate its mRNA level in the rat and human hepatoma cell line, respectively. Chronic inflammation has been well-documented in the setting of end-stage renal disease. Low levels of fetuin-A have been reported to be associated with rs4918 SNP, increased mortality, and vascular calcification [11]. Therefore inflammation may influence the expression of mRNA and subsequently the protein level, and the mentioned mechanism may be most outstanding in the presence of this SNP, which can be an explanation for that observed in ESRD and dialysis patients.As we did not measure the fetuin-A serum level and inflammatory proteins, the judgment is difficult and we are not sure that whether patients with rs4918 polymorphism necessarily had a lower fetuin-A serum level. The rs4918 SNP may not affect protein levels, structure, or function, and no association was observed between this SNP and the incidence of CAD and CAC in our results.Another possible explanation for the observed inconsistency in the role of rs4918 can be attributable to the post-translational modifications and different roles of fetuin-A in CAD and CAC pathology. This protein behaves differently in the presence of various risk factors, so with a similar genotype and whatever the phenotype might be, different biological responses may be observed.It is considered as an atherogenic protein as it inhibits the insulin receptor, which leads to insulin resistance, diabetes, metabolic syndrome, key promoters of atherosclerosis, and CAC. On the other hand, it inhibits vascular calcification by enhancing the calcium phosphate blood solubility and preventing apoptosis and VSMCs differentiation. Furthermore, it has anti-inflammatory activity by preventing the production and release of TNFα from activated macrophages [7], which has a central role in the occurrence of CAD. Hence it can be considered a protective protein. Thereby, its contribution to the pathogenesis of CAD may be dependent on the underlying pathologies and risk factors involved. Consistent with this explanation, in ischemic heart disease, high fetuin-A levels were associated with a lower risk of mortality only in patients with acute coronary syndrome where the exacerbation of inflammatory response is prevalent [23] and no association between baseline fetuin-A and CVD events was found in patients with stable angina [17]. Apart from its anti-inflammatory role and inhibition of calcification, fetuin-A inhibits insulin receptors. Jensen et al. reported that, while there was an inverse association between fetuin-A levels and the incidence of CVD among non-diabetics, an insignificant direct relation was observed in diabetic patients [24]. In another study, high concentrations of fetuin were directly correlated with the higher risk of CVD death only in older diabetic adults, whereas an inverse association was observed among non-diabetic individuals independent of CVD risk factors and renal function [25]. Consistent with these data, Chen et al. found that an inverse relation between fetuin-A and CVD, and all-cause mortality was limited to CAD non-diabetic patients only and disappeared in patients with diabetes mellitus [23].These findings suggest that the protective effect of fetuin-A could be masked by its insulin receptor inhibitory activity [24], or excessive fetuin-A levels may result in metabolic syndrome and insulin resistance [25], leading to a positive or no association between fetuin-A levels and CVD in diabetic patients [24].There are limitations in this study that should be considered. The number of subjects who participated in this project was limited, which reduces the statistical power. Second, because of practical and economical reasons, we did not measure the fetuin-A levels, which made it quite difficult to interpret the results and explain the observations. In addition, CRP and other inflammatory markers were not evaluated in this study. Inflammation is a major inducer of vascular calcification [26], and an inverse relationship between fetuin-A levels and inflammatory markers such as CRP in patients with CAD has been reported, which points to its anti-inflammatory activity and its role in attenuating atherosclerosis and calcification development [23]. However, it is not clear whether inflammatory factors have an impact on the transcription of a particular genotype and consequent fetuin quantity or not. Therefore, including these factors could give better insight into the activity of the rs4918 genotype specifically in situations where a flare of inflammatory response exists, such as acute coronary syndrome and MI. Finally, fetuin-A has a dual role in the pathophysiology of CVD and coronary calcification, and risk factors can influence its activity and quantity. However, in our study individuals in the control group were free of CVD and coronary risk factors, hence it would be beneficial to change the criteria for the selection of the control group and include individuals without CVD who share similar risk factors to the case group.In conclusion, there was no association between the rs4918 SNP and the coronary artery calcification score in patients with CAD, and its frequency was almost similar in healthy individuals and CAD patients, indicating that the carriers of this allele are not at increased risk of developing cardiovascular diseases as compared with those without.Z.A.: conducting experiments, data analysis, and manuscript preparation; M.M.: planning, conducting experiments, and manuscript preparation; S.N.: conducting experiments and manuscript preparation; M.A.: conduct, data analysis, and manuscript preparation; S.E.: conceptualization, design analysis, planning, data analysis, and manuscript preparation; A.H.M.: conceptualization, design analysis, planning, data analysis, and manuscript preparation. All authors have read and agreed to the published version of the manuscript.The authors are thankful for the funding of this study by the Research Council of Mashhad University of Medical Sciences.This study is part of a research thesis for a Ph.D. Degree at Mashhad University of Medical Sciences.The authors declare no conflict of interest.Comparison between the calcium scores of the main coronary arteries in the presence and absence of rs4918 SNP. Independent samples t-test was used for the analysis. * indicates a significant difference in the mean calcium score. CX: circumflex; LAD: left anterior descending; LMCA: left main coronary artery; RCA: right coronary artery; SNP: single-nucleotide polymorphism.Characteristics of the study participants.CX: circumflex; LAD: left anterior descending; LMCA: left main coronary artery; RCA: right coronary artery; SNP: single-nucleotide polymorphism; Std: standard. Values are the mean calcium score of four main coronary arteries. Independent samples t-test was used for the analysis.Calcium scores of the main coronary arteries in the presence and absence of rs4918 SNP.p values in bold indicate statistically significant—that is, p ≤ 0.05. CACS indicates coronary artery calcium score; SNP, single-nucleotide polymorphism. The frequency of genotype was expressed as a percentage.Comparison of the frequency of the rs4918 SNP between different calcium groups.Chi-squared statistic was used for the analysis. p ≤ 0.05 indicates statistically significant difference.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/cardiogenetics/cardiogenetics-10-02-00008.txt

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+European Reference Network for Rare and Complex Diseases of the Heart (http://guardheart.ern-net.eu).“A new era in Cardiogenetics”. This was the title of my opening editorial when we started the fantastic journey of Cardiogenetics [1].Back in 2011, when new techniques like next generation sequencing were just emerging in the genomic panorama [2]. Exome sequencing, noncoding DNA, bioinfomatics, big data, micro-RNA, long-noncoding DNA, epigenetics, entered our daily vocabulary, highlighting the importance of forming a “cardiogenetics team” working side by side, bringing the lab to the patients’ bed, and vice-versa [2,3,4,5]. Inherited and rare cardiovascular disease became a peculiar and important entity in the panorama of cardiovascular disease. Thanks to the new therapeutics opportunities, an increasing number of cardiologists are converging their interest in the field [6,7].Here in 2020, Covid-19, a tsunami on the health systems worldwide, but also on the capacity of scientists to develop and give priority to new projects, unrelated to SARS-CoV2. Inherited and rare disease clinical services have changed their perspectives, favoring teleconsulting/telemedicine approaches [8].Can we imagine a new era “post Covid-19"? We must learn from mistakes and good things (misleading scientific and media information on one side, use of technologies on the other side), and build new global and regional priorities on this ground. We are all anxious to open our drawer to find that our mask and hand sanitizer are not there anymore, that our fight with the virus has been won, and our priorities as clinicians and scientists are again “Covid-19 free”.In this difficult and confusing time MDPI, an organization with more than two decades of experience in online, Open Access science publishing, and dedicated editorial office staff who have a science background, took over Cardiogenetics from PAGEPress, betting on the renewed interest of the genetics and the cardiology communities [9].I am happy share this venture with Prof. Lia Crotti and Prof. Perry Elliott, who represent recognized world-wide leaders in the field of cardiogenetics. Cardiogenetics publishes reviews, original and research articles, short and case reports.A strong Editorial Board of active clinicians and scientists will support this new experience. Rapid and high-level peer review, fast online publication, new special issue proposals, waived fees for 2020, and other promoting initiative will help to increase Cardiogenetics visibility and readers’ appetites.Ready to start. Join us on MDPI (https://www.mdpi.com/journal/cardiogenetics).Giuseppe Limongelli, MD, PhD, FESC. Dr. Limongelli graduated (1997) and completed his Cardiology Training (2001) from the Second University of Naples (SUN). In 2001, he attended the “Master in Nephrology” (SUN). In 2001, he won a four years PhD course in Cardiovascular and Thoracic Sciences and Associated Biotechnology (SUN).From 2002 to 2003, he was attached to St George’s Hospital Medical School (London, UK) as Research Fellow of The Genotyping Lab & Heart Failure Clinic. From 2003 to 2004, he was attached to The University College of London (London, UK) as Research Fellow of The Cobbold Lab (Middlesex Hospital) & Heart Failure Clinic (The Heart Hospital). During his stay in St George’s Hospital, he attended a 6 months “Bioinformatics Half Module” at the Department of Basic Sciences. In 2003, he started an “European Doctorate in Biotechnology” course (HeduBT, UK), with a project on “Clinical and Molecular Aspects of Cardiomyopathies”. He was appointed with the title of “European Doctor in Biotechnology” in 2007.He is currently an Associate professor of Cardiology (since november 2017) at the Department of Translational Science, Università della Campania “Luigi VanvitellI”-Monaldi Hospital (AORN Colli), and Honorary Senior Lecturer (since May 2017) at the the Institute of Cardiovascular Sciences, University College of London, London, UK. His main research interests are clinical and genetic mechanisms of cardiomyopathies and heart failure, congenital, inherited, and rare disease, and athlete’s heart. Lia Crotti, MD, PhD, FESC. Lia Crotti is associate professor of Cardiology at the University Milano Bicocca in Milan and she is the head of the Cardiomyopathy Unit in the IRCCS Istituto Auxologico Italiano in Milan, Italy. Furthermore, in the same Institute she also has a leading role in the Center for Cardiac Arrhythmias of Genetic Origin and Laboratory of Cardiovascular Genetics.Lia Crotti is an internationally renowned expert in channelopathies and cardiomyopathies and her research interests are mainly focused on the genetic basis of sudden cardiac death, in genetically transmitted arrhythmogenic diseases. She identified a number of genetic modifiers of the clinical severity of the Long QT Syndrome. She discovered through whole exome sequencing that calmodulin genes were novel genetic substrate for severe form of Long QT Syndrome and through the creation of an international Calmodulinopathy Registry she better characterized this new clinical entity. She also identified CDH2 as a new gene responsible for Arrhythmogenic Cardiomyopathy and recently characterized some specific features. Furthermore, her research is focused on the improvement of the clinical management and treatment of patients affected by arrhythmogenic diseases and through the use of cardiomyocyte-derived IPs cells new drugs identified, are now under clinical evaluation. She regularly reviews manuscripts for all leading journals in cardiology and she is author or co-author of 146 peer reviewed papers and 17 book chapters. Her total Impact factor is 1151, with a total H-index of 41. She is vice-chair of the European Cardiac Arrhythmia Genetics (ECGen) focus group within EHRA. Besides her home Country (Italy) she has been spending research periods in the USA, South Africa, and Germany. Perry Elliott, MBBS, MD, FRCP, FESC, FACC. Perry Elliott (H-index 105) is Professor of Cardiovascular Medicine at University College London (UCL) and a Senior Investigator of the UK National Institute for Health Research (NIHR). He is director of the UCL Centre for Heart Muscle Disease, Head of Clinical Research at the Institute of Cardiovascular Science UCL and a consultant cardiologist in the Centre for Inherited Cardiovascular Disease at the Bart’s Heart Centre, St. Bartholomew’s Hospital London, UK. He is Chairman of the ESC Heart Academy and the ESC Council on Cardiovascular Genomics, past Chairman of the ESC Working Group on Myocardial and Pericardial Diseases (2010–2012) and the Executive Committee for the European Outcomes Research Programme registry on cardiomyopathies, chair of the ESC Guideline Task Force on Hypertrophic Cardiomyopathy, member of the Congress Programme Committee 2018–2020 and a member of the ESC Managerial Council. He is President of Cardiomyopathy UK, Europe’s foremost charity for patients with heart muscle disease. He is past Deputy Editor of The Heart Journal and the International Journal of Cardiology. He is currently an executive Editor for the European Heart Journal. Over the past 25 years, Prof. Elliott has established an international reputation in the field of heart muscle disease, authoring more than 500 peer-reviewed papers on the subject. He develops diagnostic standards, risk stratification tools and clinical service models based on some of Europe’s largest inherited heart disease cohorts, fostering industry collaborations in sequence interpretation, therapeutic trials and multicentre research partnerships.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+These authors contributed equally.We describe a 55 year old male diagnosed with cardiomyopathy due to Fabry disease. Biochemical testing of blood spot and plasma showed low-normal alpha-galactosidase A (α-Gal A) levels. Genetic testing revealed somatic mosaicism for GLA c.901C>T, p.(Arg301Ter). Usually, males with Fabry disease due to loss of function variants in GLA show symptoms of the multisystemic features of the condition early in life, and have very low levels of the α-Gal A enzyme. This demonstrates that the diagnosis of Fabry disease in males with cardiomyopathy should still be considered even in the context of a normal plasma enzyme assay.Fabry disease is a rare X-linked lysosomal storage disorder (MIM 301500). It is an inborn error of glycosphingolipid metabolism, resulting from a deficiency or total absence of the alpha-galactosidase A (α-Gal A) enzyme [1]. This enzyme deficiency results in the accumulation of globotriaosylceramide (Gb3) in a number of cell types, leading to complex, progressive, multisystem disease. In affected males, the usual presentation is of episodic crises in childhood of severe pain in the extremities (acroparesthesia), angiokeratomas (vascular cutaneous lesions), sweating abnormalities, and characteristic corneal and lenticular opacities [2,3]. Gradual deterioration of renal function to end-stage renal disease (ESRD) usually occurs in the third to fifth decade [2]. Cardiac involvement in Fabry disease is common, and typically cardiac hypertrophy develops, often with a concentric non-obstructive pattern mimicking hypertrophic cardiomyopathy (HCM) due to variants in genes encoding components of the sarcomere [4]. The precise mechanism resulting in the degree of cardiac hypertrophy is unknown. Estimates of incidence range from as high as 1 in 40,000 to 1 in 117,000 live male births [5,6].Males with Fabry disease are hemizygous for a loss of function GLA allele [7]. Females are heterozygous for a loss of function allele and are cellular mosaics due to random X-chromosome inactivation. This mosaicism results in a wide spectrum of phenotype with a later age at the onset of symptoms, and frequently milder disease [8]. Often manifesting carrier females present with cardiac involvement [8].Males with greater than 1% α-Gal A activity may have different clinical presentations with the development of left ventricular hypertrophy and arrhythmia later in life [2,3].The case presented here is of a middle aged male presenting with arm pain, which triggered an electrocardiogram (ECG) that ultimately revealed co-incidental cardiac hypertrophy and normal α-Gal A enzyme levels. Genetic studies revealed a mosaic pathogenic variant in GLA consistent with Fabry disease. The mosaicism explains the late onset milder disease presentation and low to normal enzyme activity in blood spot and plasma, and this suggests that α-Gal A activity in the normal range does not exclude a diagnosis of Fabry disease.A 55-year-old (II-3), previously a fit, healthy, and normotensive male, presented to the emergency department with an episode of sudden onset left arm pain. He was an active smoker, but had no other past medical history or cardiac risk factors. There was no family history of cardiac disease (Figure 1). On arrival to the emergency department, his ECG demonstrated sinus rhythm, normal axis, preterminal negative T waves in I, avL, V4-V6, RS conversion in V3/V4 and ST-segment depression in V5 and V6 reflecting anterolateral repolarisation abnormalities (Figure 2). There was no abnormality of conduction and no dynamic changes on serial ECGs (Figure 2). Serial cardiac troponin levels were borderline elevated at 0.13 ng/mL and 0.14 ng/mL (normal < 0.03 ng/mL) and so an echocardiogram was performed.The echocardiogram demonstrated overall moderate, concentric left ventricular hypertrophy (LVH) with septum predisposition. The interventricular septal diameter was 2.2 cm (normal ≤ 1.2 cm, severe hypertrophy ≥ 2.0 cm), and the posterior wall diameter was 1.5 cm (normal ≤ 1.2 cm, mild hypertrophy 1.3–1.5 cm). Left ventricular mass was calculated at 315 g (normal range 96–200 g, severely elevated ≥ 254 g) and mass index at 158.4 g/m2 (normal range 50–102 g/m2, severely elevated ≥ 130 g/m2). The right ventricle had normal size and function and was reported as showing no clear evidence of right ventricular hypertrophy (RVH). Diastolic function was impaired, but left ventricular size and systolic function were within normal limits. There was no evidence of left ventricular outflow tract obstruction at rest, and no evidence of systolic anterior motion of the mitral valve.In view of a mild troponin rise, he underwent a coronary angiogram, via the right radial approach demonstrating smooth coronary arteries in a conventional distribution. The provisional diagnosis in the absence of a past medical history of hypertension was hypertrophic cardiomyopathy (HCM) pending further investigation.A cardiac MRI scan was performed on a 1.5 Tesla MAGNETOM Avanto Siemens (Erlangen, Germany) MRI scanner using 0.1 mmol/kg gadobutrol (Gadovist, Bayer) as a contrast agent to facilitate late enhancement imaging in order to determine the nature of the myocardial injury responsible for the small troponin leak. In addition to standard volumetric analysis and late gadolinium enhancement imaging, native, and post-contrast T1 mapping was performed (Figure 3). This demonstrated bi-ventricular hypertrophy with concentric LVH and a maximal left ventricular wall thickness diameter of 2.3 cm. There was evidence of RVH with a maximal right ventricular free wall diameter of 7.5 mm. Late gadolinium enhancement imaging demonstrated patchy hyperenhancement in the basal/mid-lateral and inferior and inferolateral wall segments as well as the RV insertion point and apex. Volumetric analysis indicated a normal 4-chamber size and normal aortic dimensions. Biventricular function was normal; left ventricular ejection fraction was 72% (normal 58–76%). Native T1 mapping highlighted markedly reduced T1 levels in the inferoseptum (basal segment 880 ms, midsegment 870 ms, which was against local reference values of 950–1030 ms). The triad of concentric hypertrophy, typical pattern of late gadolinium enhancement, and low native septal T1 is characteristic of advanced Fabry disease cardiomyopathy [4].Following an initial phenotype based diagnosis of HCM, further evaluation of underlying aetiology included enzyme assays for Fabry disease, all of which were within normal limits. The patient had normal dried blood spot α-Gal A enzyme activity of 7.84 picomol/punch/h (range 6.3–47). His plasma α-Gal A level was 3.0 micromol/L/h (range 3.0–20), not indicative of a diagnosis of Fabry disease.He provided written consent (ref 11/H10003/3) to undergo genetic testing by next generation sequencing (NGS) of a panel of 21 genes associated with hypertrophic cardiomyopathy (Supplemental Table S1). Enrichment was performed with a custom designed SureSelect target enrichment kit (Agilent) following the manufacturer’s protocols. The target enrichment design consists of the coding region of transcripts, including the immediate splice sites (+/−5 bases) for genes associated with inherited cardiac disorders. The samples were sequenced using a NextSeq 500 (Illumina), according to the manufacturer’s protocols. Sequence data were aligned to hg19 human genome using BWA-AIn vO.6.2 and BWA-Sampe vO.6.2. For genes associated with HCM, variant calling was completed using GenomeAnalysisToolKitUte-v2.0.39 (GATK) (SNVs and indels), Pindel v0.2.4.t (large indels), and DeCON v1.0.1 to detect copy number variation (CNV) (in-house bioinformatics pipeline version 1.15.1). Analytical validation of variants detected by NGS was confirmed by Sanger sequencing. Variant classification was performed according to ACGS guidelines [9]. Here, 99.9% of the target coding region of the genes was covered to a minimum read depth of 50×.NGS testing identified that he had evidence of both a wild type (25% of reads) and the variant c.901C>T, p.(Arg301Ter) allele (75%) in GLA (Table 1, Figure 4). This variant is predicted to result in a premature termination of translation of GLA at a position expected to result in nonsense mediated decay. The variant has been reported multiple times to be associated with Fabry disease [10,11,12,13,14,15]. A multiplex quantitative fluorescence-PCR (QF-PCR) assay was used to exclude an X chromosome aneuploidy. This assay uses primer sets to target a mixture of polymorphic and nonpolymorphic microsatellite markers [16]. The primers target six X-specific markers, two Y-specific markers, an Xp22 marker located within the pseudoautosomal region (PAR2) of the X and Y chromosomes that indicated the number of either sex chromosome, a marker within the AMELX at Xp22.2 (105 bp) and Yp11.2 (110 bp) indicating the ratio of X to Y chromosomes, and a TAF9 marker at Xq21.1 and 3p24.2 indicating the ratio of X chromosomes to autosomes. Analysis was undertaken according to the ACGS best practice guidelines [17].A subsequent genetic analysis was undertaken on samples of DNA extracted from blood, urine, and saliva to examine tissues of different embryonic origin to determine whether somatic mosaicism was present or whether the mosaicism was confined to blood. The analysis by an NGS and Sanger sequencing of GLA (Table 1) determined that the wild type (cytosine, C) allele was present at a minor allele frequency of between 25 and 41% in the samples of a different origin. Lower peak heights in the Sanger sequencing traces for the C nucleotide at position 901 compared to 900 of GLA in the patient samples, and a lower height in position 901 between the control and patient samples were consistent with mosaicism (Table 2).To determine more fully the biochemical effects of the genotype, the biochemical testing was repeated. Plasma α-Gal A level was below the normal range at 1.4 micromol/L/h, as was the leukocyte level at 1.4 micromol/L/h (range 10–50). Urine globotriaosylceramide (Gb3) 0.86 mg/mmol creatinine (normal 0–0.03) and plasma globotriaosylsphingosine (lyso-Gb3) 20.7 ng/mL (normal 0–1.8) were measured. Both were elevated, consistent with a diagnosis of Fabry disease.A reverse phenotyping of the proband established that he had experienced occasional episodes of non-limiting acroparaesthesia in his left hand. These had been attributed to his occupation as a truck driver. There was no evidence of angiokeratoma. His renal function revealed CKD stage 2 with a glomerular filtration rate (GFR) of 86 mL/min/1.73 m2, a normal urine albumin creatinine ratio (urine albumin < 3.0 mg/L, urine creatinine 5.4 mmol/L, ratio < 0.56 g/mol normal reference range 0.00–2.5 g/mol) and an elevated baseline troponin of 338 ng/L (normal reference range < 40 ng/L). Cerebral MRI demonstrated widespread deep white matter hyperdensities but no infarction or space occupying lesion. Ophthalmological and audiological assessments were unremarkable.In the presence of the now-established Fabry cardiomyopathy, a decision was made to treat with enzyme replacement therapy (ERT). Chaperone therapy was not an option as the GLA c.901C>T, p.(Arg301Ter) variant is not amenable to this approach [18]. ERT was well tolerated, and no infusion related complications were reported over the next 18 months. At the six month follow-up, multiple clinical parameters showed improvement (Table 3). Lyso-Gb3 levels had declined from 20.7 to 6.2 ng/mL, and the estimated GFR had decreased from 86 to 68 mL/min/1.73 m2; urine albumin creatinine ratio was within normal limits at 0.31 g/mol (urine albumin 4.0 mg/L, urine creatinine 13 mmol/L, ratio 0.31 g/mol, normal reference range 0.00–2.5 g/mol). Consequently a low dose ACE-inhibitor was commenced to manage early renal complications of Fabry disease. A repeat echocardiogram at 18 months demonstrated a reduction in left ventricular hypertrophy. The interventricular septal diameter had reduced in thickness from 2.2 to 1.9 cm, while the posterior wall diameter had reduced from 1.5 to 1.4 cm (normal ≤ 1.2 cm, mild hypertrophy 1.3–1.5 cm). Left ventricular mass reduced from 315 to 293 g (normal range 96–200 g, severely elevated 254 g) and mass index had reduced from 158.4 to 146.8 g/m2 (normal range 50–102 g/m2, severely elevated ≥ 130 g/m2). As seen previously, the left ventricular size and ejection fraction was within normal limits, but the left atrium appeared mildly dilated, and right ventricular hypertrophy was noted on visual assessment. Furthermore, a repeat ECG was performed demonstrating new first degree heart block (PR interval increased from 179 to 213 ms), and a subsequent 24-h tape demonstrated an isolated salvo of four ventricular ectopics as well as infrequent ventricular and supra ventricular ectopics. The clinical decision was therefore made to implant a loop recorder for further monitoring. ERT was continued and continues to be well tolerated.Here, we describe the diagnosis of Fabry disease due to a somatic pathogenic variant in GLA in a man with low, but normal levels of alpha-galactosidase. The diagnosis in the affected individual is vital, both in terms of effective treatment and determining the level of risk to his close relatives. He commenced ERT following the genetic diagnosis which would not have been available to him based on his enzyme assay results, with improvement to his cardiac function 18 months since commencing therapy. The accurate diagnosis facilitated renal, ophthalmological and skin screening, and early stage asymptomatic renal disease has been discovered and treated.The GLA c.901C>T pathogenic variant has been reported previously [10,11,12,13,14,15] and on a number of occasions in a public sequence variant database [19]. Where clinical information has been provided, individuals hemizygous for this variant have been affected with multisystem features of Fabry disease, including joint pain and kidney disease [11], skin, ophthalmological, renal, neuropathic and cardiac involvement [12], and a predominantly renal disease presentation with cardiac involvement [14]. One male with this variant who presented with renal disease had symptomatic heterozygote female relatives [15]. His mother experienced acroparesthesia and neuropathic pain in her thirties, with angiokeratoma, and cornea verticillata and significant left ventricular hypertrophy, without renal involvement. His sister had more extensive features with skin, ophthalmological, renal and neurological evidence of disease and marked left ventricular hypertrophy [15]. This series of clinical reports illustrates the broad phenotypic variability expressed in both male and females with the GLA c.901C>T variant. There is no evidence to indicate that this variant results in a predominantly or exclusively cardiac phenotype as has been shown for some GLA variants. The predominant cardiac presentation in the proband is more similar to the females reported to be symptomatic heterozygotes for this variant [15] and parallels the cellular mosaicism present in him that is present in heterozygote females.Somatic mosaicism for a known pathogenic loss of function GLA variant was reported previously in a 58 year old male who presented with features consistent with hypertrophic cardiomyopathy, proteinuria and mild renal impairment [20]. The similarity in the presentation and age of onset in this individual highlights that somatic mosaicism for pathogenic GLA variants may preferentially present with hypertrophic cardiomyopathy. In this mosaic individual, the alpha-galactosidase level in blood was below the normal range at 0.7 mmol/L/h (2.0–11.7) [20].The diagnosis of Fabry disease in the proband excludes any increased risk for his son and the need for clinical or genetic testing in him. It is important to note that if he had a daughter she would have been at increased—but not at obligate—risk of inheriting the variant allele as he is mosaic for this. The absence of a family history of Fabry disease is consistent with the identification of mosaicism, a de novo post zygotic event, in the proband. We excluded the proband being a chimera (a single organism composed of two or more different populations of genetically distinct cells that originated in different zygotes) compared to being a mosaic (a mixture of two cell lines in one organism originating in one zygote), as there was no evidence of biallelic variants of LAMP2 and FHL1, the other X-linked genes on the gene panel.We considered the possibility of revertant mosaicism, i.e., that the proband inherited the pathogenic variant from his mother, but that the variant allele was replaced by the wild type allele in a proportion of cells following fertilization [21]. To date, revertant mosaicism has not been reported in Fabry disease. Despite no history of heart problems or clinical features consistent with Fabry disease in the mother, we undertook genetic testing in one of the proband’s female siblings (II-2), which was a wild type for the variant. An alternative explanation for the identification of two GLA alleles in a male resulting in a milder clinical phenotype would be sex chromosome aneuploidy, but this was excluded by QF-PCR.Generally, the preferred approach for diagnosis of Fabry disease in males if there is no previous family history, is enzyme and biomarker analysis followed by DNA sequencing of GLA if high clinical suspicion remains. However, if there is a known familial genotype, then a DNA analysis of GLA is recommended as the first line test. Enzyme level analysis is not the preferred approach to diagnose Fabry disease in females, as the levels can often be normal despite obligate affected status. This may be due to variable X-inactivation in some tissues. Our proband exhibits a phenotype more in keeping with a heterozygote female where single organ disease is more common, compared to male hemizygotes who exhibit younger age of symptom onset and a broader spectrum of organ system involvement [2,8].We recommend that males with cardiomyopathy, especially with imaging changes characteristic of Fabry disease, should not have the diagnosis excluded entirely on the basis of a normal enzyme assay, but should consider more detailed biochemical testing, including lyso-Gb3 or genetic testing to determine if mosaicism is present. Such diagnoses will have a profound impact for the affected individuals and their family members.The following are available online at https://www.mdpi.com/2035-8148/11/1/1/s1, Table S1: Genes associated with HCM screened by NGS, Video S1: Cardiac MRI functional assessment using Steady State Free Precession (SSFP) cine in the three chamber view. Video S2: Cardiac MRI functional assessment using Steady State Free Precession (SSFP) cine in the horizontal long axis view, Video S3: Cardiac MRI functional assessment using Steady State Free Precession (SSFP) cine in left ventricular mid-ventricular short axis view.Conceptualization, W.G.N.; methodology, M.X., C.O., J.E., A.W., H.J.C., K.T., M.S., and W.G.N.; formal analysis, J.E., A.W., H.J.C., and K.T.; investigation, S.D., C.C., P.W., C.M., M.S., and A.J.; data curation M.X., C.O., J.E., A.W., H.J.C., K.T., M.S., A.J., and W.G.N.; writing—original draft preparation, M.X., C.O., and W.G.N.; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the South Manchester Ethics Committee (11/H10003/3, approved 2011).Informed consent was obtained from all subjects involved in the study.The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical restrictions.We thank the patient for his agreement to participate. W.G.N. is supported by the Manchester NIHR BRC (IS-BRC-1215-20007).The authors declare no conflict of interest.Family pedigree.A 12-lead electrocardiogram demonstrating sinus rhythm, normal axis, preterminal negative T waves in I, avL, V4-V6, RS conversion in V3/V4 and ST-segment depression in V5 and V6, reflecting anterolateral repolarisation abnormalities.Panel of cardiac MRI images. Image (a), vertical long axis view of the left ventricle acquired using a balanced steady-state free procession sequence in end-diastole demonstrating significant left ventricular hypertrophy. Image (b), short axis view acquired in the late phase following Gadolinium administration using a phase sensitive inversion recovery imaging acquisition. The arrow demonstrates significant non-ischaemic enhancement and fibrosis of the basal lateral and basal inferolateral wall segments. Image (c), three chamber view acquired using a balanced steady-state free procession imaging sequence in end-diastole demonstrating significant left ventricular hypertrophy. Image (d), native T1 map acquired in the basal short axis using a modified Look-Locker Imaging (MOLLI) sequence. Arrow demonstrates an area of elevated native T1 values in the region of the basal inferolateral wall segments (1019 ms) compared to the low T1 native valves in the basal inferoseptum (880 ms).(a). NGS reads demonstrating biallelic presence of the variant (reverse strand read, i.e., G > A). (b). Sanger sequence trace with GLA c.901C>T variant (arrow).Assessment of levels of C (wild type) allele at GLA position 901 by NGS assay and Sanger sequencing in DNA derived from blood, saliva, and urine.Analysis of peak heights for the cytosine (C) nucleotide at positions 900 and 901 demonstrating the lower height of the wild type nucleotide at position 901, consistent with mosaicism.Peak heights at position c.900 are comparable between the proband (II-3) and a normal control. Peak heights are expressed in relative fluorescent units as reported by Sequencing Analysis Software (Applied Biosystems).Assessment of clinical parameters before and after a six month course of enzyme replacement therapy (ERT).Note: IVSd = interventricular septal dimension; LVPWd = left ventricular posterior wall dimension.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Heart involvement in Cardiac Amyloidosis (CA) results in a worsening of the prognosis in almost all patients with both light-chain (AL) and transthyretin amyloidosis (ATTR). The mainstream CA is a restrictive cardiomyopathy with hypertrophic phenotype at cardiac imaging that clinically leads to heart failure with preserved ejection fraction (HFpEF). An early diagnosis is essential to reduce cardiac damage and to improve the prognosis. Many therapies are available, but most of them have late benefits to cardiac function; for this reason, novel therapies are going to come soon.Amyloidosis is a rare condition caused by an abnormal extracellular deposition of a misfolded protein, called amyloid, potentially affecting any organs and tissues.Among the different types of this clinical disorder, more than 95% of all cardiac amyloidosis (CA) are associated with immunoglobulin light-chain amyloidosis (AL) and transthyretin amyloidosis (ATTR) [1].AL, previously known as primary amyloidosis, is related to the misfolding of antibody light chain fragments produced by a plasma-cell clone. The estimated incidence is 3–12 cases per million persons per year, with a prevalence of 30,000 to 45,000 affected in the United States and European Union [2].ATTR is related to the abnormal production of a type of amyloid called transthyretin (TTR, previously called prealbumin), which is primarily made by the liver, and it can be either acquired or congenital. The acquired wild-type variant (ATTRwt) typically affects older males, and is therefore named “senile amyloidosis”. The mutant variant (ATTRm or Hattr—hereditary) derives from mutations in the TTR gene and it may present mostly with cardiac symptoms, neurological symptoms, or both.Chronic amyloid fibril deposition in myocardium leads to the development of hypertrophic cardiomyopathy and diastolic dysfunction. Dyspnea on exertion and atrial fibrillation with cardioembolic events are the most frequent onset symptoms. Low extremity edema and ascites may also occur in patients with prevalent right-heart involvement [3]. Other findings are bundle branch block and complete atrioventricular block. A deep evaluation of clinical symptoms, cardiac and systemic involvement (biomarkers and cardiac imaging, serum, urine testing, biopsy) can help to diagnose amyloidosis and to differentiate it into AL or ATTR.Treatment of CA aims to improve symptoms and limit further production of amyloid protein. Specific treatments vary depending on the type of amyloidosis (see Figure 1 and Figure 2) [4].Conventional therapy of heart failure, such as ACEi/ARB, diuretics and beta-blockers, may be poorly tolerated [5,6]. Poor ventricular compliance implicates higher filling pressure to reach a pre-load fitting. Diuretic therapy needs attention because of the impairment of preload, which can reduce systolic output and cause cerebral hypoperfusion. Furthermore, mostly in cases of restrictive pathophysiology, cardiac output is strictly related to heart rate. In addition to beta-blockers, digoxin is an option, but it should be used with caution, because of the binding to the amyloid fibrils and the consequent rise in their levels [7,8,9].Progressive deterioration of ejection fraction and heart failure, but also bradyarrhythmia and electrical mechanical dissociation, can increase sudden cardiac death (SCD) risk [10]. A prophylactic placement of an implantable cardioverter-defibrillator (ICD) should be considered when syncope and complex non-sustained ventricular arrhythmias occur [11].Subcutaneous devices (s-ICD) can reduce the defibrillation threshold and result in successful defibrillation [12]. More trials are needed to better understand and recognize predictors of arrhythmia associated with SCD in CA.Early insertion of a pacemaker may help to treat symptomatic bradyarrhythmias. In familial amyloidosis, selected electrophysiological criteria have been published to guide pacemaker placement: His-ventricular interval ≥ 70 ms, His-ventricular interval > 55 ms (if associated to fascicular block), Wenckebach anterograde point ≤ 100 beats/min. In fact, in these conditions, there is a high-degree atrioventricular block risk [13].Without treatment, AL amyloidosis has a progressive course due to uncontrolled organ damage. Thus, patients with systemic disease should immediately initiate therapy, and there are now multiple novel therapeutics approved or under development [14,15,16].For primary amyloidosis, treatments include the same agents used to treat multiple myeloma (MM), with adaptations for dosages and schedule [17]. In patients with good cardiac, liver and renal function, the first-line therapy is usually a combination of high-dose melphalan (HDM) and autologous stem cell transplantation (ASCT). An induction therapy before HDM/ASCT can be assessed with Cyclophosphamide, Vincristine and Dacarbazine (CVD) combination treatment.If ASCT is not a suitable option for the patient, an oral Melphalan and Dexamethasone (MDex)-based regimen is considered an effective and well-tolerated therapeutic scheme [18].A milestone for the treatment has been represented by the introduction of proteasome inhibitors. Bortezomib is a reversible inhibitor of the 26S proteasome involved in the degradation of ubiquitous proteins. It can rapidly reduce misfolded light chains’ concentration and it represents a standard of care in non-transplant-eligible patients [19].Ixazomib is a second-generation oral proteasome inhibitor authorized for the treatment of MM combined with Lenalidomide and Desametasone. Several randomized studies are currently ongoing, comparing Ixazomib with standard of care.Effectiveness and safety of Carfilzomib has been evaluated in a phase I/II study, in patients with relapsed/refractory AL amyloidosis, showing an acceptable response rate, despite an increased cardiopulmonary toxicity [20].A good clinical efficacy has been shown for relapsed or refractory disease by Thalidomide, Lenalidomide and Pomalidomide, even if their mechanism of action is not completely understood [21] and the toxicity profile is not insignificant. Thalidomide is more effective when used in association, but it causes neurological toxicity and it is teratogenic. Lenalidomide is more tolerated, although it needs renal function monitoring. Pomalidomide is a novel immunomodulating drug for the AL amyloidosis and is now under study in a phase 2 trial.Increasing research into new therapeutic strategies is essential, since most patients do not reach a complete hematological response to the standard therapy.Alkylating agents causing DNA damages, such as Bendamustine, are used for the treatment of MM and Waldenström macroglobulinemia [22]. A survival benefit and an efficient hematological response have been proven in a recent study evaluating Bendamustine and Prednisone association [23]. The safety and efficacy of Bendamustine and dexamethasone are under evaluation in patients with relapsed AL amyloidosis and advanced disease stages.New therapies for relapsed/refractory MM in the advanced cardiac AL amyloidosis include Daratumumab, a human IgG1k monoclonal antibody targeting the CD38 antigen on plasma cells (also expressed in AL amyloidosis) [24,25]. Venetoclax is a BH3-mimetic used for chronic lymphocytic leukemia (CLL), and has a proven role in patients with translocation t(11;14), most commonly associated with AL amyloidosis.New therapeutic strategies targeting light-chain (LC) aggregation aims to prevent their cardiotoxic effects and prevent the further formation of amyloid fibrils.Doxycycline seems to play a role on both fronts [26]. In a phase II open label trial (DUAL trial), oral doxycycline is administered together with plasma cell-directed drugs [27]. Bortezomib, Cyclophosphamide and Dexamethasone are being evaluated in association with Doxycycline in a randomized phase II/III trial [28].Epigallocatechin-3-gallate (EGCG), a polyphenol able to convert amyloid fibers into unstructured oligomers, is proven to prevent amyloid formation in vitro [29]. Extracellular LC deposition causes contractile dysfunction by promoting apoptosis through a mitogen-activated protein kinase (MAPK) pathway. In this regard, SB203580, a selective p38 MAPK inhibitor, has been shown to reduce oxidative stress and apoptosis induced by extracellular LC in AL amyloidosis-cultured cardiomyocytes [30]. Moreover, LC-mediated cardiotoxicity seems to be sustained by a dysregulation of the autophagic pathway, which could be restored by Rapamycin (Sirolimus), but the modulation of this pathway could not be considered for new therapies to date.Systemic amyloid deposits are typically not surrounded by an inflammatory reaction [31], and their rapid elimination is necessary to restore vital organ function. Therefore, new therapeutic strategies have the aim to trigger an immune response against amyloid fibrils. Thus, the potential use and efficacy of a serum amyloid P component (SAP) binding molecule, R-(1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-exanoyl]-pyrroli-dine-2-carboxylicacid (CPHPC) [32] will be clarified in further studies.Passive immunotherapy is an additional promising pharmaceutical strategy using specific antibodies targeting the misfolded LC proteins.A humanized murine monoclonal antibody, NEOD001, has been shown to react with LC aggregates, thus increasing AL amyloid clearance in a murine model [33], but two phase II/III trials have been interrupted due to negative results.Many new therapies for TTR amyloidosis have been developed in recent years, in addition to conventional supportive therapy.Liver transplantation (LTx) was the conventional first-line therapy for patients with mutant ATTR [34] since 1990. Data contained in The Familial Amyloidotic Polyneuropathy World Transplant Registry (FAPWTR) show an excellent long-term survival in well-selected patients, underlying that an optimal nutritional status, an early onset and a short duration of disease at the time of LTx are independent factors for survival. Furthermore, non-TTR Val30Met patients have a worse outcome compared to TTR Val30Met patients (Figure 3), as demonstrated by the continuing production of fibrils derived from wild- type TTR after LTx in some autopsy studies [35].Heart transplantation can be feasible in patients with ATTR-amyloidosis and heart failure earlier than LTx, because the slowness of cardiac amyloid production and deposition. Combined heart and liver transplantation in younger patients has better prognosis than cardiac transplantation alone, probably because cardiac graft does not contain preformed amyloid deposits (nidus) to facilitate the addition of wild-type ATTR.LTx has a positive impact on patient prognosis, but it is reserved to a restricted range of patients because of the procedural limitations and its uncertain benefits, except for Val30Met mutation patients [36].Patisiran (ONPATTRO™) is the first small-interfering RNA-based drug approved for the treatment of adults with ATTRv-related polyneuropathy both in USA and in European Union. It specifically acts on a genetically stored sequence in the untranslated 3′ region of the entire mutant and wild-type TTR mRNA. It is formulated in the form of lipid nanoparticles, administrated through intravenous infusion, and it causes the catalytic degradation of TTR mRNA in the liver, resulting in reduced serum levels of the TTR protein. The efficacy and tolerability of Patisiran have been evaluated in a randomized double-blind trial, the APOLLO trial, in which patients were randomized 2:1 to receive Patisiran or placebo via intravenous infusion once every 3 weeks for 18 months. Patisiran-treated patients have been shown a better outcome at month 18, as demonstrated by decreased mean left ventricular wall thickness, decreased N-terminal prohormone of brain natriuretic peptide (NT-proBNP) levels, and by promoting a reverse remodelling, improving clinical and cardiac manifestations of ATTR. In a phase II trial, Patisiran has shown an effective reduction in both mutant and wild-type TTR levels in patients with ATTR—familial polyneuropathy [37].Inotersen (TEGSEDI™) is an antisense oligonucleotide, approved by FDA and EMA for ATTRv-related polyneuropathy patients, which inhibits transthyretin production by binding the TTR mRNA. Its effectiveness has been assessed in a randomized, double-blind phase 3 trial, performed in 172 patients with stage 1 (ambulatory patients) or stage 2 (ambulatory patients with assistance) hereditary transthyretin amyloidosis with polyneuropathy. Inotersen was proven to reduce clinical manifestations of polyneuropathy and to improve the quality of life. Only 3% of patients underwent glomerulonephritis or thrombocytopenia as major adverse events, with one death associated with grade 4 thrombocytopenia [32]. Patients who completed a phase III trial were enrolled in NEURO-TTR trial in which patients with ATTRv and polyneuropathy were randomized to Inotersen (300 mg weekly) or placebo. The Inotersen group showed a good drug tolerance and a slower neurological decline during the follow-up [38].Targeted therapies have focused on small molecules that are able to stabilize TTR-tetramer, preventing TTR amyloid fibril formation.Tafamidis (VYNDAQEL®), an approved drug for the treatment of wild-type or hereditary ATTR in adult patients with cardiomyopathy and polyneuropathy, binds selectively to the tiroxine binding sites, stabilizing the tetramer and preventing the dissociation into monomers [39,40].Its effectiveness has been proven in a randomized double-blind study, the ATTR-ACT trial, in which 441 patients with ATTR-amyloid cardiomyopathy were randomized to receive Tafamidis or placebo for 30 months [41]. A reduction in all-cause mortality rate was observed in Tafamidis group compared to the placebo (78 of 264 (29.5%) vs. 76 of 177 (42.9%); hazard ratio 0.70, 95% confidence interval (CI) 0.51–0.96) and a lower rate of hospitalizations for cardiovascular events. Most benefits regarding mortality were seen after 18 months of follow-up treatment. The drug was well-tolerated and no differences in adverse events were reported between placebo and Tafamidis groups [26].New therapeutic approaches are aimed at targeting serum amyloid P (SAP), involved in amyloidosis pathogenesis (both AL and ATTR).Miridesap is a small molecule that causes a rapid depletion of circulating SAP via hepatic clearance. A decrease of more than 90% of SAP circulating levels, in addition to a depletion of SAP within amyloid deposits in histological samples, was observed in seven patients with systemic amyloidosis after Miridesap-based treatment [42,43]. The treatment with anti-SAP antibodies after Miridesap infusion was shown to improve liver function and deplete amyloid tissue deposits in a phase I trial including patients with systemic amyloidosis. However, cardiac disease was not an inclusion criterion. Another trial showed no significant improvement or cardiac-adverse effect after Miridesap in patients with cardiac damage [44].Anti-SAP antibody therapy is no longer evaluated. Considerable interest is focused on monoclonal antibodies directed towards the aggregates forms of ATTR to facilitate their uptake into macrophages. These antibodies show a rapid amyloid deposit removal and degradation of amyloid deposition with an improvement of the cardiac performance in a novel in vivo models of wtATTR [45].Future research is focused on combinations of drugs with different mechanisms of action for a synergy approach, to prevent amyloid deposition and to improve clearance of amyloid deposits.Many advances and novel opportunities in the treatment of CA have become available in recent years, and a disease with a poor prognosis has been revalued as a more manageable and possibly curable condition. The list of therapeutic options is rapidly expanding, with new options for CA targeting the multiple phases of amyloid cascade. Therefore, clinical competence and cardiologist’s awareness are crucial to get an early diagnosis and to obtain a better prognosis.Conceptualization, F.I. and A.E.; methodology, M.C.; software, M.C.; validation, M.C., A.E. and F.I.; formal analysis, M.D.M.; investigation, A.I.; resources, R.P.; data curation, M.D.M.; writing—original draft preparation, F.I.; writing—review and editing, M.G.M.; visualization, M.D.M.; supervision, A.E. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.Not applicable.The authors declare no conflict of interest.Targets for treatment in cardiac light-chain (AL) amyloidosis [4].Amyloid-specific pharmacotherapies.Patient survival between 1990 and 2010. Comparison between TTR Val30Met/non-TTR Val30Met mutations and early or late onset of disease.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Purpose: To evaluate the clinical features, laboratory and instrumental tests results and the effectiveness of complex treatment in a patient with multiple etiologies of dilated cardiomyopathy (DCM) with a high risk of sudden cardiac death. Methods: Female patient was 34 years old. Follow up period was seven years. Since the age of 23 (after a respiratory infection), chest pains and shortness of breath appeared. Coronary arteries were intact. After syncope in 2013, Holter-ECG was performed: 2048 premature ventricular beats (PVBs)/day and episode of sustained ventricular tachycardia (VT, 1 min) were registered. MRI was performed, and a cardioverter defibrillator (ICD) was implanted. Results: ECG showed low QRS voltage and negative T waves in leads V2-V6, III, aVF. In signal-averaged ECG, late potentials were detected. Echocardiography (EchoCG) demonstrated left and right ventricular dilatation, diffuse reduction of left ventricular (LV) contractility and multiple pseudochordae in LV. MRI showed LV noncompaction (LVNC), thickening of the epicardial fat and hypo-/dyskinesia of the anterior wall of the right ventricular (RV), dilatation of both ventricles with decrease of their ejection fraction and subepicardial gadolinium enhancement in the early and late phase in the LV, intraventricular septum and the free walls of the RV. The presence of LVNC was confirmed by cardiac computed tomography (CT). Late contrast enhancement in the middle and subendocardial layer of the LV was observed as well. The level of anticardiac antibodies was high (1:160–1:320). The reasons for statement of a possible diagnosis of myocarditis in this case were the connection of the onset of symptoms with viral infection, high titers of anticardiac antibodies, and early and late subepicardial contrast enhancement by MRI and CT. The endomyocardial biopsy was obtained, and subendocardial lipomatosis, separation of myocardium by fibrous septa, lymphocytic infiltrates (more than 14 cells/mm2) and vasculitis were found. Viral genome in myocardium was not detected. A new splicing mutation in the desmoplakin (DSP) gene was found (NM_004415.4: c.1141-2A>G/N (rs794728111)). Combination of arrhythmogenic right ventricular cardiomyopathy (ARVC), LVNC and myocarditis was diagnosed. Immunosuppressive therapy (prednisone and azathioprine) was prescribed, LV ejection fraction stabilized at the level of 40%. The appropriate shocks of the ICD due to sustainedVT (HR 210/min) with transformation into ventricular fibrillation were recorded twice. For this reason, sotalol was temporarily replaced with amiodarone. After the suppression of myocarditis activity, sustained VT and ICD interventions were not observed. Conclusions: In a young patient with arrhythmogenic syncope and DCM syndrome, a combination of ARVC (two major and three minor criteria, definite diagnosis) and LVNC with the biopsy proved virus-negative chronic myocarditis was diagnosed. DCM as a syndrome can have multiple causes, and the combination of myocarditis and primary cardiomyopathy is not rare. LVNC can be observed in patients with typical desmosomal protein mutations. The use of immunosuppressive therapy led to the stabilization of heart failure and decreased the risk of arrhythmic events.The problem of diagnostics and treatment of myocardial diseases in the practice of modern cardiology is of great importance. Even more of an actual issue, is the diagnosis of a combination of several myocardial diseases. This paper will discuss the combination of three nosological entities simultaneously: arrhythmogenic right ventricular cardiomyopathy (ARVC), left ventricular noncompaction (LVNC) and myocarditis.ARVC is an inherited myocardial disease characterized by right ventricle (RV) fibrofatty replacement, ventricular arrhythmias and a high risk of sudden cardiac death (SCD) [1]. ARVC was described in 1977 and, at that time, was considered to be a rare disease; however, later, as a result of improvement of diagnostic criteria [2], including the development of MRI criteria, information about its prevalence has changed. At present, the incidence of this cardiomyopathy varies depending on the population, from 1:1000 to 1:5000 [3,4].LVNC is characterized by intensively developed ventricular trabeculae combined with deep intertrabecular lacunas lined with endocardium, not connected with coronary blood flow. It is generally accepted that LVNC syndrome in adults was first described by Chin et al. in 1990 [5], but LVNC was included in cardiomyopathy classification only in 2008. [6]. The incidence of LVNC, according to different data, varies from 0.014% to 1.3% [7]. The diagnosis of this cardiomyopathy is based on three imaging techniques: echocardiography (EchoCG), cardiac magnetic resonance imaging (MRI) and cardiac multispiral computed tomography (CT). Diagnostic criteria have been developed for each of these methods [5,8,9,10].Myocarditis is defined as an inflammatory myocardial disease, diagnosed on the basis of established histological, immunological and immunohistochemical criteria [11]. It is difficult to estimate its prevalence in the population, since endomyocardial biopsy (EMB), the “gold” standard of in vivo diagnosis of myocarditis, is performed infrequently. The incidence of morphologically verified myocarditis among young patients who died suddenly reaches 42%, and among adults and children with dilated cardiomyopathy (DCM), myocarditis in EMB is detected in 16% and 46% of patients, respectively [12]. Primary genetically determined cardiomyopathy was thought to be a favorable background for superimposed myocarditis; moreover, the inflammatory component could contribute to realization of the abnormal genetic program [13,14].Patient I, 34 years old, was admitted to the Vinogradov Faculty Therapeutic Clinic (FTC; Sechenov University) on 2 December 2013. She complained of palpitations, dyspnea provoked by ordinary physical activity, recurrent leg edema and general weakness.The patient is a philologist and journalist. She does not smoke or abuse alcohol. There is no family history of cardiomyopathies; her mother (64 years old) has rheumatoid arthritis, Sjögren’s syndrome and autoimmune pancreatitis.The patient had thrombocytopenic purpura at the age of two, she was treated with prednisolone and complete remission was achieved. At the age of 18, she was diagnosed with chronic autoimmune thyroiditis, hypothyroidism, for which she receives hormone replacement therapy (L-thyroxine).Patient noticed onset of chest pain in 2000 (20 years old), she did not undergo any investigation and did not receive any treatment (Figure 1). In 2003, after an upper respiratory tract infection with a prolonged (up to one month) period of subfebrile fever, chest pain became more intensive and dyspnea appeared. EchoCG in 2005 revealed mitral valve prolapse with first degree regurgitation and left ventricular (LV) dilatation, with decreased ejection fraction (EF) to 42%. After these findings, she underwent EchoCG annually and the cardiac contractility remained on the same level.The deterioration of her condition started in September 2007. On the back of psycho-emotional stress and fatigue, she was experiencing constant stabbing pain in the left side of her chest, which was self-limited, and increased dyspnea. In April 2008, she was hospitalized at the A.N.Bakoulev Center for Cardiovascular Surgery. EF remained on the same level (43%), and myocardial radionuclide perfusion scan revealed decreased accumulation in the area of interventricular septum (IVS). During 24 h ECG monitoring, 865 premature ventricular beats (PVBs) of three morphologies were registered. During coronary angiogram, no hemodynamically significant stenoses were found. Myocardial positron emission tomography was performed, and moderate decrease in perfusion and glucose metabolism in the middle sections of the IVS was registered, which was most likely caused by local thinning or local myocardial fibrosis. Diagnosis was formulated as dilated cardiomyopathy (DCM). Patient was prescribed perindopril 8 mg/day, bisoprolol 1.25 mg/day, trimetazidine 70 mg/day, furosemide 40 mg/day, spironolactone 25 mg/day and L-thyroxine 50 mg/day. Her condition remained stable. EchoCG performed in 2009, 2012 and 2013 showed no significant dynamics. EF varied from 38% to 40%.The next deterioration of well-being was in the summer of 2013, when pre-syncope appeared. In September 2013, Holter monitoring recorded 2048 PVBs of three morphologies and an episode of sustained ventricular tachycardia (VT) lasting 1 min, accompanied by loss of consciousness. She was hospitalized at the A.N.Bakoulev Center for Cardiovascular Surgery, where she underwent cardiac MRI. The MRI revealed the epicardial fat thickening along the anterior wall of RV and posterior wall of LV with signs of “crawling” on myocardium, RV (end-diastolic diameter (EDD) 48 mm) and LV (EDD 66 mm) dilatation with decrease of EF of both ventricles (RV EF 25% and LV EF 41%), dilatation of LV outflow tract (20 mm), edema of LV and prominent fibrous changes of non-ischemic genesis of RV and LV. The condition was thought to be chronic myocarditis, but ARVC and LVNC were not ruled out either. On 30 October 2013, a cardioverter-defibrillator (ICD) was implanted. After upper respiratory tract infection in November 2013, the patient noticed increase of chest pain, dyspnea and leg edema. She was admitted to the Department of Cardiology, FTC, for further examination and choice of treatment strategy.Heart sounds were regular, clear, heart rate 60 per min, blood preassure 90/60 mm Hg. Lungs auscultation revealed vesicular breathing, no rales, respiratory rate 20/min. Liver and spleen were not enlarged. Moderate legs edema was observed.Full blood count, biochemical panel, coagulogram and urinalysis showed no abnormalities. Anticardiac antibodies (Ab) test was performed to verify possible chronic myocarditis, showing specific antinuclear factor to cardiomyocyte nuclei (ANF) 1:320 (normally negative), Ab to endothelial antigens (AbE) 1: 160 (N ≤ 1:40), Ab to cardiomyocyte antigens (AbC) 1:160 (N ≤ 1:40), Ab to smooth muscle antigens (AbSM) 1:160 (N ≤ 1:40), and Ab to cardiac conductive fibers antigens (AbCF) 1:320 (N ≤ 1:40). No genome of cardiotropic viruses (herpetic group viruses, parvovirus B19 and cytomegalovirus) were detected in the blood.ECG on admission (Figure 2) showed low QRS voltages in the limb leads, and negative T waves in the left precordials (minor ARVC criterion) and in the inferior leads (indicating LV involvement). The signal-averaged ECG revealed late potentials (minor ARVC criterion): filtered QRS = 137 ms (N < 114 ms), low-amplitude signal duration = 40 ms (N < 38 ms) and root-mean-square voltage of terminal 40 ms = 26 µV (N > 20 µV). On 24 h ECG monitoring (treatment: sotalol 160 mg/day) 1700 PVBs (minor ARCV criterion), 54 couplets and one triplet were registered.EchoCG showed left and right ventricular dilatation (EDD 6.2 and 4.0 cm, respectively) and diffuse reduction of LV contractility (EF 37%), with normal VTI 15.4 cm. Multiple pseudochordae were visualized in LV, but there were no convincing echocardiographic signs of LVNC. To verify the presence of LVNC, the patient underwent cardiac computed tomography (CT); coronary arteries were intact, LV myocardium had increased trabecularity in the apical-lateral and posterior walls, and the ratio of noncompact and compact layers was 3:1. Zones of late contrast agent accumulation in the middle and subendocardial layer of LV were visualized. In addition, a myocardial radionuclide perfusion scan was performed, which showed indicator inclusions in LV myocardium with diffusely nonuniform distribution, with areas of relative indicator hypoaccumulation more pronounced in the IVS and apical and middle sections of LV anterior wall. Such pattern is nonspecific, but typical for myocardial diseases. Cardiac MRI CD-disk was reanalyzed by Professor Sinitsyn. MRI findings were not typical for postinflammatory changes or DCM; the picture of ARVC (according to TFC-2010 [2]) in combination with LVNC (Figure 3) was found. Thus, the presence of LVNC was verified by cardiac CT and MRI.Thus, according to the results of the complex examination, the patient had one major (indexed RV volume according to MRI over 100 mL, combined with dyskinesias and decrease of EF up to 25%) and three minor (sustained VT, negative T waves in the left precordial leads and late potentials) criteria of ARVC, signs of LVNC according to cardiac MRI and CT. At the same time, there was an episode of prolonged fever after acute respiratory infections without adequate treatment in the disease onset, decompensation of heart failure provoked by upper respiratory tract infection, angina pain with unchanged coronary arteries, extensive areas of late gadolinium enhancement on MRI (interpreted ambiguously), as well as late contrast agent enhancement on CT (in the middle and subendocardial layer of the LV) and significant increase in titers of anticardiac antibodies (including specific ANF 1: 160). All these features required exclusion of active myocarditis as a cause of systolic dysfunction, along with two verified cardiomyopathies.To verify myocarditis endomyocardial, biopsy from the free wall of RV was obtained: endocardium was thin and contained adipocytes in subendocardial area (less than 10%); cardiomyocytes were irregularly hypertrophied, with dystrophic changes in cytoplasm, separated by fibrous septa with focal lymphohistiocytic infiltrates (>14 cells/mm2); Tebizia vessels with edematous endothelium and vasculitis phenomena. No genome of cardiotropic viruses (herpetic group viruses, parvovirus B19 and cytomegalovirus) was found in myocardium. Thus, in addition to the combination of ARVC with LVNC, active chronic infectious-immune myocarditis was confirmed.The patient was consulted by a geneticist. DNA-diagnostic using ARVC and sarcomeric panels was recommended. A new genetic variant (NM_004415.4: c.1141-2A>G/N (rs794728111)) was identified in the DSP (desmoplakin) gene. According to bioinformatic analysis, it is a pathogenic splice mutation, which is another major criterion of ARVC.Antiarrhythmic (sotalol 160 mg/day), cardiotropic (bisoprolol 5 mg/day, perindopril 2.5 mg/day) and diuretic therapy (thorasemide 5 mg/day, spironolactone 50 mg/day) were prescribed, on the basis of which the patient noted significant decrease of dyspnea, regression of leg edema and improvement of general well-being. Due to an episode of tachycardia in the ICD memory, which did not allow unequivocal judgement about presence or absence of atrial fibrillation; the existence of LVNC, which is a risk factor for thrombosis; and a decrease of LV EF to 37%, anticoagulant therapy with rivaroxaban 20 mg/day was prescribed. Because of the patient’s family circumstances, immunosuppressive therapy of myocarditis (IST) was not started immediately; spontaneous normalization of Ab titers was observed in March 2014 (Table 1).Due to the absence of clinical deterioration, IST was not administered until spring 2015, when dyspnea increased significantly, leg edema appeared again, LV EF decreased to 30% and anticardiac Ab titers increased. Besides, twice appropriate ICD shocks because of VT with heart rate 210/min (Figure 4) with transformation into ventricular fibrillation were registered, and PVBs number according to 24 h ECG monitoring exceeded 4000/day.The condition was considered as exacerbation of chronic myocarditis. Active IST was started (low doses of methylprednisolone—4 mg/day combined with azathioprine 150 mg/day) and sotalol was temporarily replaced by amiodarone (permanent treatment with amiodarone is impossible, due to photosensitization while taking amiodarone). As a result, a significant clinical and laboratory effect was achieved: dyspnea decreased, leg edema regressed, LV EF stabilized at 40%, VT was completely suppressed, blood tests for anticardiac Ab in January 2016 showed almost no signs of myocarditis immunologic activity. The patient continues to be monitored annually in FTC and her condition is determined not so much by ARVC progression, but by the activity of superimposed myocarditis.In review of the literature, we encountered only a few cases of the combination of ARVC and LVNC. In 2006 and 2015, Turkish scientists published two such cases (two young men, in one of them, the presence of LVNC was verified only by EchoCG) [15,16]. One case (a woman who underwent heart transplantation due to the development of refractory heart failure) was described in Italy [17]. A group of Polish scientists in 2009 described nine patients with signs of ARVC in whom a more detailed examination revealed LVNC, which, according to the researchers’ opinion, mimicked the clinic of ARVC [18]. This publication has been widely discussed in the scientific community, including by experts such as Finsterer and Stöllberger [19]. In this paper, the criteria for diagnosis of LVNC on MRI and the frequency of confirmation of LVNC by EchoCG and by MRI were not clear. Besides, the presence of LVNC did not explain dilatation of RV in all nine patients, and there were no data on DNA diagnoses of both ARVC and LVNC. It is possible that some patients had a true combination of the two cardiomyopathies, which the authors do not admit. There are data on combinations of ARVC with RVNC, but criteria of “RV noncompaction” have not been developed, so it is considered that increased RV trabecularity is a variant of normal [20,21]. At the same time, none of the above-mentioned publications contains any data on the presence or absence of superimposed myocarditis in patients.In 2018, we published an article in which the combination of ARVC and LVNC was described in eight patients, and we considered it as a special clinical variant of ARVC [22]. Currently, we have nine such patients in our registry, representing 15.5% of all our patients with ARVC (the ARVC registry includes 58 patients) and 7.2% of patients with LVNC (the LVNC registry contains of 125 patients). Superimposed myocarditis was diagnosed in eight out of nine patients (88.9%), which allows consideration of the combination of these two cardiomyopathies as a favorable background for myocarditis. Patients with a combination of ARVC and LVNC revealed peculiarities of the clinical course of the disease, which distinguished them from the patients with isolated ARVC or LVNC. Life-threatening ventricular arrhythmias, resistant to antiarrhythmic drugs and leading to appropriate ICD interventions (shocks were recorded in 100% of patients with ICD), were noteworthy. Dilation of RV according to EchoCG, low QRS voltages on ECG, AV block and absence of signs of LV hypertrophy on ECG allowed us to suspect ARVC in patients with verified LVNC. On the opposite hand, in patients with verified ARVC, LV dilatation with decreased LV EF can indicate the presence of LVNC, although it can be observed in patients with biventricular variant of ARVC as well. Nevertheless, the biventricular variant of ARVC by itself does not exclude the presence of LVNC. In 2020, a group of Italian scientists from Padua proposed updated criteria for ARVC that are more sensitive to detect biventricular and dominant-left ARVC variants [23]. According to the criteria they propose, the patient described in this paper has classical signs of biventricular ARVC: morphofunctional and structural changes of both RV and LV.Another indication for the possible presence of LVNC in patients with ARVC with LV involvement could be the detection of mutations in the DSP gene, as changes in this gene were detected in one third of patients with a combination of ARVC and LVNC in our cohort (22.2%—potentially pathogenic variants and 11.1%—variant of uncertain significance). A group of Spanish scientists also described a terminating mutation c.1339C > T in the DSP gene detected in three probands and 15 family members of patients with dominant-left ARVC; LVNC was detected in five of them [24].The prevention of sudden cardiac death (SCD) is particularly important in the management of patients with combination of ARVC and LVNC, since all these patients, despite adequate antiarrhythmic therapy, have a high risk of SCD. Thus, in our patients, there were two appropriate ICD shocks due to sustained VT with transformation into ventricular fibrillation. Diagnostics and treatment of superimposed myocarditis are of great importance, since it aggravates the existing rhythm disorders (sustained VT in the described clinical case was observed only in periods of high activity of myocarditis), as well as contributes to progression of heart failure. There is an opinion that LVNC in patients with myocarditis reflects only secondary rearrangement of myocardial structure due to its inflammatory dysfunction, but the presented observation clearly demonstrates the possibility of combination of genuine myocarditis (verified by EMB and responding to IST) and true LVNC with ARVC caused by mutation in DSP gene.The combination of ARVC and LVNC is not rare and represents a special form of cardiomyopathy, which is a favorable background for superimposed myocarditis development. Various imaging modalities, in particular, cardiac MRI and CT, play a vital role in the diagnosis. However, verification of myocarditis and determination of indications for its treatment in patients with primary cardiomyopathies is impossible without myocardial biopsy. The primary importance in the management of such patients is the prevention of SCD, since they are at risk of life-threatening ventricular rhythm abnormalities. In addition, prompt diagnosis and treatment of superimposed myocarditis are necessary, since its presence has a significant impact on the clinical course of the disease and prognosis.Conceptualization (idea, treatment and follow-up of the patient described in the case), Y.L. and O.B.; methodology, O.B.; genetic consulting and testing, A.S. and E.Z.; formal analysis, Y.L. and N.V.; investigation, S.A. (cardiac MRI), N.G. (cardiac CT), E.K. (morphologist who analyzed EMB) and V.S. (EchoCG); data curation, Y.L., O.B. and N.V.; writing—original draft preparation, Y.L.; writing—review and editing, Y.L., O.B. and A.N.; supervision, O.B.; project administration, E.Z. and A.N.; funding acquisition, E.Z. All authors have read and agreed to the published version of the manuscript.This study (performing DNA diagnostics) was supported by Grant No. 16-15-10421 of the Russian Science Foundation.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of I.M. Sechenov First Moscow State Medical University (Sechenov University) (protocol code 11-15, date of approval 16 February 2015).Informed consent was obtained from all subjects involved in the study.The data presented in this study are available on request from the corresponding author. The data are not publicly available because this publication describes a clinical case and the name of the patient should not be disclosed.We thank Valentin Sinitsyn for meticulous reanalysis of cardiac MRI disk of the patient.The authors declare no conflict of interest. Scheme of the medical history (explanation in the text). ARVC—arrhythmogenic right ventricular cardiomyopathy; DCM—dilated cardiomyopathy; ECG—electrocardiography; EchoCG—echocardiography; ICD—implantation of cardioverter-defibrillator; LVEF—left ventricular ejection fraction; LVNC—left ventricular noncompaction; MRI—magnetic resonance imaging; PET—positron emission tomography; PVCs—premature ventricular contractions; URTI—upper respiratory tract infection.Patient’s ECG. Low QRS voltages in limb leads, negative T wave in left precordial leads (minor ARVC criterion) and in inferior leads.Cardiac MRI: left ventricular (LV) end-diastolic diameter (EDD) 66 mm, LV end diastolic volume EDV 243 mL, EF 41%, RV EDD 48 mm, RV EDV 115 mL/m2, EF 25%, areas of hypo/diskinesia of the front wall of RV, in the early and late phases—subepicardial areas of late gadolinium enhancement throughout LV, in IVS from the side of RV and along the walls of RV.Paroxism of sustained ventricular tachycardia.Follow-up of patient I.Ab—antibodies; ANF—specific antinuclear factor to cardiomyocyte nuclei (normally negative); AbE—Ab to endothelial antigens (N ≤ 1:40); AbC—Ab to cardiomyocyte antigens (N ≤ 1:40); AbSM—Ab to smooth muscle antigens (N ≤ 1:40); AbCF—Ab to cardiac conductive fibers antigens (N ≤ 1:40); ICD—implantable cardioverter defibrillator; LV EF—left ventricular ejection fraction.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Since late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease 2019 (COVID-19) have become a worldwide threat to public health [1,2]. SARS-CoV-2 is characterized by an extremely strong inflammatory response that can lead to severe manifestations such as adult respiratory syndrome, sepsis and potentially fatal coagulopathy [3]. These symptoms occur mainly in elderly subjects, males, and patients with preexisting serious comorbidities like cardiovascular, pulmonary, and renal diseases [4].In addition to significant mortality, another key problem of COVID-19, is the exponential increase of infections and the very large number of patients admitted to hospitals. Even more confusing is that not all infected patients develop a severe respiratory illness. Currently, the reason for these large inter-individual variations in disease severity is not clear.So, what makes some people more vulnerable than others to SARS-CoV-2? The answer to this question could be found in interpersonal genetic variability, which determines how the individual responds to the virus [5]. Currently, there is no proven effective therapy.In this regard, Hugozeberg and Svante Pääbo had an article published in Nature [6] in which they considered a recent study about genetic susceptibility to COVID-19. In another recent work [7], the authors conducted a genome-wide association study involving 1980 patients with COVID-19 during which they highlighted two chromosomal regions associated with severe forms of the disease. The gene clusters under consideration are six genes present on chromosome 3 (3p21.31 position) and the region associated with AB0 blood groups on chromosome 9 (9q34. 2 position). In this study, it was understood that only the gene cluster present on chromosome 3 is associated with the more severe forms of COVID-19 at the genome-wide level. This gene cluster on chromosome 3 has a high linkage disequilibrium (LD). Furthermore, these genes are highly associated in the population (r2 > 0.98) and are 49.4 kb long. The haplotype appears to have been inherited from Neanderthals or the Denisovans [8,9]. The aim of the study, therefore, was to understand how these genes came down to us. Many of them have been found homozygous in the Vindija33.19 Neanderthal genome found in Croatia.From the Neanderthals to the present day, there is probably an ancestry, but due to recombination in each generation, the haplotype gradually shrank into smaller and smaller fragments. All this has been confirmed by studies concerning the genetic flow from Neanderthals to modern mankind [10].These studies compared the Neanderthal genome with the genome present in the 1000 Genomes Project data. The result was that 253 haplotypes of today’s people contain 450 variable positions and exhibit a different frequency among different populations. In fact, the 49.4 kb haplotypes are almost absent in African and East Asian populations, but have a 30% frequency in South Asian and about an 8% frequency in European populations. The population that has the highest frequency is in Bangladesh (63%), where subjects are carriers of at least one copy of the Neanderthal-risk haplotype, whereas 13% are homozygous for the haplotype [10].From this it can be deduced that carriers of the Neanderthal haplotype could be a contributing factor to the dangerousness of COVID-19 in some populations, especially among those of an advanced age. To confirm this, individuals of Bangladeshi origin, present in the U.K., have about twice the risk of dying from COVID-19 compared to the general population (Hazzard ratio 95% CI:1.7–2.4) [11].Similarly, in the study of A. Nguyen [12], gene susceptibility to the more severe form of COVID-19 was also analysed. He focused on the genetic variables of the major complex of histocompatibility (MCH) class I, in particular on Human Leukocyte Antigen (HLA) alleles. It is known that there is an association between the HLA genotype and the severity of some diseases including COVID-19. A comparison of the binding affinity between MHC class I and 145 different HLAs against SARS-CoV-2 revealed that HLA-B*46:01 had less affinity for the virus, suggesting that individuals with this allele may be particularly vulnerable to COVID-19. A similar result had previously been obtained for SARS [13]. Conversely, it was observed that HLA-B*15:03 has a greater compatibility with the peptides of the virus, thus giving greater protection to those who possess it. This shelter is tied to conserved and common peptides between SARS-CoV-2 and other common human coronaviruses. Who has the HLA-B*15:03 overexpression gains greater protection due to the T-cell based immunity obtained by past infections of less harmful coronaviruses [14].In conclusion, knowing individual genetic variation can help explain different susceptibility to SARS-CoV-2. Such knowledge could therefore help to identify individuals who have a higher risk of contracting the disease. Of course, this would also help create a vaccine or make it possible to find adequate, if not preventive, therapy for those who have a greater chance of meeting the most serious form of the virus, thus preventing hospitalization or death. For this reason, it is very important to stimulate further exploration of current findings for their usefulness in clinical risk-profiling of patients with COVID-19 and toward a mechanistic understanding of the underlying pathophysiology.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Hypertrophic cardiomyopathy and left ventricular noncompaction commonly occur as separate disorders with distinct clinical and pathoanatomical features. However, these cardiomyopathies may have a similar genetic origin with mutations encoding sarcomeric proteins. The described case report demonstrates an example in which phenotypic expression of both diseases occurred in the same patient, who has two different alterations; one of them is a likely pathogenic variant in the MYL3 gene (MIM#160790) and the second variant in the MYH6 gene (MIM#160710) of unknown significance so far. To better understand associations between specific genetic variants and phenotypical expression of these genetic alterations and to stratify patient risk and decide on the most appropriate treatment, a comprehensive multimodality imaging approach and experienced multidisciplinary cardiomyopathy team decisions are warranted. In the clinical routine, awareness of the existence of complex cardiomyopathy phenotypes should be paid more attention during echocardiographic examination and should encourage a broader use of cardiovascular magnetic resonance.Hereditary cardiomyopathies (CMs) represent a very large and heterogeneous group of inherited heart disorders. According to the results of instrumental evaluation, many types of the disease have been characterized: hypertrophic CM (HCM), left ventricular noncompaction (LVNC), dilated CM, and arrhythmogenic right ventricle CM to mention the most frequent entities. LVNC (ORPHA:54260) and HCM (ORPHA:217569) commonly occur as separate disorders with distinct clinical and pathoanatomical features [1,2]. However, in some patients, overlapping or mixed phenotypes are diagnosed only based on the use of sophisticated imaging modalities, especially cardiovascular magnetic resonance (CMR). Unfortunately, sometimes the phenotypic manifestation of overlapping phenotypes poses some difficulties in determination of the particular disorder which is essential in the treatment and surveillance of the patient. Moreover, the same genes may be implicated in the pathogenesis of different CMs, making diagnostics even more complicated. Pathogenic variants in MYBPC3 and MYH7 genes are responsible for the development of most non-syndromic HCMs and a big part of LVNC cases, although more than 30 genes are currently associated with the pathogenesis of these disorders.In this case report, the authors present two patients from one nuclear family suffering with hereditary heart disorders possessing variants in the MYL3 gene (MIM#160790) and the MYH6 gene (MIM#160710). Although we know that the pathogenic variants of the MYL3 gene have been associated with familial HCM, the alteration in the MYH6 gene NM_002471.3:c.169G>A, NP_002462.2:p.(Gly57Ser) has not been described in the scientific literature previously. The genetic change was classified as variant of unknown significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) criteria. Based on the case presented, we postulate that the presence of this double mutation causes an overlapping hypertrophic–noncompaction phenotype. Additionally, we emphasize the utility of multimodality imaging for the phenotypical assessment of cardiomyopathy patients, as well as a cardiomyopathy team approach to choose the most suitable treatment.Next-generation sequencing analysis of genomic DNA isolated from two patients’ peripheral blood was performed using TruSight Cardio Sequencing panel (Illumina Inc., San Diego, CA, USA). A total of 174 genes (coding exons) were analyzed, including the main genes associated with cardiomyopathies (ABCC9, ACTC1, ACTN2, ANKRD1, BRAF, CAV3, CBL, CRYAB, CSRP3, DES, DSC2, DSG2, DSP, DTNA, GAA, GLA, HFE, HRAS, JUP, KRAS, LAMA4, LAMP2, LDB3, LMNA, MAP2K1, MAP2K2, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOZ2, MYPN, NEXN, NRAS, PKP2, PLN, PRDM16, PRKAG2, PTPN11, RAF1, RBM20, RYR2, SCN5A, SGCD, SHOC2, SOS1, TAZ, TCAP, TGFB3, TMEM43, TNNC1, TNNI3, TNNT2, TPM1, TTN, TTR, VCL). Prepared DNA libraries were sequenced on the Illumina MiSeq system. The combined coverage was 572 kbp in sequence length. Data analysis was performed using standard Illumina bioinformatic workflow. Detected gene variants were analyzed and annotated using the VariantStudio 3.0 software. Synonymous or intronic variants and variants with a minor allele frequency of less than 2% were excluded. In silico analysis of missense mutations was performed using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/ (accessed on 4 August2019)), SIFT Human Protein (http://sift.jcvi.org/ (accessed on 4 August2019)), and Mutation Taster (www.mutationtaster.org/ (accessed on 4 August2019)).Polymerase chain reactions (PCR) of gDNA sequences flanking variant NM_000258.2:c.382G>T, NP_000249.1:p.(Gly128Cys), rs199474704 of the MYL3 gene and variant NM_002471.3:c.169G>A, NP_002462.2:p.(Gly57Ser) of the MYH6 gene were performed using specific primers designed with the Primer Blast tool [3,4]. The PCR products were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waldham, MA, USA) and the ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific, Waldham, MA, USA). The sequences were aligned with the reference sequence of the MYL3 (NCBI: NM_000258.2) and MYH6 (NCBI: NM_002471.3) genes.Two-dimensional transthoracic echocardiography (TTE) using an ultrasonic system equipped with 1.5–4.5 MHz transducer (GE Vivid E9, GE Healthcare, New York, NY, USA) and 1.5 T cardiovascular magnetic resonance (CMR) (Siemens Avanto, Erlangen, Germany) was used to assess the specific CM phenotype.A 39-year-old male consulted a cardiologist on an outpatient basis due to nonanginal chest pain episodes five years ago and was diagnosed with isolated apical HCM by using TTE. At the time of follow-up consultation, the patient was asymptomatic with an unremarkable personal history and a family history of unspecified congenital heart disease and sudden cardiac death of his sister and his aunt. His physical examination was without any pathological findings. Electrocardiogram showed sinus rhythm, 80 bpm, hypertrophy of left ventricle (LV), and deep negative T-waves in leads I, aVL, V4–6. Blood biochemistry was normal. TTE was performed and demonstrated isolated apical HCM with apical micro-aneurysm. Interestingly, blood flow was registered in the projection of hypertrophied midventricular segments, raising the question about the correctness of the previous diagnosis. CMR was scheduled for clarification of the heart’s morphology and function, as well as for additional risk stratification of the patient. The CMR revealed hyperkinetic LV with a hyperkinetic ejection fraction around 84% and overlapping phenotypical pattern of LV myocardium with hypertrophy of compacted apical segments (maximum wall thickness up to 16 mm (Figure 1E,F) and hypertrabeculation of midventricular segments with a ratio of non-compacted and compacted myocardium up to 2.8 at end-diastole (Figure 1C,D; Supplementary Videos S1–S5). Additionally, LV apical micro-aneurysm (Figure 1A,B) was detected with transmural late gadolinium enhancement (LGE) in its wall, showing transmural fibrotic changes (Figure 2A). To exclude any coronary artery anomalies or underlying coronary artery disease coronary, computed tomography (CT) angiography was performed which demonstrated normal coronary arteries without any atherosclerotic changes or anomalies with morphological changes of LV consistent with the CMR findings (Figure 2B). Then, 24 h ECG monitoring was performed and revealed four sporadic ventricular premature beats.The patient underwent genetic consultation and testing. Phenotypic evaluation revealed a non-syndromic type of HCM. Genealogy analysis showed multiple individuals affected with cardiac disorder in the family (Supplementary Figure S1). The father died of myocardial infarction at the age of 72 years; the mother suffered from heart rhythm disorder and died at the age of 64 years. The sister of the patient experienced sudden cardiac death at the age of 5 years, having an unspecified “congenital” heart defect. The genetic testing of the patient revealed a heterozygous missense type MYL3 gene variant NM_000258.2c.382G>T, NP_000249.1:p.(Gly128Cys), rs199474704 and a heterozygous missense type MYH6 gene variant NM_002471.3:c.169G>A, NP_002462.2:p.(Gly57Ser).Additionally, the patient’s 15-year-old daughter was invited for cardiological examination and genetic consultation and testing. CMR revealed normal LV systolic function without evidence of LV hypertrophy. However, hypertrabeculation of the apical to midventricular segments was observed, with a ratio of non-compacted to compacted myocardium up to 2.0, which was not diagnostic for left ventricular noncompaction (see Supplementary Videos 6–7). Subsequently, 24 h ECG monitoring was performed and revealed one sporadic supraventricular premature beat. Sanger sequencing confirmed that the patient had passed both MYL3 gene variant c.382G>T and MYH6 gene variant c.169G>A to his affected daughter (Supplementary Figures S2 and S3).The above-described genetic alterations were discussed among a dedicated cardiomyopathy team, taking into account the presence of double genetic variants and LV apical micro-aneurysm with fibrotic changes and considering the patient’s preferences. The decision was to schedule the patient for implantation of a cardioverter-defibrillator (ICD) for primary prevention of sudden cardiac death. Treatment with beta-blockers (47.5 mg of metoprolol succinate) was prescribed, and the patient was referred for regular cardiological follow-up. Two years following, the patient remains asymptomatic without any events registered by the ICD events so far. The patient’s daughter was scheduled for a cardiological follow-up.With this case report, the authors present two patients from one nuclear family suffering from hereditary heart disorders possessing variants in the MYL3 gene (MIM#160790) and the MYH6 gene (MIM#160710). Based on the case description, we postulate that the presence of the particular double mutation causes an overlapping hypertrophic–noncompaction phenotype.The MYL3 gene (MIM#160790) encodes myosin light chain 3 protein, belonging to the myosin family [5]. Myosins are essential in maintaining the structural integrity and shape of the cell. Moreover, the interaction with actins plays major role in myocyte contractility [6]. The alteration of the MYL3 protein (either hereditary or secondary due to the activation of caspase) results in the disruption of sarcomeres and eventually in contractile dysfunction due to the disorganized actin–myosin bonds in cardiomyocytes [7]. Pathogenic variants of the MYL3 gene have been associated with familial HCM, type 8 (MIM#608751) inherited in autosomal dominant or autosomal recessive manner. To date, 41 different pathogenic variants have been identified in individuals affected with HCM [8]. The alteration NM_000258.2c.382G>T, NP_000249.1:p.(Gly128Cys), rs199474704 has been reported in patients with end-stage HCM [9]. The variant leads to amino acid change in a conservative position (EF–hand domain/EF–hand domain pair domain of the protein), and the biochemical difference between glycine and cysteine is high (Grantham score 159). In silico analysis results: SIFT—deleterious (score 0.01), PolyPhen2—probably damaging (score 0.927), Mutation taster—disease causing (score 0.99). The variant is listed in The Human Mutation Database (CM117919), and the frequencies in the 1000 Genomes Project and the ExAC project are 0.0 and 0.0, respectively. Functional analyses have not been performed. Based on these observations, the alteration was classified as possibly pathogenic according to the ACMG criteria, and segregation analysis in the family was recommended.Another heterozygous missense type MYH6 gene variant (NM_002471.3:c.169G>A, NP_002462.2:p.(Gly57Ser)) was also identified in the patient. The MYH6 gene (MIM#160710) encodes an alpha heavy chain myosin, functioning as a fast ATPase in cardiac muscle and participating in the contraction of myocytes [10]. The alteration of alpha-MHC protein may lead to the switch of expression to the MYH7 gene located upstream of the MYH6 gene, thus accelerating the development of HCM [11]. Pathogenic variants of the MYH6 gene have been associated with atrial septal defect type 3 (MIM#614089), dilated CM type 1EE (MIM#613252), HCM type 14 (MIM#613251), and sick sinus syndrome type 3 (MIM#614090) [12]. The alteration in MYH6 gene NM_002471.3:c.169G>A, NP_002462.2:p.(Gly57Ser) has not been described in scientific literature previously. The variant causes amino acid change in a semiconservative position (SH3-like/P-loop containing nucleoside triphosphate hydrolase domain at the N-terminal of myosin), but the biochemical difference between glycine and serine is moderate (Grantham score 56). In silico analysis results: SIFT—deleterious (score 0.024), PolyPhen2—benign (score 0.256), Mutation taster—disease causing (score 0.99). The frequency of the variant in the 1000 Genomes Project is 0.0; ExAC project—0.0. Functional analyses have not been performed. The genetic change was classified as a variant of unknown significance (VUS) according to the ACMG criteria.The segregation analysis performed on the daughter of the patient revealed the same variants of MYL3 and MYH6 genes as her father. These findings uncovered the nature of the variants, making them clinically important. However, we currently are not able to discriminate how much they would influence disease development if they acted separately, and at which age we will see clear phenotypical CM expression. Nevertheless, the possession of two very rare alterations in CM genes might be associated with faster development of the disorder and with the diverse manifestation of structural rearrangements (both HCM and LVNC are present in our patient).The overlapping phenotype in the adult patient described was diagnosed based on the established diagnostic criteria of both CM. The best imaging modality to prove phenotypical features is definitely CMR imaging. In our adult patient, HCM was diagnosed based on the European Society of Cardiology (ESC) guidelines for the diagnosis and management of HCM. In the latter guidelines, HCM is defined by a wall thickness ≥15 mm in one or more LV myocardial segments—as measured by any imaging technique (echocardiography, CMR or CT)—which is not explained solely by loading conditions [2]. In our patient, apical segments proximal to the apical microaneurysm were measured to be up to 16 mm in diastole, which is consistent with diagnostic criteria for HCM (Figure 1A,B,E,F). Additionally, next to the hypertrophied compact part of LV, we noticed a noncompaction area, which was more prominent at the level of midventricular segments. In order to diagnose noncompaction, we used the CMR criteria proposed by Petersen et al. [13]. Using the latter criteria, a diastolic non-compacted to compacted ratio >2.3 identifies pathological noncompaction with values for sensitivity, specificity, and positive and negative predictions of 86%, 99%, 75%, and 99%, respectively. Thus, a diastolic non-compacted to compacted myocardial ratio equal to 2.8 was consistent with the diagnosis of left ventricular noncompaction at the level of midventricular segments (Figure 1C,D). Taking into account two phenotypical features in our patient, we considered our patient to have an overlapping hypertrophic and noncompaction phenotype.From a clinical point of view, clinicians should take into account the presence of red flags (RFs) known to be associated with specific systemic disease in a patient with HCM and other CMs. In our patient, we found a couple of features consistent with a non-syndromic type of CM. First of all, the patient’s daughter has the same variants of MYL3 and MYH6 as her father. Additionally, the proband has multiple individuals affected with unspecified cardiac disorder in his family. Other clues were LV hypertrophy, repolarization abnormalities on the ECG, and LV apical hypertrophy with apical micro-aneurysm on cardiac imaging. We did not find any RF associated with non-sarcomeric (syndromic, skeletal myopathy, or infiltrative phenotype) HCM. According to the literature, the presence of RFs shows a high negative predictive value to exclude any specific (non-sarcomeric) HCM disease (98% [95%CI 94–99%]) [14].The choice of the best treatment and surveillance strategy in patients with CM requires a personalized, specific, and experienced approach. Therefore, we involved a multi-disciplinary dedicated cardiomyopathy team composed of a clinical cardiologist, an interventional cardiologist, a cardiac surgeon, and an electrophysiologist in the decision-making regarding the further treatment strategy for our patient. The strategic role of cardiomyopathy teams working in experienced centers has been suggested as a critical step in the management of cardiomyopathy patients [15].There are currently no trials or predictive models to guide ICD insertion specifically for apical HCM or overlapping cardiomyopathy phenotypes. The ESC five-year HCM sudden cardiac death (SCD) risk score [16,17] was based on all HCM morphological subtypes without breakdown for apical HCM [16]. Maron’s group has proposed an enhanced American College of Cardiology/American Heart Association (ACC/AHA) guideline-based risk factor algorithm strategy for HCM patients fulfilling one or more major risk factors for SCD. This novel risk prediction strategy includes novel high-risk markers, such as CMR LGE demonstration of extensive fibrosis comprising ≥15% of LV mass by quantification or “extensive and diffuse” by visual estimation, as well as the presence of LV apical aneurysm, independent of size, with associated regional scarring [18]. Compared with enhanced ACC/AHA risk factors, the ESC risk score [16,17] retrospectively applied to the study patients was much less sensitive than the ACC/AHA criteria (34% [95% CI, 22–44] vs. 95% [95% CI, 89–99]), consistent with recognizing fewer high-risk patients. Additionally, the main indications for ICD implantation for the primary prevention of sudden cardiac death that may apply to patients with LV noncompaction are patients with CM and LV ejection fraction ≤35% and high-risk HCM with LVNC [19]. Thus, we took into account the history of sudden cardiac death in the first-degree relative under 40 years of age (the proband’s sister) and the presence of LV apical micro-aneurysm with associated transmural LGE (a major risk marker) to justify the recommendation for prophylactic ICDs in our patient.In summary, our case suggests that the phenotypical expression of both LVNC and HCM can occur in the same patient having two different alterations (one of them likely pathogenic and the second is a VUS so far). This case also highlights the need for comprehensive multimodality imaging for simultaneous morphological and functional evaluation of the heart in order to assess the correct phenotype and to stratify the patient’s risk. In the clinical routine, awareness of the existence of complex cardiomyopathy phenotypes should be raised during echocardiographic examination and should encourage the broader use of CMR.The following are available online at https://www.mdpi.com/2035-8148/11/1/5/s1, Video S1: Magnetic resonance four chamber cine heart view. Video S2: Magnetic resonance two chamber cine heart view. Video S3: Magnetic resonance three chamber cine heart view. Video S4: Magnetic resonance midventricular short axis cine heart view. Video S5: Magnetic resonance apical short axis cine heart view. Video S6: Magnetic resonance two chamber heart cine view of patient’s daughter: Video S7: Magnetic resonance apical short axis cine view of patient’s daughter. Figure S1: Genealogy of the family (black symbols denote patients with detected variants in MYL3 and MYH6 genes. Half-filled symbol in II-3 denotes congenital heart defect. I-1 and 1–2 family members were affected with not specified cardiac disorders). Figure S2: The electropherogram of Sanger sequencing of MYL3 gene variant NM_000258.2:c.382G>T, NP_000249.1:p.(Gly128Cys), rs199474704 (reverse strand). Figure S3: The electropherogram of Sanger sequencing of MYH6 gene variant NM_002471.3:c.169G>A, NP_002462.2:p.(Gly57Ser) (forward strand).Conceptualization, S.G. and N.R.V.; writing—original draft preparation, S.G., E.P. and V.M.; writing—review and editing, S.G., V.M., E.P., R.N., R.J., and N.R.V.; visualization, S.G.; supervision, S.G. and N.R.V. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Informed consent was obtained from the patient.Password protected data supporting reported results can be found at https://eli.santa.lt/ (accessed on 4 August 2019) (could be provided anonymized data upon request by authors).The authors declare no conflict of interest.Cine magnetic resonance (steady state free precession sequence) heart views in diastole (left-sided column) and systole (right-sided column). (A,B) Four-chamber cine views; (C,D) midventricular short axis cine views; (E,F) apical short axis cine views. LA—left atrium; LV—left ventricle; RA—right atrium; RV—right ventricle. * Blood pool.(A) Magnetic resonance four chamber heart view obtained with inversion recovery sequence 10 min after injection of gadolinium-based contrast agent (late gadolinium enhancement (LGE) image) with apical fibrotic changes denoted with a white arrow. (B) Four-chamber computed tomography heart view representing the morphologic findings of the heart seen on magnetic resonance images. LA—left atrium; LV—left ventricle; RA—right atrium; RV—right ventricle.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Cardiovascular disorders are the main complication in autosomal dominant polycystic kidney disease (ADPKD). contributing to both morbidity and mortality. This review considers clinical studies unveiling cardiovascular features in patients with ADPKD. Additionally, it focuses on basic science studies addressing the dysfunction of the polycystin proteins located in the cardiovascular system as a contributing factor to cardiovascular abnormalities. In particular, the effects of polycystin proteins’ deficiency on the cardiomyocyte function have been considered.Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic renal disorder, implies a progressive loss of renal function and accounts for 4% to 10% of patients with end-stage renal disease (ESRD) [1,2]. The disease, which occurs worldwide with an estimated prevalence of 1:400 to 1:100, is characterized by the age-dependent growth of renal cysts such that ESRD typically ensues during mid adulthood [3,4,5].Mutations in Polycystic Kidney Disease 1 and 2 (PKD-1 and PKD-2, respectively) genes have a well-defined role in uncontrolled cystogenesis in both kidneys, the hallmark of the disease, as well as in the liver, and less frequently in the pancreas, seminal vesicles and arachnoid membrane [1,2,6,7,8,9,10,11,12,13,14]. Approximately 85% of ADPKD cases are caused by mutations in the PKD1 gene [MIM 601313], which is located on chromosome 16p13.3, while the remaining cases are due to mutations in PKD2 [MIM 173910], located on chromosome 4q21 [9,10].The protein products of PKD1 and PKD2, polycystin-1 (PC1) and polycystin-2 (PC2), are membrane proteins that probably form a functional complex [15,16,17,18,19]. They have been shown to localize predominantly, although not exclusively, in primary cilia and mediate the sensitivity of kidney epithelial cells to fluid shear stress [6,7,8]. PC1 has been proposed to be a mechanosensitive molecule, given its large extracellular domain and remarkable mechanical strength [1]. The distribution of the polycystin proteins at different subcellular locations is required for them to orchestrate a network of signaling pathways that have been implicated in the pathogenesis of PKD [8,20].PC1 or PC2 dysfunction may result in decreased intracellular calcium inflow with increased 3′, 5′-cyclic adenosinemonophosphate (cAMP) production and the activation of mechanisms which contribute to cyst cell growth by stimulating both epithelial cell proliferation and transepithelial fluid secretion [21]. Indeed, relevant downstream responses of the changed calcium signaling ultimately lead to increased proliferation and increased apoptosis [22]. Factors impacting upon disease progression include the level of PKD protein expression and the penetrance of pathogenic alleles [23,24]. However, both ADPKD type 1 and type 2 share the full spectrum of renal and extrarenal manifestations, although type 2 has a delayed onset compared to type 1 [1,2,25,26,27]. In type 2 patients, the common complications of ADPKD, such as hypertension, hematuria and urinary-tract infection, seem to be milder than in type 1 patients at the same age. The median age at death or onset of end-stage renal disease has been reported as 53 years in type 1 patients and 69 years in type 2 [25].The renal phenotype in patients with ADPKD ranges from elderly patients without renal failure to rare cases of enlarged kidneys that are detected in utero [28]. The disease course is highly variable, as a significant minority of patients do not reach ESRD even in old age, while a small number exhibit early-onset disease, with a diagnosis made in infancy by the identification of enlarged echogenic kidneys.Renal cyst expansion results from aberrant proliferation of the cyst wall epithelial cells and the accumulation of fluid within the cavity of the cyst. There is increased extracellular matrix remodeling as the cyst invades the adjacent parenchyma, leading to abnormal matrix deposition and fibrosis. The gradually growing renal cysts start to develop in utero and can originate from all areas of the kidneys, although cysts usually form in the distal regions of the nephron and the collecting duct [29,30,31,32,33].Impaired urinary concentrating capacity has been well documented as common even in early stages [1,26,27,28].Another important feature of ADPKD is the increase in plasma vasopressin concentrations. This defect is often attributed to the disruption of the medullary architecture, because its presence and severity correlate with the extent of the cystic disease. There is substantial evidence that the urinary concentrating defect and raised vasopressin concentrations could contribute to cystogenesis [34,35,36]. They might also contribute to the glomerular hyperfiltration seen in children and young adults, the development of hypertension and chronic kidney-disease progression [37].Another early functional defect is a reduction in renal blood flow, likely due to the development of cysts with changes in intrarenal pressures, and to neuro humoral or local mediators [38,39,40]. As mentioned above, the identity of the mutated gene in patients with ADPKD explains part of the phenotypic variability observed in clinical practice, so that patients with a mutation in PKD1 have earlier-onset ESRD, lower glomerular filtration rate (GFR) and larger kidney volumes than patients with a mutation in PKD2 [1,25,41].Even though enlarged cystic kidneys are the most obvious phenotype in ADPKD, cardiovascular disorders are the main complication contributing to both morbidity and mortality [42]. Systemic arterial hypertension is an early finding, occurring in about 60% of patients prior to a significant decline in GFR [3]. In particular, hypertension is observed in patients with ADPKD about a decade earlier than in the general population and even before the loss of renal function [43,44,45]. Although the mechanism underlying hypertension is not completely understood, the up-regulation of the renin angiotensin aldosterone system (RAAS), due to cystic compression of the renal vasculature, is considered a key mechanism in the pathogenesis of hypertension in ADPKD, as it leads to an increase in vascular resistance, and contributes to cardiac hypertrophy [46,47]. The RAAS pathway is the subject of clinical trials in patients with ADPKD in the HALT study, a US multicenter trial conducted on about 1000 ADPKD patients for 5.5 years. The benefits of blood-pressure control by antihypertensive drugs on patients with ADPKD have been largely demonstrated [48]. In patients with eGFR above 60 mL/min/1.73 m2, a target blood pressure of 110/70 mm Hg was suggested, especially in young patients with a high progression risk [49], while a target blood pressure below 120/80 mm Hg was recommended in chronic kidney disease stage 3 [50]. Thus, the aggressive control of hypertension in ADPKD patients appears to be clinically necessary and practically relevant. Nevertheless, as hypertension is likely dependent on the interaction of hemodynamic, endocrine and neurogenic factors, it has been postulated that mechanisms including increased sympathetic nerve activity [51], increased plasma endothelin-1 concentrations [52] and insulin resistance [53,54] contribute to the pathogenesis of hypertension in ADPKD. These theories can account for the high prevalence of hypertension but fail to explain why hypertension is frequently observed in patients with ADPKD, including children, even before the onset of renal insufficiency [55,56].The PKD genes are expressed in a wide range of tissues beyond the kidney, and their expression is developmentally regulated in most of these tissues [57,58]. The fact that PC1 and PC2 are expressed in vascular smooth muscle and endothelial cells suggests that a reduced polycystin protein function in the vasculature could play a primary role in the early development of hypertension [59,60].The primary dysfunction of the PC1 and PC2 complex results in decreased intracellular calcium inflow, with enhanced vascular smooth muscle contractility in response to adrenergic stimulation [61], increased muscular cell proliferation and apoptosis [62], and impaired endothelial-dependent vasorelaxation [63], functional abnormalities that might play a role in vascular remodeling and the early development of hypertension [64].The reduced expression of the polycystin proteins (haploinsufficiency) in the endothelial cells and vascular smooth muscle cells of most blood vessels, including the aorta and cerebral arteries, are likely to cause the vascular abnormalities in patients with ADPKD [59,65].The functions of PC1 and PC2 in the vasculature indicate that they have a crucial role in mechano-sensation [66,67,68,69]. In endothelial cells, the proteins are involved in fluid-shear stress sensing, thereby regulating calcium signaling, and there is NO release or vasodilatation in response to the increased blood flow [70,71,72,73,74,75,76,77]. In vascular smooth muscle cells, the polycystins regulate pressure sensing by modulating the activity of the stretch-activated cation channels and myogenic contraction [78,79,80]. A loss of myogenic tone may contribute to aneurysm formation owing to an increase in arterial wall stress [81,82].Indeed, the risk of cerebral aneurysms is higher in families with a positive family history of ADPKD than in those without, probably because of modifying genes [83,84]. Moreover, patients with ADPKD are more prone to coronary arteries aneurisms and acute dissection, which are a source of coronary syndromes and death [85,86,87]. Likewise, primary defects in vascular structure may lead to acute aortic dissection, that occurs in ADPKD patients more frequently than in the general population [88,89,90].ADPKD is a systemic disease associated with several extrarenal manifestations, including aortic root dilatation and cardiac valvular abnormalities, mostly mitral valve prolapse [91,92]. The early development of hypertension leads to LV hypertrophy, a major cardiovascular risk factor, and to LV diastolic dysfunction [67]. The occurrence of advanced kidney failure contributes to the increased frequency of cardiovascular events seen in ADPKD, as it is a well-known key factor in the development of cardiac function impairment.The relevance of LV hypertrophy in ADPKD has been established by clinical studies [93,94,95,96,97,98]. Among 116 consecutive ADPKD patients, LVH was found in 46% of males and 37% of females, and the LV mass, as measured by two-dimensional echocardiography and indexed by body surface area, correlated significantly with the blood pressure level. An early onset of hypertension and a frequently inadequate treatment may account for the role of blood pressure as a contributing factor to LV hypertrophy in ADPKD patients. In the HALTPKD study (NCT0028368), the rigorous blood pressure control obtained with the use of RAAS inhibitors was able to achieve a significant reduction of LV mass as measured by MR imaging [48]. It is worth noting that LV hypertrophy can also be observed in normotensive ADPKD patients [56,91,92,93,94]. In a single-site study [99] including 126 ADPKD patients (78% with hypertension), the prevalence of LV hypertrophy was not influenced by the co-diagnosis of hypertension (21% vs. 17%). Factors other than hypertension, including anemia, obesity and sodium intake, as well as the increased activity of the RAAS, vitamin D deficiency [100] and secondary hyperparathyroidism, might be involved in the increase of the LV mass in ADPKD. Note that the vitamin D receptor is expressed in renal juxtaglomerular cells, vascular smooth muscle cells and, most interestingly, cardiac myocytes. Insulin resistance could be a further factor responsible for the onset of cardiac hypertrophy in ADPKD patients [53,54]. Endothelial dysfunction, oxidative stress and nitric oxide deficiency are also related to cardiac hypertrophy owing to arterial hypertension. Alterations in fluid shear stress mechanosensitivity are evident early on in ADPKD [71,72,73,74,77]. Normotensive ADPKD patients display a loss of nitric oxide release and an associated reduction in the endothelium-dependent dilation of conduit arteries during sustained flow increase (hand skin heating), notwithstanding a preserved flow mediated dilation during transient flow stimulation (postischemic hyperemia) [77]. However, the occurrence of LV hypertrophy in young adult patients before the onset of hypertension [56,94,95] and even in children [101] suggests a role of ADPKD’s specific mechanisms in the pathogenesis of cardiac involvement. It has been reported that cardiac diastolic dysfunction is an early feature of ADPKD, as it is often present prior to the loss of kidney function and even in normotensive individuals. Bi-ventricular diastolic dysfunction has been documented even in young ADPKD patients with still normal blood pressure [102].A single-site study utilized speckle-tracking echocardiography to assess the LV function in a cohort of ADPKD patients with normal ejection fraction as compared to matched healthy subjects as well as to matched chronic kidney disease (other than ADPKD) patients [103,104]. ADPKD patients were found to have subclinical systolic dysfunction with reduced LV global longitudinal strain and twist in association with LV diastolic dysfunction. Interestingly, the impairment of the torsional function was associated to the increase in diastolic filling pressures as evaluated by Doppler measures of E/e’, that is, the velocity of the mitral valve inflow compared to the mitral annulus velocity. The subclinical changes in the LV function were considered as due to myocardial cell dysfunction, which was directly related to the disease rather than to confounding factors such as hypertension or renal insufficiency.Understanding the mechanisms involved in ADPKD-associated cardiac manifestations may open up specific therapeutic perspectives; nevertheless, the precise pathways and interactions remain largely unknown. Several in vivo studies have attempted to clarify the role of polycystin proteins in heart cells. To gain insight into the disease mechanisms and pathogenesis of ADPKD, various mouse models have been genetically engineered, including inducible, conditional knockout mice and mouse models with hypomorphic or hypermutable alleles of PKD1 or PKD2. Otherwise, the ablation of both alleles of PKD1 or PKD2 results in embryonic lethality, and heterozygous mice develop only a very mild form of the disease [105,106,107].PC1 and PC2, the PKD1 andPKD2 products, are essential for the development of the heart. One of the major sites of PKD1 expression is in the developing and mature cardiovascular system [105]. Moreover, PC1 has been found to regulate L-type calcium channel protein levels through the AKT pathway and cardiomyocyte contractility in a mouse model, suggesting a crucial role for this protein in cardiac function [108,109,110]. PC1 has been shown to act as a mechanosensor in cardiomyocytes, required for stretch-induced cardiomyocyte hypertrophy as well as for pressure overload-induced hypertrophy [108]. PC1 is a critical modulator of apoptosis and fibrosis. Cardiomyocyte PC1 plays a pivotal role in regulating fibroblast-to-myofibroblast differentiation, with implications for cardiac fibrosis and remodeling [111]. Also noteworthy is the fact that mice harboring a cardiomyocyte-selective conditional silencing of PC1 do not develop fibrosis after being subjected to transverse aortic constriction [108], supporting the role of this protein in cardiac remodeling. Moreover, PC1 mitigates cardiac damage during ischemia/reperfusion, likely through AKT activation, and regulates the connective tissue growth factor expression in cardiomyocytes [111]. The cardiomyocyte-specific deletion of PKD1 impairs the systolic and diastolic functions in mice [112].PC2 is involved in the cardiomyocyte regulation of ion transport and calcium signaling [113,114,115,116]. Heart function has been shown to depend on PKD2 activity, as evidenced by the fact that a lack of PC2 in zebrafish led to signs of heart failure. Moreover, patients with ADPKD due to reduced PC2 activity are particularly prone to develop idiopathic dilated cardiomyopathy [113]. While PC2 regulates intracellular calcium cycling, a follow-up report showed that PKD2-haploinsufficient mice present a desensitized calcium contraction coupling in cardiomyocytes and an altered response to adrenergic stimulus [114]. PC2 is also important for the regulation of ryanodine receptors’ RyR2 function, and, thus, the loss of RyR2 regulation that occurs when PC2 is mutated results in altered calcium signaling in the heart [107]. Moreover, cardiomyocyte specific PC2 knockout mice manifest an impaired autophagic flux in the setting of nutrient deprivation, due to the altered control of intracellular calcium homeostasis [115].The role of polycystin proteins for autophagy, through which the cell can remove the buildup of deleterious protein components, might affect cardiomyocytes’ sarcomeric function and impair the ability of the cardiomyocytes to contract, and ultimately results in a stiffer and less compliant heart [117]. This mechanism may in part explain the decreased torsional compliance of ADPKD patients’ hearts [104].A study that used ADPKD patient-specific induced pluripotent stem cells differentiated toward ventricular-like cardiomyocytes, confirmed the PC1 and PC2 cardiomyocyte expression and showed that the PKD mutation per se is a cause of cardiomyocyte calcium cycling abnormality and is proarrhythmogenic [118]. These findings are consistent with previous observations in mouse models. The most relevant result was the close correspondence between the iPSC-derived cardiomyocytes behavior and the donor patient’s clinical phenotype.Nevertheless, unraveling the organ-specific pathophysiology of a human disease with multiple organ involvement such as ADPKD is very challenging. Figure 1 provides an overview of the mechanisms of cardiac involvement.A relatively frequent coexistence of ADPKD and inherited cardiomyopathies such as idiopathic dilated cardiomyopathy and hypertrophic obstructive cardiomyopathy has been found out by reviewing the ADPKD database of a large tertiary center [119]. A genetic interaction between the PKD genes and the genes mutated in inherited cardiomyopathies can be hypothesized, rather than ADPKD being the direct cause of the cardiomyopathies.Taken together, data from clinical and experimental studies provide compelling evidence that the cardiovascular complications of ADPKD are due at least in part to primary manifestations of the mutant proteins. Considering the importance of the polycystin proteins in cardiac development and myocardial function, the heart should be regarded as a target organ in ADPKD patients. Understanding the pathophysiologic mechanisms of heart involvement may be the foundation of novel therapy development.L.S.: proposed the idea, proposed the structure of the paper, wrote the paper; G.G.: provided a substantial contribution to the interpretation of data from the research literature; G.E.: critically appraised the paper, made final suggestions. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.Not applicable.The authors declare no conflict of interest.Autosomal dominant polycystic kidney disease and the heart. Current understanding of pathophysiological mechanisms underlying cardiac involvement in autosomal dominant polycystic kidney disease. Activation of RAAS and other mechanisms, including the increased activity of the sympathetic nervous system, the decline of renal function, insulin resistance, disturbances in the fine- tuning of vascular tone and arterial hypertension, are responsible for cardiac hypertrophy and left ventricular diastolic dysfunction. A reduced activity of cardiomyocyte PC1 or PC2 is thought to reduce myocardial contractility and contribute to diastolic dysfunction. PC, polycystin; RAAS, renin–angiotensin–aldosterone system; SERCA, sarco/endoplasmic reticulum calcium-ATPase; PLB, phospholamban.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Systems biology is established as an integrative computational analysis methodology with practical and theoretical applications in clinical cardiology. The integration of genetic and molecular components of a disease produces interacting networks, modules and phenotypes with clinical applications in complex cardiovascular entities. With the holistic principle of systems biology, some of the features of complexity and natural progression of cardiac diseases are approached and explained. Two important interrelated holistic concepts of systems biology are described; the emerging field of personalized medicine and the constraint-based thinking with downward causation. Constraints in cardiovascular diseases embrace three scientific fields related to clinical cardiology: biological and medical constraints; constraints due to limitations of current technology; and constraints of general resources for better medical coverage. Systems healthcare and personalized medicine are connected to the related scientific fields of: ethics and legal status; data integration; taxonomic revisions; policy decisions; and organization of human genomic data.The term “constraints” was described in biology in the context of natural selection and organism survival. Biological networks are constrained by a variety of factors such as the biological, environmental and physicochemical. Biological constraints could be self-imposed and produced by regulatory networks while hard constraints are imposed by other factors (e.g., environment) [1]. Constraint-based analysis methods are being used to study genome-scale models and the biological properties of whole organisms. The constraint-based concept is used widely in systems biology (SB) from genomes to clinical phenotypes, and it is related to personalized medicine and clinical guidelines having an impact on current clinical practice and diseases. Cardiologists following patients with complex cardiovascular diseases (CVDs), such as coronary artery disease (CAD) and heart failure (HF), are complying with current clinical practice, but they are also familiarized with the clinical constraints hypothesis incorporated in medical guidelines directions.Traditional healthcare systems are using a reductionist approach explaining and managing complex diseases as they reduce—through a very simple manner—the medical problem to an isolated organ-problem or biochemical fault [2]. In traditional medicine the term “disease” designates that specific molecular systems or organs of the human body are unnaturally functioning and, therefore, subvert human health [3]. With the reductionist approach significant advances in diagnosis and therapy of CAD were applied in everyday clinical practice, with a decrease of cardiac events and symptoms and an increase in longevity. Despite these successes in clinical management, complex CVDs continue to be the leading cause of mortality and morbidity, while chronic progression of the atherosclerotic process continues. Over the past few years it has become obvious that medical issues from molecular to clinical reasoning necessitate a new scientific approach requiring cooperation between medicine and interrelated sciences. Medicine cannot be considered in isolation from other systemic sciences, and a holistic approach is needed for complex diseases. Chronic complex systemic medical problems should be addressed with the holistic approach as it needs interdisciplinary integration and study of dynamical interactions between organs’ complex networks involving genetic, epigenetic and environmental factors. Many of the medical issues are interconnected with other systemic sciences including SB. The SB approach should be regarded as the science that combines biology with physics, mathematics, medicine and many other sciences like ecology and sociology. Systems healthcare (systems medicine) is the holistic approach to health based on the holistic principle of SB and to current clinical medical practice. Systems healthcare integrates data from molecules to phenotypes and from societies to environment, extending to the disciplines of economics, ethics and law [4].The present paper discusses the constraint-based concept of SB as it is applicable to clinical cardiology and to the important clinical limitations that are present in the current practice of personalized medicine [5]. Constraints’ application to clinical decisions augments robustness to the unremittingly progressive clinical course of chronic cardiac diseases. In order to overcome some important inherent limitations in contemporary clinical cardiology, the interdisciplinary approach between a constraint-based concept and personalized medicine is underlined. In clinical practice, cardiologists, unknowingly to themselves, are practicing in conformity with the concept of constraints and that is in accordance with current medical guidelines. The concept of constraint-thinking is a significant decision-making clinical tool which constantly and in a timely fashion is revised by medical advances in prophylaxis and clinical management. Complex cardiovascular diseases like CAD and HF are multifarious in clinical presentation revealing, also, a progressively advancing natural clinical course. Cardiovascular diseases are complex biological entities produced by a combination of genetic and environmental factors. Furthermore, personal reaction to pharmaceutical therapy and drug effectiveness and toxicity are the result of the interactions between genetic profiles and environmental factors [6]. The holistic approach is based on the integration of a network components and functions (e.g., genetic, molecular or environmental) during the different stages of disease progression. Personalized medicine and clinical constraints are considered essential concepts of the holistic principle of SB in following complexity of the disease states and clinical progression.The traditional biological description of the living world is based on the nonlinear interactions of molecular processes without explanation for their functional interconnections [7]. The advances of molecular biology proved successful in studying isolated molecules and some of their interconnections, but were unsuccessful in experimentally predicting complex phenomena such as complex disease progression. Traditional and molecular biology are based on the classical reductionist understanding that differs from the SB holistic perception. Reductionist understanding of the biological phenomena “is mostly understood as a means to explain phenomena generated by systems in terms of the properties of their parts, often when considered in isolation” [7]. With the holistic approach, complex wholes are understood from the properties they possess as “whole systems” and not from the behavior of the isolated parts. The holistic principle analyzes the structural organization and regulation of biological networks, deciphers complex signaling mechanisms and interconnections and discloses positive or negative feedback mechanisms [8]. From cellular mechanisms to phenotypes, functional properties are “emerging” through a self-organized procedure which complies with the foundations of a hierarchical multileveled system. Systems biology is regarded as the science that somehow “replaced” molecular biology with emphasis on the construction and explanation of bigger biological systems such as networks and signaling information [9].The aim of SB is to ascertain how function and behavior of living organisms are justified by the interaction of their constituents [10]. Thus SB is a scientific discipline that analyses living organisms and biological networks (systems) from the level of genomics and molecules to phenotypes. The biological networks demonstrate hierarchical structuring composed of collaborating biological components (nodes). The complex interactions (detectable or unnoticed) existing between nodes constructs a network system with new emergent properties that reflect health related behaviors or different disease states [11]. Systems biology methodology with its integrative computational analysis constructs interacting and integrating network processes and models of clinical phenotypes having an impact to disease progression and therapy [12]. The SB approach, besides the concept of network construction, uses two potential directions for studying and explaining complex diseases: the bottom-up direction (indicates progression from genes to phenotypes) and the top-down direction (indicates decomposition from phenotypes to genes) [13] (Figure 1).The bottom-up direction is important to personalized medicine, as upon the emergence of new properties in each level of the disease progression the possibility of exploration for new diagnostic biomarkers and drugs is increased. The top-down direction causes the enforcement of constraints (forcing boundaries) imposed by the higher order phenotypes or modules to the molecular or genomic lower level, independently of lower order changes [14,15].Dunbar [16] underlines that “causal reasoning in science is not a unitary cognitive process, but a combination of very specific cognitive processes that are coordinated to achieve a causal explanation”. In clinical medicine the causal dependency between stages of disease’s progression and the extraction of useful information in downward direction is recognized. The extracted information is useful to practicing physicians to impose diagnostic and therapeutic constraints. In a downward direction the physician has decreased degrees of freedom, but he selects the most available and clinically suitable diagnostic and therapeutic applications.During progression of a complex disease, while the genes produce proteins for tissues and organs in a bottom-up direction, it is the disease’s phenotype in a downward causation that determines the kind of proteins that are needed. The downward causation of SB is a holistic principle that objects the classical reductionist position of biology. Based on the holistic principle, behavior of the lower level is regulated by the behavior of the higher level, which in the case of complex CVDs imposes constraints on clinical progression and management. The top-down constraints demonstrate also the impact that the hierarchical higher level of phenotype has on modular clinical level, a fact with significant repercussions for progression and mode of treatment for complex diseases.The SB emerges as a discipline to expound the complexities of human physiological status and diseases such as CVDs. From the perspective of clinical phenotype it is essential the construction of appropriate networks in each step of complexity, from the genomic, molecular, modular to phenotypic level (Figure 2).The specific organizational properties and behavior of each complex biological network should be explored. Networks should be constructed not only separately in each level of complexity of the biological ladder, but also between the successive levels of the complex disease that will facilitate the integration of each molecular component or clinical finding in the “appropriate” network position. This approach will reduce chances for “wrong” positioning of data, and the biomedical research will not be isolated or “indifferent” from the clinical momentum.The bidirectional transfer of information, together with the system of interconnected networks, increases the possibility of scientific communication and interexchange of ideas between groups of people with diverse talents and scientific fields. The challenge remains to construct that kind of networks by a multidisciplinary participation of researchers and clinicians. In the mind of a clinician this approach of integration of facts from conceptual networks and the simultaneous integration of bidirectional information is more than relevant and clinically fruitful. In reality, the interplay of biological and clinical factors reflects a multileveled system of networks, models and their interrelationships. This approach increases the necessity for SB application in the complex clinical medical field. In order to decipher clinical entities, the interexchange of ideas should be completed by including the concept of network medicine. In the interconnected whole, small networks are lodged or connected to larger networks in order to have a coherent entity. Complex disease does not behave like a well organized biological machine, but should be visualized as a complex network system. In a network system the biological components occupy specific network positions, and the interactions between the components formulate “logical” higher functional networks and models. The recent advent of network medicine as a tool of clinical research gives a new perspective to the “nature” of complex diseases [17]. The network concept is important for translating in a more meaningful way interconnections of collected health and clinical data. Complex disease should be visualized as an aggregate of malfunctioning complex cellular and molecular networks that induce organs’ failure and in the end specific phenotypes. Fiandaca et al. [4] suggest that “the diseased organ… produces a cascade of dysregulated networks, resulting in associated co-morbidities”… while “in the state of wellness, networks are precisely regulated via complex homeostatic mechanisms”; and that specific therapeutic intervention “requires aggregating multi-dimensional datasets…high-performance computation and analytics” while “the goal is to determine interventions that target abnormal networks and promote systems level improvements”.The application of constraints in medicine and clinical cardiology presumes that the constraint-based hypothesis, as it is implemented in biological and medical domains, should reflect a systems’ robustness. Also, the concept of constraints should be considered as a regulatory mechanism not only for molecular or other biological networks but also it is a decisive factor for medical decisions. Based on these assumptions the application of constraints encompasses three fields related to clinical cardiology: biological and medical constraints applied to cardiovascular diseases; constraints due to limitations of current technology; constraints of general resources for better medical coverage (Table 1).The constraint-based thinking constitutes a holistic concept with downward causation interpreting many complex features of biology and diseases. In biology the term “constraints” is referred to antagonistic biological processes addressed to every evolutionary change in order to strengthen natural selection. Also, the constraint-based reasoning was applied to experimental biology to strengthen robustness of some experimental biological models [18].Green and Jones [19] believe that the constraint-based interpretation of biological entities differs from mechanistic thinking of “change-relating causal features”, while the “constraint-based explanations emphasize formal dependencies and generic organizational features that are relatively independent of lower-level changes in causal details”. Furthermore, SB evaluates functional properties of living organisms and explores structured biological entities of genetic regulatory and metabolic networks, and the dynamics of enlarged networks in the form of modules (discrete functional regulatory networks) and phenotypes [13]. It seems that the term “constraints” implies the presence of scale-dependency and close connection between biological systems functioning at different levels with strong downward causation (top-down effect). The top-down direction is crucial at first for information extraction from the lower levels and secondly for the capacity of the higher level to enforce constraints to lower levels with decreased degrees of freedom. The downward causation is interpreted as a regulatory constraining process that modifies and, in the end, determines behavior of lower level variables. The applied constraints limit some behaviors at the lower stage and simultaneously allow or “authorize” alternative behaviors to be released [14,15,20].For example, the boundary of cardiac cell geometrical structure generates both cellular membrane potential and cardiac rhythm. The imposed constraints of cellular membrane boundary structure and the triggered cardiac action potential through downward causation are responsible for the appearance and maintenance of cardiac rhythm [15]. The above example indicates the limits of the reductionist position as cellular membrane, action potential and cardiac rhythm do not related directly to the genetic scale. The whole cellular membrane construction and function belong to a higher level of cardiac cellular construction.Multileveled complex CVDs progression can be translated as a staged (leveled) structure with downward causation and constraints application from the top (phenotype) to the lower step (genome) of the disease. The top-down constraints’ application actually represents downward causation “imposing” some “biological behavior” or “order” to the lower level of the disease (pathological, diagnostic or therapeutic). The concept of constraint-based reasoning is proposed as a significant scientific tool for cardiovascular questioning and clinical research organization. Implementation of constraints in clinical cardiology has an impact to explain some properties of disease complexity and, also, to elucidate the unrelenting progression towards final disease stages. In fact, medical guidelines are founded on the constraint-based concept having downward causation. The cardiologist has decreased degrees of freedom, as only specific diagnostic tests and therapeutic procedures are available. In a way, the cardiologist “selects” the appropriate methodology according to current clinical guidelines using diagnostic and therapeutic constraints in a downward direction. In the realm of SB thinking, the applied constraints increase robustness of the regulatory processes for the stability of the unsteady metabolic networks and, also, for the variability of clinical complex entities [5,21,22].In this paper, it is proposed that the constraint-based thinking could be used not only as a concept for metabolic networks, but also as a fundamental clinical tool deciphering progressiveness of a disease’s clinical course. Both cardiac atherosclerotic process and CVDs are considered complex entities that follow a downward direction and causation in pathogenesis or in clinical management [11]. For example, the size of a myocardial infarction and particularly the location and the importance of the myocardial area involved would induce a post-infarction myocardial dysfunction alongside of some compensatory mechanisms such as myocardial remodeling and growth of local coronary collaterals. According to SB approach, the size of myocardial infarction (phenotype) will impose constraints with downward direction on the degree of myocardial compensation and will dictate the most suitable medical and/or coronary invasive therapy, percutaneous coronary intervention (PCI) or bypass surgery.In another example, patients with cardiac ischemic high-risk features are recommended for clinically indicated PCI and treated with dual antiplatelet therapy (DAPT) in the post-PCI period according to 2017 DAPT guidelines [23]. Some patients demonstrated an increased number of ischemic and bleeding episodes following PCI which influenced the decision for intensity and duration of the DAPT in the post-PCI period. Therefore, pre-PCI constraints should be used in some of these patients with high-risk features if the invasive procedure is unavoidable or the DAPT regimen should be revised. A more personal approach with constraints in medical decisions and DAPT application is obligatory to some of these patients.Human HF is a complex cardiac disease characterized by chronic clinical progression that involves participation of intrinsic compensatory or regulatory mechanisms [13]. The SB methodology, to unravel potential causes of HF progression from early stages of myocardial dysfunction to more advanced phases of myocardial failure, integrates genes, epigenetic mechanisms and molecules, deciphers molecular networks or modular functional elements, and clarifies the interconnection of myocardial mechanical dysfunction with cardiac remodeling and other compensatory mechanisms [24]. Heart failure should be addressed as a biological complex entity that is unstable, adaptive and self organized through its regulatory mechanisms (Figure 3).The regulatory mechanisms incorporate neurohumoral and remodeling systems that intend to compensate failing myocardium and change the unstable clinical equilibrium to a more stable clinical equilibrium status. The size of myocardial dysfunction in HF patients is related to the degree of compensation by the regulatory mechanisms. Thus, activation of the compensatory mechanisms (neurohumoral and remodeling) represents a constraint-based control (downward causation) of the degree of the compensation from the higher level (HF phenotype) to lower level (regulatory mechanisms). However, the activation of the regulatory mechanisms is often the cause of unwanted effects (symptoms) and clinical deterioration needing specific treatment to improve clinical status. The degree of clinical deterioration (by the regulatory mechanisms) in each stage of a disease’s progression dictates appropriate personalized treatment. Information and communication technologies could help through collection of related clinical data mined from published papers to improve management of HF patients. This could be achieved by the identification of the related biological networks that connect data in each level of HF progression and, after monitoring, the activated compensatory regulatory mechanisms from genome to clinical phenotypes can be clarified (Figure 4).In clinical practice, constraints may arise for diagnostic and therapeutic interventions due to limited facilities of current technological status.New technologies require “cost effectiveness studies in the presence of health care input constraints” and crucial adjustments of conventional incremental cost effectiveness ratios (ICERs), because “without such adjustments the cost effectiveness analysis might lead to health losses” [25] (Figure 5).For example, novel techniques based on computer simulation, a process of mathematical modeling, are designed to predict atherosclerotic changes in coronary arteries. Such techniques are: positron emission tomography (PET), with the use of 18F-fluorodeoxyglucose, can label metabolically active areas in the myocardium and arteries; magnetic resonance imaging (MRI) can produce a molecular imaging of the cardiovascular system; cardiac computed tomography (CT) or CT angiography can be used for coronary calcium scoring and to measure subclinical or asymptomatic obstructive CAD; optical coherence tomography (OCT) for atherosclerotic plaque composition and stability.In coronary artery areas with bifurcations or curbs there seems to exist a strong connection between concentrations of circulating plasma low density lipoprotein and turbulent flow with the development of atherosclerosis. Digitized images of coronary arterial post-mortem segments were analyzed with a computational fluid dynamic analysis, and the critical role of the local low wall static pressure was underlined for coronary wall thickening as a precipitating factor in the pathogenesis of coronary atherosclerosis [26]. Another example of new technology is the Heart Flow Analysis, a system based on cloud services that offers non-invasively detailed information of coronary arteries and is used instead of an invasive cardiac procedure. The Heart Flow Analysis is scheduled to support the functional evaluation of CAD. It produces a personalized 3D model of coronary arteries using computed tomography (CT) images constructing a fluid dynamic model of the coronary blood flow. It identifies and calculates the size of coronary obstruction and advises cardiologists for further management. All new technologies have a positive cost-effective value for health care systems as they diminish current constraints for medical risks, and also reduce the high cost on health spending.Digital health technologies aim to increase health care decision-making and improve health management. They represent actually “a broad spectrum of measurement technologies that include personal wearable devices and internal devices as well as sensors…but the current state of technology development and deployment requires… a cautionary note” [27]. Also, digital health technology can “identify health risks and assist with diagnosis, treatment, and monitoring of health and disease conditions” [28]. To some of the patients, digital health applications can offer new diagnoses and chances for novel treatment, but worldwide use of new digital health devices will need clinical trials to prove their usefulness [28]. Therefore, the existent constraints for the full value of digital health technologies will be retracted when the new technologies become standardized and interoperable during clinical trials, and when new clinical guidelines have incorporated digital health devices [27].Gaveikaite et al. [29] suggest that telehealth services can increase the “quality of health services for chronic obstructive pulmonary disease (COPD) management” and in those patients “complex interactions between multiple variables influence the adoption of telehealth services” for COPD by different healthcare professionals. Moreover, some constraints remain as “key variables were identified that require attention to ensure success of telehealth services” but “there is no consensus where self-management services should be positioned in the COPD care pathway” [29]. Thus, medical practitioners or researchers from other fields, such as pulmonary diseases’ practitioners, computer science and network science researchers, can contribute their expertise to a common cause to explore further complex and interrelated human disease conditions.Significant constraints are raised when the genetic base of chronic atherosclerotic disease is explored with modern genetic technologies. In complex cardiovascular atherosclerotic disorder the importance of genetics is elusive, as the disease is multifaceted and is not explained by single-gene mutations. In reality, the diverse phenotypes of CAD represent integrated clinical wholes with clinical behavior continuously changing due to the progressive nature of atherosclerotic process. Current understanding of genetics and genomics, as well as genome-wide association studies (GWAS), are inadequate alone to explain the natural course of cardiac atherosclerosis. It seems that “the genetic risk variants of atherosclerosis are activated concurrently with functionally active specific environmental risk factors” and that cardiac atherosclerosis could be studied only as a unified complex entity [11]. The GWAS approach was expected to trace statistically significant interrelationships between single nucleotide polymorphisms (SNPs) and atherosclerosis. It was expected that SNPs related to atherosclerosis to be more frequently present in CAD patients than in control individuals, but, in contrast, a variety of genomic DNA markers were detected in individuals without CAD. Also, it was found that genomic technologies as SNP array, gene expression microarray and micro-RNA array were unable to demonstrate accurately the genetic atherosclerotic profile of CAD people. With the GWAS approach, it was found that only 10.6% of individuals with atherosclerosis possessed a probable heritable genetic factor. More important was that large-scale association analysis identified new risk loci for CAD, and that DNA methylation-mediated epigenetic downregulations and histone modifications triggered by lifestyle features play a vital role in atherosclerosis [30,31,32]. The GWAS approach, based only on genetic variability, does not identify and clarify the vessel wall pathological changes or the clinical progressive nature of CAD phenotypes [21].The World Health Organization determines that a biomarker is “any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease” [33]. The role of genetic biomarkers is limited and the genetic variation is of uncertain significance in clinical “whole exome sequencing” (WES), requiring continuous revision when clinical interpretation is demanded [34,35]. Moreover, WES technologies are evolving using new diagnostic tools and medical clinicians and laboratory scientists can increase further their knowledge for complex diseases and tailor unique therapies for individual patients. Timmerman [34] stresses the importance of standardization in laboratory research and argues that “the match between phenotype and genotype is circumscribed by the team’s reliance on specific standards”. As an example, he describes how a “clinical exome sequencing team” elects the time when to trust standards and a clinical exome sequencing technology will make “the transition from a laboratory research tool to a routine clinical technique used to diagnose patients”.Emerging technologies are developing for “multi-omics studies and an increasing shift toward proteomics-going straight to the heart of biology that represents actual disease state and progression” and “to gain insight into the pathophysiology of disease and to identify proteins that are causally associated with disease, providing new targets for effective drug development” [36]. Regardless of the advances in metabolomic methodologies that succeeded to produce thousands of molecules or biomarkers—some of those related to cardiology—many more are needed to give a new description of clinical phenotypes. It seems that more imaginative holistic approaches and new methodologies are needed to define novel clinical cardiac phenotypes. New multimodal systems of “omics”, metabolic pathways, environmental impacts and sophisticated disease-related networks are required to be integrated and provide a new holistic and realistic picture.Health economic evaluations and the results of cost-effectiveness analyses studies are helpful for decision makers to confront the main economic constraint, the health care budget. In reality, besides the health care budget, there are “multiple other resource constraints that are involved relating, for instance, to health care inputs such as a shortage of skilled labor” [25]. There are, also, other constraints involved, “consisting of supply-side (e.g., workforce shortages), demand-side (e.g., obstacles of access to healthcare) and healthcare system constraints (e.g., regulatory constraints)” [25]. Complex CVDs swiftly increase their complexity changing pathology and course while clinical stages are overlapping. Moreover, alterations of the clinical course are approached differently in each phase of the disease. Clinical approach is modified in each step of the disease guided by current clinical, diagnostic and therapeutic constraints. Both, health system and practicing cardiologists are responsible for the wise use of the available resources and, also, to increase patients’ longevity. In view of the chronic and progressive course of the CVDs and in order to eliminate health disparities between underserved communities health authorities should define specific medical strategies and remove imposed constraints [37].The pre-hospital management strategy for patients with acute coronary syndromes (ACS) is a strong example from a medical or ethical point of view that requires coherent relations among patients and cardiologists. Patients with chest pain need immediate medical attention in an emergency cardiology department for further management that includes probable admission in acute coronary unit and PCI. However, there are worldwide limitations to further management of these patients due to restricted resources and insufficient organization (constraints). Patient’s transfer to the nearer medical center should be imperative following standard procedures and avoiding unnecessary bureaucratic retardations. Invasive procedures such as PCI and bypass surgery vary on their outcome from medical institution to another due to differences in expertise and resources. Thus, local medical circumstances can limit (constrain) medical decisions and practices diverging from current medical guidelines.Systems biology was followed by a new concept termed “systems medicine”, a global and holistic approach that collects diverse longitudinal data for each individual [38]. The collected data can be used to explain the complexity of human biology and disease after evaluation of both genetic and environmental determinants [38]. Price et al. [39] comment on to the above data “as personal, dense, dynamic data clouds: personal, because each data cloud is unique to an individual; dense, because of the high number of measurements; and dynamic, because we monitor longitudinally” and these “data clouds embody the essence of precision medicine” [39].The Hundred Person Wellness Project (HPWP) is a 10-month pilot study of 100 “well” individuals and it is focused on “optimizing wellness through longitudinal data collection, integration and mining of individual data clouds” and to “identify markers for wellness to early disease transitions for most common diseases” [40]. The term “personalized medicine” (precision medicine in USA) “describes the ability to tailor diagnosis, prognosis, and therapy-ideally to individual patients, but at the very least to stratified patient groups” [41]. The National Institutes of Health (USA) defines precision medicine as “an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person” [42].The term of personalized medicine refers to the holistic principle for a tailored and exclusive medical care approach for refined personalized therapy and superior drug safety and efficacy. The SB concept aimed to an extensive cognitive change and a reappraisal of human diseases, and, also, contributed to evolution of novel technologies with more sophisticated explanation of human complexity that motivated the emergence of individualized medicine [43]. In personalized medicine patients are examined as “persons” and not only for symptoms and clinical signs. Cardiologists are not concentrated to angina only as a symptom but they are inquiring about personal or family medical history as well as patients’ preferences for their mode of treatment. Consequently, personalized medicine and medical guidelines are not equivalent conceptions in clinical thinking and practicing, especially for chronic CVDs. Guidelines are addressed to a specific group of patients as an ensemble of persons but disregard the “individual” patient with his personal or family medical history and his preferences for further disease management. Preferences for invasive or noninvasive therapy and variations in therapeutic effectiveness of pharmacologic agents are medical aspects to be decided between practicing cardiologist and patient.Personalized medicine, clinical guidelines and constraints are considered interrelated scientific fields for management and follow-up. Patients with chronic CVDs progressively deteriorate and need specific management in each stage of the disease. It is mandatory for a cross-disciplinary collaboration between scientists and clinical experts from the biomedical and clinical research fields in order to interexchange knowledge and skills and answer the multiple questions for unmet medical needs encountered by clinicians. Aboab et al. [44], proposed a model of data analysis to increase the reliability of published biomedical and clinical research. That is an important and welcome step for health care and research. The integrating, inter-professional and interdisciplinary collaboration of scientists with clinical experts, for meticulous analysis of health data, is a step forward to a more effective use of clinical and research findings. Complex diseases are chronic processes with an extended interplay of variables and reliable ideas [45].However, in the field of complex diseases or complex clinical situations, there remains the problem of clinical application for biomedical research findings, and the cooperation between researchers and non-researchers is more complicated. That involves the design of a complex disease modeling with a methodology that translates research findings and connects different pieces of knowledge and compares evidence. It is common knowledge that complex diseases are incomplete and inconclusive in their conception.The constraints’ concept is relevant to health services and medical technology, and available management procedures should be implemented accurately in all stages of CVDs knowing their chronic progressive nature as well as advances on the technological availability [46]. The field of digital medicine is promising but requires continuous validation based on large randomized clinical trials. In patients with CVDs, clinical evaluation using telemedicine with external sensors application to track-down important clinical data should be further assessed [47]. Constraints should be applied to clinical approval for digital medical information obtained from outpatients having a complex clinical picture. Digital medical information without further verification with well controlled randomized clinical trials should not be accepted. Medical guidelines are based on large randomized clinical trials (RCTS), but to establish effectiveness of procedural cardiac interventions precise restrictions (constraints) are implemented in the designed trials [48]. Furthermore, in everyday clinical practice, literal interpretation of the results of RCTS could be hazardous, with high risk, if medical proficiency and local technical support are not appropriate [49].Systemic medical approach requires different national health policy in accord with national laws and international consensus. A new policy includes the use of new cost-effective technology and data collection, while mandatory is patients’ cooperation. The “individualistic autonomy” notion is the dominant thinking in clinical practice and research, as well as between patients, medical practitioners and health authorities. With this term it is acknowledged that patient’s absolute prerogative is the choice of his medical management. This assumption is medically inappropriate considering complexities and constraints involved in medical management of patients with diverse cardiac clinical phenotypes. Medical guidelines and common sense support the notion that medical practitioners are strongly “related” to medical decisions and, thus, a more realistic position would be the “relational autonomy” of a patient’s decision [50]. The “relational autonomy” position is associated with the concept of constraints proposing restrictions to the individualistic approach while encourages patient-medical practitioner cooperation. Furthermore, the psychology to implicate guidelines and modern technology to clinical practice requires common sense from both patients and medical practitioners, while at the same time evaluates risks and benefits [51,52]. Sometimes, an unreasonable attitude from both patients and doctors who doubt and enquire negatively the value of modern medicine has consequences to rational application of constraints in clinical decisions. If the medical knowledge that is related to scientific data and technologies is shared between clinicians and patients, then ethical transparency for disease’s detection and treatment will improve [53].In personalized medicine a lot of ethical and moral arguments are raised by both worried and anxious patients and medical authorities [54]. It is understandable that new genome editing technologies which have the capability to introduce targeted genomic sequence changes can transfer them to next human generations [55]. These technologies not only can transform biological research and develop novel molecular therapeutics for human diseases, but they also produce ethical problems. Genome editing is a personal decision, but some ethical problems arise in the society for unwanted accidental gene mutations, and for the cost of genetic testing. The new editing technologies should secure the highest standards of research, data collection and applicability ethics.Numerous national healthcare organizations are implicated to collect, store, analyze and interpret human genomic data for biomedical research and medical application. However, in top-quality digitized health care systems deviations are observed in coding and collecting data due to different regional clinical practices. Collection and management of human genomic data give rise to some concerns about ethical, privacy and legal problems, or for unauthorized access or misuse of data [56,57]. Constraints emerge when private information and sensitive healthcare data are unprotected due to deficient measures taken for privacy and security [58]. The use of advanced technology for the prediction of future individual healthcare requirements needs data protection as inadequate security can affect a large number of people [59]. Electronic health records, sensors and servers contain a growing volume of digital data for use in healthcare and disease management [60]. The extensive use of big data on healthcare organizations are “ranging from single-physician offices and multi-provider groups to large hospital networks and care organizations” [61]. Bossen et al. [62] argue “that useful data require encounters between people, technologies, and data… routed in particular places and particular times and require effort on the part of the people involved”. To harness emerging disease data, a committee from the National Academies of Science [63] suggested a framework for an information system called a Knowledge Network of disease that “integrates the rapidly expanding range of information on the causes of disease” enabling researchers, medical practitioners and the public to get knowledge of the produced information. The Knowledge Network will help researchers to conceive disease mechanisms, and medical practitioners to initiate new treatments based on distinctive disease characteristics and adapted to each patient [63].Under the definition of personalized medicine, large number of data are gathered and integrated from different sources namely sequencing genomes, molecular banks, accumulated “omics” data, and references from clinical studies, bioinformatics and current guidelines. In patients with complex CVDs, data for comorbidities, environmental parameters, socioeconomic status nutrition and social habits are assembled, integrated and conceived as a whole [64].The SB approach is used in current medical research to reveal hidden biological pathways and also identify unseen biomarkers and design novel drugs. Moreover, SB is helpful to comprehend disease progressiveness and complexity, and clarify drug safety profiles and efficacy [65]. It is argued that novel therapies are nevertheless in early stages due to “limited accessibility of robust and affordable molecular systems biology platforms” [65]. In future, general public health and current clinical medicine will change with the application of specific preventive and therapeutic programs focused on the individual patient [66]. This approach would be successful through interconnection of the available electronic medical records with new discoveries in the fields of biomarkers and pharmacotherapy [5].A bioinformatics team (Biochemical Pharmacology Discussion Group, BPDG) scheduled a robust biomarker strategy to identify disease-related biomarkers and provide drug candidates [67]. The BPDG reported two main methods for designing pharmaceutical drugs: the traditional drug discovery (TDD) and the phenotypic drug discovery (PDD) [67]. Thus, for drug discovery, biomarker-based mechanisms are targeted (TDD method) or biological compounds are tested until final improvement of the phenotype became evident without considering the responsible molecular mechanisms (PDD method).Pharmaceutical companies helped by one or more AI-based (Artificial Intelligence) drug development companies were able to give priority for interaction to some of the hundreds of implicated proteins. In CVDs, the target is more than one protein, a network of interacting proteins is needed. In this aspect, AI plays a significant role as it matches the properties of thousands of molecules having pharmaceutical potential to the properties of proteins involved in a complex medical disorder, thus disclosing molecules able to bind to a protein-target.A new conception of disease taxonomy with patients’ stratification based on precise individual biological status is needed. Revision of current disease taxonomy to a personalized oriented medicine is a difficult task and requires important changes to structure and management of the whole healthcare system that implicates organizational and political issues. Personalized medicine to be established needs to overcome significant constraints implemented in different fields of clinical medicine and public health system. At the same moment, existing medical knowledge and practices should be revised with new data related to diagnostic biomarkers (innovating disease causation), phenotypic categorization, treatment revision and reorganization of healthcare system. Medical and social determinants that a personalized approach would include, needs a multi-disciplinary contribution. Some of the constraints and regulatory challenges are analyzed below and should be embraced by both the society and medical health system. Personalized medicine provides further rationalization to current guidelines-based clinical medicine. With personalized medicine it is expected that there will be an overhaul to some of the constraints present inside guidelines and to propose reorganization of existent disease classification with a new taxonomy.In an important “expert consensus report” a committee of experts appointed by the National Academies of Science [63], emphasized that “a new data network that integrates emerging research on the molecular makeup of diseases with clinical data on individual patients could drive the development of a more accurate classification of disease”. Also, in the above report was stressed that biomedical research data will help to develop a “New Taxonomy” which will define disease “based on underlying molecular and environmental causes, rather than on physical signs and symptoms” [63]. Furthermore, together with improvement of health status the biomedical research will advance because of the access to the patient’s information “through electronic health records, while still protecting patient rights” [63].A new taxonomy of human complex CVDs could restructure diseases’ diagnosis, therapy, mode of progression, and critical clinical directions towards to more individualized management.Green et al. [68], mentioning personalized medicine argue for “an urgent need for finer-grained disease categories and faster taxonomic revision, through integration of genomic and phenotypic data” while their analysis is associated with the Danish National Genome Center and its endeavor “to bring Denmark to the forefront of personalized medicine”. They mention, also, “how persistent tensions in medicine between variation and standardization, and between change and continuity, remain obstacles for the production as well as the evaluation of genomics-based taxonomies of difference” [68]. Green et al. [68], delineated how “the new taxonomy is supposed to be developed” and have proposed a meta-taxonomy of taxonomy revisions as a basis for discussions. In this meta-taxonomy field they included four new, “fine-grained disease categories” based on: (a) stratification into subgroups of diseases; (b) reclassification of previous categories, “merging previously distinct categories according to shared molecular characteristics”; (c) clustering of disease and risk groups, “based on a network of risk factors and observed co-morbidities”; (d) expansion of current disease categories.To explore new disease taxonomy will inevitably trigger some constraints of acceptance by the political and academic systems due to prevalent and deep-rooted problems. Change of disease taxonomy needs modifications to the entire health care system, a difficult problem for countries having fewer centralized and digitalized facilities. National personalized medical projects have been developed aiming to upgrade diagnosis and therapies, particularly for chronic complex diseases, giving rise to ethical and administrative problems. Persuasive national strategies for personalized medicine are those of the 100,000 Genomes Project in the UK and the All of Us Research Program in the USA [69,70].These national strategies include: data accumulation, creation of infrastructure, and organizing interexchange of data with physicians, nurses and genetic counselors [71]. Policy strategies by health authorities and implementation of personalized medicine in clinical practice are promising ambitions. Political, economic and scientific interests for personalized medicine are challenged by the shortage or absence of convincing evidence for wide clinical application of genomic data. It appears that not enough data have been collected yet and there are some uncertainties for clinical application [35]. It is acknowledged the urge for new health technologies and the additional investment to tackle the constraints in healthcare systems. These healthcare constraints include “a shortage of health workers, ineffective supply chains, or inadequate information systems, or organizational constraints such as weak incentives and poor service integration” [72].The committee of experts appointed by the National Academies of Science (USA) outlined a course of action for the construction of a Knowledge Network of disease with the potential to develop a New Taxonomy defining disease [63]. A Knowledge Network based on a centralized database “continuously revises and validates new disease categories” after the integration of genomic and health data, but “integrating data… are very challenging due to the existence of diverse practices for diagnosis and coding” and needs “regulatory amendments, such as data standardization” [68].In a recent Comment about ���better governance of human genomic data” it was argued that for “the collection, storage and curation of human genomic data for biomedical research”, some “genomic data repositories and consortia have adopted governance frameworks to both enable wide access and protect against possible harms” [73]. The authors of this important Comment explain, also, that purpose of this document is “the identification of the functions that governance of genomic data should fulfill”, and also to demonstrate the governance frameworks of six large-scale international genomic projects [73]. The information presented through the six genomic projects is intended primarily to demonstrate the identified governance functions and describe the differences in transparency concerning the information they produce about their governance approaches. There are constraints when access to genomic data is required for biomedical research. There is limited access to health resources or “unequal opportunities for researchers to access and analyze data… because of limitations in human capital, fiscal resources and technological sophistication” [73]. Constraints are applied when genomic data between groups are interpreted, as there are differences in the capacity to benefit from generating genomic data. For example, in the GWAS, which comprise the main source of information for genetic reference databases, 88% (2017) of the genomes still belong to people of European descent with 72% of participants recruited from three countries (USA, UK, Iceland) [74].The governance frameworks have been initiated to assist biomedical research but systems’ persistent inequalities obscure “the contributions and the important role of different data providers” [75]. It is recommended that “governance of science becomes more transparent, representative and responsive to the voices of many constituencies by conducting public consultations about data-sharing” [75]. An example of effective governance framework is the Human Heredity and Health in Africa (H3Africa) Initiative (https://h3africa.org/ accessed on 9 February 2021) that underlines the genomic research that benefits African populations [73]. The H3Africa promotes research of genomics and environmental factors of common diseases, with the objective to improve the health status of African populations, generating new data [73].Coronary artery disease and heart failure are complex and self-organized chronic and progressive entities. Human disease complexity can be explored with the holistic principle of systems biology. Two important interrelated holistic concepts are described, the emerging field of personalized medicine and the constraint-based thinking with downward causation from the phenotype to molecules and genomes. Constraints (limitations) in cardiovascular diseases include limitations to the biological and medical field, constraints to the use of current technology and constraints in health expenditures. A more aggressive healthcare cost-effective approach is required that integrates into the system any economic-based constraints for new technologies. Personalized medicine requires taxonomic revision, data integration and policy decisions concerning ethics and legal status in social regulations and economic issues. There is an evidence-based reality for personalized medicine, but this requires a determined decision-making medical and political personnel.Both the authors contributed equally to the preparation and submission of the research. Both authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.The authors report that they have no financial or other relations that could lead to a conflict of interest.Concepts of systems biology: bottom-up and top-down directions; disciplines (complex networks); emergence of new properties; constraints application (robustness). (Revised from: [5]).Coronary artery disease: The network complexity between systems biology discipline levels and environmental factors. (Revised from: [11]).Progression of complex heart diseases: Relationship between emergent properties and constraints outlines progression of complex heart diseases. CAD (Coronary Artery Disease), HFpEF (Heart Failure with Preserved Ejection Fraction), HFrEF (Heart Failure with Reduced Ejection Fraction). (Revised from: [5]).Progression of coronary artery disease (CAD) and heart failure (HF) explained by systems biology approach. HFpEF (Heart Failure with Preserved Ejection Fraction), HFrEF (Heart Failure with Reduced Ejection Fraction), MI (Myocardial Infarction), ACS (Acute Coronary Syndromes), SA (Stable Angina).Constraints in three fields related to clinical cardiology; they behave as regulatory mechanisms for complex disease networks and medical decisions.Constraints and personalized medicine in cardiovascular diseases.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Sudden death, especially at a young age, may be caused by an underlying genetic cause. Hereditary conditions with an increased risk of sudden death at a young age include cardiomyopathies, arrhythmia syndromes, and hereditary thoracic aortic aneurysms and dissections. The identification of a genetic cause allows for genetic testing and cardiological surveillance in at-risk relatives. Three sudden death cases from our hospital illustrate the value of autopsy, genetic, and cardiological screening in relatives following a sudden death. On autopsy, histology consistent with hereditary cardiomyopathy is a reason for the referral of relatives. In addition, in the absence of an identifiable cause of death by autopsy in young sudden death patients, arrhythmia syndrome should be considered as a potential genetic cause. Sudden death is defined as a nontraumatic, unexpected fatal event occurring within one hour of the onset of symptoms in an apparently healthy subject or, if death is not witnessed, the definition applies when the victim was in good health 24 h before the event [1]. Sudden cardiac death, especially at a young age, warrants consideration of an underlying genetic cause. On autopsy, histological observations consistent with hereditary cardiac disease, such as myocardial disarray, are an indication to discuss genetic testing and cardiological screening with relatives irrespective of the age and circumstances at demise. Examples of histologically recognizable disorders, with an often identifiable genetic cause, include cardiomyopathies and dissections of the thoracic aorta [2,3]. In the absence of an identifiable cause of sudden cardiac death at a young age, which is termed sudden arrhythmic death syndrome (SADS), hereditary arrhythmia syndromes must be considered as a potential cause.In a recent study among 302 cases of SADS in the young, next-generation sequencing of an extended gene panel revealed a genetic cause in 13% of cases, mainly in genes associated with long QT syndrome (LQTS, KCNQ1, KCNH2, and SCN5A), catecholaminergic, polymorphic ventricular tachycardia (CPVT, RYR2 gene) and, to a lesser extent, genes associated with cardiomyopathies. Combining genetic testing with cardiological screening in the first degree relatives resulted in an increase to 39% of cases [4]. It is important that pathologists and other health care providers involved in the care for patients with sudden cardiac death are aware of the increasing possibilities of genetic testing in these disorders [5]. Postmortem genetic testing in these patients may be lifesaving for their relatives when a pathogenic variant in a high-risk gene is identified. In this report, we present three recent cases illustrating the diverse clinical presentations of hereditary cardiac diseases and the value of DNA testing in these patients. Case 1. An autopsy was performed on a 60-year-old woman that died due to progressive bilateral pulmonary vein thrombosis of unknown cause. Her cardiovascular history was negative for syncope or palpitations. Family history was negative for sudden cardiac death. Extensive bilateral pulmonary vein thrombosis was confirmed on autopsy. In addition, in the right ventricle of the heart extensive fatty changes of the myocardium were observed, partly subendocardial with minor fibrosis (Figure 1).The histology was consistent with arrhythmogenic cardiomyopathy (ACM), a hereditary heart disease [6]. Following the recommendation by the pathologist in the autopsy report, a first-degree relative was referred for genetic counselling by the treating pulmonary physician of the patient. DNA was isolated from paraffin-embedded tissue and 56 cardiomyopathy associated genes (ACTC1, ACTN2, ALPK3, ANKRD1, BAG3, CALR3, CAV3, CDH2, CRYAB, CSRP3, CTNNA3, DES, DSC2, DSG2, DSP, EMD, FHL1, FHL2, FKRP, FLNC, GLA, HCN4, JPH2, JUP, LAMA4, LAMP2, LDB3, LMNA, MIB1, MYBPC3, MYH6, MYH7, MYL2, MYLK3, MYL3, MYOZ2, MYPN, NEXN, PKP2, PLN, PPA2, PRDM16, PRKAG2, RBM20, SCN5A, TAZ, TCAP, TMEM43, TNNC1, TNNI3, TNNI3K, TNNT2, TPM1, TTN, TTR, VCL) were analysed using next-generation sequencing on the MiSeq (in solution capture of candidate genes (SeqCap EZ Choice, Nimblegen) were paired-end sequenced (2 × 150 bp) on the MiSeq and mapped to GRCh37/hg19 reference genome using BWA-MEM (0.7.12-r1039). Variants were identified using the HaplotypeCaller from GATK version 3.8 (Genome Analysis Toolkit, Broad Institute, Cambridge, MA, USA) along with Picard tools version 1.89 and Alissa Interpret v.5.2.4). A pathogenic (class 5) variant, c.2265+5G>A in the FLNC gene (NM_001458.4) was identified. No other (likely) pathogenic variants were found. The FLNC variant was previously identified in a patient with dilated cardiomyopathy in our lab. RNA analysis in blood showed altered splicing because intron 14 was not spliced out, resulting in a truncated protein p.(Asn757Argfs*51). The variant is rare in the general population (2/247,832 gnomAD alleles see http://gnomad.broadinsitute.org). Truncating variants in this gene are a known cause of dilated cardiomyopathy and ACM associated with an increased risk of ventricular arrhythmias [6,7]. Several relatives carried the FLNC variant and were referred for cardiological screening. One of them was recently diagnosed with ACM. Nonsymptomatic carriers were recommended to remain under cardiological surveillance. Case 2. A previously healthy 33 years old male presented with out-of-hospital cardiac arrest due to persistent ventricular fibrillation. Resuscitation was unsuccessful. Health and family history of the mother were uneventful. The biological father of the patient was unknown. On autopsy, extensive fatty and fibrotic changes of the myocardium of the left ventricle were observed (Figure 2A,B). DNA isolated from material derived from autopsy was sequenced on the MiSeq using the above-mentioned 56 cardiomyopathy panel. This revealed a causal, pathogenic (class 5) variant in the FLNC gene, c.554G>A p. (Trp185*). No other (likely) pathogenic variants were detected. The variant was not found in gnomAD alleles. The observations are consistent with reports of predominant left ventricular involvement in FLNC associated ACM [8].Case 3. A 41-year-old woman was found dead at home. She was diagnosed with severe, therapy-resistant, (tonic–clonic) atypical epilepsy from the age of 14 years on. Seizures were often at night and were characterised by apnea with discoloration. Apart from a benign cyst on the pituitary gland, cerebral MRI revealed no abnormalities. She had sometimes fainted and suffered from cardiac palpitations, but cardiological evaluation had not yet been performed. She used venlafaxine for episodes of depression which had been increased in dosage (from 75 mg/day to 150 mg/day) a few weeks prior to her death. This drug is associated with QTc prolongation when serum levels exceed the therapeutic range [9]. Postmortal toxicological examination showed elevated plasma levels of venlafaxine, a QTc prolonging drug (1137 µg/L, upper limit of 1000 µg/L). The autopsy revealed diffuse cerebral ischemia possibly associated with her epilepsy, but no clear cause of death was identified. Histological evaluation of cardiac tissue showed no abnormalities. Given the young age, genetic counselling of relatives was recommended by the general practitioner. DNA was isolated from paraffin-embedded tissue and the 54 arrhythmia associated genes (ABCC9, AKAP9, ANK2, ASPH, CACNA1C, CACNA1D, CACNA2D1, CACNB2, CALM1, CALM2, CALM3, CASQ2, CAV3, DPP6 (NM_001936, only position c.-340), GJA5, GNB2, GPD1L, HCN4, JPH2, KCNA5, KCND3, KCNE1, KCNE5, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNJ8, KCNQ1, LAMP2, LMNA, MYL4, NKX2-5, NPPA, PKP2, PLN, PPA2, PRKAG2, RANGRF, RYR2, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SCN10A, SLMAP, SNTA1, TECRL, TNNI3K, TNNT2, TRDN, TRPM4) were sequenced on the MiSeq using the method, described above. A known pathogenic (class 5) variant, c.2467C > T p.(Arg823Trp), in the KCNH2 gene (NM_000238.3), associated with long QT syndrome type 2 (LQTS 2), was identified. No other (likely) pathogenic variants were found. The causal KCNH2 variant is rare in gnomAD (allele frequency of 1/251,454), has often been reported in LQT patients (PMID: 10973849, 11854117, 16831322, 19695459, 19716085, 23158531, 23631430) and functional studies showed inactivation of the potassium channel ((PMID: 11741928, 16432067, 11278781, 23303164). It is likely that the ‘atypical epileptic seizures’ were caused by cardiac arrhythmias. Genetic testing in relatives revealed that the variant was de novo. With the description of three cases, the authors hope to have pointed out how the multidisciplinary approach in a specialised centre aids in diagnosing rare inherited cardiac diseases in sudden unexpected death in the young. All diagnoses were made by use of gene panels. In case of negative findings in sudden unexpected death in children, trio-whole-exome sequencing (WES) may be considered after counselling by a clinical geneticist.Conceptualization, S.N.v.d.C., A.C.H., A.M.C.V., C.v.d.W.; writing—original draft preparation, all authors; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable. The study was conducted according to the guidelines of the Declaration of Helsinki. Written informed consent was obtained from the families presented in the paper.The authors would like to thank A.A.M. Wilde for critically reading the manuscript.The authors declare no conflict of interest.Hematoxylin- and Eosin-stained section of the right ventricle of the heart with extensive fatty changes of the myocardium, partly subendocardial, with only minor fibrosis. Asterisk indicates fatty tissue.Hematoxylin- and Eosin-stained sections of the left ventricle of the heart (A) with extensive fatty and fibrotic changes of the myocardium and the right ventricle of the heart and (B) with only epicardial fat. Asterisk indicates fatty tissue. Arrows indicate fibrosis.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/cardiogenetics/cardiogenetics-11-02-00009.txt

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+Genetic variants in MYBPC3 are one of the most common causes of hypertrophic cardiomyopathy (HCM). While variants in MYBPC3 affecting canonical splice site dinucleotides are a well-characterised cause of HCM, only recently has work begun to investigate the pathogenicity of more deeply intronic variants. Here, we present three patients with HCM and intronic splice-affecting MYBPC3 variants and analyse the impact of variants on splicing using in vitro minigene assays. We show that the three variants, a novel c.927-8G>A variant and the previously reported c.1624+4A>T and c.3815-10T>G variants, result in MYBPC3 splicing errors. Analysis of blood-derived patient RNA for the c.3815-10T>G variant revealed only wild type spliced product, indicating that mis-spliced transcripts from the mutant allele are degraded. These data indicate that the c.927-8G>A variant of uncertain significance and likely benign c.3815-10T>G should be reclassified as likely pathogenic. Furthermore, we find shortcomings in commonly applied bioinformatics strategies to prioritise variants impacting MYBPC3 splicing and re-emphasise the need for functional assessment of variants of uncertain significance in diagnostic testing.Hypertrophic cardiomyopathy (HCM) is a relatively common genetic disorder (with a prevalence of 1:500) characterised by left ventricular cardiomyopathy, a non-dilated left ventricle and normal, or increased, ejection fraction, and is associated with myocardial fibre disarray [1,2,3]. HCM is usually asymmetrical and develops in the absence of an identifiable secondary cause such as hypertension or aortic valvular stenosis. Patients with HCM usually have a relatively benign clinical course, although HCM can cause sudden cardiac death particularly in adolescents and young adults [3]. Risk factors such as severe cardiac hypertrophy, family history of sudden cardiac death, syncope and non-sustained ventricular tachycardia have been associated with sudden cardiac death in HCM patients, and high-risk patients may be offered implantable cardioverter-defibrillators (ICDs) to ameliorate their risk [2,3,4,5,6,7].HCM usually shows an autosomal dominant mode of inheritance, although very rare cases of autosomal recessive and X-linked modes of inheritance have been described [3,8,9,10]. Incomplete penetrance and variable clinical expression are common confounding phenomena. The phenotypic variability is thought to arise, at least in part, from interactions between pathogenic variants and genetic and non-genetic modifiers [3,11,12,13]. Variants in at least 12 genes encoding sarcomere and sarcomere-associated proteins cause HCM, the two most common of which (accounting for approximately 50% of families with HCM) are β-myosin heavy chain (MYH7) and myosin-binding protein C3 (MYBPC3) [2,3,14,15,16,17,18,19]. The β-myosin heavy chain has the ATPase enzymatic activity in the myosin head required for force generation in muscle fibres [20]. The myosin-binding protein C3 interacts with both actin and myosin, as well as titin, and regulates cardiac contraction [21,22]. While the majority of variants causing HCM in MYH7 and other less common causative genes are missense variants, pathogenic variants in MYBPC3 tend to be frameshift, nonsense or splice site variants, which result in premature termination codons (PTCs) and predicted loss of function [3,14,16,23]. PTC-containing MYBPC3 transcripts may result in truncated MYBPC3 protein upon translation; alternatively, mutant transcripts may be degraded by the nonsense-mediated decay pathway, causing allelic loss-of-function and reduced expression [23,24]. Approximately 15% of MYBPC3 variants in HCM are non-truncating, including missense and short in-frame insertion/deletion variants. The pathogenic mechanisms of these non-truncating variants are largely unknown, although it has been shown that disease severity and clinical outcome, while highly variable, is largely independent of whether the pathogenic MYBPC3 variant is truncating or non-truncating [23,25,26].While genetic testing is now common in patients with HCM, the diagnostic yield is only 40–50%, and the causative genotype is unknown for at least 40% of patients, even for those with family histories of the disease [27,28]. There are many factors which may explain this low genetic testing yield. For example, phenocopying occurs in other syndromic conditions such as Noonan syndrome and storage disorders, including Anderson–Fabry disease, while in other cases complex and as-yet unknown genetic mechanisms may cause HCM [27,29,30,31,32]. However, conventional genetic testing methods may also fail to identify pathogenic non-coding variants, in particular cryptic splice variants (i.e., intronic variants out-with the canonical splice acceptor and splice donor dinucleotides which alter pre-mRNA splicing) in known HCM genes, which may be a major contributor to the missing heritability in HCM. For example, several recent publications have identified novel intronic cryptic splice variants within MYBPC3 in cohorts of HCM patients, which would not have been detected as pathogenic variants by standard genetic testing [27,33,34,35,36,37]. These intronic variants were shown to alter MYBPC3 splicing and result in allelic loss-of-function and haploinsufficiency.Here, we present three patients with HCM presenting with intronic MYBPC3 splice variants out-with the invariant splice donor/acceptor dinucleotides. We use in vitro minigene splicing assays in human cell lines, combined with direct analysis of patient RNA in one case, to confirm the aberrant effect of these variants on MYBPC3 splicing. This study supports the importance of sequencing intronic regions in MYBPC3 to increase the detection of pathogenic variants causing HCMThe three probands were recruited by the Manchester Centre for Genomic Medicine. For proband 1, clinical DNA sequencing by NGS was undertaken of the entire coding sequence of four genes—MYH7, MYBPC3, TNNT2 and TNNI3. For probands 2 and 3, a panel of >20 genes associated with HCM was screened by NGS, as previously described [38].In silico pathogenicity prediction with SpliceAI v1.3.1 and Human Splicing Finder v3.1 were used to score cryptic splice-affecting variants in MYBPC3 [39,40]. Population frequency of MYBPC3 variants was analysed using gnomAD v.3.1 (https://gnomad.broadinstitute.org/) (accessed 12 September 2020) [41].Human K562 cells were cultured under standard tissue culture conditions in RPMI-1640 medium (Sigma) supplemented with 10% Fetal Bovine Serum (FBS) (Sigma) and incubated in 5% CO2 at 37 °C. Original K562 cell stocks were supplied at passage 10.Fragments of MYBPC3 (reference transcript NM_000256) were amplified by polymerase chain reaction (PCR) from commercially available reference genomic DNA (Promega) using either wildtype or mutagenic primers (Table S1) using Phusion High Fidelity DNA Polymerase (NEB) according to the manufacturer’s recommendations. For the c.927-8G>A variant, the total MYBPC3 fragment length was 502bp, containing the 164 bp exon 12 plus 170 bp of intron 11 and 168 bp of intron 12 of MYBPC3 (Figure 1(ai)). For the c.1624+4A>T variant, the total MYBPC3 fragment length was 332bp, containing the 167 bp exon 17 plus 65 bp of intron 16 and 100 bp of intron 17 (Figure 1(aii)). For the c.3815-10T>G variant, the MYBPC3 fragment was 657 bp and contained the 187 bp exon 33 and 37 bp exon 34, plus 120 bp of intron 32, the 190 bp intron 33, and 123 bp of intron 34 (Figure 1(aiii)). The wildtype and mutagenic MYBPC3 fragments were cloned into the SK3 plasmid (a derivative of the pSpliceExpress minigene splice reporter vector, gifted from Stefan Stamm, Addgene #32485) using the Gibson method [42,43]. Constructs were transformed into competent bacteria and candidate colonies were cultured and vector DNA isolated using the GenElute Plasmid Miniprep kit (Sigma, St. Louis, MO, USA). Sequences of the minigene vector constructs were verified by Sanger sequencing (performed by Eurofins genomics).Human K562 cells were plated in 2 mL RPMI-1640 medium (Sigma) supplemented with 10% Fetal Bovine Serum (FBS) (Sigma) at approximately 80% confluency in 6-well tissue culture plates (Corning) on the day of transfection. Cells were transiently transfected with 2.5 µg plasmid DNA using Lipofectamine LTX (Invitrogen) following the manufacturer’s protocol and incubated for 18–24 h at 37 °C with 5% CO2. The splicing minigene assays were performed twice independently.For the splicing minigene assay, total RNA was extracted from K562 cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The resulting RNA was cleaned up using the RNeasy Mini RNA Isolation Kit (Qiagen, Hilden, Germany), including an on-column DNA digestion step. An equal amount of RNA for each sample was converted to cDNA using Superscript IV (Invitrogen, Carlsbad, CA, USA) with random hexamers (Invitrogen) according to the supplier’s recommendations. cDNA was amplified using Phusion polymerase and primers listed in Table S2. PCR products were separated by agarose gel electrophoresis supplemented with SafeView nucleic acid stain (NBS Biologicals, Huntingdon, UK) and visualised under a blue-light transilluminator. Products were extracted using QIAquick gel extraction kit (Qiagen) and sequenced by Sanger sequencing to confirm their identity.For proband 3 with the MYBPC3 c.3815-10T>G variant, an RNA sample (Paxgene) was derived from blood following the manufacturer’s instructions. A cDNA synthesis was performed as above using patient RNA as a template and the cDNA amplified using Phusion polymerase and primers listed in Table S2. PCR products were separated, gel extracted and sequenced, as described above.Three patients with a clinical diagnosis of HCM, but no known genetic cause underwent genetic and molecular screening by next-generation sequencing (NGS) to establish the genetic basis for their specific cardiac phenotype.The proband in family 1 (Figure 2a) was a 77-year-old woman with a history of persistent atrial fibrillation. An echocardiogram revealed severe asymmetrical left ventricular hypertrophy with a septum thickness of 2 cm. The proband’s father was reported to have died suddenly at the age of 53; however, there was no other family history consistent with a diagnosis of cardiomyopathy. Genetic testing revealed a heterozygous c.927-8G>A variant in intron 11 of MYBPC3 (Figure 1b). The affected nucleotide is 8 base pairs (bp) upstream of the canonical 3′ splice site in intron 11, and potentially creates a new cryptic 3′ splice site AG. This c.927-8G>A variant has not been described previously in HCM patients in the literature, is not present in the gnomAD population genomic sequence database and is classified as a variant of uncertain significance. However, this variant has been identified in seven additional unrelated individuals with HCM in diagnostic laboratories performing cardiomyopathy testing in the United Kingdom. Additionally, a variant in the adjacent residue (c.927-9G>A) has been reported as likely pathogenic, although analysis of patient RNA derived from blood for this adjacent variant did not confirm any splicing errors caused by the variant [37,44]. The c.927-8G>A variant was not predicted to have any significant impact on splicing signals by the Human Splicing Finder. In agreement with the Human Splicing Finder, the SpliceAI score for this variant was 0.11 (acceptor loss). Commonly applied SpliceAI scores of 0.2 as potentially splice-altering and 0.5 as likely splice-altering for variant prioritisation would (falsely) exclude this variant from prioritisation [39,45].The proband in family 2 (Figure 2b) was a 46-year-old man who presented with palpitations and shortness of breath on exertion. A cardiac MRI scan showed moderate to severe concentric left ventricular hypertrophy with septal predominance (basal anteroseptum measured 2.4 cm). There was also evidence of mid-layer fibrosis involving the hypertrophied regions. The proband had type 1 diabetes mellitus but no history of hypertension or other significant medical issues. There was no known family history of cardiomyopathy or sudden cardiac death. The proband was identified as having a heterozygous c.1624+4A>T variant in intron 17 of MYBPC3 (Figure 1b). The affected nucleotide is 4 bp downstream of the 5′ splice site but still within the splice donor consensus sequence. This c.1624+4A>T variant is present in three of 224,880 alleles in gnomAD and is predicted by the Human Splicing Finder to affect splicing by abrogating the splice donor site at the 5′ end of intron 17, although the SpliceAI score for the variant is only 0.16 (donor loss) again suggesting no impact on splicing. The c.1624+4A>T variant is classified as pathogenic, having been reported in a number of HCM patients, both within and outside of the UK, and previous blood-derived RNA analysis from these patients revealed the variant causes exon 17 skipping, which would result in a PTC and loss of the C-terminal region of the protein upon translation removing the major myosin and titin binding sites [24,37,46,47,48,49].The proband in family 3 (Figure 2c) was diagnosed with HCM after presenting with chest pain whilst walking in his fifth decade. He had a history of paroxysmal atrial fibrillation and had a stroke at the age of 58 years. A cardiac MRI scan performed shortly after his stroke revealed a maximal wall thickness of 2.6 cm and left ventricular outflow tract obstruction, consistent with HCM. On gadolinium imaging, there was evidence of very extensive enhancement involving most of the left ventricle. One of the proband’s brothers was reported to have died in his sleep at the age of 66 years and one of the proband’s sisters was identified as having mild HCM on screening shortly after his diagnosis. Genetic testing in the proband revealed a heterozygous c.3815-10T>G variant in intron 33 of MYBPC3 (Figure 1b). The variant is 10 nucleotides upstream of the 3′ splice site. This c.3815-10T>G variant has been reported once in 242,620 alleles in gnomAD and has been reported previously in one HCM patient in ClinVar, although it is currently classified as a likely benign variant [33]. In silico pathogenicity prediction by Human Splicing Finder suggested no impact of this variant on splicing signals, while the variant had a SpliceAI score of 0.37 (acceptor loss), indicating possible impact on splicing. Genetic testing also revealed a heterozygous MYL2 c.37G>A, p.Ala13Thr variant in this proband.To test the effects of the identified MYBPC3 intronic variants on MYBPC3 pre-mRNA splicing, we conducted in vitro minigene splicing assays in human cell lines. The assays compared spliced RNA transcripts extracted from human K562 cells following transfection with pairs of wild type and variant-containing minigene constructs containing fragments of MYBPC3 (Figure 1a).For the c.927-8G>A variant, we found that while the wild type sequence led to canonical splicing of MYBPC3 only, the variant resulted in loss of the wild type splice acceptor and the use of an upstream cryptic splice acceptor site instead, resulting in the inclusion of an additional 19 bp at the 5′ end of exon 12 (Figure 1c). This 19 bp exon extension would result in a frameshift and the creation of a PTC in exon 13.For the previously characterised c.1624+4A>T variant, the wild type sequence resulted in a primary product containing canonically spliced exon 17, and a small fraction of product with exon 17 skipping. However, the mutant sequence resulted in complete exon 17 skipping (due to mutation of the wild type splice donor consensus sequence) and no canonically spliced product, in agreement with previous reports (Figure 1c and Figure S1) [33,37].Finally, for the c.3815-10T>G variant, the wild type sequence resulted in two spliced products, one containing exon 34 and one with exon 34 skipped. However, for the mutant construct, there was complete skipping of exon 34 and no exon 34 retention, suggesting the variant led to complete loss of splice acceptor recognition at the 3′ end of intron 33 (Figure 1c). Interestingly, exon 34 (of 35 total exons for the NM_000256 reference transcript used in this study) is the final coding exon of MYBPC3, and so the skipping event observed here would result in loss of the normal stop codon. RNA from blood was available for this patient, and direct analysis of the spliced products in blood-derived patient RNA revealed only a wild type product, suggesting that mutant transcripts produced from the variant-containing allele were degraded (data not shown). This finding suggests the c.3815-10T>G variant results in decreased MYBPC3 expression, in agreement with the known disease mechanism of haploinsufficiency.In this study, we present three patients with MYBPC3 intronic variants and assess the effects of these variants on MYBPC3 splicing in minigene assays in vitro. Confirming the pathogenicity of these intronic splice variants makes it possible to utilise the findings clinically and offer targeted genetic testing to at-risk family members. For the c.927-8G>A and c.3815-10T>G variants, we can now apply PS3 from the American College of Medical Genetics (ACMG) guidelines for assigning pathogenicity to sequence variants as we have shown, using a well-established in vitro functional study, the damaging effect on splicing of these two variants [50]. Patient-derived RNA is the ideal analyte to determine the effect of sequence variants on splicing, but where this is not available, we propose that in vitro minigene splicing assays can generate supportive evidence, which can be used as part of the variant classification [51].While the MYBPC3 c.1624+4A>T variant has been well-characterised and previously classified as pathogenic, we included it in our minigene assays as a positive control to validate our methodology and confirm the sensitivity of our in vitro assay for detecting splice-altering variant effects [24,37,46,47,48,49]. In our system, this variant resulted in complete MYBPC3 exon 17 skipping, in agreement with these previous analyses.The novel c.927-8G>A variant, described for the first time in proband 1 of our study, had a clear impact on MYBPC3 splicing, activates a cryptic splice site upstream of the canonical 3′ splice site at intron 11, resulting in the extension of exon 12 by 19 bp at the 5′ end, leading to a frameshift and a PTC. It is interesting that the report of a variant in the adjacent nucleotide (c.927-9G>A) could not confirm aberrant splicing in patient RNA samples by RT-PCR; the authors only observed the wild type spliced product [37]. We postulate that the adjacent variant may have the same effect on MYBPC3 splicing as our c.927-8G>A variant as both variants affect the same canonical splice site in intron 11 and likely activate the same cryptic upstream 3′ splice site. It is likely that transcripts from the mutant allele are degraded by nonsense-mediated decay (NMD), such that they are not observed by RT-PCR of patient RNA. Quantitative analysis of the MYBPC3 expression by qPCR and/or heterozygous variant ratios to assess whether transcripts from the mutant allele are present in patient RNA samples could be used to confirm this mechanism, as well as assessing the variant in a minigene assay.Finally, the previously described c.3815-10T>G variant in proband 3 of our study had a clear effect on splicing in our minigene assay, resulting in total loss of the splice donor recognition at the 3′ end of intron 33 leading to complete exon 34 skipping and loss of the normal stop codon. The nucleotide sequence of exon 35 codes for 46 amino acids in-frame with exon 33 with an in-frame stop codon and so the mis-splicing event associated with the variant could result in a change to the 3′ end of the encoded protein. This event would not be detected from our minigene assay because the construct only contained MYBPC3 exons 33 and 34 and not exon 35. However, RT-PCR analysis of patient RNA indicated that this event causes NMD of transcripts from the mutant allele, as opposed to production of a protein with an altered C-terminal. Two points of interest are worth noting here. Firstly, even for our wild type minigene construct, there is a relatively strong RT-PCR product (approximately the same intensity of band on the gel as the canonically spliced RT-PCR product) corresponding to the exon 34-skipped splice product, and so the variant appears to shift the ratio of splice isoforms towards an exon 34-skipped product, which is already normally observed. In contrast, while a very faint band corresponding to the exon 17-skipped splice product is also present for the wild type construct for the c.1624+4A>T variant, this band is much weaker than the canonically spliced product. MYBPC3 exon 34 is a very short exon (37bp), and previous studies have indicated shorter exon length is associated with more frequently skipped exons, which may explain the relatively high prevalence of the shorter splice isoform even for the wild type minigene constructs [52,53]. The second point of interest with the c.3815-10T>G variant is that this individual also carries a heterozygous MYL2 c.37G>A, p.Ala13Thr variant. This amino acid substitution has been shown to have an important effect on calcium binding and subsequent myosin binding of the encoded protein and has been previously reported in patients with HCM, particularly associated with left ventricular obstruction. This observation is consistent with the phenotype of left ventricular outflow tract obstruction in our patient, but the high minor allele frequency means that the clinical significance of this variant is unclear [54,55,56,57,58]. It may be that because the MYBPC3 c.3815-10T>G splice variant enhances an exon skipping event, which is normally observed at a relatively high frequency, this variant must be combined with a second variant in another sarcomere-associated gene and/or must occur in the presence of a background of particular genetic modifiers to be fully penetrant. Another non-mutually exclusive possibility is that the common MYL2 variant in this patient is only pathogenic in the presence of a second variant in a sarcomere-associated gene (in this case, the MYBPC3 c.3815-10T>G variant), explaining the link between the variant and specific cardiac phenotypes even though its population frequency is relatively high. It is not clear whether the previously reported individuals with the MYBPC3 c.3815-10T>G variant also carried the MYL2 c.37G>A variant [33]. One of this proband’s sisters was identified as having a mild HCM phenotype. Genetic testing will now be extended to the affected sibling.A further point of interest is that none of the intronic splice-affecting variants presented in this study had particularly high SpliceAI scores and only the c.1624+4A>T variant was predicted by Human Splicing Finder to affect MYBPC3 splicing. Indeed, for this variant, the SpliceAI score was only 0.16, and yet the variant resulted in total loss of the intron 17 splice donor and complete exon 17 skipping and is already classified as pathogenic on the basis of previous RNA analysis. Similarly, the c.927-8G>A variant had a SpliceAI score of just 0.11, and yet had a very clear impact on the MYBPC3 exon 12 splice acceptor site, while the c.3815-10T>G had a SpliceAI score of 0.37 but had a major impact in shifting MYBPC3 splicing towards exon 34 skipping. In general, our laboratory policy is to consider variants with SpliceAI scores >0.2 as potentially splice-altering, while scores >0.5 are considered as very likely to be splice-altering, which is similar to previously described SpliceAI cut-offs used for variant prioritisation [39,45]. However, some reports investigating MYBPC3 intronic splice variants have used very stringent SpliceAI cut-offs of ≥0.9 to initially prioritise variants [27]. Nonetheless, when these studies expanded their inclusion criteria to include variants with lower SpliceAI scores, an MYBPC3 c.1898-23A>G variant with a SpliceAI score of just 0.04 was identified; assessment of patient RNA using RT-PCR showed the inclusion of intron 19, leading to a premature stop codon and presumed NMD [27]. In our study, we further demonstrate that intronic variants beneath commonly applied SpliceAI filters do have a significant impact on splicing, and these data re-emphasise that functional investigation alongside in silico assessments remains an important consideration for diagnostic testing in the context of splicing.Intronic MYBPC3 cryptic splice variants are an important genetic cause of HCM and may not be accurately predicted by in silico bioinformatic methods. Analysis of intronic MYBPC3 splice variants increases the diagnostic yield in HCM patients.The following are available online at https://www.mdpi.com/article/10.3390/cardiogenetics11020009/s1. Table S1: Primers used for generating MYBPC3 minigene constructs. Table S2: Primers used for reverse-transcription polymerase chain reaction (RT-PCR) experiments. Figure S1: Sequence traces showing the 19 bp insertion at the 5′ end of MYBPC3 exon 12 for the c.927-8G>A variant in the minigene assay.Conceptualization, W.G.N.; methodology, H.B.T. and K.A.W.; software, J.M.E.; validation, K.A.W., H.B.T. and J.M.E.; formal Analysis, K.A.W., H.B.T. and J.M.E.; investigation, K.A.W. and J.M.E.; resources, W.G.N. and R.T.O.; data curation, K.A.W., J.M.E. and C.H.; writing—original draft preparation, K.A.W. and C.H.; writing—review and editing, H.B.T., J.M.E., J.E., R.T.O. and W.G.N.; visualization, K.A.W.; supervision, W.G.N. and R.T.O.; project administration, W.G.N.; funding acquisition, W.G.N., R.T.O. and J.M.E. All authors have read and agreed to the published version of the manuscript.K.A.W. is funded by a Medical Research PhD Council studentship (1916606). W.G.N. is supported by the Manchester NIHR BRC (IS-BRC-1215-20007). C.H. is a clinical lecturer supported by the NIHR. R.T.O. and H.B.T. are supported by the BBSRC (BB/N000258/1). J.M.E. is supported by the Health Education England Genomics Education Programme.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the South Manchester Research Ethics Committee (11/H10003/3).Informed consent was obtained from all subjects involved in this study.The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the sensitivity of patient information.The authors acknowledge the NW Genomic Laboratory Hub for genetic screening by NGS of patient samples.The authors declare no conflict of interest.MYBPC3 intronic splice variants in three probands with hypertrophic cardiomyopathy. (a) Schematic of the minigene constructs used to test the pathogenicity of MYBPC3 splice variants i. c.927-8G>A, ii. c.1624+4A>T and iii. c.3815-10T>G. MYBPC3 exons are shown in purple. Endogenous minigene exons are shown in light blue. The locations of the splice site variants are indicated by red stars. Ex = exon, In = intron. (b) Location of MYBPC3 splice variants analysed in this paper and the probands in which the variants were identified, using the reference transcript NM_000256. Purple boxes refer to exons (Ex), black lines refer to introns. (c) Representative RT-PCR results showing the outcome of the minigene assays. Identities of RT-PCR products were confirmed by Sanger sequencing. High molecular weight bands above 500 bp correspond to unspliced vector DNA contamination. WT = wild type, Var = variant. n = 2.Family pedigrees for the three probands with HCM presented in this study. Pedigrees for (a) family 1, (b) family 2, and (c) family 3. Arrow indicates the probands subject to genetic testing in this study. Filled shapes indicate clinically affected individuals.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Background. Each year, approximately 5000 New Zealanders are admitted to hospital with first-time acute coronary syndrome (ACS). The Multi-Ethnic New Zealand Study of Acute Coronary Syndromes (MENZACS) is a prospective longitudinal cohort study embedded within the All New Zealand Acute Coronary Syndrome Quality Improvement (ANZACS-QI) registry in six hospitals. The objective of MENZACS is to examine the relationship between clinical, genomic, and cardiometabolic markers in relation to presentation and outcomes post-ACS. Methods. Patients with first-time ACS are enrolled and study-specific research data is collected alongside the ANZACS-QI registry. The research blood samples are stored for future genetic/biomarker assays. Dietary information is collected with a food frequency questionnaire and information about physical activity, smoking, and stress is also collected via questionnaire. Detailed family history, ancestry, and ethnicity data are recorded on all participants. Results. During the period between 2015 and 2019, there were 2015 patients enrolled. The mean age was 61 years, with 60% of patients aged <65 years and 21% were female. Ethnicity and cardiovascular (CV) risk factor distribution was similar to ANZACS-QI: 13% Māori, 5% Pacific, 5% Indian, and 74% NZ European. In terms of CV risk factors, 56% were ex-/current smokers, 42% had hypertension, and 19% had diabetes. ACS subtype was ST elevation myocardial infarction (STEMI) in 41%, non-ST elevation myocardial infarction (NSTEM) in 54%, and unstable angina in 5%. Ninety-nine percent of MENZACS participants underwent coronary angiography and 90% had revascularization; there were high rates of prescription of secondary prevention medications upon discharge from hospital. Conclusion. MENZACS represents a cohort with optimal contemporary management and will be a significant epidemiological bioresource for the study of environmental and genetic factors contributing to ACS in New Zealand’s multi-ethnic environment. The study will utilise clinical, nutritional, lifestyle, genomic, and biomarker analyses to explore factors influencing the progression of coronary disease and develop risk prediction models for health outcomes.In New Zealand, 15% of all deaths annually are caused by ischaemic heart disease [1] and one in four major coronary events are fatal [2]. Although age standardised mortality rates for ischaemic heart disease have fallen dramatically since the late 1960s, New Zealand’s mortality rates are still higher than many other western countries with persistent disparities based on ethnic group and social deprivation. Māori (the indigenous population of New Zealand) and Pacific peoples typically present with disease at a younger age, have higher readmission rates, and incur approximately double the European age-standardised mortality rate [3,4]. Whilst there have been considerable advances in the management of acute coronary syndromes (ACS), there are important knowledge and practice gaps in optimal risk stratification, treatment, and long-term outcomes for patients with cardiovascular disease (CVD) in New Zealand [5,6,7]. Recurrent event rates remain high, with a recent follow-up analysis of patients admitted with a first-time ACS showing that 15% experienced a non-fatal cardiovascular readmission and 16% had died within a year [8]. Identification of individuals or groups at high residual risk of further events, despite contemporary therapies, could lead to more targeted strategies to improve inequitable clinical outcomes. For patients admitted with a first-time ACS in New Zealand, there is a high incidence of premature disease with 25% being aged less than 55 years [4] and a very high burden of risk factors: half are current smokers, half have a BMI > 30 kg/m2, and 16% have diabetes. Along with established clinical risk factors [9], it is becoming widely accepted that CVD risk prediction is improved by incorporating environmental and sociodemographic variables and their interactions with genetic and other omics markers [10].However, genetic risk markers identified in international genome-wide association studies (GWAS) for risk assessment may not be ideal for translation to New Zealand’s multi-ethnic population, since it is estimated that approximately 80% of all participants in GWAS are of European ancestry despite this group representing only 16% of the global population [11]. Population profiles of GWAS for coronary artery disease follow a similar pattern and are also primarily of European descent [11]. The transferability of this knowledge to other populations is now known to be problematic since populations vary in terms of allele frequency, effect size of risk variants [12,13], and having unique ethnic-specific genetic variants associated with disease risk. Moreover, genetic variants influence how different populations metabolise drugs [14,15,16] and this leads to disparate outcomes between ethnic groups, even when under the same treatment regimes. The primary aim of the Multi Ethnic New Zealand study of Acute Coronary Syndromes (MENZACS) is to define the extent to which environmental and genetic factors contribute to the overall burden of ACS in New Zealand’s ethnically diverse population presenting with first-time ACS. The study will utilise clinical, nutritional, lifestyle, genomic, and biomarker analyses to explore aetiological factors and to develop risk prediction models for outcomes. The broad themes of the proposed research are as follows: (1) to explore the role of genetic variation in the progression of coronary disease in a contemporary cohort of New Zealanders with ACS; (2) to refine screening strategies in certain high-risk populations to enhance secondary risk prediction and early intervention using a combination of clinical risk factors, genomics, and biomarkers; and (3) to define how the response to therapies for ACS differs by ethnicity across New Zealand’s diverse ethnic population groups. The protocol and structure of the study are reported along with a description of the initial cohort.MENZACS was established in 2015 in New Zealand by the Heart Health Research Group, University of Auckland, in collaboration with the Christchurch Heart Institute, University of Otago. The study received national ethics approval in April 2015 from the Health and Disability Ethics Committee (Ref: 15/NTB/59), with each participant providing written informed consent, and the protocol is registered at the Australian New Zealand Clinical Trials Registry (ACTRN12615000676516).MENZACS is a prospective longitudinal cohort study linked to the All New Zealand Acute Coronary Syndrome Quality Improvement (ANZACS-QI) electronic registry by using a common web-based platform, which records detailed clinical information and routine laboratory test results on over 95% of patients admitted with suspected ACS undergoing coronary angiography across hospitals in New Zealand [17]. The primary aim of ANZACS-QI is to support evidence-based management of ACS regardless of age, sex, ethnicity, socioeconomic status, or geographical domicile [18]. Anonymised linkage of registry data with national routinely collected data on hospitalisations, mortality, medication dispensing, and other administrative health data will enable extensive phenotyping of this patient cohort (Figure 1).A Māori Governance Group (MGG) was established at the beginning of this study. The MGG interact regularly with the research team and are kaitiaki (custodians) for Māori participants by providing advice and guidance using the principles of Te Mata Ira—Cultural Guidelines for Biobanking & Genomic Research [19] to ensure best practice. The patient information sheet and consent forms have been translated into Te Reo Māori, Tongan, and Samoan to help increase recruitment and engagement with Māori and Pacific patients. In addition, a kaupapa Māori information sheet is also available that more fully describes the cultural processes involved in the collection, storage, transportation, and destruction of samples.Patients aged >18 years with a clinical diagnosis of ACS are identified during the index hospital admission. Those fulfilling the criteria for a Type 1 myocardial infarction [20] or unstable angina [21] are eligible for enrolment. Patients are excluded if there is an elevation of troponin and/or if ECG changes are not thought to be due to an ACS, the patient has end stage renal failure (eGFR < 15 mL/min/m2 or is receiving or planned to receive renal replacement therapy), is unable to give informed consent, or is not a New Zealand resident.Patients admitted to the coronary care units of Auckland City, Middlemore, Christchurch, Waikato, North Shore, and Tauranga Hospitals are screened for eligibility into the study by a research nurse according to the criteria listed above. Eligible patients are invited to participate and are given the appropriate participant information and consent forms.The routine clinically available data relating to the acute admission is extracted from the ANZACS-QI registry dataset and completed on all patients as part of the clinical workflow. These data include demographics, primary diagnosis, key cardiovascular risk factors, past cardiovascular history and admission vital signs, blood results, ECG, echocardiogram, in-hospital management, and coronary angiogram findings. MENZACS research-specific data are captured using a purpose-built module linked to the web-based ANZACS-QI platform, allowing efficient and standardised data collection at all participating sites. Data on physical activity was collated using the World Health Organisation Global Physical Activity Questionnaire [22] and information on stress and mood was gathered from a previously validated questionnaire of patients with stable coronary disease [23]. Other risk factors and clinical variables including smoking, history of gout, marijuana use, admission medication, and waist circumference were also captured.Research and routine clinical data obtained by using the platform are linked to externally analysed research data on biomarkers, lipidomics, genetics, epigenetics, and diet. Specialised centres for analysis include the Christchurch Heart Institute-NT-proBNP and GDF-15, AgResearch (Invermay, Dunedin)-genotyping and the epigenetics analysis, AgResearch (Lincoln)-lipidomic analysis, and the Liggins Institute (University of Auckland)-Lp(a) concentrations. The results of these sample analyses are then sent to the MENZACS central data science management group at the University of Auckland for linkage to the main dataset accompanied with appropriate encryption processes. These data are then linked to national routinely collected data on past and future hospitalisations, dispensed medications, and death provided by the national Ministry of Health and curated by the Vascular Information and the Web (VIEW) programme of cardiovascular research (University of Auckland). Individualised patient data linkage was enabled by matching each patient’s unique National Health Index (NHI) number to an encrypted NHI using a well-established protocol of de-identification, data security, and management that is central to the ongoing processes of the VIEW and ANZACS-QI programmes [18,24].This process allows long term follow up of 100% of the cohort that remained as residents in New Zealand.Given the multi-ethnic emphasis of the study, in-depth data about family history, ancestry, ethnic background, iwi (tribal) affiliation, participants’ and parents’ country of birth, and grandparents’ ethnicities are obtained. A dietary food frequency questionnaire (FFQ), with the wording of the food groups based on that used in the EPIC-Heart study [25] and adapted to the modern multi-ethnic New Zealand environment, was administered to all participants to survey dietary habits. This is a contemporary and culturally-appropriate questionnaire, which has been validated and shown to be reproducible in New Zealand adults [26], and is designed to enable comparison between cohorts and investigate the relationship between diet and chronic disease.A total of 40 mL of blood for genomic and biomarker analysis is drawn from each participant in a non-fasting state and seated. The time of acquisition of this blood sample in relation to the time of admission was recorded. The samples are centrifuged (at 4 °C, 4000 rpm for 10 min) and separated as required into cryogenic tubes and frozen within 30 min of collection. The resulting EDTA and lithium heparin plasma, serum, and whole blood samples are stored at −80 °C in a secure dedicated facility in the University of Auckland School of Medical Sciences under the aegis of Te Ira Kāwai Auckland Regional Biobank framework (https://www.aucklandregionaltissuebank.ac.nz/ accessed on 19 April 2021) or at the Christchurch Heart Institute’s Health and Disability Ethics Committee (HDEC) accredited tissue bank. All samples are logged in a specimen tracking and storage system. This is via OpenSpecimen (Krishagni Solutions Pvt Ltd., Pune, India) in Auckland or STARLims (Abbott Informatics, Hollywood, FL, USA) in Christchurch.For Māori, the separation of body parts, tissues, or fluids from the person is acknowledged as an important cultural consideration and appropriate protocols are involved. In terms of using tissue or fluid samples for research it is accepted that those samples and the DNA extracted from them are taonga (gifts) and are therefore tapu (sacred). A kaupapa Māori research protocol information sheet has been created by the Māori Governance Group which outlines the appropriate tikanga (Māori custom) processes developed to ensure that the blood samples are treated with care and respect. There are processes around karakia (blessing) that is usually performed prior to a sample being destroyed or sent away for analysis.Laboratory results from tests undertaken as part of routine clinical care are available to the study and this includes creatinine, high sensitivity troponin, and lipid profiles. These have been recorded for >98% of the cohort. HbA1c is assayed when clinically indicated and has been recorded in 79% of the cohort. Additional research assays will measure other established and emerging risk markers and will include N-terminal pro B-type natriuretic peptide (NT-proBNP), Growth Differentiation Factor 15 (GDF-15), lipoprotein(a), and untargeted lipidomics using a mass spectrometry based lipidomics platform. This will measure more than 300 lipid molecules in plasma, which includes sphingolipids, phospholipids, glycerolipids, ceramides and di- and triglycerolipids, and cholesterol esters. Genomic DNA on all participants has been extracted from 1 mL frozen whole blood using an automated QiaSympony DNA extraction process. Genome-wide genotyping will be performed using the Illumina Infinium Global Screening Array (640k SNPs, Illumina Inc., San Diego, CA, USA). This platform has been selected based on its ability to process samples within New Zealand and has high imputation accuracy at minor allele frequencies of >1% across multiple populations and includes curated clinical research variants and quality control markers. Epigenetic analysis will provide DNA methylation profiles in a subset of 1015 MENZACS participants (including all Māori participants) and will be performed using the Illumina Infinium Human Methylation EPIC beadchip array.Clinical outcomes are defined from the national registries of ICD-10-AM (International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, Australian Modification) coded hospitalisations and death and ACHI (Australian Classification of Health Interventions) coded procedures. A primary outcome of interest is major adverse cardiac events (MACE) defined as coronary revascularisation, readmission for cardiovascular cause including recurrent ACS, and death. Specific secondary outcomes include fatal or non-fatal ACS, fatal or non-fatal stroke or transient ischaemic attack (TIA), cardiovascular death, and all-cause death. Cardiovascular death will be defined from the ICD-10 coded death certificate or if death had occurred within 28 days of a CV hospitalisation. This study will act as a resource for genomic discovery as well as providing a comprehensive study of the environmental, genetic, and conventional risk factors that are associated with ACS in New Zealand. Whilst the data will allow “hypothesis free” unbiased discovery studies (see below), specific research questions and themes include:What dietary, lifestyle, and socio-economic factors are associated with first time ACS and subsequent outcomes;Can risk stratification be refined using clinical, biomarker, genetic, and epigenetic factors to build on existing secondary risk equations in New Zealand [27];Association studies of genetic variants with first time ACS and subsequent outcomes;The interaction of genomics, environmental factors, and biomarkers associated with certain phenotypes (e.g., diabetics, metabolic syndrome, hypertension, and obesity);Pharmacogenetic variability and the frequency of certain known variants across a diverse New Zealand population;Ethnic variation in genomic and genetic “signatures” related to cardiovascular risk factors.What dietary, lifestyle, and socio-economic factors are associated with first time ACS and subsequent outcomes;Can risk stratification be refined using clinical, biomarker, genetic, and epigenetic factors to build on existing secondary risk equations in New Zealand [27];Association studies of genetic variants with first time ACS and subsequent outcomes;The interaction of genomics, environmental factors, and biomarkers associated with certain phenotypes (e.g., diabetics, metabolic syndrome, hypertension, and obesity);Pharmacogenetic variability and the frequency of certain known variants across a diverse New Zealand population;Ethnic variation in genomic and genetic “signatures” related to cardiovascular risk factors.A Data Science Advisory Group (DSAG) has been formed to provide specialist expertise in theoretical and applied statistics; this includes statistical genetics and will guide all analyses. Key analytical considerations include assessment of biological variation within and between data sources, reducing data dimensionality, time-to-event analyses, and the development of incremental risk scores for clinical use. Existing clinical [28] and polygenic risk scores [29,30,31,32,33] will provide a starting point for the development of new incremental risk scores. In collaboration with data source specialists in nutrition, biomarkers, lipidomics, genetics, epigenetics, clinical science, and national health data repositories, the DSAG will discuss and advise on specialty-specific approaches to data reduction and analyses. The outputs of these analyses will inform how the data sources are best represented in the model, how model performance is assessed, and what model structure will be used. All data science will have input from the MGG on the appropriate use and interpretation of data in the Māori and Pacific context.In the current report, key descriptors of the MENZACS study cohort (Table 1) have been presented as mean ± standard deviation, median (interquartile range), or frequency (percentage) as relevant. Any other analyses performed to this point have focused on the extent of missing data—for demographic and clinical variables and for the earlier quality assurance assessments of dataset linkage and the calculation of a published genetic risk score [31].The MENZACS study commenced with a pilot phase of recruitment at Auckland City Hospital (ACH) in July 2015. Middlemore and Christchurch started recruitment in March 2016, Waikato Hospital in May 2016, North Shore Hospital in May 2018, and Tauranga Hospital in July 2018. The first phase of recruitment was completed in July 2019. Of the 4846 patients who were screened and met the inclusion criteria, 229 were excluded and 2601 were not enrolled due to patient or logistic issues and one withdrew, leaving a total of 2015 patients included in this cohort (Figure 2). Fifty-seven patients did not have a diagnosis of confirmed ACS in the ANZACS-QI registry, and, thus, an adjudication process was undertaken by designated cardiologists at each centre who reviewed the clinical records and determined whether ACS occurred or not (including the type of ACS event). This resulted in 11 of these patients being excluded as confirmed non-ACS events. The baseline demographics of the initial MENZACS cohort are shown in Table 1. The median age was 61 years, with 61% aged <65 years, and the cohort was predominantly male (79%). Māori constituted 13% of the cohort, Pacific and Indian peoples each comprised 5%, and 74% of participants were of European ethnicity. Based on the ethnic distribution and demographics of a contemporaneous cohort of first-time acute coronary syndrome patients in the ANZACS-QI registry, there is unlikely to have been an ethnic or risk factor bias in enrolment status in MENZACS [4].Nineteen percent were in the most deprived quintile, 19% had diabetes, and 24% were current smokers. Almost all patients underwent angiography during the index admission and revascularisation rates were high, with 90% of the cohort undergoing either PCI or CABG during their hospital admission. These rates are consistent with the recruiting centres for MENZACS being secondary and tertiary referral centres for coronary intervention. Comprehensive information on extent of coronary artery disease, left ventricular function, GRACE score [34], and admission and discharge medication was obtained (Table 2).A quality assurance assessment using data on the first 500 participants was performed. It confirmed that all MENZACS data sources can be linked via an encrypted NHI number. This involved transferring and merging the genetic database with MENZACS and ANZACS-QI data held at the National Institute of Health Innovation, University of Auckland (NIHI) and similarly, transferring, and merging diet data. A MENZACS study encrypted NHI was applied to the final merged dataset. By using 100 samples from four sites, genomic DNA was isolated from 3 mL of frozen whole blood. All samples had DNA yields sufficient for genotyping (≥50 ng/µL) and 96% of samples had 260/280 ratios > 1.7, which indicated high purity. Genotyping was performed for 23 coronary-disease associated or gender-related single nucleotide polymorphisms (SNPs) using an Agena Bioscience MassARRAY®. A genetic risk score based on published methods [31] was able to be calculated for 97% of samples and gender was correctly identified in 100% of samples (see Supplementary Figure S1).MENZACS has been established as a significant epidemiological bioresource for the study of environmental and genetic factors contributing to ACS in New Zealand’s contemporary ethnically diverse population. Genomic research into cardiovascular disease in New Zealand with the Post-Myocardial Infarction Study and Coronary Disease Cohort Study have already contributed valuable insights into genomic risk variants associated with clinical outcome and age of onset of CVD [35,36]. Building on this experience, the MENZACS cohort will have comprehensive data to gain a better understanding of genetic influence on the varying phenotypic presentations of ACS in New Zealand’s population and will explore the clinical utility of genomics in predicting secondary outcomes. The strength of this registry-based study is in the breadth and depth of data gathered, which will enable research into clinical, nutritional, genomic, lipidomic, and epigenetic factors influencing secondary outcomes. The current MENZACS cohort reflects a population of patients who are treated intensively at the time of their index ACS admission, with 99% undergoing coronary angiography, 90% having coronary revascularization, and the vast majority prescribed statin (97%) and dual antiplatelet therapy (82%) at the time of hospital discharge. Compared to the ANZACS-QI registry cohort of patients with first-time ACS over a similar time period (January 2015 to December 2016), there were slightly more Māori participants and more patients had STEMI [8]. However, the overall demographic differences are small and reflect a referral population in the enrolment centres in the current study. Importantly, with the requirement to represent patients who undergo coronary angiography, ANZACS-QI only captures approximately 60% of New Zealanders admitted to hospital with their first ACS and there are important differences in patient characteristics and outcomes between those who are and are not included in the registry. Patients who are not captured in ANZACS-QI are older, are more commonly women with a higher comorbidity burden, and are more than twice as likely to experience death or a non-fatal CV readmission within 12 months of the index ACS admission [8].The initial phase of this research has highlighted several findings relevant to the study of acute coronary disease in New Zealand. Firstly, MENZACS has served as a “proof of concept” that research studies can be successfully embedded within a web-based electronic registry used for clinical quality improvement purposes. The level of data capture was very high with minimal missing data and successful linkage of multiple datasets; this enabled a cost effective and efficient means of running a registry-based research study in an acute care environment. Linkage to national and routinely collected health data has allowed anonymised long-term follow-up of patient outcome, rehospitalisation, and medication dispensing. One limitation of the study is that there can be no feedback of information to an individual patient due to the anonymisation process. However, participants can opt to be approached for future research studies as part of the consent process.Another limitation of the study is that a relatively high percentage of screened patients were not able to be enrolled in the study. The logistic issues related to performing a study such as MENZACS in tertiary and secondary referral centres have been significant. Patients are often transferred back to the referring hospital soon after coronary intervention at regional centres and may be sedated or are too unwell to enroll in the study. In addition, the length of hospital stay in the contemporary era of ACS management is short, resulting in a limited time period to approach patients for research during an acute index ACS admission. Overall, the willingness to participate in a study involving donating DNA has been very high and the research team has worked hard to ensure appropriate and detailed explanation is given in a culturally appropriate context. The Māori Governance Group has been integral to guiding culturally appropriate study processes, including conceptualising the study goals, facilitating recruitment strategies, developing sample handling and disposal protocols, and data governance.The vast majority of studies examining the genetic contribution to the risk of secondary events in those with established coronary artery disease have been in European populations, with the largest being the GENIUS-CHD consortium involving over 185,000 participants [37,38]. However, there are far fewer studies of non-European populations who are at particularly high risk [39,40]. The predictive value of polygenic risk scores derived from cohorts of predominantly European ancestry can be attenuated in other ethnic groups and this emphasises the need for well phenotyped studies involving indigenous populations [11,41,42]. This is a crucial goal as genomic research focussed on European populations can compound inequity when applying the results of the research, for example, in the utility of risk prediction and health prevention strategies [41,43,44,45]. The high incidence of premature CVD and worse cardiovascular health outcomes among some ethnic groups and a single national public healthcare system with a unique patient identification number renders New Zealand ideally suited to study the genetic and environmental drivers and influences on cardiometabolic outcomes.The first phase of MENZACS analyses will focus on the association of established cardiovascular risk factors with secondary cardiovascular outcomes, and the development of incremental risk prediction tools utilising genetic, biomarker, lipidomic, and epigenetic markers. Subsequent studies will examine inter-ethnic variation, nutritional, lifestyle, pharmacogenomic, and kaupapa Māori approaches to optimising outcomes following an ACS, and assist in personalisation of risk stratification, and therapeutic intervention. The following are available online at https://www.mdpi.com/article/10.3390/cardiogenetics11020010/s1, Figure S1: Distribution of 27 SNP genetic risk score by gender.Conceptualization, M.E.L., V.A.C., R.N.D., K.K.P., N.E. and A.R.; methodology, M.E.L., V.A.C., R.N.D., K.K.P., N.E. and A.R.; writing—original draft preparation M.E.L., V.A.C., R.N.D., K.K.P., N.E., A.R., review and editing, M.E.L., V.A.C., R.N.D., K.K.P., N.E., A.R., S.A., Y.C., K.E.B., C.W., R.S., A.K., W.H., G.D., R.T., A.M.R., G.P., P.G. All authors have read and agreed to the published version of the manuscript.The MENZACS study is supported by grants from the Heart Foundation (Heart Health Research Trust grant 1957), Healthier Lives National Science Challenge (Ministry of Business Innovation and Employment Reference UOOX1902), Green Lane Research and Educational Fund (17/26/4130), Freemasons Foundation, and the University of Auckland. R.N.D. is the holder of the Heart Foundation Chair of Heart Health; K.K.P. is the holder of the Heart Foundation Hynds Senior Fellowship; N.E. holds a NZ Heart Foundation Post-doctoral Research Fellowship; K.E.B. holds a Sir Charles Hercus Health Research Fellowship from the Health Research Council of New Zealand.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the New Zealand Northern B Health and Disability Ethics Committee (Ref: 15/NTB/59) 14 April 2015.Informed consent was obtained from all subjects involved in the study.Restrictions apply to the availability of these data.MENZACS Executive Group; M Legget * (Chair and Co-PI), V Cameron (Co-PI), S Aish * (Project manager), R Doughty *, N Earle *, K Poppe *, A Rolleston, C Wall. * Coordinating Centre. MENZACS Steering Group; M Legget, R Doughty, R Stewart, A Kerr, W Harrison, G Devlin, V Cameron, R Troughton, AM Richards, S Aish, K Poppe, C Wall, G Porter, and P Gladding. International Advisors. J. Danesh (Cambridge, UK), and J Howson (Oxford, UK). MENZACS Māori Governance Group; A Rolleston (Chair), K Southey, K Henare, R Stewart, C Grey, and H Wihongi. MENZACS Data Science Group; K Poppe (Chair), N Earle, J Howson, T Lumley, and A Pilbrow. Study centres; University of Auckland (M Oakes-Ter Bals, M Heath, P Shepherd, A Rykers, T Frugier, J Copedo, B Wu, Y Jiang, B Seers, A Chaptynova, C Fyfe, S Wall, and N Kluger). Auckland City Hospital (R Stewart, and K Marshall). Christchurch Hospital and Christchurch Heart Institute (V Cameron, A Pilbrow, S Prue, L Skelton, and R Troughton,). Waikato Hospital (C Nunn, G Devlin, V Pera, L Lowe, S Pilkington, and G Francis). Middlemore Hospital (A Kerr, L Pearce, M Ma, R Railton, L Sharp, P Sharma, and J Gilmore). Tauranga Hospital (G Porter, J Goodson, J Shippey, J Tisch, K Presley, T McKenzie, and CCU Staff). North Shore Hospital: T Scott, G McAnnalley, C Hulbert, K Smith, C Campbell, K Stanley, C Clow, and J Chen. AgResearch: K. Fraser. Enigma Solutions Ltd.: S Breen, and C Wiltshire. The MENZACS investigators would like to express their deepest gratitude to all the patients who have participated in the study.RND has received research grants (administered through host institution the University of Auckland) from the NZ Heart Foundation, Health Research Council of New Zealand, Roche Diagnostics and Bayer. AMR holds grants and/or aid in kind and has received speaker fees and Advisory Board fees from Roche Diagnostics, Abbot Labs, Sphingotec, Critical Diagnostics, and Thermo Fisher and has received biomarker study support in kind and/or as grants from AstraZeneca and Bristol Myers Squibb. RT has received grant funding and consulting fees from Roche Diagnostics. PG holds stock in Theranostics Lab, a molecular diagnostics company providing polygenic risk scores. All other authors declare no conflicts of interest.Schema showing the data platforms and linkages that result in the MENZACS cohort.Flow diagram of screening and enrolment into the MENZACS.Baseline demographics of the MENZACS cohort.Values are n (column percentage), median (interquartile range), or mean ± standard deviation. # Due to the nature of the registry, some variables were not mandatory and resulted in limited missing data which is represented by the denominator. NZDep = New Zealand Socioeconomic Deprivation; COPD = chronic obstructive pulmonary disease; BMI = body mass index; TC:HDL = ratio of total to high-density lipoprotein cholesterol; LDL = low-density lipoprotein; eGFR = estimated glomerular filtration rate, HbA1c = glycosylated haemoglobin. * “Other” ethnicities are non-Chinese Asian, Middle Eastern, Latin American, African, or Other.Baseline clinical characteristics of the MENZACS cohort.STEMI = ST-elevation myocardial infarction; GRACE = Global Registry of Acute Coronary Events; PCI = percutaneous coronary intervention; CABG = coronary artery bypass graft; ACEi = angiotensin-converting-enzyme inhibitor; ARB = angiotensin receptor blocker.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Cardiac amyloidosis (CA) is a common and potentially fatal infiltrative cardiomyopathy. Contrast-enhanced cardiac MRI (CMR) is used as a diagnostic tool. However, utility of CMR for the comprehensive analysis of biventricular strains and strain rates is not reported as extensively as echocardiography. In addition, RV strain analysis using CMR has not been described previously. Objectives: We sought to study the global and regional indices of biventricular strain and strain rates in endomyocardial biopsy (EMB)-proven, genotyped cases of CA. Methods: A database of 80 EMBs was curated from 2012 to 2019 based on histology. A total of 19 EMBs positive for CA were subjected to further tissue-characterization with histology, and compared with four normal biopsy specimens. Samples were genotyped for ATTR- or AL-subtypes. Five patients, with both echocardiography and contrast-enhanced CMR performed 72-h apart, were subjected to comprehensive analysis of biventricular strain and strain-rates. Results: Histology confirmed that the selected samples were indeed positive for cardiac amyloid. Echocardiography showed reduced global and regional left-ventricular (LV) longitudinal strain indices. CMR with tissue-characterization of LV showed global reductions in circumferential, radial and longitudinal strains and strain-rates, following a general trend with the echocardiographic findings. The basal right-ventricular (RV) segments had reduced circumferential strains with no changes in longitudinal strain. Conclusions: In addition to providing a clinical diagnosis of CA based on contrast clearance-dynamics, CMR can be a potent tool for accurate functional assessment of global and regional changes in strain and strain-rates involving both LV and RV. Further studies are warranted to validate and curate the strain imaging capacity of CMR in CA.Cardiac amyloidosis (CA) has poor prognosis unless identified and treated early. On histology, CA shows infiltration and deposition of abnormal non-contractile extracellular proteins within myocardial tissue [1]. This process leads to initial diastolic dysfunction and ultimately systolic dysfunction, often involving both left and right ventricles [2]. Recently, there have been significant advances in the multimodality imaging of CA, and the disease is being increasingly recognized as a cause of heart failure [2]. CA presents in two distinct forms: ATTR amyloidosis, which is due to deposition of transthyretin in myocardium, and AL amyloidosis, which is a plasma cell dyscrasia distinct from multiple myeloma [2,3].Recent clinical trials have demonstrated that CA is a treatable disease [4]. The specificity of diagnosing ATTR-CA with PYP scan is very high, whereas AL-CA can be ascertained by serum and urine tests. However, current imaging modalities for myocardial functional evaluation of CA are limited: echocardiography is the primary means of non-invasively diagnosing cardiac amyloidosis, with findings including speckling and strain imaging lending confidence to the diagnosis [5]. However, echocardiography is known to be operator-, equipment- and reader-dependent, leading to variability [6]. In addition, acoustic windows are easily affected by patient anatomy. As such, the gold standard for diagnosis of cardiac amyloidosis is an invasive endocardial biopsy, both to diagnose and correctly genotype the underlying pathology [7].Cardiac MRI (CMR) has been shown to play an increasingly important role in the detection of cardiac amyloidosis. Contrast-enhanced CMR can determine the myocardial amyloid burden [8], particularly as the infiltrative process can advance prior to reduction in LVEF [9]. The sensitivity and specificity for characteristic late gadolinium enhancement (LGE) to diagnose amyloidosis by CMR has been shown to be 85% and 92%, respectively [10]. Echocardiography is not able to perform tissue characterization in the same manner as CMR, but does reliably show abnormal strain in CA [11]. CMR also has been shown to have increasing utility in prognostication of cardiac amyloidosis, particularly CMR-derived indexed stroke volume and wall excursions [9,12].In this study, we aimed to demonstrate the feasibility of biventricular strain imaging using CMR in EMB proven cardiac amyloid, illustrate the parallels of CMR strain imaging with echocardiographic strain imaging, and provide an initial foray into CMR based evaluation of right ventricular strain [13].This is a multimodality retrospective feasibility study in a select group of patients with biopsy-proven cardiac amyloidosis. All patients were selected from a single, tertiary care center, Buffalo General Hospital (BGH) and Gates Vascular Institute (GVI) in Buffalo, New York. The study was approved by the University at Buffalo Institutional Review Board. An institutional database of 80 endomyocardial biopsies (EMB) from 2012 through 2019 was reviewed to identify 19 patients with cardiac amyloidosis on histology, with corresponding controls. From this cohort, 5 patients and corresponding controls were further identified to have echocardiography and contrast enhanced CMR done 72 h apart.EMBs were initially evaluated at BGH by hematoxylin–eosin (H&E) staining. Tissue sections showing homogeneous eosinophilic material on H&E were subjected to Congo red staining. Briefly, 10-micron paraffin embedded sections were deparaffinized, hydrated, and stained with Congo red. These sections were counterstained with hematoxylin, washed, dehydrated in 100% alcohol, cleared in xylene and mounted. Amyloid deposition was determined with bright field microscopy under polarized light. Amyloid typing was performed with liquid chromatography tandem mass spectrometry (LC MS/MS) on peptide extracted from Congo-red positive microdissected areas of the paraffin embedded specimens at Mayo Clinic (Rochester, MN, USA), and confirmatory genetic testing was performed for transthyretin cardiac amyloidosis (ATTR-CA).A GE 1.5-T scanner with technical parameters recommended by the manufacturer was used. After acquiring the localizer/scout images in coronal, sagittal and axial planes, fast spin-echo (FSE) axial slices, short-axis and two, three, and four-chamber steady-state free precession (SSFP) sequences, T2-weighted triple-inversion recovery images and T1-weighted FSE sequence were obtained. Then, delayed enhanced images were obtained 2–10 min after intravenous (IV) gadolinium injection (Omniscan 0.1 mmol/kg) using previously validated inversion recovery pulse sequence. The TI Cardiac function and strain imaging was performed by using Segment version 3.1 R8123 (http://segment.heiberg.se accessed on 15 June 2020), as described previously by our group [14,15,16,17,18].Echocardiography-Based Approach: Studies were uploaded to TomTec software module (TOMTEC USA, Chicago, IL, USA). Two-dimensional speckle tracking echocardiography was done using TomTec AutoSTRAIN, Version; Image-Com5 5.5.4.467461 to determine segmental longitudinal strain of all the visualized left ventricle segments. The endocardium was visualized and traced during end-systole and end-diastole. The software automatically tracked the region-of-interest during the cardiac cycle. The magnitude of deformation was used to generate the strain curves.Cardiac MRI-Based Approach: Strain analysis was conducted on amyloidosis patients using principles from finite strain theory, as described previously [14]. Cardiomyocyte contraction was evaluated by using one-dimensional Lagrangian strains (ε) along the circumferential, radial and longitudinal directions, which are defined by the following formula [19]:(1)ε=L−L0L0
+        where L is the length of the myocardial segment and L0 is the original length at end diastole. Cardiac contraction and relaxation can be measured using Eulerian strain rates (SR), which are based on using the myocardial velocity gradient as shown below [19]:(2)SR=v2−v1L=1LdLdt=1ε+1dεdt
+        where v1 and v2 are myocardial velocities. Circumferential and radial strains were measured by using three short-axis slices at the level of basal, midventricular and apical regions. Longitudinal strains were evaluated by using 2, 3 and 4-chamber long-axis images. Strains were determined by drawing the left ventricular (LV) and right ventricular (RV) borders at end diastole, after which the analysis module created the contours automatically. Quality control was achieved by manual adjustment of the initial contour of end-diastolic image. Quantitative analyses for strains were performed at end-systole, while quantitative analyses for the strain rate were done at peak-systole (SRS) and peak-diastole (SRD), which represent the two peaks seen in the strain curves, respectively [14]. Both LV and RV strains and strain rates were evaluated globally, as well as in basal, midventricular, apical and septal regions. Additionally, RV lateral strains and strain rates were also measured to evaluate RV free wall deformation.Quantitative findings were summarized within groups, using the mean and standard error of the mean (SEM). Quantitative regional comparisons were done using unpaired Student’s t-tests and Analysis of Variance (ANOVA). All tests were 2-sided, and p < 0.05 was considered significant.The mean age of 19 subjects with endomyocardial biopsy proven cardiac amyloidosis was 71 years, with a sex distribution 74% males. Out of 19 subjects, 10 had ATTR and 8 had AL cardiac amyloidosis, with one patient having an insufficient sample for subtyping (Supplementary Table S1).LC MS/MS analyses sub-classified the amyloid to ATTR and AL subtypes. Congo red-stained deposits are orange-red with bright field microscopy and display apple-green birefringence under polarized light. (Figure 1).A summary of comparative differences in the longitudinal strains measured by speckle tracking echocardiography is provided in Table 1. Representative echocardiograms of cardiac amyloidosis are shown in Figure 2. The global longitudinal strain (GLS) of the LV with cardiac amyloidosis was −7.96 ± 1.45% when compared to the GLS of a LV with normal group, which was −19.44 ± 0.33% (p < 0.01). There was significant reduction in LV segmental longitudinal strain of all the LV segments except in the basal inferoseptal, apical-inferior, apical-anterior and apical-lateral segments. The longitudinal strain of the amyloid apical septal myocardium was −14.50 ± 2.83%, which was lower than that of the normal apical septal myocardium at −20.86 ± 1.29%.First, the amyloidosis pattern was visualized based on diffuse myocardial late enhancement affecting the entire ventricle with a pathological gadolinium clearance (Figure 3). Strain analysis was conducted on five biopsy-proven cardiac amyloidosis cases with five corresponding age-matched controls. The control group exhibited preserved strain patterns across the circumferential, radial and longitudinal axes in all myocardial segments (basal, mid, apical and global) when compared with controls and previously published values [20]. Representative curves illustrating LV changes in longitudinal strain and strain rates for a normal patient, an AL amyloidosis patient, and an ATTR amyloidosis patient are shown in Figure 4A,B.Compared to controls, amyloidosis patients had exhibited significantly reduced circumferential, radial and longitudinal strains in the basal, midventricular, and apical segments (Figure 5, Table 2). These strain parameters in the LV septum were also significantly lower in amyloidosis patients. Amyloidosis patients also had an overall reduction in global circumferential strain (Table 2).Compared to normal patients, amyloidosis patients had reduced peak-systolic and diastolic circumferential strain rates in the basal and midventricular segments. These patients had an overall reduction in global peak systolic and diastolic circumferential strain rates (Table 2). The comparison of radial and longitudinal strain rates in different myocardial territories measured during both end-systole and end-diastole are shown in Table 2.RV strains were calculated in a fashion similar to that of LV strains, but radial values could not be reported due to the physical structure of the RV. The basal right-ventricular (RV) segments had reduced circumferential strains with no changes in longitudinal strain. The RV septal segments showed significant reductions in circumferential and longitudinal strains. Representative curves and quantitative comparison illustrating RV changes in circumferential and longitudinal strain and strain rates for a normal patient, an AL amyloidosis patient, and an ATTR amyloidosis patient are shown in Figure 4C,D and Figure 5 and Table 3.Our study further underscores the ability of CMR to identify patients with CA. While it is known that CMR is clinically indicated as a diagnostic modality to identify CA using LGE, we describe another diagnostic dimension of CMR that exhibits parallel trends with echocardiography: strain imaging. Our findings suggest that detection of abnormal gadolinium clearance kinetics and quantification of global and regional deformation by contrast-enhanced CMR can be complementary diagnostic tools for cardiac amyloid.When evaluating CA, echocardiography has long been the standard for non-invasive imaging techniques, with recent advances including strain rate imaging and speckle tracking improving its efficacy [21,22]. Echocardiography with high frame rates is known to be effective in strain imaging. However, echocardiography may have challenges with image quality, which can be a reflection of a patient’s body habitus, technical limitations or user variability (for instance, appropriate placement of the probe to minimize the angle between the beam and the LV wall if tissue doppler-based strain imaging techniques are employed) [23,24]. In addition, the LV apex is typically omitted on echocardiograms due to image shortening [25].Another diagnostic pathway utilizes CMR. Technological improvements have greatly improved its efficacy in measuring LVEF and ventricular volumes [26]. CMR is mostly not affected by body habitus, and standardized protocols have been developed for comprehensive myocardial imaging. CMR has the benefit of tissue characterization, which cannot be completed using ultrasound-based techniques [27]. In this study, we were able to analyze EMB proven cardiac amyloidosis with CMR, and focused on the often-underutilized strain imaging capability of CMR, as tissue characterization with LGE is well described in the literature. EMB proven CA was essential to directly link CMR based strain imaging to CA, in order to remove the risk of false-positive and false-negative diagnoses, which can occur when solely relying on non-invasive imaging.The results from our CMR analysis have shown mild-apical sparing in LV peak systolic circumferential strain rate. Instead, there was a consistent reduction in circumferential, radial and longitudinal strains in the basal, midventricular and apical regions. These results echoed the trends seen in echocardiographic based strain imaging; statistical analysis, namely regression analysis, could not be conducted during this study due to its small sample size; this should be explored in future larger, prospective studies. An interesting finding is that both the LV and RV septal segments showed significant reductions in systolic strain and strain rates, which suggests that cardiac amyloidosis might affect the contractility of the interventricular septum. A repository of standardized CMR strain values must be curated, and validated by established strain imaging echocardiography, to provide clinicians with a reference by which to characterize myocardial strain using CMR in patients with amyloid myocardium [28,29]. For the purposes of this study, our CA samples and corresponding analyses were compared to controls without any major structural or infiltrative heart diseases; in addition, there is no consensus for normal strain rate values even when using speckle tracking echocardiography [8].When assessing the RV, circumferential and longitudinal strains were analyzed, as the thin RV myocardium does not make assessing radial strain possible. Apical sparing was observed in the RV systolic circumferential strain, while RV systolic longitudinal strain was reduced in the apical region. Both the RV circumferential and longitudinal strains did not show significant differences in the midventricular and lateral RV segments, which could suggest possible sparing of these regions. It is known that RV involvement is common in CA. Current literature reports, however, have only recently studied RV-LGE, but these have not yet been validated as a prognostic tool [30,31]. In order to provide a more complete diagnostic picture for CA, strain imaging and LGE both need to be taken into consideration when using non-invasive imaging methods such as CMR [31]. To date, there has only been minimal study of the RV using strain imaging techniques, both with echocardiography and CMR.Strain rate analysis also shows promise in the diagnosis of CA. Early diastolic LV strain rate was determined to be an independent predictor of outcomes in patients with preserved EF [32]. However, there is no current literature that describes changes in RV strain rate. Our study shows a mild apical sparing in RV peak-diastolic circumferential strain rate, but this apical sparing pattern is not seen in other RV strain rates. Previous studies have primarily looked at LV strain while neglecting to determine systolic strain rate [28]. However, further larger studies need to be done to validate these results.Limitations: CMR has some limitations and may not be the most suitable imaging modality particularly in patients with renal dysfunction and presence of MRI-non-conditional devices. However, the advent of new contrast agents that are substantially less nephrotoxic and the shift in implantable devices to those that are MRI compatible will allow for more broader application of CMR to a larger population of patients [33]. Presently, acquisition time of CMR images is far longer than conventional echocardiography, although with advances in MRI technology these shortcomings should improve. Our study has a few other limitations which can be overcome with future research. This is a retrospective study with a small sample size which aims to illustrate the feasibility of using CMR to determine myocardial strain in CA; as a result, determining sensitivity and specificity of myocardial strain is difficult. Regression analysis between echocardiography and CMR is also limited due to the small sample size; as a result, complete validation of our CMR based strain with echocardiography has not been done and rather, general trends have been reported. In addition, delineation between ATTR and AL amyloidosis using CMR strain imaging was not conducted due to the small sample size.CMR can be a powerful non-invasive diagnostic modality for myocardial tissue characterization and strain imaging, providing a potential one-stop shop for non-invasive imaging in infiltrative cardiac disease. However, further large prospective studies are needed to validate strain findings with parallel modalities such as echocardiography and apply them to a more inclusive patient population.The following are available online at https://www.mdpi.com/article/10.3390/cardiogenetics11030011/s1, Table S1: Demographics and baseline clinical characteristics of the patients included for histological and imaging analysis.A.R. collected the clinical data and wrote the manuscript draft; V.S. cross-checked the data presentation and provided additional inputs on the conceptual aspects; B.K. performed MRI analyses; L.J. quantified histological parameters; S.K. (Silva Kristo) obtained the histological images from the myocardial biopsy specimens; S.K. (Sharma Kattel) performed the statistical analyses; R.A. performed echocardiographic strain analyses; S.P. conceptualized the project and supervised the genotypic and histological aspects; and U.C.S. conceptualized the project, edited manuscript, provided funding support and was the overall supervisor of this project. All authors have read and agreed to the published version of the manuscript.This research was supported by NIH UL1TR001412 and P30CA016056. UCS received support from K08HL131987 and SP received support from R21 HL138555.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of University at Buffalo (protocol code 3490, date of approval 18 November 2019).Patient consent was waived due to a retrospective nature of this study, which did not require patient identification or the genetic tracking of the family members.The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the sensitivity of patient information. The authors declare no conflict of interest.Bright field microscopy (A–C) and polarized light (D–F) display of Congo red-stained myocardial sections. Amyloid deposits are orange-red on Congo red staining with bright field microscopy and display apple-green birefringence under polarized light. (A,D) ATTR amyloid, (B,E) AL amyloid, and (C,F) Control myocardium.Left ventricular (LV) longitudinal strain imaging of cardiac amyloidosis using speckle tracking echocardiography. The basal segments are noted to exhibit dyskinetic motion, as observed by the outward movement of the basal inferior, basal anteroseptal and basal anterolateral segments. The apical segments are more contractile, but still relatively hypokinetic.Cardiac MRI (CMR) visualization of cardiac amyloidosis using late gadolinium enhancement (LGE) and strain imaging. (A–C) Lack of myocardial nulling and characteristic speckled appearance are noted after gadolinium injection in cardiac amyloidosis. (A) Representative LGE-CMR short-axis image of a normal patient. (B) Representative LGE-CMR short-axis image of an ATTR amyloidosis patient. (C) Representative LGE-CMR short-axis image of an AL amyloidosis patient. (D,E) Visualization of left and right ventricular longitudinal strains under 4 chamber (4CH) long-axis view. (D) Representative 4CH-CMR image of a normal patient. (E) Representative 4CH-CMR image of an amyloidosis patient.Strain curves illustrating left ventricular (LV) and right ventricular (RV) changes in global longitudinal strain and strain rate over the course of one cardiac cycle. Representative strain curves were used from a normal patient, an AL amyloidosis patient, and an ATTR amyloidosis patient. (A) LV longitudinal strain curves; (B) LV longitudinal strain rate curves; (C) RV longitudinal strain curves; (D) RV longitudinal strain rate curves.Strain curves illustrating right ventricular (RV) global changes in strain and strain rate over the course of one cardiac cycle. Representative strain curves were used from a normal patient, an AL amyloidosis patient, and an ATTR amyloidosis patient. (A) RV circumferential strain curves; (B) RV circumferential strain rate curves; (C) RV longitudinal strain curves; (D) RV longitudinal strain rate curves. SRS = peak systolic strain rate; SRD = peak diastolic strain rate.Left ventricle longitudinal strain characteristics of amyloid patients compared to normal subjects using speckle tracking echocardiography.Abbreviations: GLS = Global longitudinal strain; SEM = Standard error of mean; (*) indicates p < 0.05.Left ventricular (LV) strain and strain rate characteristics of amyloidosis patients compared to controls using cardiac MRI.Values are presented as mean ± standard error of mean (SEM). (*) indicates p value < 0.05 for amyloidosis patients compared to controls.Right ventricular (RV) strain and strain rate characteristics of amyloidosis patients compared to controls using cardiac MRI.Values are presented as mean ± standard error of mean (SEM). (*) indicates p value < 0.05 for amyloidosis patients compared to controls.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Amyloidosis is a group of diseases in which amyloid fibrils build up in tissues, leading to organ dysfunction. Cardiac involvement is observed in immunoglobulin light chain amyloidosis (AL) and transthyretin amyloidosis (ATTR) and, when it occurs, the prognosis worsens. Cardiac tissue infiltration can lead to restrictive cardiomyopathy with clinical signs of diastolic heart failure, without reduction of ejection fraction (HFpEF). The aim of multiple and less invasive diagnostic tests is to discern peculiar characteristics and reach the diagnosis without performing an invasive endomyocardial biopsy. These diagnostic tools allow early diagnosis, and they are crucial to best benefit from target therapy. In this review article, we describe the mechanism behind amyloid fibril formation, infiltration of tissues, and consequent clinical signs, focusing on the diagnostic tools and the red flags to obtain a diagnosis.Amyloidosis is a rare protein deposition disease that occurs when the amyloid, an abnormal protein formed from other precursors’ misfolding, assembles into oligomers and fibrils that deposit in the organs, interfering with their normal functions. Amyloid fibrils show typical features on electron microscopy and a pathognomonic “apple-green” birefringence on polarized light microscopy (related to Congo red staining’s affinity for ß-pleated sheets) [1,2,3].Among several types of amyloid diseases, two of them account for almost all cardiac amyloidosis (CA): transthyretin amyloidosis (ATTR) and immunoglobulin light chain amyloidosis (AL) [4].AL, also named primary amyloidosis, is related to the deposit of immunoglobulin light chain fragments. It can be defined as a rare disease [5], with an estimated incidence and prevalence of about 3–12 cases per million persons per year and 30,000–45,000, respectively [6]. It is strongly associated with multiple myeloma (MM), and in about 10% of cases, the two diseases are concomitant. The median age at diagnosis is 63 years old, although it can also present in patients aged between 30 and 40. It is an aggressive disease (more so than ATTR) that can affect all organs, in particular the kidneys, heart, and nervous system. The heart’s involvement decreases survival, especially if heart failure is present [7]. For this reason, an early diagnosis of cardiac disease is essential in order to start treatment and reduce the otherwise high mortality.In ATTR amyloidosis, there is a misfolding of transthyretin (TTR), a liver-derived protein involved in the carriage of thyroid hormone and vitamin A in the blood. There is an acquired wild-type variant (ATTRwt) and a hereditary mutant variant (ATTRm or ATTRv), differentiated by genetic testing for mutations of the TTR gene [8].ATTRwt is also named “senile CA” because it typically occurs in older age. The most common clinical manifestations are carpal tunnel syndrome (almost always bilateral), spinal stenosis, and—if there is extensive heart involvement—hypertrophic restrictive cardiomyopathy with diastolic heart failure, often unrecognized in the elderly [9]. Considering the aging population, the ATTRwt variant will become the most common form of amyloidosis. It is much more common in males than in females.The ATTRm variant is related to TTR gene point mutations. The clinical manifestations are mainly related to heart and nervous system involvement, and usually both are implicated. In some cases, there is a clear prevalence of one of them, with a more evident vertical transmission, as in familial amyloid polyneuropathy (FAP) [10] and familial amyloid cardiomyopathy. The most common of the more than 100 possible gene mutations is V122I, in which an isoleucine substitutes a valine at the 122nd amino acid position. This mutation causes extensive heart involvement with late-onset restrictive cardiomyopathy, often unrecognized and confused with hypertensive heart disease, and with minimal neuropathy. Although the prognosis is usually better than that of AL, without treatment, the median survival for V122I ATTRm-CA is about 2 years.Regardless of which type of precursor protein misfolds, in both AL-CA and ATTR-CA, there is a large amyloid fibril organ deposition. This causes the thickening of both ventricles’ walls, with a different pattern in the two types of CA; in AL-CA, the involvement is more disseminate and usually subendocardial, while in ATTR-CA, the deposits can involve more the interventricular septum, mimicking hypertrophic cardiomyopathy, with more transmural involvement [11]. CA leads to an increased ventricular wall thickness and stiffness in both ventricles, trademarks of restrictive cardiomyopathy. In CA, the amyloid deposits are usually interstitial, surrounding the myocytes, but they can also be intramural, causing stenosis of coronary arteries and angina or even myocardial infarction. Amyloid deposits can be found by taking a biopsy of myocardial tissue (endomyocardial biopsy, EMB), which is the most sensitive diagnostic tool for CA diagnosis (almost 100%). This is the main difference from other deposition diseases such as sarcoidosis, in which a biopsy cannot give any diagnostic information. In AL-CA, other than the damage related to fibril deposition, cardiac dysfunction may also be related to direct cell damage mediated by light chains [12].The different types of CA can have various clinical manifestations and different prognoses and treatments. Key “red flags” for possible systemic amyloidosis can be evaluated (Figure 1) [4].Cardiac involvement is usually asymptomatic at diagnosis. The most typical cardiac manifestation in CA is heart failure with preserved ejection fraction (HFpEF—diastolic heart failure). Symptoms related to low cardiac output (dyspnea on exertion, fatigue, and weakness) are common and are often the cause of initial misdiagnosis. In other cases, the first manifestation could be atrial fibrillation or cardioembolic stroke. Atrial fibrillation has a high prevalence and is more common in ATTRwt-CA [13]. For years, it was not considered a sign of CA. The risk of thromboembolism can also be increased by electromechanical dissociation secondary to atrial infiltration, even without atrial fibrillation. More so in ATTR-CA than in AL-CA, amyloid infiltration can lead to bundle branch block and third-degree atrioventricular block. Some patients develop symptoms such as ascites and lower extremity edema related to right-sided heart failure [14]. Simil-ischemic manifestations (angina with normal coronary arteries, cardiogenic shock) and low flow, low gradient aortic stenosis are less common. The normalization of blood pressure values in previously hypertensive patients can be a sign of possible CA. Other symptoms can raise suspicion of CA if considered in a specific clinical context. Carpal tunnel syndrome, almost always bilateral, and spinal stenosis can precede for years the symptoms of heart failure. They can be present in about 50% of patients with ATTRwt [7], with a higher specificity than in AL-CA. Peripheral and autonomic neuropathy can occur in both forms, but they are uncommon in ATTRwt. Nevertheless, some point mutations in the TTR gene can be associated with predominant neurological symptoms. This is the case with the Val30Met mutation, a cause of prevailing peripheral nervous system involvement (hereditary amyloid transthyretin amyloidosis with polyneuropathy). Other signs and symptoms typical of AL are macroglossia and periorbital purpura, which are pathognomonic when occurring together but infrequent, and signs of renal and gastrointestinal involvement, such as proteinuria, diarrhea, and weight loss. ATTRv amyloidosis, related to gene mutations, is differentiated into early- or late-onset if symptoms occur before or after 50 years old. Late-onset is more frequently sporadic and aggressive, with predominant peripheral neuropathy [15].It is often difficult to obtain an early diagnosis of CA, which has consequences for the prognosis. This is mainly related to the different possible clinical manifestations of the disease and the need for a histological demonstration requiring endomyocardial biopsy. A complete evaluation of CA includes consideration of clinical symptoms, cardiac involvement, and systemic amyloidosis, followed by the differentiation of the amyloid deposits into AL or ATTR; last, in the case of ATTR, there is the need to find the specific genetic mutation.Endomyocardial biopsy could be a gold standard considering its high sensitivity (100%), but it is not practical as a screening test for CA because of the procedural risk and the requirement of high procedure and disease knowledge. Furthermore, because it samples only some heart areas, it cannot quantify whole-heart involvement or the extra-cardiac burden, and it has a limited ability to evaluate the response to therapy. Thus, there is a need for a multi-imaging approach with contemporary imaging techniques, including CMR, radionuclide imaging, and echocardiography with longitudinal strain quantification. These are now becoming the main features to diagnose and manage CA [16].A diagnostic approach using biomarkers is possible only for AL-CA, in which immunofixation has a high sensitivity to detect and quantify free light chains. By contrast, there is no blood test that can actually diagnose ATTR-CA by identifying TTR oligomers. However, it has to be considered that abnormal levels of free light chains alone are not specific for the diagnosis of AL amyloidosis, considering the incidence in older age groups of monoclonal gammopathy of undetermined significance (MGUS)—up to 5% of the population over 65 years old. Elderly patients with ATTR-CA and MGUS could show high levels of light chains, leading to a misdiagnosis of AL-CA. Patients with chronic kidney failure could have increased serum concentrations of free light chains filtered by the renal glomeruli. Natriuretic peptides are usually observed in AL-CA, often disproportionate to the symptoms [17,18]. Elevated levels of troponins are also common, related to a toxic effect of the amyloid, and this can lead to false diagnoses of acute coronary syndrome. Laboratory tests can also predict the prognosis in CA. In AL-CA, a combination of NT-pro-BNP, Troponin T, and the difference between kappa and lambda free light chains has been used for a staging system by the Mayo Clinic; patients with a marked elevation of one or more of these parameters tend to have a worse prognosis. At the same time, NT-pro-BNP reduction predicts the clinical outcome and survival independently of the type of therapy.Gene sequencing is essential to differentiate acquired from hereditary amyloidosis, and it is recommended in all clinical settings when there is a high suspicion of TTR-related amyloidosis. For incomplete and late penetrance, it is unusual to find a family history indicating autosomal dominant inheritance. DNA sequencing is a valuable approach to confirm or exclude ATTRv diagnosis in these cases when ATTR variants alone cannot confirm the diagnosis. Thus, transthyretin gene sequencing is recommended in cases in which mass spectroscopy is positive for hereditary TTR or negative but with a high probability of disease. Gene sequencing is also essential for the diagnosis of ATTR with polyneuropathy to search for specific TTR gene amyloidogenic variants (Val30Met).Several electrocardiographic patterns can be present in CA. The most typical is related to low QRS voltages (height <5 mm in all limb leads). In almost 50% of patients with AL-CA, there is a pseudoinfarction pattern associated with poor R wave progression in the chest leads (Figure 2). As mentioned, one of the most common arrhythmias in CA is atrial fibrillation (about 20% of patients), but amyloid infiltrations can also involve the conduction system in different degrees of severity, from first-degree atrioventricular block (about 20%) to third-degree atrioventricular block. Other less common manifestations are left and right bundle branch block and ventricular tachycardia [19,20]. Holter ECG monitoring helps to identify asymptomatic arrhythmias in more than 75% of AL-CA patients, mainly supraventricular tachyarrhythmias and some nonsustained ventricular tachycardias. Some ECG patterns are more frequent in AL than in ATTR-CA, such as low QRS voltages. Conversely, atrial fibrillation, left bundle branch block, and complete heart block are more common in ATTR-CA. Conflicting data between ECG and echocardiography could sometimes raise the suspicion of CA. For instance, a left ventricular wall thickening can usually be associated with high QRS voltages. If there are low or normal QRS voltages, CA should be considered. A useful instrument to help diagnose CA is the ratio of ECG voltage to LV wall thickness [21]. Echocardiography plays a fundamental role in the non-invasive diagnosis of CA, studying heart function and structure in patients with cardiac symptoms or even before symptoms appear. The main focus in CA is to evaluate the thickening of LV walls and the exclusion of other likely causes of LV hypertrophy, such as severe hypertension and moderate to severe aortic stenosis (Figure 3) [22]. However, a clear distinction could be challenging. The suspicion of CA can be increased by other echocardiographic features: biatrial enlargement with a small or normal LV cavity size, presence of thrombi in the left atrium or left atrial appendage, thickening of the cardiac valves, thickening of the right ventricular wall, pericardial effusion, and a restrictive transmitral Doppler filling pattern. In particular, the typical increased wall thickness (>12 mm) with a reduced fractional shortening (<30%) has a significant impact on diagnosis and prognosis [23]. A granular sparkling of the myocardial walls can also be recognized, especially in the interventricular septum, but it does not have a high specificity. Other than the classical evaluation of left ventricular systolic function with LV ejection fraction (LVEF), tissue Doppler imaging (TDI) and speckle-tracking echocardiography (STE) have refined the evaluation of longitudinal systolic function and consequently have increased the probability of an early diagnosis of CA [24,25]. The study of cardiac function with global longitudinal strain (GLS) can help in the diagnosis of CA; despite a preserved LVEF, there is an early reduction of longitudinal shortening, absent in other causes of increased LV wall thickness. In both AL-CA and ATTR-CA, there is a typical apical sparing pattern in STE-derived longitudinal strain; apical LV segments are commonly unaffected, while there is severe impairment in basal and mid-cavity segments (Figure 4). This pattern does not affect patients with other causes of LV hypertrophy, in which the areas with the impairment of the LV longitudinal strain are those with maximal hypertrophy. Despite this peculiarity, it could be difficult to exclude or confirm a diagnosis of amyloidosis in patients with increased heart wall thickness [26]. A multiparametric echocardiographic approach has been proposed, focusing on relative wall thickness, global longitudinal strain (with apical sparing pattern), TAPSE, and E/E’. To guide the diagnostic algorithm, it is often necessary to use highly specific or sensitive cutoffs in patients with a hypertrophic phenotype to avoid unnecessary tests and to limit the time to diagnosis. This approach could be useful to restrict the use of other imaging techniques, such as CMR or endomyocardial biopsy, to patients for whom there is an intermediate to high probability of disease, despite the uncertainty of the first-level diagnostic modalities [27]. Other echocardiographic parameters can be useful. The myocardial contraction fraction (MCF) is the ratio of stroke volume to myocardial volume, and it is helpful in the evaluation of volumetric shortening (correlated with LV longitudinal strain) independent of LV size. Abnormalities beyond the left ventricle can also suggest CA [28]. Stroke volume index has prognostic value in predicting survival in AL-CA; similar to LV strain, it is routinely calculated and easier to achieve than STE. Left atrial dysfunction can also be documented, resulting in both reservoir and pump function impairment with strain because of the increased LV filling pressure and direct amyloid infiltration. This dysfunction may cause the formation of thrombi directly in the atrium or in the appendages (mostly the left appendage), increasing cardioembolic stroke risk even if the patient maintains sinus rhythm [29,30]. The right chambers’ involvement, as documented by CMR studies, contributes to predicting the prognosis in CA. The right ventricle is often affected because of direct amyloid infiltration (as with the left atrium) and the increased afterload from pulmonary hypertension. The result is an impairment of right ventricle systolic function, measured as reduced tricuspid annular plane systolic excursion (TAPSE), tissue Doppler systolic velocity (Sm wave), and longitudinal strain [31,32]. Even with the most modern echocardiographic techniques, the diagnosis of the right chambers’ involvement in CA often depends on the tissue characterization provided by CMR. The combination of echocardiographic parameters with other findings (clinical, biomarkers, and electrocardiography) can increase diagnostic accuracy [33].Cardiac magnetic resonance (CMR) has a central role in the non-invasive diagnosis of CA because of its capacity to give a precise tissue characterization and to differentiate CA from other causes of LV thickening. An extensive CMR evaluation of the four chambers is essential to provide information on ventricle thickness and function, as well as for the study of the atria and the research of potential thrombi. An extensive evaluation with CMR involves cine imaging, assessment of8 native T1 signal, late gadolinium enhancement (LGE), and extracellular volume (ECV) [34,35]. Amyloid deposition has a typical appearance of global and subendocardial LGE, and the distribution strictly correlates with prognosis [36]. However, the enhancement can sometimes be more transmural or, conversely, localized and spotty. To make scans less operator-dependent and to reduce false negatives, it is possible to use phase-sensitive inversion recovery (PSIR) sequences. T1 mapping, which compares scans after contrast administration with the basal images (native T1), is a useful technique to take a quantitative approach in the evaluation of myocardial involvement. In CA, there is an increase in native T1 in both AL and ATTR-CA (higher in AL-CA). In the case of renal impairment, it is possible to use only native T1, requiring no contrast administration, and it may be abnormal before the thickening of the left ventricular wall becomes evident [37]. ECV evaluation before and after contrast administration and combined with native T1 mapping is useful to measure the amyloid burden and myocardial edema—it is usually higher in ATTR-CA. This approach is also helpful in following the progression of the disease and the response to therapy [38,39,40,41,42].Radionuclide imaging plays a key role in the non-invasive diagnosis of CA. Cardiac involvement is measured by the evaluation of the uptake of diphosphonate radiotracers, such as 99mTechnetium-pyrophosphate (99mTc-PYP), 99mTechnetium-3,3-diphosphono-1,2propanodicarboxy-lic (99mTc-DPD), and 99mTechnetium-hydroxymethylene diphosphonate (99mTc-HMDP). Myocardial uptake for these tracers is remarkably sensitive for ATTR-CA, although not completely specific (Figure 5) [43]. Collectively, uptake in AL-CA is almost absent, and this is the most significant difference from ATTR-CA, which has a high affinity for bone tracers. This differential uptake seems to be related to preferential binding to ATTR because of a higher calcium content [44]. The affinity of the bone tracers in ATTR-CA is useful for the early identification of the disease, and it can help to identify cardiac ATTR deposits in asymptomatic patients at an early stage when the other diagnostic findings might still be absent. Nevertheless, the uptake of 99mTc-DPD can occur in some patients with AL-CA (about 30%); in these cases, the use of 99mTc-DPD SPECT-CT can help to distinguish the two types of CA, having high sensitivity for ATTR-CA. Its tracing has also been evaluated as a potential target for diagnosis and screening [45,46]. The role of 99mTc-YP/DPD/HMDP cardiac scintigraphy was highlighted in the recently developed consensus algorithm for the non-invasive diagnosis of CA (Figure 6) [47].This algorithm gives an approach to the evaluation of patients with CA considering the probability of CA. The diagnosis of ATTR-CA can be highly suspected, avoiding the need for endomyocardial biopsy, if there are not clonal abnormalities and there is a high level of uptake of 99mTc-PYP/DPD/HMDP in the radionuclide imaging. On the other side, patients with biomarker abnormalities, such as high light chain levels in serum/urine immunofixation, have a high probability of AL-CA, and they should be evaluated by hematologists. Cardiac involvement can be evaluated with the aforementioned non-invasive imaging techniques or with endomyocardial biopsy.Endomyocardial biopsy (EMB) was previously considered the diagnostic gold standard to evaluate cardiac amyloid deposition. Typically, the pathognomonic apple-green birefringence under cross-polarized light microscopy confirms the presence of amyloid fibrils. Today, it cannot be considered the optimal diagnostic choice to obtain a diagnosis of CA because of the need for an invasive approach and the potential risks. For the evaluation of systemic burden, the diagnostic accuracy of an extra-cardiac biopsy, i.e., the abdominal fat pad, depends on the examined tissue and the type of amyloidosis. The accuracy is higher for AL-CA, in which the yield of a fat pad biopsy is >70%, and it is strongly associated with whole-body amyloid load [48]. For the relatively low invasiveness, a fat pad biopsy is the preferred initial site, but a negative result is not sufficient to rule out a diagnosis of CA. If there is a high suspicion of disease, an EMB should be performed despite a negative extra-cardiac biopsy.Cardiac involvement is variable in the different types of amyloidosis, but it has a major impact on prognosis [49]. Cardiac amyloidosis has been revalued as a more treatable and possibly curable condition thanks to the recent improvements in diagnostic and therapeutic strategies. Nevertheless, morbidity and mortality remain high. For this reason, more advancements are needed to obtain an early diagnosis and to enhance prognoses in these patients.Conceptualization, M.G.M. and F.I.; methodology, M.D.M.; software, A.I.; validation, M.C. and A.E.; formal analysis, R.P.; investigation, F.I.; resources, M.D.M.; data curation, A.I.; writing—original draft preparation, M.G.M.; writing—review and editing, A.E.; visualization, M.C.; supervision, A.E.; project administration, R.P.; funding acquisition, A.I. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The authors declare no conflict of interest.Red Flags for cardiac amyloidosis.ECG of a patient with cardiac AL amyloidosis showing the pseudoinfarction pattern in anterior leads and small QRS voltages predominantly in the limb leads.Sequence of still images showing typical echocardiographic features of cardiac amyloidosis. (A): Two-dimensional (2-D) parasternal long axis showing an increase of left ventricular wall thickness—in both the interventricular septum and the posterior wall (concentric hypertrophy)—and left atrial dilatation. (B): Two-dimensional parasternal short axis. (C): Two-dimensional apical four-chamber view. In B and C, it is possible to highlight not only the left ventricular hypertrophy but also the biatrial dilatation. (D): Pulsed wave Doppler of mitral inflow showing a restrictive pattern at transmitral flow: increase in E/A ratio and normal E wave deceleration time with a marked reduction in transmitral A wave velocity. (E): Pulsed wave Doppler of pulmonary vein inflow showing marked diastolic prominence and increased duration and peak velocity of atrial reversal compared with the transmitral signal. (F): Pulsed tissue Doppler of the lateral mitral annulus showing marked reduction in apical systolic and diastolic velocities (normal velocities: >6 cm/s and >8 cm/s, respectively). Images courtesy of Professor Elliott, University College London, UK.(Left panel) Segmental color coding in the apical four-chamber view showing the typical apical sparing pattern, with the more negative strain segment in the apex (darker red), compared with a lesser negative strain in the basal segments (pink). (Middle panel) Individual segmental strain for the same view. (Right panel) A bull’s-eye plot derived from the three apical views, showing sparing of apical strain (center of plot) with impaired mid-cavity and basal strain.A positive 99mTc-DPD scan for TTR cardiac amyloid (left) showing uptake in the heart (arrow) and reduced bone uptake. The right-hand panel shows a fused CT/SPECT image showing myocardial uptake, with greater uptake in the septum.Consensus algorithm for non-invasive diagnosis of cardiac amyloidosis.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Background: AARS2 encodes the mitochondrial protein alanyl-tRNA synthetase 2 (MT-AlaRS), an important enzyme in oxidative phosphorylation. Variants in AARS2 have previously been associated with infantile cardiomyopathy. Case summary: A 4-year-old girl died of infantile-onset dilated cardiomyopathy (DCM) in 1996. Fifteen years later, her 21-year-old brother was diagnosed with DCM and ultimately underwent heart transplantation. Initial sequencing of 15 genes discovered no pathogenic variants in the brother at the time of his diagnosis. However, 9 years later re-screening in an updated screening panel of 129 genes identified a homozygous AARS2 (c.1774C > T) variant. Sanger sequencing of the deceased girl confirmed her to be homozygous for the AARS2 variant, while both parents and a third sibling were all found to be unaffected heterozygous carriers of the AARS2 variant. Discussion: This report underlines the importance of repeated and extended genetic screening of elusive families with suspected hereditary cardiomyopathies, as our knowledge of disease-causing mutations continuously grows. Although identification of the genetic etiology in the reported family would not have changed the clinical management, the genetic finding allows genetic counselling and holds substantial value in identifying at-risk relatives.Genetic variants in the nuclear gene AARS2 have previously been associated with recessively inherited infantile mitochondrial cardiomyopathy [1,2,3,4]. AARS2 encodes the mitochondrial protein alanyl-tRNA synthetase 2 (MT-AlaRS), an important enzyme in oxidative phosphorylation contributing to normal functioning of respiratory chain complexes I, III, and IV1.Typically, genetic variants in AARS2 have been identified in one tissue-specific disease, most commonly in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy [5].A 21-year-old Caucasian male diagnosed with Asperger Syndrome, was admitted (2011) to the emergency department with progressive dyspnea and lower limb edemas. The patient was the second of three children from healthy nonconsanguineous Danish parents. Due to a family history of dilated cardiomyopathy (Figure 1), an echocardiography at age 6 years (1996) had been performed with no abnormal findings, and no follow-up was planned at that point.At the time of admission in 2011, the patient was hemodynamically stable (blood pressure 116/69, heart rate 100) and presented with a fever leading to initiation of antibiotic treatment due to suspicion of pneumonia. Routine blood testing including cardiac troponins, hematological, and kidney biomarkers were unremarkable. However, an ECG showed intraventricular conduction defect with a QRS-duration of 150 ms (Figure 2). A chest radiograph revealed cardiomegaly (Figure 3A). Subsequently, an echocardiography demonstrated severe left ventricular (LV) dilation with an end-diastolic diameter of 82 mm (indexed 41 mm/m2) and a LV ejection fraction (EF) of 10% (Figure 3B). These findings led to the initiation of anti-congestive medical treatment and later implantation of a cardiac resynchronization therapy-defibrillator. Further diagnostic work-up included genetic screening, right heart catheterization, and coronary angiography. The right heart catheterization showed a reduced cardiac index (1.8 L/min/m²) and elevated cardiac chamber filling pressures (right atrial pressure of 12 mmHg, mean pulmonary artery pressure of 47 mmHg, and wedge pressure of 31 mmHg). The coronary angiogram was normal, and the myocardial biopsy showed histologic features consistent with dilated cardiomyopathy with severely hypertrophic cardiomyocytes (Figure 4A–C).The patient was hospitalized for two weeks, and subsequently followed closely as an outpatient in the heart failure clinic. Despite heart failure medication and cardiac resynchronization therapy, the patient remained severely symptomatic with no signs of left ventricular reverse remodeling or recovery of LVEF. Five months later, a left ventricular assist device was therefore implanted as a bridge to heart transplantation. Transplantation was performed without complications 40 months after the initial diagnosis. Today, eight years post-transplantation the patient is alive and doing well.Fifteen years prior (1996) to the events described above, the patient’s 4-year old sister was admitted with dyspnea and fever to a local hospital. Initially, the patient was suspected to have pneumonia. The patient’s mother had noticed a reduced appetite and onset of mild dyspnea a week prior to admission and prior to admission the girl was described to have a short stature and physically weak compared to her peers with some circumstantial evidence of delayed neuro-cognitive development. Symptoms of dyspnea progressed and was accompanied by fever (40 °C) and incessant coughing with intermittent mucous vomit on the day of admission. On clinical examination the patient was found to be cyanotic, have massive jugular venous distension (10–12 cm), and peripheral edema. A chest X-ray did not identify pneumonia, but cardiac enlargement was observed. An echocardiography showed severe LV dilation, a LVEF of 5% and severe universal hypokinesia of the LV. At this point, the patient was suspected to suffer from acute fulminant myocarditis and was transferred to a tertiary hospital. During transport, the patient developed cardiogenic shock with pulmonary edema, respiratory acidosis and was admitted directly to an intensive care unit (ICU). At the ICU, the patient received invasive mechanical ventilation along with vasopressors, intravenous immunoglobulin, and other supportive care. Treatment led to some initial clinical improvement although development of kidney failure required hemodialysis. During the admission, no subsequent improvement in systolic functioning was observed and the patient died of terminal heart failure after four weeks of hospitalization. An autopsy was performed with histologic features strongly suggestive of cardiomyopathy compatible with dilated cardiomyopathy (Figure 4D–F).Initially, genome based genetic sequencing was performed in a standard screening panel, which in 2011 consisted of 15 genes (MYH7, MYBPC3, TNNT2, TNNI3, TPM1, ACTC, MYL2, MYL3, CSRP3, SCN5A, LAMP2, PRKAG2, GLA, LMNA, and SLC225A). No disease-associated genetic variants were discovered. At a consultation nine years later in the outpatient clinic, re-screening with an updated screening panel including 129 genes was requested. Two genetic variants were identified: a homozygous variant in AARS2 (NM_020745.3:c.1774C>T, p.(Arg592Trp)) classified as pathogenic, and a heterozygous variant in ACTN2 (NM:001103.3:c.1445G>A, p.(Arg482Gln)) classified as a variant of unknown significance.The timing of the patient’s disease (1996) predated the era of genetic screening. Following the genetic findings in patient 1, the patient’s Guthrie card (routinely collected in all infants in Denmark since 1981) [6] was analyzed. DNA was extracted, and Sanger sequencing confirmed that patient 2 was a homozygous carrier of the AARS2 variant but did not carry the ACTN2 variant.Sanger sequencing of both parents (with no familial relation) and the third sibling found all to be unaffected, heterozygous carriers of the AARS2 variant (Figure 1). Only the mother carried the ACTN2 variant. The AARS2 variant is classified as pathogenic in homozygous carriers, causing infantile mitochondrial cardiomyopathy in homozygous or compound heterozygous carriers, however the genetic causes are poorly known Ref. [1]. A significant reduction in oxidative phosphorylation complexes in the heart but also in the skeletal muscle and brain has earlier been observed. Therefore, the AARS2 is an example of how mutations in the same gene can lead to very different diseases with dissimilar tissue involvement such as leukoencephalopathy and ovarian failure. The tissue-specific defects affect distinct domains of the synthetase but compromise the aminoacylation with different outcomes [5].This variant has an allele frequency of 0.04% (52 of 128,596) in the European population of the Genome Aggregation Database (gnomAD v2.1.1). Heterozygote carriers are phenotypically unaffected [7,8,9]. There are no homozygotes carriers in gnomAD. The ACTN2 variant is a rare variant of uncertain significance, the variant has been reported in 4 of 129146 control alleles in gnomAD.The case presented above underlines the importance of re-assessing whether sufficient genetic information is available to perform qualified diagnostic work-up and genetic consultation in families with familial dilated cardiomyopathy.In index patients from families with familial dilated cardiomyopathy, a genetic etiology is currently identified in 25–40% of cases [10]. This leaves a large proportion of patients with familial disease without a genetic explanation. While multiple reasons for this observation exist, a substantial subset of patients, who were screened prior to broad clinical application of Next Generation Sequencing could potentially benefit from re-sequencing as our knowledge of disease-causing mutations continuously expands [11].Knowledge of the genetic etiology of disease would not have changed the clinical outcomes in the family described above. However, the discovery of the AARS2 variant as the underlying cause of heart failure had great impact on the clinical management of the remaining family members who could be reassured that they were not at risk of developing heart failure and that further screening could be ceased. Furthermore, patient 1 and his healthy sister (being a heterozygous carrier) may be offered genetic counselling in case of a planned future pregnancy since the AARS2 variant occurs in 1:2500 in the general population.Finally, the evolving understanding of specific genotype-phenotype correlations could potentially provide a more personalized approach towards future management of genotype-positive patients [12,13,14].In conclusion, extended genetic screening in families with an initial negative result in small genetic screening panels can provide substantial value in providing correct risk-assessment, treatment, and follow-up.All authors have contributed equally. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Written informed consent was obtained from the patient for publication of this case report.The authors declare no conflict of interest.Pedigree showing recessive inheritance of dilated cardiomyopathy (DCM). Individuals with DCM are illustrated by black filling.ECG of patient 1 at admission time showing an intraventricular conduction defect.(A) Chest radiograph of patient 1 at admission time showing cardiomegaly. (B) Echocardiography of patient 1 at admission time showing severe left ventricular dilation.(A–C) Histological study of the myocardial biopsy of patient 1. In both deep (A) and subendocardial myocardium (B) substantial numbers of cardiomyocytes are replaced with fibrosis consisting of irregularly arranged fine collagen fibrils with entrapped myocytes in the periphery. Severe hypertrophy (myocyte diameter up to 50 µm) as well as irregularly shaped and enlarged nuclei (diameter typically 15 µm) are observed in the remaining myocytes. In addition, fragmentation and loss of central myofibrils occurs focally. No obvious myocyte disarray was observed. The features are consistent with cardiomyopathy and compatible with the clinically observed dilated cardiomyopathy, although hypertrophic cardiomyopathy is a histological differential diagnosis. (D–F) Histological study of the myocardial biopsy of patient 2. Appearance of variably solidified fibrotic areas in both central (D) and subendocardial myocardium (E) replacing few cardiomyocytes. The caliber is lightly variably with a diameter around 20 µm and nuclei appearing slightly enlarged (typically 10 µm in diameter) and often slightly irregular in shape. Focally perinuclear myofibrils seem fragmented and diminished in occurrence. The features are similar to those observed in patient 1, although less severely manifested, and consistent with cardiomyopathy compatible with the clinically observed dilated cardiomyopathy.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+A spontaneous coronary artery dissection as the sole presenting feature of vascular Ehlers-Danlos syndrome is an uncommon finding. We present a 33-year-old woman with sudden onset chest pain caused by a spontaneous coronary artery dissection. Genetic testing revealed vascular Ehlers-Danlos syndrome as the underlying cause. Specifically, we show the value of genetic testing, which in some patients may be the only way of establishing a diagnosis.Spontaneous coronary artery dissection (SCAD) is a rare cause of acute coronary syndrome and sudden cardiac death. SCAD is the result of the formation of a hematoma in the coronary arterial vascular wall, in the absence of a traumatic or iatrogenic cause [1]. The most common cause is atherosclerosis. In addition, SCAD is associated with pregnancy, fibromuscular dysplasia, and vigorous exercise. In approximately 3% of SCAD cases a genetic defect can be identified [2]. Genetic causes include polycystic kidney disease and connective tissue disorders. Establishing a genetic diagnosis allows for disease specific follow up for the patient. In addition, pre-symptomatic testing and preventive measures in relatives may be lifesaving. Here, we present the case of a previously healthy woman diagnosed with SCAD. Genetic testing resulted in the diagnosis of vascular Ehlers-Danlos syndrome (vEDS). vEDS is an autosomal dominantly inherited connective tissue disorder caused by pathogenic variants in the COL3A1 gene encoding type III procollagen. vEDS patients are at risk for arterial, bowel, and uterine rupture and pneumothorax. In addition, other recognizable phenotypic features are often present. These include thin, translucent skin, easy bruising, characteristic facial appearance, joint hypermobility, and acrogeria. The reported prevalence is 1:50,000–1:200,000. However, this is likely to be an underestimation since prevalence estimates in rare genetic disorders are often based on only the ‘classical’ severe phenotypes of the disorders [3]. This is not surprising as only a few years ago DNA testing was very time consuming and expensive and was only performed in patients with a high prior risk of a genetic disease. In 2017, criteria suggestive of vEDS were proposed. Arterial rupture or dissection in individuals younger than 40 years is one of the major criteria [4]. This report underlines that SCAD may be the sole presenting feature of vEDS in the absence of other features associated with the disorder. In addition, this report shows that genetic testing may be the only way of establishing a diagnosis.Case. A 33-year-old previously healthy woman presented with sudden onset chest pain. She was diagnosed with a myocardial infarction caused by SCAD. She was treated successfully by percutaneous coronary intervention with placement of four stents (Figure 1). Since in a minority of patients a spontaneous arterial dissection is caused by an underlying genetic cause, she was referred for genetic counselling. Her medical history (including two uncomplicated pregnancies), family history, and physical examination did not reveal any further signs indicating a connective tissue disorder or hereditary kidney disease. The prior risk of a genetic cause therefore was low. A previously reported heterozygous pathogenic variant, c.1744G >A p.(Gly582Ser) in the COL3A1 gene was identified using targeted next generation sequencing analysis of 21 genes associated with aortic dilatation and Marfan-like syndromes, confirming the diagnosis of vEDS. Establishing the diagnosis allowed for disease specific recommendations including vascular imaging, strict regulation of blood pressure, and additional vascular imaging. Regulation of blood pressure was first attempted with celiprolol, as suggested by the BBEST study, but was switched to atenolol due to side effects (frequent headaches) [5]. MRA was performed and no other arterial aneurysms or dissections were present. The patient remains under regular surveillance. Relatives were offered the option of pre symptomatic genetic testing.This case underlines that isolated SCAD can be the sole presenting feature of vEDS and further illustrates the clinical variability that can be associated with pathogenic COL3A1 variants. The role of genetic testing in SCAD patients remains to be established. In a recent study, among 384 SCAD survivors from the UK a pathogenic variant was detected in 14 patients (3.6%), including two COL3A1 variants [2]. In many cases, the presence of recognizable skeletal features, age at presentation, or family history will provide additional clues for an underlying genetic disease. However, these clues are easily missed, and they may be completely absent as illustrated by this report [6]. In some patients, DNA testing can therefore be the only way to diagnose these genetic disorders. In families and patients without the classical presentation of a genetic predisposition, it is likely that DNA testing is not performed, resulting in preventable morbidity and mortality. This is illustrated by a recent study by our group in 810 patients referred for genetic testing after diagnosis of a thoracic aortic aneurysm or dissection. A (likely) pathogenic variant was detected in 66 of 810 patients (8.1%). The mean age at DNA testing in the group of patients with an identifiable genetic cause was 11 years younger than the mean age in the group without a genetic cause. However, 10 of the 66 patients carrying a pathogenic or likely pathogenic variant (15.2%) were over the age of 60 years at the time of DNA testing. Of these, three had a negative family history for aortic disease and no systemic features of a connective tissue disorder [7]. These observations indicate the need for increasing awareness of these genetic disorders and improvement of evidence-based guidelines for DNA testing. The time and costs associated with DNA testing have decreased rapidly over recent years. In addition, the availability of large genomic population databases has facilitated the interpretation of detected variants. When combined, these factors have resulted in the possibility of genetic testing at lower thresholds in many countries. Evaluation of existing guidelines in breast cancer patients indicated that nearly half the (likely) pathogenic variants are missed using current guidelines for DNA testing. The option of offering all breast cancer patients the possibility of genetic testing is currently being debated by some experts [8,9]. This debate is likely to include other diseases, possibly including SCAD in the near future.Conceptualization, E.O. and A.C.H.; writing—original draft preparation, all authors; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Amsterdam University Medical Centers (NL70190.018.19, approved 5 August 2019).Informed consent for publication was obtained from the patient reported in this study.The data presented in this study are available on request from the corresponding author.The authors declare no conflict of interest.(a) Type F coronary dissection, caused by a Type 4 SCAD, of the right coronary artery (RCA) before intervention; TIMI flow grade 0. (b) Above Restoration of flow after wiring, however widespread coronary dissection and diminished coronary flow remain. Below Result after percutaneous coronary intervention with four drug-eluting stents with restoration of coronary flow.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Co-first authors.Thoracic aortic aneurysms (TAAs) that progress to acute thoracic aortic dissections (TADs) are life-threatening vascular events that have been associated with altered transforming growth factor (TGF) β signaling. In addition to TAA, multiple genetic vascular disorders, including hereditary hemorrhagic telangiectasia (HHT), involve altered TGFβ signaling and vascular malformations. Due to the importance of TGFβ, genomic variant databases have been curated for activin receptor-like kinase 1 (ALK1) and endoglin (ENG). This case report details seven variants in SMAD4 that are associated with either heritable or early-onset aortic dissections and compares them to pathogenic exon variants in gnomAD v2.1.1. The TAA and TAD variants were identified through whole exome sequencing of 346 families with unrelated heritable thoracic aortic disease (HTAD) and 355 individuals with early-onset (age ≤ 56 years old) thoracic aortic dissection (ESTAD). An allele frequency filter of less than 0.05% was applied in the Genome Aggregation Database (gnomAD exome v2.1.1) with a combined annotation-dependent depletion score (CADD) greater than 20. These seven variants also have a higher REVEL score (>0.2), indicating pathogenic potential. Further in vivo and in vitro analysis is needed to evaluate how these variants affect SMAD4 mRNA stability and protein activity in association with thoracic aortic disease.Transforming growth factor β (TGFβ) plays a critical role in vascular development. Many vascular disorders, such as hereditary hemorrhagic telangiectasia (HHT), Marfan syndrome, and thoracic aortic aneurysm and dissection (TAA/TAD) have been associated with disruption of the TGF β signaling axis. Mutations in many proteins that are involved in this pathway have been identified. For example, pathogenic variants that underlie HHT are found in multiple genes, including endoglin (ENG) associated with HHT1, activin receptor-like kinase 1 (ALK1) associated with HHT2, mothers against decapentaplegic homolog 4 (SMAD4) associated with HHT3, and bone morphogenetic protein 9 (BMP9) associated with HHT5. The majority of HHT cases result from variants in ENG or ALK1, and genomic variant databases have been established and maintained that elegantly catalogue numerous ENG and ALK1 genetic variants [1]. Such databases are critical for curating genetic variants associated with a disorder and can assist early clinical diagnoses. To date, no such resource exists for SMAD4 or BMP9 variants. Herein, we report seven confirmed SMAD4 variants associated with heritable or early-onset TAA.Aortic aneurysms are enlargements of the aorta. Thoracic aortic aneurysms are typically asymptomatic and may lead to sudden death due to acute aortic dissections. TAAs are less prevalent and occur in younger patients when compared to abdominal aortic aneurysms. TAAs can result from single-gene pathogenic variants that confer a high risk of TAA, termed heritable thoracic aortic disease (HTAD)  [2,3]. Early detection and clinical management of TAAs are critical to prevent deaths due to TAD. SMAD4 protein is a central molecule in TGFβ signal transduction through the canonical arm of the TGFβ signaling pathway. The recognition of the SMAD4 variants reported herein was facilitated by GeneMatcher and MyGene2, nodes of the MatchMaker Exchange platform which serves to connect investigators with an overlapping interest in a registered gene, providing an interface to clinicians and scientists that supports the discovery of genes underlying rare diseases  [4]. Herein, we report SMAD4 variants that were identified through a screen of exome sequencing data from individuals with HTAD or TAD performed in the Milewicz lab which employed whole exome sequencing [5,6]. This report increases the accessibility of the identified SMAD4 variants and may be utilized in future efforts that aim to build a SMAD4 variant database for genetic disorders.Whole exome sequencing data were obtained for affected probands and family members from 346 unrelated heritable thoracic aortic disease families (HTAD) and 355 individuals with early-onset (age ≤ 56 years old) thoracic aortic dissection (ESTAD) from 2000 to 2019. Blood or saliva samples were collected after obtaining approval from the Institutional Review Board at the University of Texas Health Science Center at Houston. Informed consent was obtained from all participants.Exome sequences were captured by SeqCap EZ Exome probes version 2.0 (Roche) and recovered according to the manufacturer’s directions. Enriched libraries were then sequenced on an Illumina GAIIx using manufacturer protocols. Reads were mapped to the reference human genome (UCSC hg19) with BWA (Burrows–Wheeler Aligner), and variant detection and genotyping were performed using the UnifiedGenotyper (UG) tool from GATK. Annotation of variants was performed using the SeattleSeq server (http://gvs.gs.washington.edu/SeattleSeqAnnotation, SeattleSeq v.151, accessed on 17 August 2018) and Annovar variant annotation (https://annovar.openbioinformatics.org/ accessed on 17 August 2018). Sanger DNA sequencing assay was performed to validate SMAD4 variants identified by exome sequencing.To identify potential pathogenic or likely pathogenic SMAD4 (NM_005359.6) variants, whole exome sequencing data were filtered based on three criteria: (1) variants that altered amino acids, including nonsynonymous, stop-loss, stop-gain, coding indel, frameshift, and splice site variants; (2) variants with a minor allele frequency less than 0.05% in the Genome Aggregation Database (gnomAD exome v2.1.1) and a combined annotation-dependent depletion score (CADD) that was larger than 20, and (3) variants that segregated with thoracic aortic disease in HTAD families. The Sorting Intolerant from Tolerant for Genome (SIFT4G) database was used to predict possible damaging variants.SMAD4 variants in the gnomAD structural variant (SV) v2.1 were examined and compared to TTA-associated genes. The gnomAD is based on more than 141,000 exomes and genomes from unrelated individuals sequenced as part of various disease-specific and population genetic studies and is aligned against the GRCh37 reference. The referenced data are derived from a whole exome sequencing database described by Karczewski et al. and Collins et al. [7,8].Exome sequencing analysis identified seven rare variants in SMAD4 with a CADD score of more than 20 that are predicted to result in amino acid substitutions. SMAD4 protein is comprised of two functional domains, named MH1 and MH2, which are held together by a linker. The MH1 domain complexes with DNA  [9,10,11], while the MH2 domain located at the C-terminal interacts with other proteins, including other SMAD proteins. For these variants, two are located in each MH1 and MH2 domain, while the remaining three are located in the linker domain (Figure 1A). The variants M24V, R97L, and P246V, indicated in the Figure by white arrows, have been previously reported  [5]. The R97L mutation in the MH1 domain had the highest CADD score (32) and a Rare Exome Variant Ensemble Learner (REVEL) score (0.938), suggesting a more severe phenotype. Further analysis of these variants using the SIFT4G database identified possible damaging (D) or tolerant (T) variants (Table 1). All variants were validated by Sanger sequencing (Figure 1B). Subsequent analysis showed that R97L is characterized by decreased SMAD4 stability and reduced TGFβ signaling  [5]. Two other variants, M24V and P246T, were also associated with TAD [5]. R97L and I525V were identified in unrelated HTAD families; R97L segregated with disease, and I525V was shared by two affected cousins. With the exception of I525V, which was found in two unrelated ESTAD families, the remaining variants were identified in only one ESTAD family. The GenomeAD v2.1.1 database also identified three more variants, viz., R445X, R496C, and I500V (black arrows). Taken together, we report seven novel variants of SMAD4 identified in individuals with either earl- onset or familial TAA.Many TAA are asymptomatic and only diagnosed when a life-threatening TAD occurs. Several parameters are used for early diagnosis of TAA, so that monitoring and clinical interventions can be pursued to prevent TAD. These include a family history of TAA or TAD, genetic testing, and familial screening. There are over 20 genes with evidence that variants within the gene predispose to TAA  [12], and sufficient evidence of TAA disease-causing for 11 of these genes  [13]. Genetic variants that predispose to TAA lead to decreased smooth muscle cell (SMC) contraction and survival, altered extracellular matrix (ECM) integrity, and decreased canonical TGFβ signaling [2,14,15,16]. Thus, variants in genes involved in TGFβ signaling, that regulate many processes associated with vascular development and repair, are associated with TAA  [12,17,18].SMAD4 protein plays a critical role in TGFβ signal transduction  [12,17,18]. It binds to phosphorylated (i.e., activated) SMAD2/3, and in turn this complex translocates to the nucleus, where it alters target gene expression in concert with other transcription factors  [19]. We report rare, predicted damaging SMAD4 variants identified through exome sequencing of a large cohort of patients with TAA. Exome sequencing identified seven variants distinct from the three variants reported in gnomAD v2.1.1 with potential pathogenic or likely pathogenic consequences. These variants are located within regions of SMAD4 that encode the MH1, MH2, and linker domains of SMAD4 protein. It remains unclear whether specific SMAD4 variants associate with AVM formations as seen in HHT, underscoring the need for a SMAD4 variant database. Further analysis of individual variants is necessary to establish disease specificity. Together with available data, the identification of these variants contributes to our understanding of how SMAD4 may protect against vascular disorders.Research with the global Smad4 knockout mouse model has revealed critical roles in embryogenesis and in the development of cancer  [20,21,22]. Smad4 plays a key role in blood vessel angiogenesis, and establishing its importance in vascular development remains a very active area of research. To understand the importance of Smad4 in embryonic blood vessel development and disease, elegant studies have been conducted with animal models of inducible, tissue-specific Smad4 loss. Total loss of Smad4 in mice is embryonically lethal due to the inability of the embryo to complete gastrulation, supporting a critical role for Smad4 in embryonic development  [20]. Endothelial cell-specific deletion is also embryonically lethal due to an angiogenesis failure and impaired recruitment of smooth muscle cells  [21]. In adult and neonatal mice, inducible Smad4 conditional knockout models have been used to understand the role of Smad4 in adult tissues. Inducible endothelial cell-specific Smad4 knockout mice develop AVMs and vascular defects following tamoxifen injection at postnatal day 1 (PN1), recapitulating, in part, the AVMs that form in patients with HHT  [23]. Importantly, endothelial cell-specific inducible deletion of Smad4 has uncovered that AVM formation is associated with reduced signaling through TEK due to increased expression of its antagonistic ligand angiopoietin-2  [24]. This phenotype can be rescued through inhibition of angiopoietin 2, supporting the development of new therapeutic approaches to treat AVMs  [24]. While complete loss of Smad4 has been shown to result in early embryonic lethality, the variants identified here were sufficient to support human development but are associated with early onset of familial TAA. These data raise the possibility that specific SMAD4 variants may partially disrupt vascular development and/or exacerbate angiogenic mechanisms that contribute to thoracic aortic disease risk.In addition to the critical roles of Smad4 in embryonic development in mice, human SMAD4 variants have been identified and are associated with several human diseases. Variants in the SMAD4 gene have been associated with juvenile polyposis syndrome (JPS) and also in association with HHT and Myhre syndrome. More specifically, variants that encode the MH2 domain associate with JPS and HHT and result in decreased activity of SMAD4 protein due to increased ubiquitination-mediated degradation  [25]. Additional variants in the MH2 domain that increase SMAD4 activity are also associated with a different disorder, Myhre syndrome  [26]. Vascular malformations are a common factor amongst HHT, Myhre syndrome, and TAA, yet only a limited number of specific TAA/TAD-associated SMAD4 variants have been identified. A report by Lifei Wu suggests that an S271N mutation in non-MH regions of the protein may not be directly causal to TAA but may contribute to TAA in combination with other risk factors  [27]. More recently, an R97L mutation in SMAD4 has also been associated with TAA in the absence of HHT or JPS [5]. Herein, we report results from exome sequencing of a cohort of thoracic aortic disease patients without HHT or JPS and the identification of novel SMAD4 rare and damaging variants, in addition to the previously reported R97L [5]. Together with available data, the knowledge of these variants contributes to our understanding of how SMAD4 variants may contribute to vascular diseases.With the exception of R97L  [5], further investigation and mechanistic validation are needed to determine the physiological relevance of each variant and to examine the mechanistic relationship between SMAD4 and vascular diseases, including thoracic aortic disease, HHT, and Myhre syndrome. Additional research is needed to pursue how these variants affect SMAD4 expression and activity, such as a reduction of SMAD4 mRNA levels, altered protein structure, or allosteric inhibition of the endogenous, normal SMAD4 protein. Finally, the comparison of variants identified through exon sequencing of TAA tissue with potential pathogenic and likely pathogenic variants in gnomAD strengthens the significance of exon sequences encoding the MH2 domain. Taken together, the SMAD4 variants identified in ESTAD and HTAD patients in this report may have utility in future work aimed at generating a detailed database for human SMAD4 variants with possible pathogenic potential. Future investigation in vivo and in vitro of the reported variants is now needed to pursue their molecular functions and advance our understanding of the interaction between the reported SMAD4 variants and vascular health.D.G. and D.M.M. planned and executed all the experiments. M.J.B. and D.A.N. executed the exome sequencing. D.G. and M.C.W. facilitated the use of GeneMatcher and MatchMaker Exchange. S.A.B. and D.G. contributed the majority of the original writing. All authors contributed to data analysis and interpretation and writing of the manuscript. All authors have read and agreed to the published version of the manuscript.The Milewicz Lab is supported by NIH R01HL109942, the John Ritter Foundation, and Remembrin’ Benjamin. Exome sequencing was provided by the University of Washington Center for Mendelian Genomics (UW-CMG) and was funded by NHGRI and NHLBI grants UM1 HG006493 and U24 HG008956 and by the Office of the Director, NIH under Award Number S10OD021553. The Wallingford Lab is supported by NIH/NICHD K99/R00HD090198 (M. Wallingford), AHA 19CDA34660038 (M. Wallingford), and the Forbes Family Foundation.This study was approved by the Institutional Review Board at the University of Texas Health Science Center at Houston (Protocol code HSC-MS-01-251 approved on 20th October 2020).Informed consent was obtained from all the participants involved in the study.The availability of exome data is depended on individuals’ consent. For individuals who signed agreement to deposit their exome data on the NCBI database of Genotypes and Phenotypes (dbGaP), the exome data are available on the dbGaP phs000693.The authors declare no conflict of interest.SMAD4 protein map and locations of the identified variants. (A) SMAD4 protein map indicating domains that harbor the identified variants; MH domains are indicated in orange. Arrows indicate the approximate location of variants identified by exon sequencing which were reported in Duan et al. 2019 (white), were novel (red), or identified as pathogenic or likely pathogenic variants in gnomAD v2.1.1 (black). (B) Representative Sanger sequencing results of seven variants that were identified by whole exome sequencing of TAA patient samples.Human SMAD4 Genomic Variants Identified by Thoracic Aortic Aneurysm Screening. A list of variants with MAF ≤ 0.001 and CADD score ≥ 20 are included. The ESTAD and HTAD columns denote the number of unrelated cases for each variant. Abbreviations: Alternate amino acid (Aa alt), Rare Exome Variant Ensemble Learner (REVEL), Combined annotation-dependent depletion score (CADD), Sorting Intolerant from Tolerant for Genome (SIFT4G) prediction, Damaging (D), Tolerant (T), Clinical significance of the Variant (ClinVar), Genome Aggregation Database allele frequency (gnomAD exome v2.1.1), early-onset thoracic aortic dissection (ESTAD), and unrelated heritable thoracic aortic disease (HTAD).Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+First two authors contributed equally to the paper.Brugada syndrome (BrS) is an inherited disorder with high allelic and genetic heterogeneity clinically characterized by typical coved-type ST segment elevation at the electrocardiogram (ECG), which may occur either spontaneously or after provocative drug testing. BrS is classically described as an arrhythmic condition occurring in a structurally normal heart and is associated with the risk of ventricular fibrillation and sudden cardiac death (SCD). We studied five patients with spontaneous or drug-induced type 1 ECG pattern, variably associated with symptoms and a positive family history through a Next Generation Sequencing panels approach, which includes genes of both channelopathies and cardiomyopathies. We identified variants in MYBPC3 and in MYH7, hypertrophic cardiomyopathy (HCM) genes (MYBPC3: p.Lys1065Glnfs*12 and c.1458-1G > A, MYH7: p.Arg783His, p.Val1213Met, p.Lys744Thr). Our data propose that Brugada type 1 ECG may be an early electrocardiographic marker of a concealed structural heart disease, possibly enlarging the genotypic overlap between Brugada syndrome and cardiomyopathies.A typical ECG pattern, characterized by a coved ST-segment elevation ≥ 2 mm in at least one right precordial lead followed by a concave or straight ST segment elevation and a negative symmetric T-wave [1], is associated with Brugada syndrome, an inherited disease characterized by an increased risk of ventricular arrhythmias and sudden cardiac death (SCD) in a structurally normal heart [2]. Nowadays a pathogenic genetic variant is detected in up to 30% of subjects with BrS, usually transmitted in an autosomal-dominant manner [3,4]. The gene most frequently involved, and the sole one considered as definitively associated with the syndrome [5], is SCN5A, which encodes the alpha subunit of the cardiac sodium channel Nav1.5 and is responsible for nearly 20–30% of all cases [6]. Several other genes have been proposed as underlying BrS, but with incomplete, often disputed evidence [7]; more and more evidence suggest a possible oligogenic or polygenic inheritance for inheritable cardiac disorders, including BrS [8,9,10]. Genetic counseling in BrS is complicated by many confounding factors: genetic and allelic heterogeneity, variants of uncertain significance [11], incomplete penetrance [12], phenotypic overlaps [6] and new evidence of complex inheritance [12] and mutation load [13], thus overcoming the one gene–one disease paradigm [10]. Furthermore, the concept of BrS as a pure electric condition, occurring in an otherwise structurally normal heart, has been contradicted by several observations. Indeed, the overlap between BrS and arrhythmogenic cardiomyopathy (ACM) is well known in the literature, so much that ACM and BrS could be seen as two different entities belonging to the same disease spectrum [10,14].Sarcomeric genes, encoding for components of the contractile unit in the cardiomyocyte (the sarcomere), are characteristically associated with structural cardiomyopathies [15]. MYBPC3 and MYH7, encoding for the cardiac myosin binding protein C and the cardiac beta-myosin heavy-chain, respectively, represent the genes most frequently involved in hypertrophic cardiomyopathy (HCM), accounting for 50% and 33% of cases with a positive genetic test result, respectively [16]. In five patients with spontaneous or drug-induced type 1 ECG pattern (BrP), negative for mutations in SCN5A and other putative BrS genes, we found likely pathogenic/pathogenic variants in MYBPC3 and MYH7, structural cardiomyopathy genes. This observation adds evidence to the genotypic overlap between arrhythmic and structural heart diseases and supports Brugada type 1 ECG as an early electrical sign of an upcoming structural disease.Written informed consent for genetic testing was obtained from all enrolled subjects (local ethical committee approval 26/7/2012, P. 7/2012). NGS analysis was performed on MiSeq™Dx Instrument using the commercial gene panel Trusight Cardio Sequencing Kit by Illumina (San Diego, CA, USA, www.illumina.com accessed on 21 September 2020), which includes both structural and arrhythmogenic cardiomyopathies genes. Data were analyzed and filtered using Sophia Genetics DDMR software (https://dropgen.sophiagenetics.com accessed on 21 September 2020). The minimum depth of coverage for variant calling was 20×. The Exome Variant Server (ESP), the Exome Aggregation Consortium (ExAC) and the gnomAD database (version 2.1.1) with a frequency greater than 0.1% were used to filter out common variants. Potential disease-causing missense variants were assessed using Mutation Taster, Polyphen2 and SIFT. VarSome (https://varsome.com/ accessed on 15 July 2021) and/or ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ accessed on 15 July 2021) were used as a tool to sum up actual knowledge about the variants. All identified variants were classified according to American College of Medical Genetics and Genomics (ACMG) guidelines [11]. The molecular confirmation of variants was performed by standard Sanger sequencing on an automated analyzer (ABI PRISM® 3130). The MLPA technique was used to detect deletions or duplications in SCN5A by using MRC-Holland probemix P108 kit (Amsterdam, The Netherlands, https://www.mrcholland.com/ accessed on 16 July 2021).Among a cohort of 51 patients with BrP tested by the gene panel including both structural and arrhythmogenic cardiomyopathies, we identified five patients carrying a pathogenic/likely pathogenic variation in sarcomeric genes. Table 1 summarizes clinical and genetic evaluations, Figure 1 shows the pedigree of the five patients and Figure 2 shows their ECGs.P1 is an adult male with a spontaneous intermittent type 1 BrP, which was detected at 34 years old as an incidental finding during a routine visit prior to sport activity. ECG showed: PR = 172 ms; QTc = 440 ms at a heart rate of 56 bpm. His family history was negative for cardiovascular disease (CVD) or sudden cardiac death (SCD).He experienced recurrent palpitations (at least two times a week, duration 5 to 20 min); symptoms were documented several times by an external loop recorder which detected an atrio-ventricular nodal reentrant tachycardia that was subsequently successfully treated by radiofrequency ablation. No programmed electrical stimulation (PES) was performed.His echocardiogram showed no signs of structural abnormalities. He performed a cardiac magnetic resonance (MR), which shows absence of right ventricle (RV) bulging, normal RV function, preserved LV volume, myocardial mass and function, no fibro-fatty involvement of both ventricles, normal perfusion scan and no abnormalities in late-gadolinium-enhancement (LGE) study.P2 is an adult male who had an incidental finding of spontaneous type 1 BrP at 56 years old during a routine visit, without any other symptoms, except for sporadic extrasystoles. ECG showed: PR 148 ms, QTc 416 ms at a heart rate of 68 bpm.His father died suddenly at 75 years old (no previous history of cardiac disease). He performed an echocardiogram that showed left ventricular hypertrophy (LVH), more evident in the mid-basal septum and in the posteroinferior wall (maximum thickness: 16 mm) and a cardiac MR that showed left ventricular hypertrophy with a maximum wall thickness of 18 mm in the mid basal septum (Figure 3). His HCM risk-SCD score was calculated as 1.97% at five years. No PES was performed.P3 is a 26-year-old woman with a history of syncope and a drug-induced type 1 BrP. ECG showed: PR = 148 ms, QTc = 420 ms at a heart rate of 68 bpm. Her father, who was a known carrier of a type 1 BrP, died suddenly during sleep at 45 years old (a moderate left ventricular hypertrophy was found before during cardiac ultrasound and at the autopsy). Her echocardiogram showed no signs of structural abnormalities; she also performed a cardiac MR, which showed normal biventricular dimension, wall thickness, volume and function and negative LGE sequences. Because of her family history of SCD and the history of recurrent syncope, she received a subcutaneous implantable cardioverter defibrillator (S-ICD) in primary prevention.P3 has an 18-year-old brother, who also had a drug-induced type 1 BrP and no sign of structural heart disease at echocardiography and MR scan. ECG showed: PR = 158 ms, QTc = 380 ms at a heart rate of 60 bpm. He also received an S-ICD in primary prevention.The defibrillation was effective with 30 and 25 J, in P3 and her brother, respectively. No PES was performed in both siblings.P4 is an adult male who had an ECG finding of spontaneous type 1 BrP at 61 years old during a routine visit. ECG showed: PR = 160 ms, QTc = 433 ms at a heart rate of 86 bpm. He did not have any symptoms of palpitations or syncope. He had a positive familial history of SCD, which occurred in his father at 59 years old during sleep. His echocardiogram shows no evidence of structural abnormalities and preserved biventricular function. No PES was performed.P5 is an adult male who had an incidental finding of spontaneous type 1 BrP at 46 years old during an episode of palpitations and abdominal pain. ECG showed: PR = 160 ms, QTc = 372 ms at a heart rate of 70 bpm. His echocardiogram showed no signs of structural cardiomyopathy. His family history was negative for CVD or SCD. No PES was performed.In the five patients, neither variations nor deletions/duplications were identified in SCN5A and no variations were identified in other minor BrS genes. We detected heterozygous variations in sarcomeric genes (Table 1). All of these mutations, but the last one in P5, are known in literature as associated with HCM [17,18,19,20].P1, P2 and P3 are heterozygous, respectively, for the frameshifting mutation c.3192dupC (p.Lys1065Glnfs*12) in exon 30 in MYBPC3, the splicing mutation c.1458-1G > A in intron 16 in MYBPC3 and the missense variation c.2348G > A (p.Arg783His) in exon 21 in MYH7. All these changes are known in literature as associated with HCM [17,18,19] and are classified as pathogenic or likely pathogenic according to ACMG criteria. The MYH7 variant detected in P3 segregated in her 18-year-old brother.P4 is heterozygous for the missense variation c.3637G > A (p.Val1213Met) in exon 27 in MYH7, classified as VUS/LP by prediction tools and databases. Nonetheless, this variant is absent from general population and it has been described several times in literature as associated with HCM [20].P5 is heterozygous for the missense variation c.2231A > C (p.Lys744Thr) in exon 20 in MYH7. This sequence change has not been described in literature in affected subjects and is absent from controls (PM2 criterion according to ACMG). In silico prediction tools support its pathogenicity and the variant is currently classified as LP (Table 1).In BrS patients, gross structural alterations are not usually detected at routine investigation and BrS is considered a primary electrical disease. Nonetheless, there is growing evidence that patients with BrS may exhibit subtle cardiac abnormalities; thus, the idea of a ‘Brugada cardiomyopathy’ has been proposed [21]. The phenotypic and genotypic overlap between BrS and arrhythmogenic cardiomyopathy (ACM) is well-known in literature and it is supported by the identification of both conduction abnormalities and structural alterations in the right ventricular outflow tract (RVOT) in BrS patients [14,22,23,24]. Nademanee, in particular, reported fibro-fatty replacement of the RVOT in both autoptic and in vivo collected cardiac samples from BrS patients, who showed no cardiac structural abnormality at ecocardiography and/or cardiac MRI, thus suggesting that BrS pattern could be a very early sign of disease, anticipating manifest myocardium alterations [22]. By using Doppler tissue imaging (DTI), we recently demonstrated a contraction delay at the RVOT in individuals with spontaneous type 1 Brugada ECG pattern [25].Mutations in ACM desmosomal genes have been associated with BrS and studies have shown that deficiencies in PKP2 (one of the structural components of the cardiac desmosome and a major ACM gene) determine downstream effects, both on the integrity of gap junctions and on the sodium channel function, with consequences on electrical coupling and on sodium current [10].Further supporting type 1 BrP as a possible marker of an occult cardiomyopathy, we detected variants in sarcomeric genes in five patients presenting spontaneous or drug induced BrS pattern.MYBPC3 encodes the cardiac myosin binding protein C and MYH7 encodes the cardiac beta-myosin heavy-chain, two proteins typically involved in the structural function of the cardiac sarcomere. Mutations in the two genes are mainly associated with hypertrophic cardiomyopathy (HCM) [15]. In recent years, some authors have suggested a possible link between BrS and HCM, although with still limited evidence. In 2015 Di Resta and colleagues detected a borderline significant association for mutations in MYH7 gene in BrS patients [5/91 (5.5%) patients vs. 0/91 controls] [26]. In 2016, a family with four members showing both HCM and BrS was described and an integrated linkage analysis and NGS approach identified a missense mutation within the sarcomeric TPM1 gene [27]. More recently, Pappone et al. reported a family in which a MYBPC3 mutation segregated in the father, who was affected by HCM, and in his daughter and son, who were affected by BrS [28]. The presence of left ventricular hypertrophy in autopsy data of BrS subjects has also been described [29].In our case series, all patients shared a type 1 BrP (spontaneous or drug-induced) and carried a heterozygous variation in a sarcomeric gene, but only one patient (P2, carrying a pathogenic splicing mutation in MYBPC3), presented instrumental signs of HCM. The other patients showed a pure electrical phenotype, variably associated with a positive family history for BrS and/or sudden death. Nevertheless, it has to be taken into account that these patients are relatively young and HCM penetrance is incomplete and age dependent.Despite the lack of segregation analysis, which was possible only in two individuals in one family, available data support the identified mutations in sarcomeric genes as disease causing. Indeed, except for the missense variation c.2231A > C (p.Lys744Thr) in exon 20 in MYH7 in P5, the mutations we identified have been reported in several individuals in the literature, all affected by structural cardiomyopathy [17,18,19,20], and all these variations are absent in population control databases. Based on the increasing knowledge in literature of a strong structural/electrical overlap [26,27,28,29], it may be speculated that these sarcomeric mutations may justify Brugada type 1 ECG in our patients, which is that the Brugada type 1 ECG is an early sign of a structural disease. Further evidence is needed to confirm this hypothesis and, most of all, how defects in sarcomeric genes can lead to BrS-related electrical manifestation remains to be clarified. Notably, Baudenbacher et al. have shown that increased myofilament Ca2+ sensitivity determined arrhythmia susceptibility in mice expressing troponin T mutants, even without anatomical abnormalities [30]. Furthermore, an abnormal SCN5A mRNA splicing with reduction of the full-length transcript has been observed in HCM patients [31], suggesting that a reduction in SCN5A may contribute to the arrhythmic risk in HCM.In conclusion, data obtained from this case series provide further evidence of the genotypic overlap between BrS-related electrical phenotype and structural cardiomyopathies and support the concept that individuals presenting with either primary cardiomyopathy or arrhythmias should be tested for a comprehensive cardiomyopathy/arrhythmia panel. Based on our observation, the type 1 BrP may represent an early sign of a structural disease that would remain concealed unless investigated, with implications in clinical management and genetic counseling of BrS patients and overlapping phenotypes.M.F.: data acquisition, processing and interpretation; writing of the manuscript. F.G., C.B.: study concept and supervision, data interpretation, critical revision of manuscript. A.F.: study concept and critical revision of manuscript. C.B., E.D.M., M.B. (Mauro Biffi), C.R., P.I., M.B. (Matteo Bertini): data acquisition and interpretation, critical revision of manuscript. M.F., A.D.D., M.D.R., F.G.: data acquisition and clinical evaluation. A.M., R.S.: genetic analysis, data interpretation. All authors have read and agreed to the published version of the manuscript.No financial assistance was received in support of the study.Written informed consent was obtained from the patients (Ferrara local ethical committee approval 26/7/2012, P. 7/2012).Informed consent was obtained from all subjects involved in the study.The data that support the findings of this study are available from the corresponding Authors upon reasonable request.Thanks to the “Associazione Voglio Volare-Davide Barbi” for supporting this study.The authors have no conflict of interest to declare.Pedigrees of the five patients. The legend is shown in the figure. For clinical and familial details, see text in Results.ECG images of patients P1, P3, P4 and P5. (A) ECG of patient P1 showing type 1 BrP in V1–V2 recorded at second, third and fourth intercostal space; (B) ECG of patient P3 showing type 1 BrP in V1–V2 recorded at fourth intercostal space; (C) ECG of patient P4 showing type 1 BrP in V1–V2 recorded at fourth intercostal space; (D) ECG of patient P5 showing type 1 BrP in V1–V2 recorded at fourth intercostal space.ECG and cardiac MRI of the BrS patient P2 carrying the MYBPC3 c.1458-1G > A splicing mutation. (A) ECG of patient P2 showing type 1 BrP in V1-V2 recorded at fourth intercostal space. (B) MRI imaging of the same patient showing left ventricular hypertrophy.Patients with Type 1 ECG Brugada pattern carrying heterozygous variants in hypertrophic cardiomyopathy genes.F: Female, M: Male; ex: Exon; int: Intron; AVNRT: Atrio-Ventricular Nodal Reentrant Tachycardia; HCM: Hypertrophic cardiomyopathy; VUS: Variant of uncertain significance; LP: Likely pathogenic; P: Pathogenic. Data from Clinvar and VarSome tools and ACMG criteria are updated 15 July 2021. Accession numbers: MYBPC3: NG_007667.1(NM_000256.3), MYH7: NM_000257.2. Exons are numbered according to LOVD database (https://databases.lovd.nl/ accessed on 21 September 2020). * Sudden death in his father at 75 years old. ** Sudden death in her father at 45 years old, a known carrier of a type 1 Brugada pattern and with moderate left ventricular hypertrophy. Younger brother (carrying the same mutation) with drug-induced type 1 Brugada pattern and Premature Ventricular Contractions. *** Sudden death in his father at 59 years old.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Thoracic aortic aneurysm (TAA) is a heritable aortopathy with significant morbidity and mortality, affecting children and adults. Genetic causes, pathobiological mechanisms, and prognostic markers are incompletely understood. In 2015, the Collaborative Human Aortopathy Repository (CHAR) was created to address these fundamental gaps. Patients with thoracic aortopathy, associated genetic diagnoses, or aortic valve disease are eligible for prospective enrollment. Family members and controls are also enrolled. Detailed clinical and family data are collected, and blood and aortic tissue biospecimens are processed for broad usage. A total of 1047 participants were enrolled. The mean age in 834 affected participants was 47 ± 22 (range <1 to 88) years and 580 were male (70%). A total of 156 (19%) were under the age of 21 years. Connective tissue diagnoses such as Marfan syndrome were present in 123 (15%). Unaffected participants included relatives (N = 176) and healthy aorta tissue controls (N = 37). Aortic or aortic valve biospecimens were acquired from over 290 and 110 participants, respectively. RNA and protein were extracted from cultured aortic smooth muscle cells (SMCs) for 90 participants. Over 1000 aliquots of aortic SMCs were cryopreserved. The CHAR’s breadth, robust biospecimen processing, and phenotyping create a unique, multipronged resource to accelerate our understanding of human aortopathy.Thoracic aortic aneurysm (TAA) is an aortopathy that predisposes to aortic dissection, a life-threatening emergency. There is a strong heritable basis for TAA [1]. For example, Mendelian autosomal dominant connective tissue disorders including Marfan syndrome (MFS), Loeys–Dietz syndrome (LDS), and others have strong TAA associations. In addition, autosomal dominant causes of familial TAA have been identified in patients without syndromic characteristics. Turner syndrome (TS) is a genomic disorder that is commonly associated with TAA, and copy number variants (microdeletions or microduplications) account for a proportion of TAA [2,3]. In addition, bicuspid aortic valve (BAV) is the most common form of congenital heart disease and is frequently associated with TAA. Components of TAA pathogenesis have been elucidated from human genetics discoveries and mouse models [4]. Over time, molecular testing has begun facilitating organized approaches to prognosis and clinical decision making. Despite substantial progress, there remain key knowledge gaps in the areas of (1) genetic cause, (2) pathophysiology, and (3) clinical risk classification.The Collaborative Human Aortopathy Repository (CHAR) study was created in late 2015 at Indiana University School of Medicine (IUSM). The study leverages the large, statewide clinical network of IUSM and existence of subspecialty programs dedicated to aortopathy care across all age ranges. The key objectives in the CHAR design were to (1) prospectively enroll patients with aortopathy for collection of human blood and aortic tissue biospecimens, (2) process and store biospecimens for multidimensional uses, and (3) acquire detailed clinical and family data to optimize the utility of biospecimens. By so doing, the CHAR is a unique, aortopathy-dedicated platform for investigations that seek to advance genetic understanding of TAA progression and cause, investigate molecular and cellular disease mechanisms, and develop clinical studies. In this report, we describe the CHAR study’s protocol and enrollment figures to date. We then discuss the basic and clinical questions that can be investigated using this platform, including current and future applications.An overview of the CHAR study design is provided in Figure 1. The protocol was developed through discussions with a diverse array of clinical providers including cardiothoracic surgeons, medical geneticists, and cardiologists. Discussions were also held with clinical researchers with expertise in biobanking, technical directors of core laboratories, and other translational scientists. This study was approved by the Indiana University Institutional Review Board (Committee Reference Number: IRB00000219).In this prospective study, eligible patients are primarily identified via electronic medical record review at IUSM. Study personnel routinely review cardiothoracic surgery, genetics, and cardiology clinic rosters, as well as lists of admitted patients and operating room schedules, among the IUSM tertiary care facilities of Methodist Hospital, University Hospital, and Riley Hospital for Children. At Riley Hospital, a multidisciplinary aortopathy clinic was created and has expanded over the past 6 years to provide specialized, destination care to patients of all ages. The clinic’s providers are a highly integrated team of cardiologists, medical geneticists, and genetic counselors. A small number of participants (<5) have been enrolled outside of IUSM through public research avenues including clinicaltrials.gov and All IN for Health. The eligibility criteria are as follows:Diagnosis of aortic disease including TAA or dissection, aortic tortuosity, or aortic hypoplasia/stenosis.Diagnosis of a syndrome or genetic abnormality that poses risk for the development of aortopathy.Diagnosis of aortic valve disease.Family members of eligible subjects.Control subjects having aortic tissue removed during a surgical procedure, such as during heart transplantation.Organ donors who have authorized the use of their specimens for research.Diagnosis of aortic disease including TAA or dissection, aortic tortuosity, or aortic hypoplasia/stenosis.Diagnosis of a syndrome or genetic abnormality that poses risk for the development of aortopathy.Diagnosis of aortic valve disease.Family members of eligible subjects.Control subjects having aortic tissue removed during a surgical procedure, such as during heart transplantation.Organ donors who have authorized the use of their specimens for research.In this study, TAA is defined by an absolute aortic diameter ≥ 3.7 cm or body surface area-adjusted Z-score value ≥ 2 by cardiac imaging that may include echocardiography, computed tomography (CT), magnetic resonance imaging (MRI), or conventional angiography. Syndromic diagnoses that meet eligibility criteria include, but are not limited to, MFS, LDS, TS, and vascular Ehlers–Danlos syndrome (vEDS). Aortic valve disease includes bicuspid, unicuspid, or tricuspid disease.Eligible patients are approached in person or via telephone for opportunity to consent. Telephone consents follow a standardized script, to ensure that all study elements are discussed with the patient. Upon consent, study procedures are initiated. Sometimes patients have a medical emergency that requires immediate surgical intervention prior to the study staff being alerted of their medical condition. For these cases, the study staff provisionally acquire their biospecimens and basic clinical data. These patients are approached for consent and enrollment when they have recovered from their procedure and possess consent capacity. If the patient declines enrollment, their biospecimens and data are destroyed. If the patient dies prior to having the opportunity to obtain consent, the patient is permitted to remain enrolled. In the event that an enrolled participant dies, study staff may continue to collect data from the electronic medical record for research use.Table 1 summarizes the collected data categories and the main components of each. A customized database for clinical data was created in REDCap (Research Electronic Data Capture) for the purpose of this study [5,6]. It is an important priority of the study for data to be collected via a structured interview that occurs directly between clinical research staff and participants or their close family relatives (e.g., parents of children). This interview is accomplished in person or via phone. The database format is used to structure the interview. Data are entered directly into the REDCap database in real time using a tablet or computer. Any data that are uncertain or unavailable through the direct interviews are acquired in the electronic medical record, which is an integrated system across IUSM recruitment locations.The cardiovascular-related data that are collected specifically are shown in Table 2. These include diagnosis and procedure history. The medication history data that are specified in the database to be recorded focus on cardiac medications. In non-cardiovascular data (Table 3), the components of a routine connective tissue evaluation comprise the majority of the characteristics that are collected. The non-cardiovascular characteristics of connective disorders MFS, LDS, and vEDS are included. This includes multiple systemic criteria for MFS in the revised Ghent nosology [7]. Characteristics that have been reported to be relatively frequent in LDS include bone fracture, osteoporosis, club foot, osteoarthritis, and cleft palate [8,9]. Characteristics associated with vEDS include bowel rupture, uterine rupture, recurrent hernia, and atrophic scarring [10]. Not all participants have undergone a genetics evaluation clinically. Therefore, the connective tissue characteristics that were thought to require a formal dysmorphology examination, such as facial features, pes planus, and hindfoot deformity, are not included in the interview.A comprehensive family history is collected for each participant and entered as a three-generation pedigree. Similar to clinical data collection, the family history is acquired in person or over phone and utilizes a script. The script includes specific questions regarding cardiovascular history and risk, genetic testing and findings, and age/cause of death for deceased relatives. Non-cardiac family information, such as family members who have connective tissue characteristics, provided by the participant at the time of the interview is recorded. If a pedigree has been obtained clinically by a genetics provider, then that information is entered for this study. Any missing or new data since the time that the pedigree was last entered clinically are updated for this study.The results of all prior CT scans that include imaging of the aorta (chest, abdomen, pelvis, and occasionally neck) are collected. The data are collected directly from radiology reports and entered into a formatted spreadsheet. The procedures for collection of data from CT scans were recorded as an instructional video made available to research staff. For each scan, the body segments imaged and whether contrast was administered are recorded. Height and weight are recorded. The recorded vascular diameters are collected from all reported segments. A nomenclature for aortic segmental anatomy was adopted to encompass the range of reporting variation. Most collected scans were performed within the IU system, which minimizes such variation. Any diameter measured within a segment that has been previously repaired, replaced, or contains endovascular graft is denoted accordingly. The presence of arterial tortuosity and its specific location are recorded. Non-aortic arterial dilation/ectasia/enlargement, location, and diameters are collected. Other findings that are specifically recorded include elongation of transverse arch and venous ectasia. Additional cardiovascular abnormalities specified in reports, such as vascular/valvular calcification or atherosclerosis, are recorded. Aortic dissection is recorded and described as free text entry. Post-operative aortic changes such as pseudoaneurysms are also described. In addition to these cardiovascular data, incidental non-cardiovascular findings are also collected, such as abdominal organ cysts, hernia, and spine abnormalities. A customized downstream script was written in R (https://www.R-project.org; currently using version 4.0.4 access on 15 February 2021) to organize the spreadsheet data and automatically collate common anomalies that have been entered as free text.The diameters of the proximal aorta segments (annulus, root, sinotubular junction, ascending aorta) were directly measured by study investigators in a proportion of participants, including those who did not have CT scans completed clinically and young participants for longitudinal analysis.Ambulatory peripheral blood samples are collected in clinical laboratories. In surgical cases where a blood sample was unable to be obtained prior to surgery, the sample is instead collected in the operating room prior to initiation of cardiopulmonary bypass. Blood is routinely collected into two purple-top EDTA tubes (approximately 6 mL per tube) and a 2.5 mL tube that contains RNA preservative (PAXGene; Hombrechtikon, Switzerland). An additional third purple-top tube is selectively acquired for generation of induced pluripotent stem cells from peripheral blood mononuclear cells [11]. Blood samples are immediately delivered to a core research laboratory (Clinical and Translational Support Laboratory) via the medical center’s interconnected tubing system. One purple-top EDTA tube is immediately placed into a −80 °C freezer. The second purple-top EDTA tube is centrifuged to fractionate the components, and the plasma (2 of 1 mL aliquots) and buffy coat are collected. According to the manufacturer’s instructions, the PAXGene tube is kept upright at room temperature for 2 to 72 h and then transferred to a −80 °C freezer. Samples are transferred in batches to the study laboratory for longer-term storage. Upon receipt, DNA is extracted from buffy coat samples and spectrophotometrically analyzed for quantity and quality.In children, a blood draw may collect no more than 3 mL per kg of body weight at one time. Multiple draws may be completed in order to collect the total desired amount of blood. Rarely, additional blood samples outside of the original 21 mL would need to be drawn from a participant of any age. Possible reasons for additional draws include sample loss, exhaustion of the original sample, or technical errors. If participants require additional draws, study staff will ask the participant to re-consent to the study. Participants who enroll into the study but are not willing to provide a blood sample are selectively offered the alternative to provide a saliva sample.When participants are scheduled for cardiac surgery that includes the removal of aortic and/or aortic valve tissue, tissue that will not be used clinically (i.e., sent to pathology) are collected for study in the operating room. Cardiothoracic surgeons and operating room staff were instructed on the protocol for processing explanted tissues. The study supplies for tissue processing are provided to operating room staff at the beginning of the case. An instruction sheet is always provided, which specifies in detail how an explanted tissue sample should be apportioned. A tissue sample information sheet is also provided, where collection data are recorded, including the time of explant and the time that the sample is processed.Each aortic tissue sample is apportioned in four ways: formalin fixation, glutaraldehyde fixation, primary aortic cell culture, and rapid freezing. The collection is segmentally organized, such that, if a patient has more than one segment removed, then each segment is apportioned independently. The tissue samples are apportioned as quickly as possible.A 1 × 1 cm piece is submerged in 10% formalin solution in the operating room. In the study laboratory, 24 to 72 h after collection, the formalin solution is changed to 30% ethanol for 1 h, followed by 50% ethanol for 1 h, and then the sample is stored in 70% ethanol at 4 °C until the time of paraffin embedding.A small 3 × 3 mm piece is placed into a microcentrifuge tube containing 4% glutaraldehyde with phosphate buffer in the operating room. Samples are kept at 4 °C until the time of embedding.A 2 × 2 cm piece is submerged in sterile aortic biopsy medium. The recipe for the medium was described by Kwartler et al. (https://bio-protocol.org/e2045; access on 1 June 2017). This tissue piece is promptly transported to the study laboratory for cell culture. Immediately upon receipt, an explant outgrowth method is utilized to culture primary aortic smooth muscle cells (SMCs). The adventitial and intimal layers of the aorta are dissected away from the medial layer. The medial layer tissue is cut into approximately 3 to 5 mm pieces and placed onto the surface of a cell culture flask (flask surface area of 25 cm2). The flask is then turned upright, and complete SMC growth medium is placed into the bottom of the flask, maintaining the fluid level below the tissue pieces. The complete SMC growth medium is composed of MCDB 131 (Gibco, Waltham, MA, USA) supplemented with glutamine, glucose, 5% fetal bovine serum (FBS), and the growth factors and antibiotics in the SmGM-2 Smooth Muscle SingleQuots Kit (Lonza, Basel, CH, Switzerland). The flask is placed upright into a sterile, humidified, 37 °C, 5% CO2 incubator. The flask remains upright for 2 h to allow for tissue attachment to the flask surface. After 2 h, the flask is repositioned flat so that the medium covers the tissue pieces. While tissue pieces are in the flask, the medium is changed every 3 to 4 days. When the outgrowing cells cover approximately 20% of the flask, which occurs approximately 3 weeks after the initial plating, the tissue pieces are removed and discarded, and the flask-adherent cells are trypsinized and subcultured according to the Lonza protocol.Passaging, expansion, and routine subculturing steps are strictly protocolized to optimize consistency between primary SMC lines acquired from different participants. Passaging occurs every 7 days. At each passage, the cells are plated at a density of 5000 cells/cm2 into cell culture flasks (25 or 75 cm2 surface area) containing 1 mL per 5 cm2 of complete medium. Medium changes are performed the following day and then 3 days later, each time using 2 mL per 5 cm2 of fresh medium.At the time of passages 2 and 3, cells are also plated into NuncTM Cell-Culture-treated six-well plates (Thermo Scientific, Waltham, MA, USA). These cells are used for routine RNA and protein extraction. The cells are plated at concentration of 10,000 cells/cm2 in 5 mL/cm2 of complete medium per well. The medium is changed the following day with complete medium. Two days later, the complete medium is changed to low-serum medium (0.5% FBS) that does not have growth factor supplements. RNA and protein extractions are performed the following day. RNA is extracted using the RNeasy Mini kit (Qiagen, Germantown, MD, USA) from triplicate wells (yields three separate samples) and then aliquoted for storage at −80 °C. RNA samples are quantified by spectrophotometry with NanoDrop 2000 and a portion reverse-transcribed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). In parallel to RNA extractions, whole-cell protein lysates are extracted using ice-cold 1× RIPA (abcam, Cambridge, MA, USA; ab156034) supplemented with 1% Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Adherent SMCs are washed twice with ice-cold PBS. After application of RIPA, the well surface is scraped and fluid is collected into cold microcentrifuge tubes by pooling the lysates of three wells of a six-well plate, before nutating at 4 °C for 60 min and centrifuging for 15 min at 12,000 rpm and 4 °C; then, the supernatant is collected. The supernatant sample is aliquoted and stored at −80 °C. During these extractions, the cell culture medium (i.e., extracellular fluid) is collected immediately prior to the first PBS wash. The collected media is centrifuged for 5 min at 300× g and 4 °C, and then the supernatant is stored at −80 °C.At each routine passage, any excess cells not utilized for subculture or for RNA/protein sample extractions are cryopreserved in FBS and dimethyl sulfoxide. The cryovials are placed into a freezing-rate-controlled container (Mr. Frosty, Thermo Scientific) which is temporarily placed into a −80 °C freezer. Cryovials are transferred to liquid nitrogen tanks for long-term storage.In the operating room, all of the tissue that remains after the apportioning described above is placed into a sterile Cryobag (OriGen, Austin, TX, USA). The Cryobags are immediately placed into a BioT™ ULT Transporter (Biocision, Larkspur, CA, USA) that has been prefilled with dry ice for rapid freezing. The biospecimens are then transferred to a −80 °C freezer. Frozen specimens are stored long-term in a customized drawer rack manufactured locally for Cryobag storage (Mid America Manufacturing Solutions, Mooresville, IN, USA).Aortic valve tissues undergo similar processing in the operating room, except that the cell culture is not routinely performed for aortic valves.A total of 1047 participants were enrolled into this study. These included 834 affected individuals and 213 who were not affected. The latter group consisted of relatives enrolled for family-based investigation (N = 176) and participants whose aortic tissue samples were used as healthy control tissues (N = 37). Table 4 summarizes the characteristics of affected participants. Patients with aortopathy were broadly eligible, but the predominant recruitment focus to date has been patients who have TAA or a genetic predisposition such as diagnosis of MFS. Consistent with epidemiological data showing that TAA is more common in males [12], there is a greater proportion of affected male participants than female. The racial and ethnic composition is generally consistent with the statewide demographics of Indiana. Approximately 50% of participants who are under the age of 21 years have a syndromic diagnosis. As expected, BAV is the most common congenital heart malformation in TAA cases. TAA is associated with less common congenital heart lesions such as tetralogy of Fallot and other conotruncal defects; however, these patients were not targeted for enrollment.As described in Methods, a direct interview with the participant or close relative (e.g., parent) is prioritized for all participants when available. To date, a direct interview has been accomplished for approximately 80% of participants. In total, 340 aortic tissue samples have been acquired from a total of 290 unique participants. The number of aortic tissue samples are shown across the different aortic segments in Figure 2. More than one segment has been collected from 48 participants. In most of such cases, the segments were acquired during the same operation. A total 110 aortic valve samples have been collected. RNA has been extracted from primary aortic SMCs following the routine protocol at passage 2 or 3 (usually both) for 96 participants. Protein has been extracted at passage 2 or 3 (also usually both) for 90 participants. The extracellular fluid has been collected for 68 participants at the time of cellular extractions. At least one aliquot of cultured aortic SMCs has been cryopreserved for 89 participants. In total, 1006 individual aliquots of cultured SMCs have been cryopreserved.There are myriad opportunities to investigate human aortopathy in the framework of the CHAR study. The successful development and implementation of the study protocol across adult and children’s hospitals required substantial coordination and collaboration in multiple clinical areas, including cardiology, surgery, and genetics. The repository has begun serving as a key resource for multiple lines of inquiry. The repository provides a powerful, human biology-centered platform to address important gaps in knowledge in aortopathy.There are approximately 30 genes associated with TAA and dissection, and 11 of these genes were designated as definitive causes [13]. Understanding the biological roles for these genes and development of mouse models has increased the understanding of cellular and molecular mechanisms in heritable TAA [4]. Genotype–phenotype correlations have been developed over time, some of which have been adopted in clinical practice guidelines [14]. A genetic diagnosis is also important because it provides the opportunity to establish risk in family members through molecular screening. Despite significant advancements, currently known genetic causes account for fewer than 30% of familial cases. Thus, current clinical testing has limited yields, particularly in patients who do not have the signs for specific connective tissue disorders such as MFS. Inconclusive genetics evaluations commonly occur even in aortopathy patients who have connective tissue findings [15]. While there is clear evidence that congenital BAV is heritable, the known genetic causes account for a small fraction of cases [16]. Thus, there is a major need to identify novel causes of human TAA broadly, as well as associated aortic valve disease.Many participants enrolled into the CHAR study do not have a genetic diagnosis established clinically. The detailed collection of family data using formal pedigrees and targeted family member enrollment will facilitate investigations for novel genes associated with TAA. Many CHAR participants provide both blood and tissue samples, creating an opportunity to directly investigate the mechanisms of suspected novel genetic causes that are identified. The comprehensive nature of the tissue and blood sample processing permits the effective investigation of functional effects in multiple ways. For example, cultured SMCs from many participants have already had RNA and protein extracted and SMCs have been cryopreserved, all in a protocolized manner. The prospective acquisition of clinical data and tissue samples will streamline phenotypic descriptions and functional investigations of genes and variants that are identified.Clinically, variants of uncertain significance (VUSs) in genes that are known to be associated with TAA are commonly encountered [17]. We expect to identify VUSs through the course of genetic characterization of CHAR study participants. Here, too, the procurement of tissue samples matched to blood samples will facilitate direct investigation of the functional impact of VUSs. Functional evidence is part of the formal guidelines for interpretation of sequence variants from the American College of Medical Genetics and Genomics [18]. When VUSs are encountered clinically, functional studies are often unavailable or unfeasible. Instead, in silico methods are often utilized, but these rely on prediction algorithms that produce variable results, depend on a priori knowledge, and are not necessarily disease-specific. Where circumstances dictate, there is dedicated expertise in the multidisciplinary aortopathy clinic at Riley Hospital to report back and manage clinically actionable results, such as those encountered in prior aortopathy studies [19]. The integration between the CHAR investigators and personnel and the aortopathy clinic is also advantageous because many enrolled participants will have already had clinical testing, detailed phenotyping, and at-risk family members identified, as part of routine care. The application of genome sequencing in research and ultimately in clinical care will create a major need for resources to investigate the functional impact of potentially pathogenic genetic variants.Fundamental advances in the understanding of TAA pathogenesis have been achieved through the study of animal models. For example, in groundbreaking studies using a mouse model of MFS, Dietz et al. identified a novel mechanism of pathogenesis. Marked benefits of angiotensin II type 1 receptor blockade (ARB)- or transforming growth factor-β-neutralizing antibodies on aortopathy were demonstrated in genetically modified mice [20]. These results led to human clinical trials and the adoption of ARBs such as losartan as a medical therapy for patients with MFS [21]. While clinical efficacy has been demonstrated, the magnitude of the clinical effects of ARBs in humans may be less pronounced than in mice [22]. There are also instances of transgenic mouse models that do not completely recapitulate the human aortic phenotypes [23]. Interspecies variation in the pathobiology and genetic background, as well as different environmental conditions and exposures between laboratory animal experiments and human patients, may contribute to these incongruities. The mouse model work has been robust and highly informative, creating an opportunity to correlate the observations with human TAA pathogenesis. A more complete understanding of human pathogenesis will foster the identification of medical therapies and their indications, in order to ultimately cure TAA.TAA is a genetically heterogeneous disorder. The degrees to which disease mechanisms may be distinct or converge between different etiologies is not well understood. Histologically, medial degeneration is a common finding [24], which supports a hypothesis that overlapping downstream mechanisms may exist. The scope of the CHAR study provides the capacity to compare clinical and genetic subtypes in novel ways. This opportunity is furnished by the tightly controlled and consistent manner in which the biospecimens are collected and processed. In addition, frozen tissue storage will facilitate omics-based analyses of split samples to integrate and correlate pathobiological data in multiple omics domains. Moreover, aortic biospecimens are collected from multiple aortic segments from the same individual participants when available. This will facilitate comparisons of pathological processes between segments, which has been proposed to be influenced by different developmental origins of SMC progenitors [25,26].Patients with TAA or genetic diagnosis often do not have cardiovascular-related symptoms until the sudden development of a life-threatening dissection. Medical therapy and other clinical interventions, including surgery, have benefits in TAA management but also pose associated risks and costs. Thus, TAA is a disease in which a precise stratification of an individual patient’s risk for progression and dissection would significantly improve clinical decision making. Currently, risk classification is imprecise in nature. While certain genetic diagnoses are associated with increased risk, such as MFS and LDS, there is still a large degree of interindividual variation in the severity of TAA within these groups, including between relatives [27]. The factors that influence penetrance and expressivity of genetic aortopathy are not well understood. Furthermore, it is estimated that only one-half of patients with a BAV develop TAA, also for reasons that are not understood. In our center, we have observed wide variability in disease progression between patients within different TAA subgroups starting at young ages [28]. This included the identification of some patients with BAV who progressed from normal aorta to significant TAA at early ages. While medical and surgical treatments are beneficial in TAA, more precise classification of risk will optimize outcomes and minimize harm.The identification of genetic modifiers of aortopathy severity is a promising avenue toward optimizing risk stratification on a more individual basis [27,29]. Circulating biomarkers of disease progression may also help to understand and predict risk [30], but studies that include longitudinal sampling have been limited. We previously identified non-cardiovascular characteristics that were associated with the rate of aortic dilation in young TAA patients [15], which raised the possibility that systematic noncardiac phenotyping may have a role in the development of more precise prediction algorithms. Ultimately, the development of clinical prediction algorithms that incorporate genetic, molecular, and endophenotype attributes may lead to individualized clinical care and provide novel insight into pathobiological mechanisms. The design of the CHAR study is well situated to approach this fundamental gap.Primary aortic SMCs are an established in vitro model for the study of aortopathy, as SMCs have a crucial role in the maintenance of extracellular matrix and vascular tone and SMC dysregulation is common in TAA. The large number of RNA and protein samples that have been extracted prospectively from SMCs at early passage, and the substantial cryopreservation effort (>1000 cell aliquots) will facilitate novel characterizations of SMC dysregulation. Additionally, experiments that include molecular or pharmacological manipulation of human primary SMCs provide results in the context of human biology.Participants in CHAR are well-phenotyped clinically. Investigational phenotyping techniques may be pursued to complement the clinical data. Examples include computational analysis of fluid dynamics and biomechanics for cardiovascular phenotyping and digital photography for craniofacial phenotyping. As the study increases in size and timespan, development of electronic-based mechanisms for bidirectional communication with enrolled participants may improve the study’s monitoring of clinical and family status over time. Longitudinal blood sampling will enable biomarker studies for disease development or progression. Molecular characterization of the cohort with genetic testing performed on a research basis will optimize ongoing studies and prompt future investigations.The CHAR study encompasses a large biobank specifically designed to investigate human aortopathy in a multidimensional manner. It provides a platform for novel investigation into genetic causes and modifiers and the mechanisms of pathogenesis and disease progression.Conceptualization, B.J.L.; methodology, C.E.V. and B.J.L.; formal analysis, B.J.L.; resources, B.J.L.; data curation, C.E.V. and B.J.L.; writing—original draft preparation, B.J.L.; writing—review and editing, C.E.V. and B.J.L.; visualization, B.J.L.; supervision, B.J.L.; project administration, C.E.V. and B.J.L.; funding acquisition, B.J.L. Both authors have read and agreed to the published version of the manuscript.This research was funded (B.L.) by the National Institutes of Health K12 HD068371 and K23 HL141667, the Marfan Foundation Early Investigator Grant, the American Heart Association 19 CDA34660278, and the IUSM Strategic Research Initiative. The APC was funded by the journal.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board at Indiana University (protocol 1509977311; approval on 15 September 2020).Informed consent was obtained from all participants involved in the study. Organ donor tissues were included if research use was already authorized.We acknowledge the contributions of Lindsey Elmore, Lauren Frasier, Angie Seward, and Adam Oldham in clinical study coordination, recruitment, and data collection at IUSM. Gavin Needler and Amanda Smith provided technical specimen support. We thank the operating room personnel at Methodist and Riley Hospitals, including cardiothoracic surgeons Joel Corvera, Philip Hess, Lawrence Lee, Daniel Beckman, Jeffrey Everett, Mark Turrentine, Mark Rodefeld, Jeremy Herrmann, John Brown, John Fehrenbacher, Arthur Coffey, and Saila Pillai, and advanced providers Lea Glancy, NP, Rebecca Haviza, PA, and Brittany Barnett, PA. The repository and laboratory are located within the Herman B Wells Center for Pediatric Research at IUSM. The Clinical and Translational Support Laboratory (an Indiana Clinical Translational Science Initiative core facility) supported blood specimen processing. Tatiana Foroud provided important biobanking guidance. Stephanie Ware generously provided laboratory infrastructure support and mentorship for B.L. Larry Markham, Dianna Milewicz, Brett Graham, R. Mark Payne, and D. Wade Clapp also provided support and mentorship for B.L.The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.Synopsis of CHAR study’s eligibility, data collection, and processing of blood and aortic tissue biospecimens. P2: Passage 2; P3: Passage 3.Numbers of aortic and aortic valve tissue samples collected to date in participants with aortopathy or aortic valve disease and normal controls. Dashed lines demarcate the boundaries of thoracic aortic segments.Categories of data that are routinely entered in the CHAR study.Cardiovascular-related data collected through structured interviews and electronic medical record review.BAV: bicuspid aortic valve.Non-cardiovascular data collected through structured interviews and electronic medical record review.The database includes the ability to enter additional details and comment where appropriate.Characteristics of the affected participants enrolled into the CHAR study.Percentages are of available data.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Sarcolemmal membrane-associated proteins (SLMAPs) belong to the superfamily of tail-anchored membrane proteins known to regulate diverse biological processes, including protein trafficking and signal transduction. Mutations in SLMAP have been linked to Brugada and defective sodium channel Nav1.5 shuttling. The SLMAP gene is alternatively spliced to generate numerous isoforms, broadly defined as SLMAP1 (~35 kDa), SLMAP2 (~45 kDa) and SLMAP3 (~80–95 kDa), which are highly expressed in the myocardium. The SLMAP3 isoform exhibits ubiquitous expression carrying an FHA domain and is believed to negatively regulate Hippo signaling to dictate cell growth/death and differentiation. Using the αMHC-MerCreMer-flox system to target the SLMAP gene, we specifically deleted the SLMAP3 isoform in postnatal mouse hearts without any changes in the expression of SLMAP1/SLMAP2 isoforms. The in vivo analysis of mice with SLMAP3 cardiac deficiency revealed no significant changes to heart structure or function in young or aged mice without or with isoproterenol-induced stress. SLMAP3-deficient hearts revealed no obvious differences in cardiac size, function or hypertrophic response. Further, the molecular analysis indicated that SLMAP3 loss had a minor impact on sodium channel (Nav1.5) expression without affecting cardiac electrophysiology in postnatal myocardium. Surprisingly, the loss of SLMAP3 did not impact Hippo signaling in postnatal myocardium. We conclude that the FHA domain-containing SLMAP3 isoform has no impact on Hippo signaling or sodium channels in postnatal myocardium, which is able to function and respond normally to stress in its absence. Whether SLMAP1/SMAP2 isoforms can compensate for the loss of SLMAP3 in the affairs of the postnatal heart remains to be determined.Sarcolemmal Membrane Associated Proteins (SLMAPs) belong to a superfamily of tail-anchored membrane proteins, such as junctophilin, SNAP-25 and calnexin, involved in diverse functions, including E-C coupling, neurotransmitter release and ER stress response [1]. The SLMAP gene contains alternative start sites and can alternatively splice to generate many SLMAP isoforms that are highly conserved across species and expressed in a tissue-specific and developmentally regulated manner [2]. We defined SLMAP isoforms into three main subgroups based on the molecular size of the polypeptides noted: SLMAP1 (~35 kDa), SLMAP2 (~45 kDa) and SLMAP3 (~80–95 kDa) [3]. SLMAP3 is ubiquitously expressed, while SLMAP1 and SLMAP2 expression are restricted to muscle and myocardium [1,3]. All SLMAP isoforms share a common C-terminus with a single transmembrane domain, which can be alternatively spliced to target diverse subcellular membranes [4]. The SLMAPs are generally comprised of extended coiled-coils with two tandem leucine zippers with unique N-terminal fork head-associated domains (FHA) found in the SLMAP3 isoform [5,6]. The FHA domain is found in numerous kinases and phosphatases and acts as the regulator for phosphoprotein interactions, specifically with phospho-threonine residues [7]. Recently SLMAP3′s FHA has been shown to regulate the Hippo signaling pathway via the STRIPAK (striatin interacting phosphatase and kinase) complex [8,9]. Hippo signaling is believed to regulate organogenesis, tissue homeostasis, proliferation and apoptosis, including cardiac development [10,11,12]. The unregulated activity of Hippo signaling in mouse myocardium can result in lethal phenotypes but can also aid in cardiac tissue repair in post-myocardial infarction or in pressure overload-induced damage [13,14,15,16,17]. SLMAP is believed to interact with phosphorylated MST1/2 kinases via its FHA domain and recruit STRIPAK-PP2A to dephosphorylate the transcriptional coactivators YAP/TAZ and negatively regulate Hippo to increase proliferation and repress apoptosis [18,19,20,21].We have previously shown that endogenous cardiac SLMAPs co-localize with E-C coupling machinery, including the ryanodine receptor 2 (RyR2) and caveloin-3 in cardiomyocytes. The targeted expression of the SLMAP1 isoform in postnatal mouse myocardium leads to aberrations in electrophysiology and function due to the reduced expression of E-C coupling proteins [22,23]. Ishikawa and colleagues discovered a mutation in the SLMAP gene in Brugada patients, a channelopathy characterized by irregular electrical activity mainly due to abnormal expression of the sodium ion channel, NaV1.5 [24,25]. The mutated SLMAP prevented the trafficking of NaV1.5 to the cell surface in transfected HEK293 cells implicating SLMAP as a regulator of Nav1.5 protein trafficking [24]. In transgenic mice with cardiac-specific SLMAP3 expression, there was no noticeable cardiac remodeling; however, we observed a defect in cardiac function and electrophysiology due to a reduction in protein and transcript levels of Nav1.5, further implying a relationship between SLMAP and Nav1.5 [26]. Although studies have uncovered some unique properties of cardiac SLMAP, its precise molecular function needs to be fully interrogated.Thus, our aim here was to establish a method to delete the SLMAP gene in postnatal myocardium in a conditional and temporal manner to determine its precise role in young and adult mouse hearts. In this study, we develop the procedure to establish a mouse model using the αMHC-MerCreMer-lox method to delete the SLMAP gene in postnatal myocardium. Our strategy resulted in the specific deletion of the SLMAP3 isoform in postnatal mouse myocardium while the expression of SLMAP1 and SLMAP2 isoforms remained unaffected. We report the phenotypic assessment and any molecular impact of the specific loss of the SLMAP3 isoform in the postnatal heart under normal and β-adrenergic agonist, isoproterenol (ISO), induced stress.A targeting construct vector for SLMAP was developed using a neomycin insertion cassette. Exon 3 of the mouse SLMAP gene was flanked with loxP sites, and the vector was microinjected into embryonic stem cells that were implanted into C57BL/6 blastocysts. The founder (F0) flox-SLMAP mice were confirmed through genotyping and maintained through breeding with C57BL/6 mice (Cyagen Biosciences Inc, Santa Clara, CA, USA).The αMHC-MerCreMer mice were obtained from Dr. Mona Nemer and maintained by breeding with C57BL/6.The breeding scheme to generate a cre-lox mice line for SLMAP knockout is shown (Figure 1). Mice were genotyped by extracting genomic DNA from ear clips by boiling for 10 min at 95 °C in 180 μL of 50 mM NaOH per ear. The DNA solution was then neutralized using 20 μL of 1 M Tris-Cl pH 8.0. SLMAP KO/KD mice were identified by polymerase chain reaction (PCR) using DreamTaq Green PCR Master Mix 2x (Thermo Fischer Scientific, Waltham, MA, USA). Forward F2 primer (5′-CCT GGA GAG CCT CCG TGT GAG T-3′) and reverse R2 primer (5′-GTC AAC TGC CCA ATG TAC AGA AAT AGT AAG-3′) targeted loxP site 1 on the flox-SLMAP gene. Forward Cre-F (5′-ACG ACC AAG TGA CAG CAA TG-3′) and reverse Cre-R primer (5′-AAC CAG CGT TTT CGT TC-3′) detected the Cre gene. PCRs were visualized by Red Safe (Sigma, St. Louis, MO, USA.) staining on a 1% agarose gel.Mice were handled in accordance with the guidelines set by Canadian Council on Animal Care, Guide to the Care and Use of Experimental Animals, 2 vols. (Ottawa, Ont.: CCAC, 1980–1993) and Animals for Research Act, R.S.O. 1990, c.A. 22. All animal protocols and procedures were approved by the Animal Care Committee of the University of Ottawa.Tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) was administered to animals to activate MerCreMer. A total of 500 µg of tamoxifen was dissolved into 10 mL of peanut oil (Sigma-Aldrich, St. Louis, MO, USA.), aliquoted into 10 tubes and frozen at −20 °C for long-term storage. Tamoxifen was injected intraperitoneally in 5-week-old animals at a dose of 30 µg tamoxifen/g of animal for 3 days. Tamoxifen treated hearts were analyzed by PCR genotyping as described above. The forward primer F2 (5′-CCT GGA GAG CCT CCG TGT GAG T-3′) and reverse primer R1 (5′-GGA GAG ACT ATC ACA GCC ACA GGA-3′), coding for sequences between loxP sites, exon 3 and intron sequences, were used for amplification.The hearts of adult mice (8–12 weeks of age) were collected after CO2 euthanasia and immediately frozen at −80 °C. Each heart was washed with ice-cold 1× phosphate-buffered saline (PBS) and homogenized using a Fisher handheld Maximizer homogenizer (Thermo Fisher Scientific, Waltham, MA, USA.) in ice-cold lysis buffer (1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM ethylenediaminetetraacetic (EDTA), 20 mM Tris base, 1% Triton, 150 mM sodium chloride, 1× complete mini EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland) and 1× PhosSTOP (Roche, Basel, Switzerland). The suspension was centrifuged for 15 min at 12,000× g to separate the proteins from the cell debris. The supernatant containing heart protein lysate was collected in Eppendorf tubes and stored in a freezer at −80 °C.A total of 10–20 µg of protein lysate was loaded in each well of a 5–15% SDS-PAGE gel. The gels were transferred overnight on a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) in a buffer containing 25 mM Tris, 190 mM Glycine and 20% methanol. All membranes were blocked at room temperature for 1 h in Tris-buffered saline (TBST) containing 1 M Tris, 290 mM NaCl, 0.1% Tween 20, pH 7.4 and 5% nonfat dry milk. The primary antibodies (listed in Table 1) were incubated overnight at 4 °C with 5% bovine serum albumin (BSA). The membranes were washed 5 times for 5 min each in TBST prior to adding the appropriate horseradish peroxidase-labeled secondary antibody (Jackson Immuno Research, West Grove, PA, USA) at a 1:10,000 dilution in TBST with 5% nonfat dry milk. The membranes were shaken slowly at room temperature for 1 h while incubating with secondary antibody, followed by 5 washes for 5 min each with TBST. The membranes were treated with a BioRad western blotting kit (Bio-Rad, Hercules, USA) and developed using ChemiDoc machines (Bio-Rad, Hercules, USA). The bands were quantified by densitometry using Image Lab software v.6.0.0 (Bio-Rad, Hercules, CA, USA). The membranes were stripped (25 mM glycine, 10% SDS and pH 2.2 in dH2O) and reprobed with different antibodies. When using stain-free technology, stain-free gels (Bio-Rad, Hercules, CA, USA) and low-fluorescence PVDF membranes (Bio-Rad, Hercules, CA, USA) were used.The total mRNA from mouse hearts was extracted using the RNeasy Fibrous Tissue Kit (Qiagen, Hilden, Germany). The concentration and purity of the obtained mRNA were determined by measurement of 260/230 nm absorbance ratio and 280/260 absorbance ratio [27] using NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was used as a template to generate cDNA using SuperScript II reverse transcription protocol following the manufacturer’s guidelines (Thermo Fisher Scientific, Waltham, MA, USA). RT-qPCR was carried out using a BioRad CFX96 system and Luna Universal qPCR mater mix according to the manufacturer’s instructions. Equal amounts of cDNA were utilized in real-time PCR using primers for SLMAP3 cDNA (SLPN-F: 5′-GGA ATT CGA TGC CGT CAG CCT TGG C-3′, 558-R: 5′-TTG CTC GTC TTG TGA TCA AAC CAG-3′), total SLMAP cDNA (GTY-F: 5′-GAA AAG CCT ATC GAA ATC AAG TTG-3′, GTY-R: 5′-ACC TTC TTA AGC TCT TCT TGC AAA G-3′) and 18S Ribosome control (forward: 5′-AAT ACA TGC CGA CGG GCG CT-3′, Reverse: 5′AGT GGG TAA TTT GCG CGC CT-3′). The fold change was calculated using the ∆∆Ct method.All echocardiographic analysis was performed using the VEVO 2100 system (FUJIFILM VisualSonics, Toronto, Canada). Adult mice were anesthetized using 2% isoflurane and strapped onto a heated pad facing upwards, exposing the thoracic cavity. A 40 MHz probe was used to capture short-axis B-mode and M-mode images of the left ventricle. VEVO v1.6 software (FUJIFILM VisualSonics Toronto, Canada) was utilized for measuring LV wall thickness and inner diameters in diastole and systole. The formulas used by the VEVO v1.6 software to calculate EF, FS, LV mass and LV Vol;d/s are listed below:(1)EF: 100×LV Vol;d − LV Vol;sLVVol;d
+        (2)FS: 100×LVID;d − LVID;sLVID;d
+        (3)LV mass: 0.8×1.053×LVID;d + LVPW;d + IVS;d3− LVID;d3
+        (4)LV vol; d/s: (7.0/2.4 + LVID;d;s× LVID;d;s^3Isoproterenol (Sigma-Aldrich, St. Louis, MO, USA) delivery was mediated through the subcutaneous implantation of miniosmotic pumps (2001D Alzet, Cupertino, CA, USA) in 8-week-old mice. 0.9% saline or isoproterenol (dissolved in 0.9% saline) was delivered at a rate of 30 µg/g/d per animal. Cardiac function was analyzed through echocardiography before pumps were implanted and after seven days. After a week of isoproterenol administration, the animals were euthanized, and the hearts were collected for histological analysis.Hearts that were iso-treated were extracted from animals and fixed using 10% Neutral Buffered Formalin (Thermo Fisher Scientific, Waltham, MA, USA.). After fixing for 48 h, the hearts were sectioned 4 µm longitudinally per section. Sectioned hearts were stained with Masson’s trichrome to visualize the myocardium, nuclei and collagen.The mice were anesthetized with 2.5% isoflurane. A 6-lead surface ECG was recorded after a 5-min stabilization period of mice in the anesthetized state. ECG intervals and heart rate were analyzed manually from 6–18 most stable waves selected from a 2-min recorded stream. Analysis was blind to the genotype. Intervals were defined as follows: PP duration from the beginning of the P wave to the end, where the P wave returns to the isoelectric line, PR segment from the end of the P wave to the beginning of R wave, PR interval from the beginning of P wave to the beginning of the R wave, QRS duration from the beginning of the R wave to the point where the negative S wave returns to the isoelectric line and QT interval from the beginning of the R wave to the point where the negative or positive T wave returns to the isoelectric line. The Q wave was not visible. QT interval correction was based on Mitchell [28] using the following formula: QTc = QTo/(RRo/100)1/2. P, R and S wave amplitudes were quantified as the distance between the peak of the wave and the isoelectric line connecting the end of the P wave and the beginning of the R wave. Electrocardiograms were recorded using IOX2.4.2.6 (EMKA Technologies, Paris, France). RR interval, heart rate and wave amplitudes (P, R, and S) were analyzed by ecgAUTO v2.5.1.18 software (EMKA Technologies, Paris, France).The statistical analysis was performed using GraphPad Prism software version 8 for Windows (GraphPad Software, La Jolla, CA, USA.). All comparisons between wildtype and knockdown or knockout groups were analyzed using a two-tailed Student’s t-test. To analyze all three groups, we utilized ANOVA one-way analysis to evaluate the significance. All values and points on graphs represent mean values obtained from multiple experiments. All error bars presented in graphs are represented using the standard error of the mean. All sample size values (n) represent biological replicates.The mouse SLMAP gene is approximately 122 kilo-base pairs (bp) in length and contains 24 exons. Exons 2, 3, 10 and 16 are believed to contain putative start sites, and seven exons (11, 12, 13, 14, 17, 23 and 24) can alternatively splice to generate additional SLMAP isoforms (Figure 2) based on our lab findings and proteomic/genomic profiling databases [29,30,31]. SLMAP3 variants are generated by alternate start sites found at exon 2 or 3, and the alternative splicing of exons 11, 12, 13, 14 and 17 (Figure 2). SLMAP2 and SLMAP1 isoforms are generated by putative start sites at exon 10 and exon 16, respectively (Figure 2). SLMAP2 variants are similarly generated by alternative splicing of exons 11, 12, 13, 14 and 17, while SLMAP1 variants have an alternative exon 17 (Figure 2). All SLMAP isoforms house an alternative transmembrane domain that is generated by mutually exclusive splicing of exon 23 or 24 (Figure 2). We predicted that promoter sequences for SLMAP would reside upstream of exon 2; therefore, we targeted the excision of exon 3 to prevent the translation of functional SLMAP as it would cause a frameshift mutation. To generate the cardiac-specific postnatal knockout of SLMAP, the cre-lox system was chosen as we could manipulate the temporal and spatial targeting by using the αMHC-MerCreMer mice, ensuring that cre recombinase is expressed specifically in the myocardium and will remain dormant unless activated by an estrogen analog, tamoxifen [32,33]. To target exon 3, in the mouse SLMAP gene, loxP sites were introduced to flank exon 3. The floxed-SLMAP DNA was microinjected into embryonic stem cells that were transplanted into fertilized mouse ovum (detailed in materials and methods) [34]. Floxed-SLMAP mice were crossbred with αMHC-MerCreMer mice to generate offspring that would contain both a floxed-SLMAP and cre transgene to undergo a knockout (KO) or knockdown (KD) of SLMAP in postnatal mouse hearts (detailed in materials and methods) [35].PCR genotyping of ear clippings from individual mice determined the presence of floxed-SLMAP and/or αMHC-MerCreMer. Primers F2/R2 were used to identify floxed-SLMAP by detecting loxP site 1 and primers CreF/CreR, detected αMHC-MerCreMer as modeled in Figure 3A. The flox-SLMAP F2/R2 PCR product was 424 bp in length, whereas wildtype SLMAP was 282 bp in length (Figure 3B). Genotyping with CreF/CreR generated a 425 bp amplicon denoting a αMHC-MerCreMer+ mouse (Figure 3B). PCR products ran on separate lanes on 1% agarose gel to separate and size DNA amplicons to determine the presence of transgenes in each animal (Figure 3B). The lane labeled KO presented two alleles of flox-SLMAP (one DNA band at 424 bp) and the αMHC-MerCreMer transgene, signifying a KO mouse (Figure 3B). The KD lane presented one flox-SLMAP allele (424 bp), one wt-SLMAP allele (284 b bp) and the αMHC-MerCreMer transgene, signifying a KD mouse (Figure 3B). Finally, the lane that had only one transgene or none signifies a wildtype (Wt) animal, as both a flox and a cre are essential for DNA modification to take place (Figure 3B) [36].After generating flox-SLMAP/αMHC-MerCreMer+ mice, we wanted to ensure loxP sites flanking SLMAPs exon 3, and surrounding introns were correctly targeted. This was assessed through PCR amplification of DNA isolated from tamoxifen-treated hearts of Wt and KD mice. Studies have shown that the administration of 30 µg of tamoxifen/g of body weight for 3 days via intraperitoneal injections is the most effective method for inducing MerCreMer activity because it results in the highest recombination efficiency [33,37]. After tamoxifen treatment, we extracted DNA from KD and Wt hearts and performed PCR using primers F2/R1, targeting the sequence between the two loxP sites, modeled in Figure 3A. Excision of the floxed region would reduce the F2/R1 product to 400 bp while the unaffected SLMAP gene’s F2/R1 product would remain 1400 bp in size (Figure 4A). PCR products ran individually on 1% agarose gels to separate and size the DNA amplicons from Wt and KD hearts (Figure 4B). Amplicons from KD hearts produced bands at 1400 and 400 bp, demonstrating that only floxed SLMAP allele is targeted by the active cre enzyme (Figure 4B). Floxed-SLMAP/αMHC-MerCreMer- (Wt) hearts produced an amplicon at 1400 bp, signifying that cre is necessary to cleave floxed genes (Figure 4B). Therefore, the activation of αMHC-MerCreMer via tamoxifen successfully excised loxP flanked exon 3 from the floxed-SLMAP gene.Next, we wanted to assess the impact on SLMAP protein levels in KD and KO mice. Adult mice were tamoxifen-treated and left for a minimum of three weeks before cardiac lysates were evaluated for protein expression. Western blot analysis on mice designated KD had a significant reduction in the expression of SLMAP3 (−45.81% ± 9.58%, ρ < 0.05, n = 6) relative to the Wt (Figure 5A); however, no changes were noted in the expression of SLMAP1 or SLMAP2 isoforms. In KO mice, western blot analysis indicated the total loss of SLMAP3 protein (−89.31% ± −14.50%, ρ < 0.001, n = 10), but there were no changes noted in the expression of SLMAP1 (1.91% ± 31.94%, ρ = 0.955, n = 10) or SLMAP2 (12.86% ± 35.02%, ρ = 0.74, n = 10) in KO hearts when compared to Wt littermates (Figure 5A). Further quantification of western blot data indicated that the expression of SLMAP2 (11.07x greater, ρ < 0.05, n = 3) and SLMAP1 (22.91x greater, ρ < 0.05, n = 3) was much higher compared to SLMAP3 expression in Wt adult mouse myocardium (Figure 5B).The transcript levels of the SLMAP isoforms in tamoxifen-treated postnatal mouse hearts amplified using PCR with specific primers, SLPN-F/558-R and GTY-F/GTY-R, target exons at the 5′ end, sequences that are present in SLMAP3 but absent in SLMAP1 and SLMAP2 (Figure 2) [1]. We observed a significant decrease in SLMAP3 transcripts in the KD (−55.22% ± 27%, n = 19, ρ < 0.001) and KO (−93.47% ± 31.20%, n = 13, ρ < 0.001) hearts (Figure 5B). SLMAP1 and SLMAP2 sequences are conserved in SLMAP3; therefore, we are unable to amplify these distinct isoforms individually. Instead, we used GTY-F/GTY-R, a primer that targets all three isoforms, to determine if there are any differences in total SLMAP mRNA expression. Total SLMAP expression in KD (−1.29% ± 38.35%, n = 10, ρ = 0.759) and KO (−34.12% ± 40.71%, n = 9, ρ = 0.759) hearts revealed no significant differences compared to Wt (Figure 5B). Therefore, targeting exon 3 of the SLMAP gene nullifies SLMAP3 expression but not the abundantly expressed SLMAP1 or SLMAP 2 mRNA [1].Although only SLMAP3 expression was nullified in our cre-lox model, we asked about its impact in adult mouse hearts since SLMAP3 has been implicated in many roles [22,23,24,26]. Transthoracic echocardiography (ECHO) was performed on 5-week post-tamoxifen (post-TAM) mice biweekly for 19 weeks to observe any changes to cardiac structure and function by measuring left ventricle (LV) wall thickness (intraventricular septum (IVS) and left ventricular posterior wall (LVPW)) and the left ventricular intradiameter (LVID) during systole and diastole. These measurements were used to calculate cardiac function, including the ejection fraction (EF) and fractional shortening (FS), which measure a percentage of cardiac output and LV muscle contraction, respectively [38]. ECHO did not reveal any structural abnormalities in the young (5-week post-TAM) or old (24-week post-TAM) SLMAP3-deficient hearts when compared to Wt (Figure 6A). Further, analysis of LV walls revealed no significant changes to IVS or LVPW wall thickness during systole or diastole in young or old SLMAP3 deficient hearts when compared to wildtype (Supplementary Table S1). Further cardiac functional analysis indicated no significant differences in EF or FS in SLMAP3-deficient hearts (Figure 6B). Left ventricle mass to body weight comparison indicated no significant differences between KD, KO mice (n = 6, ρ = 0.5826) and wildtype (Figure 6C). The only difference noted between the age groups was an increase in LV mass that was likely due to age-related cardiac growth, which was similar among Wt and SLMAP3 null hearts (Supplementary Table S1). Therefore, a deficiency of SLMAP3 did not impact cardiac structure or function in the postnatal young or adult mouse hearts.Our next aim was to administer cardiac stressor isoproterenol (ISO) to cardiac-depleted SLMAP3 mice to evaluate the impact of stress on the myocardium [39]. ISO is a non-selective β-adrenergic agonist that can induce pathological cardiac hypertrophy [40]. Seven-day delivery of ISO (30 ug/g) or saline-control was mediated through mini-osmotic pumps that were subcutaneously implanted in tamoxifen-treated eight-week-old adult mice that were genotyped KO or Wt. ECHOs were performed before pumps were implanted (preISO) and seven days after implantation (postISO). PostISO ECHOs revealed a significant change in LV wall thickness in KO and Wt hearts (Figure 7A). LV walls, IVS (~25% increase, n = 7, p < 0.05) and LVPW (~20% increase, n = 7, ρ < 0.05) were significantly thicker in postISO mice in both Wt and KO hearts when compared to their preISO measurements (Figure 7B). Unsurprisingly, the weight of the hearts increased (~25% increase, n = 7, ρ < 0.05) as a consequence of cardiac hypertrophy. The similarity in heart weight and dimensions between the two iso-treated groups indicates the hypertrophic stress response is identical between the SLMAP3-KO and wildtype hearts (Supplementary Table S2).Hearts dosed with saline or ISO underwent histological analysis to observe changes to all chambers of the myocardium. Wt and KO hearts were fixed, sectioned longitudinally and stained with Masson’s trichrome. Masson’s trichrome stains the cardiomyocytes, nucleus and collagen; thus, we are able to visualize the myocardium and detect fibrotic cells. After a seven-day ISO treatment, SLMAP3-KO hearts displayed no obvious structural differences, nor did we detect any evidence of fibrosis when compared to Wt (Figure 8).Several studies have indicated that SLMAP3, via its FHA domain, can regulate Hippo signaling by recruiting MST1/MST2, which comprises STRIPAK. The core STRIPAK components (striatin, PP2A-A, PP2A-C) were assessed by western blotting of cardiac lysates together with MST1/2 and YAP. Striatin (n = 6, ρ = 0.58), PP2A-A (n = 6, ρ = 0.69) and PP2A-C (n = 6, ρ = 0.28) protein levels were not significantly altered in adult SLMAP3-KO hearts compared to Wt (Figure 9). The phosphorylation of MST1/2 (T183) and YAP (S127) was evaluated and quantified, and the phospho-YAP to total YAP ratio (n = 6, ρ = 0.34) and phospho-MST1 to total MST1 (n = 6, ρ = 0.36) was not significantly altered in postnatal SLMAP3-KO hearts compared to Wt (Figure 9).Mutations in the human SLMAP gene have been linked to Brugada with inappropriate Nav1.5 shuttling [24,26]. Western blot analysis indicated that there was a modest, albeit significant, decrease of Nav1.5 protein levels in KD (−9.81% ± −6.00%, ρ < 0.05, n = 3) and KO heart lysates (−16.66% ± −6.69%, ρ < 0.05, n = 3) when compared to Wt (Figure 10A). Since Nav1.5 plays a major role in initiating the E-C coupling cascade, we determined if a modest decrease of Nav1.5 can impact the electrophysiology of SLMAP3-deficient hearts [41]. A 6-lead surface electrocardiography (ECG) in mice aged up to 28 weeks showed no significant changes between Wt and SLMAP3-deficient hearts (Figure 10B). Electrical properties were quantified, and P, R, Q, S and T waves in each ECG reading are defined in Figure 10B. Our analysis showed that the PP duration, PR segment, PR interval, QRS duration and QTc interval were not significantly altered between Wt and SLMAP3-deficient hearts (Table 2). Further, P, R and S amplitudes did not significantly vary in SLMAP3-deficient hearts when compared to Wt (Table 2). Therefore, the decrease in Nav1.5 protein expression did not significantly alter cardiac electrophysiology in SLMAP3-deficient hearts.Mutations in SLMAP have been linked to Brugada, while it has also been shown to be a component of the STRIPAK complex involved in regulating Hippo signaling. To define the precise role of SLMAP within postnatal myocardium, we generated a cardiac-specific knockout of SLMAP using the cre-lox system by targeting exon 3 of the SLMAP gene with a view to nullify its expression. We employed the αMHC-MerCreMer method to define the impact of SLMAP loss on myocardium in a temporal manner, i.e., in the postnatal myocardium by using tamoxifen to activate cre and excise floxed SLMAP [33].To our surprise, cleaving exon 3 of the mouse SLMAP gene only nullified the expression of SLMAP3. The transcript and protein levels of SLMAP1 and SLMAP2 isoforms remained unchanged, while SLMAP3 levels were significantly decreased in KD and lost in KO hearts. We hypothesized that the loss of SLMAP3 would impact the growth/function of postnatal myocardium since SLMAP3 houses the FHA domain, which was shown to integrate MST1/2 to regulate Hippo signaling [18,19,20,21]. In addition, the loss of SLMAP3 may influence Nav 1.5 shuttling and the electrical properties of the myocardium [24]. ECHO analysis indicated that SLMAP3-deficient hearts showed no significant changes in cardiac structure or function in the acute or chronic loss of SLMAP3. Administration of ISO to experimental animals did not expose any underlying phenotypes to this stressor of the myocardium. ECHO and histological analysis indicated an identical hypertrophic phenotype in iso-treated KO and Wt hearts implying that SLMAP3 loss had no effect on the stress response. These results suggest that SLMAP3 does not play a role in this aspect of cardiac structure or function, and this may be due to compensation by the abundant levels of SLMAP1 and SLMAP2 that remain unchanged in the SLMAP3-KO myocardium. SLMAP1 and SLMAP2 could compensate due to the significant identity between these isoforms and their robust expression in the myocardium relative to SLMAP3 [5]. We theorize that expression of SLMAP1 and SLMAP2 is not affected by the cleavage of exon 3 because they may be regulated by alternate promoters [42]. SLMAP1 and SLMAP2 are truncated variants of SLMAP3 and contain unique in-frame start codons at exon 10 and 16, and expression may be driven by promoters upstream of these start codons [2].Several studies have indicated that the expression and localization of Nav1.5 can be impacted by SLMAP3 [24,26]. Ishikawa et al. linked mutations in the human SLMAP gene at exon 8 (SLMAP3-specific) and exon 21 (all SLMAP isoforms) in Brugada patients. When mutated SLMAP3 or SLMAP1 proteins were expressed in HEK293, they inhibited the trafficking of expressed hNav1.5 to the cell surface [24]. In SLMAP3-KO hearts, there was a minor but significant decrease in Nav1.5 protein expression. However, ECG analysis of adult mice with cardiac SLMAP3-deficiency displayed no significant differences in any parameters when compared to Wt. Although Nav1.5 trafficking was not assessed, the modest decrease in Nav1.5 and lack of changes in electrophysiology indicates that the loss of SLMAP3 has no impact in this respect. SLMAP1 and SLMAP2 levels remain unchanged and potentially could compensate for SLMAP3 loss of function. However, we have shown that the SLMAP3 transgenic myocardium had decreased transcript levels of Nav1.5 and Serca2a, but this was not seen in SLMAP1 transgenic hearts. Thus SLMAP3 may uniquely modulate gene expression in vivo, further implying a relationship between SLMAP3 and Nav1.5 [26]. SLMAP isoforms can homo- and heterodimerize via coiled-coil domains [5], and the overexpression of SLMAP3 could potentially sequester endogenous SLMAPs and interacting proteins to subcellular domains, impacting the mechanism that regulates expression/trafficking of Nav1.5, although this needs to be fully interrogated. Researchers have shown that mice with ~50% decrease of Nav1.5 expression display defects in atrioventricular conduction parameters without presenting any obvious cardiac phenotypes [43]. This was also noted in our SLMAP3 overexpressing transgenic mice as Nav1.5 transcript levels were reduced by 55%, displaying atrioventricular conduction defects with no overt defects in the myocardium [26].The fork head-associated (FHA) domain in SLMAP3 has been shown to recruit MST1/2 kinase to negatively regulate the Hippo pathway via the STRIPAK complex [44,45]. Evidence indicates that loss of SLMAP3 in human cells and Drosophila positively regulates Hippo signaling i.e., increased phosphorylation of MST1/2 (T183) and YAP (S127) [18,19,20,21,46]; however, no significant differences were noted in phosphorylation of MST1/2 or YAP in SLMAP3-null hearts compared to wildtype myocardium. When Hippo signaling is upregulated in adult mouse hearts via overexpression of cardiac-specific MST1/2 and/or deletion of YAP, it leads to cardiomyopathies and heart failure [47,48]. Therefore, if the loss of SLMAP3 would have affected Hippo signaling similarly, it should result in similar phenotypes in postnatal mouse hearts. It is notable that Hippo deficiency in adult myocardium leads to dysfunction in pressure overload [49] while it reversed the systolic heart failure after infarction [50]. Although we used ISO to stress the heart, we did not observe any cardiac dysfunction or changes in Hippo signaling. Whether injury, such as myocardial infarction or pressure overload, would impact Hippo in SLMAP3 deficient hearts, and function remains to be investigated [51]. If and how the abundant expression of SLMAP1 and SLMAP2 isoforms in postnatal myocardium can compensate for SLMAP3 loss in terms of Hippo signaling and function remains to be examined. The lack of change in Hippo signaling due to SLMAP3 loss could also be due to the extremely low proliferative rates of cardiomyocytes in postnatal heart development since Hippo signaling reaches its peak as early as 10-days after birth [52,53]. Thus, the lack of phenotype in SLMAP3-deficient postnatal myocardium could be due to a lack of proliferative programming in matured cardiomyocytes [54,55]. Studies to evaluate the impact of SLMAP3 by targeting its prenatal/perinatal expression, where cardiomyocyte proliferation/growth is critical, are now in progress together with nullifying SLMAP1 and SLMAP2 expression [56,57].In conclusion, cardiac-specific loss of SLMAP3 in postnatal myocardium did not significantly alter the structure/function or activity of Hippo signaling in mouse hearts. Although a minor decrease in Nav1.5 expression was noted, no effect on cardiac electrophysiology was evident in adult SLMAP3-deficient hearts. We theorize that isoforms SLMAP1 and SLMAP2 may compensate for SLMAP3 due to their abundant expression in the adult myocardium. To further investigate in vivo roles of SLMAPs, studies are in progress to nullify the expression of all SLMAP isoforms during prenatal/perinatal cardiac development.The following are available online at www.mdpi.com/article/10.3390/cardiogenetics11040018/s1, Table S1: ECHO analysis on young and old SLMAP3-deficient hearts. Left ventricular functional analysis, measurements of wall sizes and intradiameter during diastole and systole in 5-week and 24-week old Wt, KD, and KO mice. ρ-value was calculated using ANOVA statistics analysis. n = 6. IVS; intraventricular septum, LVID; Left ventricular intradiameter, LVPW; Left ventricular posterior wall, EF; ejection fraction, FS; fractional shortening, LV mass; left ventricular mass, LV volume; left ventricular volume, d/s; diastole/systole, LV/BW; left ventricular mass/body weight.; Table S2: ECHO analysis on ISO-treated SLMAP3-KO hearts. Measurement of left ventricular mass and wall sizes during diastole and systole in Wt and KO mouse hearts before and after treatment with ISO. ρ-value was calculated using two-tailed student. n = 7. IVS; intraventricular septum, LVID; Left ventricular intradiameter, LVPW; Left ventricular posterior wall, EF; ejection fraction, FS; fractional shortening, LV mass; left ventricular mass, LV volume; left ventricular volume, d/s; diastole/systole.Conceptualization, T.R., M.S., J.B. and B.S.T.; methodology, T.R., M.S., J.B. and B.S.T.; data analysis, T.R., J.M. and B.S.T.; investigation, T.R., J.M., M.S. and J.B.; visualization: T.R. and B.S.T.; writing—original draft preparation, T.R.; Project administration: M.S. and B.S.T.; writing—review and editing, B.S.T.; supervision M.S. and B.S.T.; funding acquisition, B.S.T. All authors have read and agreed to the published version of the manuscript.This research was funded by the Canadian Institute of Health Research project grant, grant number 220996-151999, to B.S.T.Mice were handled in accordance with the guidelines set by Canadian Council on Animal Care, Guide to the Care and Use of Experimental Animals, 2 vols. (Ottawa, Ont.: CCAC, 1980–1993) and Animals for Research Act, R.S.O. 1990, c.A. 22. All animal protocols and procedures were approved by the Animal Care Committee of the University of Ottawa (Protocol #: CMM-1725 and CMM-1723, Renewed: December 2020).Not applicable.The data presented in this study are available on request from the corresponding author.We would like to thank Mona Nemer and her lab staff for providing the αMHC-MerCreMer animals, sharing breeding strategies and Rick Seymour for assisting our ECHO analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Breeding diagram to produce mice that contain floxed SLMAP alleles and cre. F0 generation indicates the hetero-flox-SLMAP (green/dark blue) and hetero-cre (red) mice. The hetero-floxed and hetero-cre mouse lines were maintained separately with Wt. The separated hetero-flox mice offspring were bred together to generate the homo-floxed mice (blue), labeled in the F1 generation. Additionally, the F1 generation indicates the production of knock down (KD) mice (dark red; heterofloxed and hetero-cre) after breeding a hetero-cre mouse with a hetero-floxed mouse. F2 generation highlights the SLMAP3-KO (orange; homofloxed and cre) mice generated by breeding a homo-floxed animal with a KD animal.Intron-exon junction of mouse SLMAP gene and its splice variants. SLMAP consists of 24 exons (black) and alternatively splices to generate numerous isoforms grouped as three isoforms, SLMAP1 (35 kDa), SLMAP2 (45 kDa) and SLMAP3 (80–95 kDa). The start codon (ATG) for SLMAP3, SLMAP2 and SLMAP1 are found at exon 2/3, 10 or 16, respectively. Alternative splicing of exons 11, 12, 13, 14 and 17 results in each isoform presenting unique variants. Exon 2 (green) codes for the FHA domain. Exons 6, 8, 9, 10, 18, 19, 20 and 21 (blue) code for the coiled-coil LZ domains. Mutually exclusive exons 23 (yellow) and 24 (red) code for transmembrane domain 1 (TM1) or transmembrane domain 2 (TM2).PCR genotyping detects floxed-SLMAP and αMHC-MerCreMer gene in mice. (A) Schematic representation of primers F2/R2 identifying loxP site 1 on flox-SLMAP gene and primers Cre-F/Cre-R identifying αMHC-MerCreMer gene. (B) PCR amplicons run on separate lanes on a 1% agarose gel labeled Wt, KD or KO identifying a wildtype, knocked-down or knockout mouse heart, respectively.Administration of tamoxifen cleaves exon 3 of floxed SLMAP gene by activating αMHC-MerCreMer in mouse hearts. (A) Schematic representing cleavage of exon 3 in a KD mouse after treatment with tamoxifen. (B) PCR genotyping of hearts treated with tamoxifen (TAM) using primers F2/R1. Wt lanes indicate unaltered SLMAP, which results in a 1400 bp band. KD lanes result in two bands being present, a cleaved SLMAP band (400 bp; grey arrow) and an unaltered SLMAP gene (1400 bp; black arrow).Protein and transcript expression of SLMAP isoforms in targeted mice. (A) Western blot with anti-SLMAP for protein expression in Wt, KD and KO hearts. Bar graphs represent normalized protein expression evaluated via densitometry analysis by using α-Tubulin as a loading control. n = 10. * ρ < 0.05, ρ value was calculated using a non-paired two-way t-test. (B) Relative intensity of protein band ratios of SLMAP2 and SLMAP1 normalized to SLMAP3 from western blots in Wt hearts. n = 3 * ρ < 0.05. (C) Transcript levels for SLMAP3 and total SLMAP in KD and KO hearts compared to Wt. Fold change was calculated using 18S-Ribosome as control. n = 9. ** ρ < 0.001, ρ value was calculated using ANOVA statistical analysis.Echocardiography and left ventricular function of SLMAP3-deficient hearts. (A) Representative short-axis m-mode images of Wt, KD and KO hearts at 5 weeks and 24 weeks post tamoxifen. (B) Bar graphs represent cardiac functional analysis via ejection fraction (%) and fractional shortening (%) of young and aged SLMAP3-deficient hearts. (C) The bar graph represents left ventricle mass to body weight ratio (LV/BW) in Wt, KD and KO mice. n = 6.Echocardiography and left ventricular function in ISO challenged Wt and SLMAP3-KO hearts. (A) Representative short-axis m-mode images of Wt and KO mice preISO and postISO. (B) Left ventricular functional analysis and changes in wall dimensions between pre-/postISO treated Wt and KO hearts. n = 7. * ρ < 0.05, ρ value was calculated using a paired two-way t-test.Histological analysis of ISO challenged Wt and SLMAP3-KO mouse hearts. Representative sectioning of Wt or KO hearts after one week of saline or ISO with Masson’s trichome stain to visualize the muscle and fibrosis. Scale bar = 500 µm × 12.5 magnification.STRIPAK complex and Hippo signaling in SLMAP3-KO hearts. Western blot of Wt and KO adult mouse heart lysates evaluating protein expression STRIPAK components (striatin, PP2A-A and PP2A-C) and phosphorylation of MST1 (T183) and YAP (S127). Bar graphs represent the expression of indicated proteins by normalizing with total protein visualized by Stain-Free™ as a loading control or to total MST1 or total YAP. n = 6.Nav1.5 expression and electrocardiography in SLMAP3-deficient hearts. (A) Western blot with anti-Nav1.5 in Wt, KD and KO adult mouse hearts lysates. Bar graphs represent quantification of Nav1.5 expression normalized to total protein loading visualized by Stain-Free™. (B) Representative electrograms of lead ii acquired by surface 6-lead ECG in Wt, KD and KO adult mice. n = 3. * ρ < 0.05, ρ value was calculated using ANOVA statistical analysis.List of antibodies used in this study. All antibodies used in this study are listed with the corresponding distributor, catalog number and dilution used for western blot.Electrocardiography of SLMAP3-deficient hearts. Parameters of electrocardiography in Wt, KD and KO adult mice (detailed in materials and methods). n = 3. ρ value was calculated using ANOVA statistical analysis.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+A 14-year-old boy with hypertrophic cardiomyopathy (HCM) diagnosed at the age of 1 year and with massive left ventricular hypertrophy suffered an episode of ventricular fibrillation during mild effort. He underwent a dual-chamber implantable cardioverter defibrillator (ICD) implantation. The defibrillation threshold testing (DFT) was ineffective. Subcutaneous multi-coli arrays tunneled into the left postero-lateral position and connected to the superior vena cava (SVC) port of the dual-chamber ICD were added to increase the myocardial mass involved in the defibrillation shock pathway. A new DFT was unsuccessful. The patient was transferred to our hospital for myectomy. An epicardial defibrillation patch was placed on the left ventricular lateral wall, but again, DFT testing was ineffective using the right ventricular (RV) coil to lateral patch as shock pathway. Another epicardial defibrillation patch was then placed on the inferior wall. In this case, DFT testing was effective with a defibrillation pathway between the two patches and the can. In November 2015, a high shock impedance alarm was recorded through remote monitoring, thus compromising the safety of the ICD shock pathway. The patient underwent the implant of a new trans-venous defibrillation coil lead in the azygos vein. After few months, the patient developed symptomatic severe aortic regurgitation and underwent an aortic valve replacement. During the operation, DFT testing was performed and was successful. Our case illustrates that azygous vein ICD lead implantation is efficacious in HCM with massive hypertrophy and high DFT, and prompts further studies to systematically investigate its efficacy in this particular subgroup of the HCM population.Hypertrophic cardiomyopathy (HCM) is the most frequent inherited heart muscle disease. The annual incidence of cardiovascular death, including sudden cardiac death (SCD), heart failure death and death due to thromboembolism, is around 1–2% in the adult population [1]. The implantable cardioverter defibrillator (ICD) has been proved efficacious in primary and secondary prevention of SCD [1]. We present a challenging case of a young patient with HCM and massive left ventricular hypertrophy (LVH) with high defibrillator threshold (DFT).A 14-year-old boy came to our medical attention for the first time in December 2008 following an episode of ventricular fibrillation (VF) during mild effort. He was previously diagnosed with HCM at the age of 1 year after a systolic murmur was detected. His growth was normal, but he developed massive LVH by the age of 13 years (maximal left ventricular wall thickness of 54 mm) and significant left ventricular outflow tract obstruction (LVOT) gradient of 50 mmHg. Patient was asymptomatic. Beta-blocker therapy was started (propranolol 20 mg three times a day). An ICD implantation was recommended but was declined by his parents. In December 2008, the patient suffered a cardiac arrest due to VF while climbing a flight of stairs. He was successfully resuscitated with external defibrillation and was transported to a local A&E department where he suffered an electric storm with multiple episodes of VF requiring orotracheal intubation. The patient was extubated after 24 hours, had no neurological complications, and underwent a dual-chamber ICD (Teligen, Boston Scientific, Inc., St Paul, MN, USA) implantation with a dual-coil defibrillation ventricular lead. The DFT was ineffective, and the induced VF was externally defibrillated. Subcutaneous multi-coli arrays (Boston Scientific, Inc., St Paul, MN) tunneled into the left postero-lateral position and connected to the superior vena cava (SVC) port of dual-chamber ICD were added to increase the myocardial mass involved in the defibrillation shock pathway (Figure 1). A new DFT was unsuccessful.The patient was transferred to our hospital for myectomy. At the end of the operation, another DFT testing was performed. This time, both ICD shocks and external defibrillation were ineffective and only defibrillation with internal paddles directly connected to the heart was successful. An epicardial defibrillation patch (Medtronic, Inc., ST Anthony, MN, USA) was placed on the left ventricular lateral wall, but again DFT testing was ineffective using the right ventricular (RV) coil to lateral patch as shock pathway. Another epicardial defibrillation patch was then placed on the inferior wall (Figure 2 left panel). In this case, DFT testing was effective with a defibrillation pathway between the two patches and the can. The subcutaneous multi-coli arrays were removed.In September 2012, the left lateral epicardial patch showed high impedance (>200 ohm), indicating it was damaged. The inferior patch–can shock pathway was then programmed.In November 2015, a high shock impedance alarm was recorded through remote monitoring, thus compromising the safety of the ICD shock pathway.Several options were considered. The first one was surgical removal and replacement of the broken epicardial patches, but this was considered too dangerous due to possible strong adhesions of the patches with the underlying myocardium, with consequent risk of myocardial rupture (Figure 2 right panel).The second option was performing a new DFT testing with the old endocardial leads, but it was considered unsafe, as it had already been ineffective during myectomy in 2008.The third option was the implantation of a subcutaneous ICD, but unfortunately, screening ECG testing failed. The last available option was to implant a defibrillation coil lead in the azygos vein. The patient underwent the implant of a new trans-venous defibrillation coil lead (Medtronic, Inc. ST Anthony, MN) in the azygos vein (Figure 3). In order to maximize the defibrillation success, a new ICD (Inventra 7 VR-T, Biotronik, Inc., Lake Oswego, OR, USA) was implanted with a high nominal energy of 45 J (40.5 J delivered) from the first shock. After this implant, the connection of the high energy included the distal coil of the first endocardial lead and the new coil in azygos.A new DFT testing was not performed immediately after the new implant. It was deemed too risky for the patient (last unsuccessful DFT required internal paddles shock); also considered were the controversy regarding the predictor value of DFT [2], the long arrhythmias event-free period after myectomy for the patient, the increased safety margin from the new device with the highest possible shock energy and the possibility to program different shock wave forms, including Bifasic-2 modality. In pre-market tests, bifasic-2 wave decreased DFT thanks to time-controlled waveform at 2 ms of the second tilt in the bifasic shock wave.ICD was programmed with a VF zone above 200 bpm and a therapy including eight shocks at 45 J, automatic alternating polarity and a Bifasic-2 wave form. The shock pathway was formed by the old endocardial distal coil; the new floating coil was placed in the azygos vein and the can.After few months, the patient developed symptomatic severe aortic regurgitation and underwent an aortic valve replacement. During the operation, DFT testing was planned with the back-up of open-chest defibrillation with internal paddles. VF induction was performed using a 1-J T-wave shock delivered after eight synchronized pacing pulses. VF detection zone was programmed above 188 bpm with a 6/8 counter criterion. Therapy included 45 J with normal polarity and Bifasic-2 waveform from the very first shock with the possibility to automatically invert the polarity in case of ineffective shock. VF was immediately induced, correctly recognized by the device and defibrillated with the first 45 J shock. The last ICD check performed in September 2020 showed stable parameters and no arrhythmic events.Our case illustrates that azygous vein ICD lead implantation is efficacious in HCM with massive hypertrophy and high DFT. ICD is highly effective in restoring normal rhythm in patients with HCM suffering life-threatening ventricular arrhythmias, with intervention rates of 10.6% per year for secondary prevention and 3.6% per year for primary prevention [3]. It has been shown that HCM patients have higher DFT and that DFT is linearly correlated with increasing left ventricular wall thickness [4], although these results have not been replicated in other studies [5]. Nevertheless, the clinical utility of DFT in predicting ICD efficacy has been questioned [2] in the general HCM population, but it is still advocated in the young and in patients with massive LVH [2]. In our HCM patient with massive LVH and previous episode of VF, high DFT required surgical implantation of two epicardial patches. When the latter broke, azygous vein ICD lead implantation allowed enough coverage of critical defibrillation mass with anterior–posterior direction to achieve a safe DFT. The azygos vein originates at the level of first/second lumbar vertebrae from tributaries of the renal and lumbar veins and runs along the rightward side of the vertebral column, joining the posterior aspect of the superior vena cava slightly superior to the upper border of the right main stem bronchus. The azygos vein has the great anatomical advantage of being posterior to the heart, and studies have demonstrated a DFT decrease with a combination shock pathway that includes a coil in the azygos vein [6,7]. Some case reports and case series [8,9,10] highlight the role of implantation of a defibrillator coil in the azygous vein as a valid strategy to reduce defibrillator threshold. This approach is reported to be feasible and safe [6]. Adding an azygos coil is successful in lowering the DFT in most cases [9]. This approach may be particularly useful with a right-side-placed ICD. It can be used alone or in combination with other external defibrillation devices such as subcutaneous arrays [10].We presented a case of a young patient with HCM and massive left ventricular hypertrophy in whom the implantation of a defibrillator coil in the azygous vein was efficacious in reducing the DFT. Our case prompts further studies to systematically investigate its efficacy in this particular subgroup of the HCM population.This research received no external funding.Ethical review and approval were waived for this study due to the absence of recognizable external morphological aspects of the patient reported in the case.Patient consent was waived due to the absence of recognizable external morphological aspects of the patient reported in the case.The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reason.The authors declare no conflict of interest.Anterior–posterior (AP) chest X ray showing dual-chamber ICD with a dual-coil defibrillation ventricular lead and subcutaneous multi-coli arrays tunneled into the left postero-lateral position and connected to the SVC port of ICD.Left panel. AP chest X-ray showing dual-chamber ICD connected to two epicardial patches (lateral and inferior). The defibrillation pathway is between the two patches and the can (red arrows). Figure 2 right panel. CT scan shows strong adhesion of the patches with the underlying myocardium.Lateral (left panel) and AP (right panel) chest X-ray after azygous vein ICD lead implantation. The shock pathway was formed by the old endocardial distal coil; the new floating coil was placed in the azygos vein and the can.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Background: Left ventricular noncompaction (LVNC) is a genetically determined cardiomyopathy that occurs following a disruption of endomyocardial morphogenesis. The purpose of this study was to identify the clinical characteristics and genetic profile of children with LVNC. Methods: From February 2008 to July 2020, a total of 32 children (median 11.5 years) with LVNC were prospectively enrolled and followed up for a median of 4.02 years. Diagnosis was made based on characteristic features of LVNC in echocardiography and cardiovascular magnetic resonance (CMR). Patients’ clinical symptoms, family history, ECG, Holter ECG, and genetic tests were also evaluated. Results: The most common presenting symptom was heart failure (31% of children). ECG abnormalities were noted in 56% of patients. The most prominent features were ventricular arrhythmias, sinus bradycardia, and paroxysmal third-degree atrioventricular block. Most of the patients (94%) met the criteria for LVNC and CMR confirmed this diagnosis in 82% of cases. The molecular etiology was found in 53% of children. Conclusion: Although heart failure and arrhythmias were very frequent in our study group, thromboembolic events and genetic syndromes were rare. For the accurate and reliable assessment of children with LVNC, it is necessary to get to know their family history and detailed clinical profile.Left ventricular noncompaction cardiomyopathy (LVNC) is a genetically determined myocardial disease, the third most common cardiomyopathy in the pediatric population (after dilated and hypertrophic cardiomyopathy) [1]. Molecular studies confirm the genetic basis in approximately 40% of LVNC patients [2]. The clinical presentation of LVNC varies widely, ranging from asymptomatic cases to severe heart failure, arrhythmias, thromboembolic complications, and sudden cardiac death [3,4]. Heart failure is the most common clinical symptom, occurring in approximately 55% of patients with LVNC [5]. The spectrum of arrhythmias is very wide, with the most common one being ventricular arrhythmia. Left ventricular (LV) systolic dysfunction, arrhythmias, and blood stasis in the recesses of the myocardium predispose to thromboembolic events [6,7,8]. The basic diagnostic examination in LVNC is echocardiography. While LVNC is generally diagnosed based on published echocardiographic criteria [9], cardiovascular magnetic resonance (CMR) can also be useful in suspected cases of LVNC and may help determine the prognosis by detecting fibrosis [10,11]. Therapeutic management includes the treatment of heart failure, cardiac arrhythmias and thromboembolic complications.This study sought to determine the clinical features and genetic causes of LVNC in children diagnosed with this disease at a single institution.An assessment of clinical presentation in a homogeneous group of pediatric patients with an isolated form of LVNC has not, to date, been conducted.Furthermore, there are still no uniform recommendations for the prevention of thromboembolic events in children with LVNC.So far, no diagnostic criteria in imaging studies developed for the pediatric population with LVNC have been established. Is echocardiography sufficient to diagnose childhood LVNC?Study patients. From March 2008 to July 2020 pediatric patients with diagnosed LVNC hospitalized in the Department of Cardiology of the Children’s Memorial Health Institute were prospectively enrolled. The criteria for inclusion in the study were age < 18 years at the time of diagnosis and echocardiographic evidence of isolated LVNC defined as: 1. The presence of a two-layer structure with a compacted (C) and noncompacted (NC) endocardial layer of the trabecular meshwork with deep endomyocardial spaces. 2. A maximal end-systolic ratio of NC/C layers of >2.3. Color Doppler evidence of deep perfused intertrabecular recesses [9].The Institutional Ethics Committee approved this study. Informed consent was obtained from all individual participants included in the study.Data collection. Patients’ demographics, clinical symptoms, family history of cardiomyopathies and sudden cardiac death (SCD), arrhythmias in family members, and treatment strategy, as well as the echocardiography, 12-lead resting ECG, 24-h Holter ECG, cardiopulmonary exercise test (CPET), and CMR results, were collected. In all children, the NYHA/Ross functional class and clinical symptoms such as chest pain, palpitations, syncope, pre-syncope, and thromboembolic events were evaluated. Serum NT-proBNP levels were assessed in all patients. Each patient underwent genetic blood tests for evaluation of the molecular basis of the disease. DNA from the peripheral blood was extracted automatically using the MagCore Nucleic acid Extractor HF16Plus. Next-generation sequencing (targeted panel of 164 cardiomyopathy-associated genes) was applied in all cases. The sequencing was performed on the HiSeq 1500 platform (Illumina). The discovered variants were analyzed and prioritized considering: 1. The minor allele frequency determined with the gnomAD, ExAC, 10UK, and in-house databases, containing data from more than 5000 individuals. 2. The pathogenicity of the variants, using up to 13 different in silico prediction algorithms (CADD, SIFT, MutationTaster, PolyPhen2 (HDIV, HVAR), MutationAssessor, LRT, MetaSVM, MetaLR, FATHMM, MaxEnt, NNSPLICE and SSF). 3. Phenotypic descriptions in OMIM, GTR, HGMD, ClinVar, Varsome, and Pubmed. Sanger sequencing was used for validation of the most interesting candidate variants and for parental segregation analysis when available.Two-dimensional, Doppler, and M-mode echocardiography were performed at rest using standard methods. Echocardiographic measurements included the LV end-diastolic dimension, the left ventricular ejection fraction (LVEF) (measured according to the Simpson method), and LV noncompaction features (measured via the calculation of the NC/C ratio and visualization of the recess filling between the trabeculae with blood flowing in from LV using the color Doppler method as reported in the literature) [9,12]. The 24-h Holter monitors and 12-lead resting ECGs were also examined for evidence of supraventricular and ventricular arrhythmia, sinus node disease, sinus bradycardia, and atrioventricular conduction block. Cooperating patients underwent a cardiopulmonary exercise test with the assessment of peak oxygen uptake (VO2peak), respiratory exchange rate (RER), heart rate at maximum exercise, blood pressure response to exercise, and the presence of exercise-induced or exerted cardiac arrhythmias.CMR was performed using a 1.5-T scanner (Sonata and Avanto fit, Siemens, Germany). Cine images were acquired by the breath-hold, electrocardiographic-gated, segmented k-space steady-state free-precession technique using 25 phases per cardiac cycle. LGE images were obtained in the long-axis and short-axis imaging planes using a breath-hold segmented inversion recovery sequence implemented 10–15 min after intravenous administration of 0.1 mmol/kg of gadobutrol (Gadovist, Bayer, Berlin, Germany); gadodiamide (Omniscan, GE Healthcare, United Kingdom) was used instead of gadobutrol if the patient was under 2 years of age. The criteria for the diagnosis of LVNC in the CMR study based on the assessment of the ratio of the NC/C layer of the LV myocardium being > 2.3 in the end-diastolic phase were used in accordance with the recommendations of the literature [10].Baseline characteristics. A total of 32 patients with echocardiographic diagnosis of LVNC were recruited between February 2008 and July 2020, with the median age being 11.5 (6–15) years, and the proportion of males being 53%. Patients were followed prospectively for a median of 4.02 (IQR 0.48–10.14) years. Clinical and demographic characteristics of the patient population are presented in Table 1.Population characteristics. In the study group, 3% of patients were under 1 year of age, 31% were between 1 and 10 years of age, while as many as 66% were over 10 years of age. In family history, as many as 17 (53%) children had cardiomyopathies in first-degree relatives (LVNC in 31% of children; HCM in 6% of patients, and DCM in 6% of patients). Family history of cardiac arrhythmias was observed in 7 (22%) patients, while sudden cardiac deaths occurred in the families of 3 (9.3%) children.Clinical presentation. NYHA/Ross functional class evaluation demonstrated grade II in the majority of patients (77%); 3% had grade IV, while 22% of children had grade I. The most common presenting symptoms were syncope (9% of patients), pre-syncope (9%), chest pain (9%), and palpitations (6%). Two patients had evidence of dysmorphic features. One child was diagnosed with Barth syndrome and one with congenital microphthalmia. Serum NT-proBNP level was increased in five (16%) patients (from 349.40 to 27,057.00 pg/mL, with the reference value of up to 320 pg/mL).Electrocardiographic findings. ECG abnormalities were noted in 18 (56%) children. The most prominent feature was ST-T changes, mainly T-wave inversion in 38% of patients, sinus bradycardia in 22%, and electrocardiographic features suggestive of LV overload in 13% of patients. Wolff–Parkinson–White syndrome (WPW) was noticed in 3% of patients (Figure 1).In 24-h ECG Holter monitoring, the most prominent features were premature ventricular and atrial contractions in 25% and 16% of patients, respectively. Other findings, including sinus bradycardia, sick sinus syndrome, paroxysmal second- and third-degree atrioventricular block, and ventricular and supraventricular tachycardia, are presented in Table 2.Two-dimensional echocardiography. Most of the studied patients (94%) met the criteria for the diagnosis of LVNC. The LV myocardial NC/C ratio ranged from 2.06 to 5.14 (Figure 2). The use of color Doppler in the parasternal short-axis and apical four-chamber views improved the visualization of the trabeculations within the LV endocardium. LV function was reduced in 31% of patients (LVEF ranged from 40 to 55%).Cardiovascular magnetic resonance. CMR was performed in 29 patients (91%); in one child, the parents did not consent to the study, and in two patients, a permanent pacemaker was implanted. CMR accurately delineated LV morphology in 24 (82%) patients, with the NC/C myocardial ratio in those children ranging from 2.3 to 6.2 (Figure 3).Cardiopulmonary exercise test (CPET). CPET (ergospirometry) was performed in 10 (31%) patients. Peak oxygen absorption (peak VO2) ranged from 15.9 mL/kg/min to 43.5 mL/kg/min, and in two patients (6%), it was below 18 mL/kg/min. The respiratory exchange rate (RER) ranged from 0.84 to 1.29 and was above 1.05 in six patients (19%). The heart rate at maximum effort ranged from 105/min to 195/min. All 10 patients had normal blood pressure responses to exercise. There were no exercise-induced arrhythmias in any of the patients.Genetics. Pathogenic/likely pathogenic variants/rare variants of unknown significance in LVNC-associated genes were detected in 17 families (53%). Autosomal dominant defects were identified in the HCN4, MYH7, RBM20, and TTN genes. ACTC1, ACTN2, DES, EYA4, HCCS, KCNQ1, and PRDM16 defects were found in single families. Syndromic LVNC (Barth syndrome and syndromic microphthalmia) was associated with TAFAZZIN and HCCS gene dysfunction in two patients, respectively. Complex genotypes (KCNQ1 and TTN) were detected only once in our group of children. To date, segregation analysis was available in seven families, thus confirming the parental origin of identified variants in six cases and one de novo case.Medical therapy. Treatment of congestive heart failure consisted of angiotensin-converting-enzyme inhibitors, spironolactone, beta-blockers, and diuretics in 19 patients (59%). Antithrombotic prophylaxis with acetylsalicylic acid was used in three (9%) patients with LVNC and significantly reduced LV systolic function. Salbutamol was used to treat sinus bradycardia in four patients (13%), and a pacemaker was implanted in two patients (6%), due to symptomatic sinus bradycardia and complete atrioventricular block, respectively. RF ablation was performed in one patient due to supraventricular tachycardia. In one child (3%), the LV mechanical support device (LVAD) was implanted; this patient died while being on the active list for heart transplantation.Today, due to advanced diagnostic tools, LVNC is a genetically determined cardiomyopathy that is being diagnosed with increasing frequency. The cause of the disease is thought to be a disruption of endomyocardial morphogenesis between the fifth and the eighth gestational week [12,13]. The variability in phenotypic expression and the lack of standard diagnostic criteria for the pediatric population make it difficult to estimate the frequency of this form of cardiomyopathy in children. In the opinion of many authors, the presence of only morphological criteria characterized by a distinct two-layer structure of the LV muscle is insufficient for the diagnosis of LVNC, and a thorough clinical evaluation of the patient is also necessary [3,12,14]. In the literature, many studies on children with LVNC [3,15] concern a heterogeneous group of patients, including children with congenital heart disease. In our study, we present the clinical and diagnostic features of LVNC in children with an isolated form of this disease.It should be emphasized that in the family history of our patients, as in the Ichida report [16], cardiomyopathies in family members occurred in 44% of children, including LVNC in as many as 31% of the studied children. Other authors also emphasize that the incidence of familial LVNC cases is higher in the pediatric population than in adults [5].It should be noted that the main reason for referring children to a cardiology center was a family history of cardiomyopathy, sinus bradycardia, cardiac arrhythmias, and sudden cardiac deaths in family members. Of 32 patients, 13 (41%) children were referred for cardiac screening due to their family history. Cardiac arrhythmias were the first symptom of the disease in 10 (31%) children. In one (3%) infant, the initial presentation of the disease was symptoms of heart failure, which were the reason for echocardiography. The patient with Barth’s syndrome was referred to perform, for the first time, cardiological diagnostics. In four (12.5%) children, postnatal echocardiography revealed left ventricular hypertrabeculation and these patients were referred to a cardiology center for comprehensive cardiological examinations. Heart murmur was the reason why three (9%) patients were referred for echocardiography with LVNC diagnosis.It is noteworthy that sudden cardiac death occurred in the families of three children. In one child, two SCDs occurred in the family—in the father at 32 years of age with previously diagnosed HCM, and in his uncle at 42 (he was not examined before death, and no postmortem examination was performed). In the second family, SCD occurred in a 2-year-old sister of a studied patient, who had been diagnosed with LVNC before her death. In the third family, SCD occurred in the patient’s aunt’s one-month-old baby; no postmortem examination was performed.Another interesting point is the association of LVNC with dysmorphic features. In our study, the genetic syndrome was diagnosed only in two children (6%), much less frequently than in the studies of the above-cited authors, where the frequency of coexistence of genetic syndromes was estimated at 14%, and even 37% and 66% [12,16,17]. In our group, one child was diagnosed with Barth syndrome with a confirmed mutation in the TAFAZZIN gene. The second patient had congenital microphthalmia and a pathogenic HCCS variant. It is extremely important that in our group of patients, as in the study by Dong X et al. [2], the molecular basis of LVNC was confirmed in 53% of cases. Corresponding with the existing literature [18], autosomal dominant inherited variants were more frequent in our cohort, with HCN4 and MYH7, RBM20, and TTN defects accounting for 18% and 12% of the cases, respectively (data not shown). Segregation analyses confirmed the origin of the mutation from an affected parent.The clinical course of LVNC in children is very diverse and may include symptoms of heart failure, cardiac arrhythmias, and thromboembolic events. According to the literature data, the most common symptom of LVNC in children is heart failure, the frequency of which is estimated at 35 to 91%, caused by LV systolic dysfunction [3,19,20,21]. Systolic dysfunction and a reduction in LVEF in patients with LVNC are associated with hypoperfusion secondary to subendocardial microvascular abnormalities and dyssynchronization between the integrated and noncompacted layers. Another possible explanation is dependence of the noncompacted area on aerobic oxidation and its sensitivity to hypoxia and the toxic effects of catecholamines [22]. In our group, symptoms of heart failure, including a reduction in the LVEF, were found in 31% of the studied children, with 16% of them having an increased serum NT-proBNP levels.Most pediatric patients with LVNC had abnormalities in resting ECG, the most frequently reported changes being intraventricular conduction disturbances, including right bundle branch block, atrioventricular block, repolarization abnormalities, and LV overload features [3,22]. As in the literature reports [12,16,23], changes in the resting ECG occurred in 56% of our patients. Most often, that is, in as many as 38% of children, repolarization abnormalities in the form of ST-T changes were found, and 13% of patients had electrocardiographic features of LV overload. Literature reports indicate that severe sinus bradycardia and sinus node dysfunction are common complications in patients with LVNC [24,25]. Our observations confirm the above reports, as sinus bradycardia was diagnosed in as many as 22% of the studied children, and sick sinus syndrome in 6%. In one of them, with symptoms of sinus bradycardia, a permanent pacemaker was implanted. The fact of frequent short episodes of paroxysmal third-degree atrioventricular block (13% of patients) in our study deserves particular emphasis. One of those patients met indications for permanent pacemaker implantation. The results of published studies show a variable frequency of ventricular arrhythmias in patients with LVNC (from 6 to 60% of cases) [5,26]. It should be emphasized that in our group of patients, arrhythmias were a common symptom: as many as 25% of children had premature ventricular beats, 16% had premature supraventricular beats, 9% had short episodes of ventricular tachycardia, and 3% had supraventricular tachycardia (in this case, successfully treated with RF ablation). The literature describes cases of coexistence of LVNC and WPW syndrome. The authors emphasize that this relationship is more common in children than in adults, and its frequency was estimated at 8–14% [27]. In our study group, LVNC and WPW syndrome coexisted less often; cooccurrence was found in only 3% of patients. Brescia et al. [28] described a case of a patient with features of WPW syndrome on a resting ECG, but no accessory atrioventricular (AV) conduction pathway was found in electrophysiology studies (EPS). Similarly, in our study, one patient (3%) had electrocardiographic features of WPW syndrome on a resting ECG, while in the EPS examination, no true accessory AV conduction pathway was found. It should be noted that this patient had episodes of atrioventricular tachycardia, while echocardiography showed normal LV systolic function. In the second patient, a single episode of paroxysmal WPW syndrome was recorded in a 24-h Holter ECG, while this patient had a completely normal resting ECG recording, without WPW syndrome features. This patient was not tested for EPS. In paper by Howard TS et al. [29], they found, however, that LVNC and true WPW syndrome coexist in most cases, which worsens the prognosis in these patients. In the opinion of these authors, the presence of an accessory atrioventricular conduction pathway in patients with LVNC increases the risk of arrhythmias and sudden cardiac death, and also contributes to the development of left ventricular dyssynchrony, which may lead to a faster development of LV systolic dysfunction. It was also emphasized that RF ablation improved the systolic function of the LV [29,30], which further confirms the negative impact of the presence of WPW in patients with LVNC [29].Systemic emboli are another important complication in patients with LVNC. Although their prevalence was as high as 38% two decades ago, a recent study reported it to be as low as 0–2%, and it was found to be 4–7% in another report [3,14]. There are no established recommendations for the use of antithrombotic prevention in children with LVNC, and the authors’ opinions are divided [12,22]. None of our patients developed systemic emboli. Antithrombotic prophylaxis with aspirin was used in only four (13%) children with significantly reduced LVEF.The first-line and standard procedure for diagnosing LVNC is 2-D Doppler echocardiography according to the criteria published in the literature [9,12,31,32]. CMR is increasingly used in the diagnostics of heart muscle diseases in the pediatric population [10,11,33,34]. It enables an accurate visualization of the heart muscle and a very reliable assessment of hemodynamic changes. It should be emphasized that, in our study, 94% of patients met the LVNC echocardiographic criteria, while the CMR study confirmed the diagnosis of the disease in 82% of children. In the remaining cases, in echocardiography and CMR, the ratio of the NC/C layer of the left ventricular muscle was borderline for the diagnosis of LVNC. Our previous research results confirm that there was also a good correlation of echocardiography with CMR in the group of patients with hypertrophic cardiomyopathy [35]. The CPET is more and more frequently performed in the analysis of hemodynamic changes and in the assessment of exercise capacity in adult patients with cardiovascular diseases. It is still a rarely performed study in the pediatric population, especially with myocardial diseases. The literature shows that peak VO2 above 18 mL/kg/min is an important prognostic factor and is associated with a good 2-year prognosis, while peak VO2 below 10 mL/kg/min is associated with 36% annual mortality [36]. In our study, CEPT was performed in as little as 31% of patients, 6% of whom showed a low peak VO2, i.e., below 18 mL/kg/min. In the remaining 22 patients, CPET was not performed due to their age being below 10 years, the lack of appropriate equipment for this age group (n = 11 children), the psychological aspect and fear of wearing a face mask in 10 patients, and a significant spine defect in one child, which was a contraindication to the test.Treatment of patients with LVNC should be directed towards three most important clinical manifestations: congestive heart failure, arrhythmias, and systemic embolic events. In our study, standard treatment of heart failure with preload and afterload reducers was started in all 31% of patients with systolic LV dysfunction and reduced LVEF. Moreover, patients with decreased LV systolic functions received a beta-blocker (bisoprolol), which has been shown to improve LV and neurohormonal dysfunction in children. Adwani et al. [37] described the case of the first successful heart transplantation in a patient with Barth syndrome. Since this report, heart transplantation has been recognized as an effective treatment for end-stage heart failure in patients with Barth syndrome. In our study, one patient diagnosed with Barth syndrome and severe heart failure (NYHA class IV) was implanted with a left ventricular assist device (LVAD) while awaiting a heart transplant. The patient died while on the waiting list for a heart transplant.The results of the studies published in the literature, as well as the results of the authors’ own research, indicate that LVNC is a myocardial disease with a varied clinical profile as well as a natural history that is not, at present, fully understood, which prompts the continuation of clinical trials involving higher numbers of patients.A diagnosis of LVNC in children is more likely in the context of a family history of cardiomyopathy.The phenotype of familial LVNC in childhood is varied and includes severe cardiac symptoms, suggesting that clinical screening should commence at a younger age.LVNC may be missed or overdiagnosed if echocardiography is the only imaging modality performed in a cardiac evaluation.Genetic evaluation (testing and counseling) is recommended in each patient with isolated or syndromic LVNC.Large-scale, multi-center, collaborative approaches to clinical and genetic evaluation in childhood LVNC are needed to develop robust standards of diagnostic and therapeutic management in this group of patients.The limitations of this study are mainly inherited by its design. The study involved single-center research with a relatively small sample size. Needless to say, the results of this study need to be confirmed in large-scale, multicenter, longitudinal studies.Although heart failure and arrhythmias were very frequent in our study group, thromboembolic events and genetic syndromes were rare.Echocardiographic examination is the gold standard for the diagnosis of LVNC. However, cardiac CMR is recommended to confirm the diagnosis, especially in uncertain cases.Our results indicate that CMR has a good correlation with echocardiography and a high sensitivity and specificity in detecting non-compacted segments.For the accurate and reliable assessment of children with LVNC, it is necessary to get to know their family history and detailed clinical profile.The high genetic yield resulted in the explanation of molecular etiology in over half (53%) of the studied children.Identifying the genetic cause allows for risk stratification and may help in the clinical management and counseling of patients and their relatives.Conceptualization: L.Z., A.P. and D.P.-A.; Methodology: L.Z., A.P., A.M.-R., D.P.-A., E.C., Ł.M. and K.B.; Validation: L.Z., D.P.-A. and E.C.; Formal Analysis: L.Z., A.P., A.M.-R., D.P.-A. and E.C.; Investigation: A.P., L.Z., A.M.-R., D.P.-A., E.C., K.B. and Ł.M.; Resources: L.Z., A.P., D.P.-A. and Ł.M.; Data Curation, A.P., L.Z., D.P.-A. and E.C.; Writing—Original Draft Preparation, A.P., L.Z., D.P.-A., A.M.-R. and Ł.M.; Writing—Review and Editing, L.Z., A.P. and D.P.-A.; Supervision, L.Z. All authors have read and agreed to the published version of the manuscript.This work was partially founded by The Children’s Memorial Health Institute (statutory grant no. S177/2018).The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of The Children’s Memorial Health Institute (protocol code 45/KBE/2018 and date of approval 24/Oct/2018).Informed consent was obtained from all subjects and their parents involved in the study.The data presented in this study are available on request from the corresponding author.The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.Twelve-lead ECG recording showing the features of the Wolff–Parkinson–White syndrome in a patient with LVNC.The 2D transthoracic echocardiography in a patient with LVNC. (A) Apical four-chamber projection: left ventricular noncompaction with deep recesses. (B) Parasternal short axis projection: the ratio of the left ventricular noncompacted to compacted layer (3:1).Cardiovascular magnetic resonance study in a patient with LVNC-short axis projection: noncompacted left ventricular layer.Patient characteristics in the whole left ventricular noncompaction cardiomyopathy (LVNC) cohort.HF—heart failure; SCD—sudden cardiac death; CTR—cardiothoracic ratio; NC/C—noncompaction to compaction layer ratio; EF—ejection fraction; RBBB—right bundle branch block; LBBB—left bundle branch block; EPS—electrophysiology study; ablation RF—radiofrequency ablation; LVAD—left ventricular assist device; LVNC—left ventricular noncompaction cardiomyopathy; HCM—hypertrophic cardiomyopathy; DCM—dilated cardiomyopathy; WPW—Wolff–Parkinson–White syndrome; AVNRT—atrioventricular nodal reentry tachycardia.Heart rhythm and conduction disturbances in the studied group of children.WPW—Wolff–Parkinson–White syndrome; a-v—atrioventricular; VT—ventricular tachycardia.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+ABC transporters are a large family of membrane proteins that transport chemically diverse substrates across the cell membrane. Disruption of transport mechanisms mediated by ABC transporters causes the development of various diseases, including atherosclerosis. Methods: A bioinformatic analysis of a dataset from Gene Expression Omnibus (GEO) was performed. A GEO dataset containing data on gene expression levels in samples of atherosclerotic lesions and control arteries without atherosclerotic lesions from carotid, femoral, and infrapopliteal arteries was used for analysis. To evaluate differentially expressed genes, a bioinformatic analysis was performed in comparison groups using the limma package in R (v. 4.0.2) and the GEO2R and Phantasus tools (v. 1.11.0). Results: The obtained data indicate the differential expression of many ABC transporters belonging to different subfamilies. The differential expressions of ABC transporter genes involved in lipid transport, mechanisms of multidrug resistance, and mechanisms of ion exchange are shown. Differences in the expression of transporters in tissue samples from different arteries are established. Conclusions: The expression of ABC transporter genes demonstrates differences in atherosclerotic samples and normal arteries, which may indicate the involvement of transporters in the pathogenesis of atherosclerosis.Atherosclerosis is an important problem for modern mankind [1]. Its medical and social importance is steadily increasing. High levels of prevalence, disability, and mortality from diseases associated with atherosclerosis carry a heavy economic burden for both patients and their families as well as for public health systems [2].Studies of the mechanisms of atherosclerosis development are particularly relevant, since early detection and early modification of disturbed processes can be an effective tool for preventing its complications. The results of numerous studies have improved our understanding of the pathogenesis of atherosclerosis. Disruption of cellular transport processes mediated by ABC transporters is known to contribute significantly to the development and progression of atherosclerosis at different stages [3].ATP-binding cassette (ABC) transporters are a large family of proteins that are involved in the movement of a wide range of substrates across cell membranes [4]. In humans, 48 ABC transporter proteins are known, which are divided into 7 subfamilies based on structural characteristics: ABCA–ABCG [5]. Currently, the role of several representatives of ABC transporters in the pathogenesis of atherosclerosis is well described [6]. The roles of the ABCA and ABCG subfamilies are best known due to the involvement of their members in lipid transport, disorders of which are an important part of the pathogenesis of atherosclerosis [4,7]. The significance of lipid metabolism disorders in the development of atherosclerosis is now sufficiently understood and supported by the results of numerous studies. Lipid accumulation in the vascular wall is considered to be one of the key stages of atherogenesis. Macrophages play an active role in this process, which is associated with their involvement in the uptake of modified lipoproteins in the arterial wall, the production of inflammatory mediators, and the secretion of metalloproteinases that contribute to plaque instability. Disruption of cholesterol homeostasis in macrophages and the accumulation of its esters in lipid droplets lead to the development of the so-called “foam cell” phenotype. At the same time, the reverse transport of cholesterol—its removal from the cells—provides protection of the arterial wall from unwanted lipid deposition. This important atheroprotective function is provided to a greater extent by ABCA1 and ABCG1 transporters. Other members of these subfamilies are also thought to be involved in providing lipid transport processes, but the biological role of many of them is currently unclear. It should be noted that cellular lipid transport can regulate many essential cellular functions. The lipid composition of plasma membranes is known to be complex, and its changes have a significant impact on both the biophysical properties of the membrane and the function of membrane proteins, including the assembly and function of inflammation-related signaling pathways. All the mechanisms that link inflammation to the development and progression of atherosclerosis are still largely unknown, but the role of cross-linking inflammation with impaired lipid metabolism and the role of ABC transporters in these processes are of great clinical interest.While members of the ABCB and ABCC subfamilies are well known for their role in the development of multidrug resistance, there is increasing evidence of their involvement in the pathogenesis of other diseases, including atherosclerosis.This multifaceted role also involves lipid transport, the functioning of ion channels, and the export of exogenous substances, which may be important in the pathogenesis of atherosclerosis.It should be noted that the biological functions of many ABC transporters are still the subject of study. In this regard, the study of their role in the pathogenesis of atherosclerosis may expand our understanding of atherogenesis.Thus, the relevance of the problem of atherosclerosis and the need for a better understanding of its pathogenesis, including the mechanisms that are associated with the disruption of transport processes in cells, determined the purpose of the present study, which is to analyze the gene expression patterns of human ABC transporters in atherosclerosis using bioinformatics analysis methods.For the analysis, data from the publicly available set GSE100927, obtained from the Gene Expression Omnibus (GEO), National Center for Biotechnological Information (NCBI), were used. The GSE100927 set contained data on gene expression in atherosclerotic lesions and control arteries without atherosclerotic lesions from the carotid, femoral, and infra-popliteal arteries from patients undergoing carotid, femoral, or infrapopliteal endarterectomy [8]. The data were obtained using the GPL17077 Agilent—039494 SurePrint G3 Human GE v2 8x60K Microchip 039381 (Probe Name version) platform. Data normalization was carried out using locally weighted scattered plot smoother analysis (LOWESS).The following information was extracted for the dataset: the platform, the number of samples obtained from patients with atherosclerosis and healthy individuals, the location in the vascular bed from which the samples were obtained (carotid, femoral, and infrapopliteal arteries), and pre-processed gene expression data. To analyze the data on gene expression, comparison groups were formed: samples obtained from patients with atherosclerosis of various localizations and a control group of samples from healthy individuals.The set included available data on gene expression in 29 samples of atherosclerotic lesions of the carotid arteries, 26 samples of atherosclerotic lesions of the femoral, and 14 samples of atherosclerotic lesions of the infrapopliteal arteries, as well as samples from control arteries without atherosclerotic lesions from 12 carotid, 12 femoral, and 11 infrapopliteal arteries.The analysis of differential gene expression in the comparison groups was carried out using GEO2R, Phantasus (v. 1.11.0), and the limma package in R (v. 4.0.2) [9,10]. Data on the differential expression of genes in the comparison groups for each set, including p value, logFC, were obtained. The statistical significance level for multiple comparisons was corrected using the Benjamini–Hochberg algorithm (FDR—false discovery rate; adj. P.Value). All p values satisfying the condition < 0.05 at FDR ≤ 5% were considered statistically significant. Visualization of gene expression levels in comparison groups is presented as box diagrams, which demonstrate the minimum value (the lower part of the vertical line), the first–third quartile (box), the median (the horizontal line inside the box), and the maximum value (the upper part of the vertical line) of the data distribution.The conducted bioinformatic analysis showed differences in the expression of ABC transporter genes in each subfamily (Table 1 and Table 2).In the ABCA subfamily, the most differentially expressed gene with upregulation in atherosclerotic samples is ABCA7 (Figure 1). The ABCA7 transporter is extensively involved in lipid transport, performing phospholipid translocation from the cytoplasm to the extracellular sheet of the plasma membrane. It also transports phosphatidylserine and cholesterol and plays a role in macrophage-mediated phagocytosis. ABCA7 demonstrates 49% amino acid sequence similarity with another member of the subfamily, ABCA1, whose differential expression was detected in datasets from the carotid artery. At the same time, ABCA8, which is also involved in lipid transport by exporting cholesterol, was found to be downregulated. ABCA10, which may play a role in lipid homeostasis and lipid transport in macrophages, was also shown to be downregulated. ABCA10 demonstrates high amino acid sequence homology with ABCA6, ABCA8, and ABCA9, known members of the ABCA6-like transporters subgroup. At the same time, ABCA6 itself also showed reduced expression in atherosclerosis. However, its function is currently largely unknown.Among the members of the ABCB subfamily, the ABCB1 transporter gene, a key participant in the mechanism of multidrug resistance due to its ability to efflux xenobiotics, was found to be upregulated in atherosclerotic samples (Figure 2). ABCB1 is well known to clinicians and researchers for its role in the pharmacokinetics of many drugs with different chemical structures. Another representative, ABCB5, also responsible for reducing drug accumulation in cells and associated with multidrug resistance, also demonstrated upregulation in atherosclerotic samples.In the ABCC subfamily, increased expression of the ABCC5, ABCC3, and ABCC6 genes was found in atherosclerotic samples (Figure 3). The ABCC5 transporter acts as a multi-specific organic anion pump that can transport nucleotide analogues. ABCC10, which is known for its role in the active release of physiological compounds such as leukotriene C4 (LTC4), as well as xenobiotics from cells, also showed increased expression. In this regard, this transporter also mediates multidrug resistance. ABCC3 provides active transport across cell membranes of various substrates, including many drugs and toxic and endogenous compounds, such as LTC4. ABCC6 actively exports xenobiotics and physiological compounds from cells; for example, it mediates the transport of glutathione conjugates, such as LTC4.At the same time, the ABCC8, ABCC9, and ABCC2 genes showed reduced differential expression. ABCC8 and ABCC9 are subunits of the ATP-sensitive potassium channel, and ABCC2 provides active transport across cell membranes of various substrates, including many drugs and toxic and endogenous compounds. The transporter is considered part of the mechanism of multidrug resistance.Among members of the ABCG subfamily, an increased regulation was found in the ABCG4 gene (Figure 4). The ABCG4 transporter may be involved in macrophage lipid homeostasis. Among members of the ABCD subfamily, increased expression was found in ABCD4 and ABCD1 (carotid artery), while decreased expression was observed in ABCD3 and ABCD1 (infra-popliteal artery) (Figure 4). ABCD1 and ABCD3 participate in cellular lipid metabolism by importing fatty acyl-Koa (coenzyme A) into peroxisomes [11,12,13]. Moreover, ABCD3 moves branched, unsaturated, long-chain dicarboxylic acids [13].Among members of the ABCE subfamily, decreased expression was found in ABCE1 (Table 2). There is currently no conclusive evidence that ABCE1 is involved in any membrane transport functions. The ABCE1 protein, also known as the RNase L inhibitor (RLI), is a type of endoribonuclease Rnase L inhibitor that is involved in many biological processes, including the response to viral infection, cell proliferation, and evasion of apoptosis [14,15,16,17]. In addition, RNase L also plays an important role in protein translation [17,18,19] and multidrug resistance, and it is an important regulator of adipogenesis [20]. The functions of the ABCF members that have shown differential expression in atherogenesis are not known (Table 1 and Table 2). The gene products of members of the ABCF subfamily do not have a transmembrane domain and are not involved in membrane transport functions.We performed bioinformatic analysis of ABC transporter gene expression in atherosclerotic samples and control samples without atherosclerotic lesions from carotid, femoral, and infrapopliteal arteries from patients undergoing carotid, femoral, or infrapopliteal endarterectomy from the GEO dataset. Using online analysis tools, we generated comparison groups: atherosclerotic samples and control samples. Our study included an analysis of the differential expression of ABC transporter genes in each of the groups (from the carotid, femoral, and infra-popliteal arteries) and in general for atherosclerotic and control samples. Statistically significant differences in gene expression levels were taken into account, which were corrected using the Benjamini–Hochberg algorithm.The analysis showed the presence of differentially expressed genes of several ABC transporters, which may indicate their involvement in cellular transport disorders in the pathogenesis of atherosclerosis. ABC transporters were chosen for analysis because they are one of the key protein families that enable the movement of many substrates across the cell membrane.Given the current information on the pathogenesis of atherosclerosis, in which lipid metabolism disorders occur throughout its natural history, the data on the differential expression of ABC transporters involved in lipid transport seem interesting. It is believed that about 20 members of the large family of ABC transporters, which in humans includes 48 members, are involved in ensuring lipid homeostasis of cells by removing lipids from the cell or transporting them inside the cell and in its plasma membrane [11].Impairment of lipid homeostasis is one of the key links in the pathogenesis of atherosclerosis. These impairments occur at different levels and involve multiple cross-linkages with inflammation, mechanoreception, and endothelial cell mechanotransduction. Excessive lipid accumulation in macrophages and lipid deposition in the vascular wall are known links in the complex chain of processes associated with the development of atherosclerosis.Lipid transporters are found in all subfamilies of ABC transporters, and the greatest role in the pathogenesis of atherosclerosis is known for members of the ABCA and ABCG subfamilies [11]. The data indicate that the highest differential expression in the ABCA subfamily in all compared groups was shown by ABCA7. ABCA7 is the closest homologue of ABCA1 and demonstrates some similar functions, carrying out lipid transport, primarily of phospholipids. ABCA1 ensures the export of cholesterol from macrophages. This function is considered one of the key ones in ensuring cholesterol homeostasis [21,22]. Decreased functional activity of ABCA1 leads to cellular accumulation of cholesterol with the formation of “foam cells”. Thus, both transporters are characterized by atheroprotective effects, enabling the reverse transport of cholesterol from macrophages via its export with high-density lipoprotein (HDL). The findings that ABCA7 and ABCA1 expression was upregulated are interesting. Similar data were obtained in another study that found elevated levels of ABCA1 mRNA in atherosclerotic plaques, with higher levels of ABCA1 mRNA found in grade II and III plaques. These data allow ABCA1 mRNA levels to be considered as markers of plaque stability. The upregulation of ABCA1 mRNA may be associated with an increase in plaque oxysterol content [23]. Interestingly, ABCA1 protein levels in the study by Heang-Fang Liu et al. were markedly reduced in plaques compared with control tissues, reflecting a negative effect on cholesterol transport in atherosclerotic plaque [23]. These data indicate the need to take into account the expression data of the gene and transporter protein to assess its disorders in atherosclerosis.In the ABCG subfamily, the ABCG4 gene showed the highest differential expression. It is known that ABCG4 demonstrates a high level of homology with the protein ABCG1, which is considered to be one of the key participants in the reverse transport of cholesterol from macrophages, preventing their transformation into “foam cells” [21]. Given this homologue, it is assumed that ABCG1 is also involved in the export of cholesterol to HDL [6,24]. However, the predominant localization of the ABCG4 transporter is not related to blood vessels. ABCG4 is found in bone marrow megakaryocyte progenitors and protects cells from sterol overload, and its role in the progression of atherosclerosis remains uncertain [6].In the ABCB subfamily, the ABCB1 and ABCB5 genes showed the most differential expression. The ABCB1 transporter (MDR1, multiple drug resistence 1) is well known for its role in the development of the mechanism of multidrug resistance. Other studies have already shown increased ABCB1 mRNA in atherosclerosis samples [21,25,26,27]. A possible role of the transporter in atherosclerotic lesions seems interesting [25]. These data allow us to reinforce the notion that the function of ABCB1 may be considered not only from the standpoint of its involvement in the export of xenobiotics and mechanisms of multidrug resistance but also in the transport of endogenous substances in some diseases. Higher levels of cholesterol esters and MDR1 mRNA and lower levels of caveolin-1 mRNA were found in smooth muscle cells from atherosclerotic lesions in arteries [25]. It is assumed that ABCB1 is involved in the intracellular accumulation of cholesterol esters detected in atherosclerotic lesions [26,27]. This is due to the role of ABCB1 in the transport of free cholesterol from the plasma membrane to the endoplasmic reticulum for its subsequent esterification by Acyl-CoA:cholesterol acyltransferase (ACAT) [27]. The upregulated expression of ABCB1 may be due to the fact that it is sensitive to activation by liver X receptor (LXR) agonists and is also activated by differentiation from monocytes to macrophages [21,28].Increased expression of ABCB5 was also previously detected in atherosclerotic plaques in another study. The role of ABCB5 in atherogenesis is not clear, but it is known that the ABCB5 protein was detected in smooth muscle cells in normal samples and in large quantities in the necrotized plaque core [29].ABCC6 is currently regarded as a key participant in ectopic calcification [30,31]. Although its role in atherogenesis is not clear, ABCC6 dysfunction increases the risk of vascular calcification and myocardial infarction [6,31,32,33]. Calcification is associated with atherosclerosis and coronary heart disease [6,32,33]. In addition, ABCC6 gene mutations are associated with generalized arterial calcification in infancy [34,35].ABCC8 (sulfonylurea receptor 1 (SUR1)) and ABCC9 (sulfonylurea receptor 2 (SUR2)) are the subunits of ATP-sensitive potassium channels K+ channels [36,37]. Their role in the pathogenesis of atherosclerosis is not clear, but through the regulation of K+ channels in vascular smooth muscle cells, they can participate in the regulation of vascular tone and blood pressure [6]. Sur2 −/− mice exhibited hypertension and coronary artery vasospasm accompanied by recurrent episodes of ST-segment elevation, during which decreased heart rate was recorded due to atrioventricular heart block and sudden cardiac death [38]. The data obtained on the downregulated expression of ABCC8 and ABCC9 in atherosclerotic samples may indicate their involvement in atherogenesis, which requires further investigation.ABCC1 (MRP1), ABCC2 (MRP2), ABCC3 (MRP3), ABCC4 (MRP4), ABCC5 (MRP5), ABCC6 (MRP6), and ABCC10 (MRP7) are members of the multidrug resistance protein (MRP) family that causes multidrug resistance [39]. Their function in the pathogenesis of atherosclerosis is not clear, but it should be considered that they carry out the outflow of many xenobiotics and endogenous substances [37]. There are also data on the direct involvement of members of ABCC transporters in atherogenesis, such as ABCC1, which is found in vascular smooth muscle cells involved in the process of atherosclerosis [40].The relationship of ABCC1, ABCC2, ABCC4, ABCC6, and ABCC10 transporters that carry out LTC 4 transport with inflammation is interesting. Leukotrienes are a group of highly effective lipid mediators that are important participants in inflammation. In this regard, the transport activity of ABCC may be involved in inflammation. Their importance in atherogenesis can also be emphasized by their participation in the transport of glutathione, oxidized glutathione, and leukotriene C4 (LTC4) [40,41,42], potentially necessary for the regulation of reactive oxygen species production in vascular cells. It is suggested that the release of LTC4 under the action of ABCC1 from vascular smooth muscle cells may be important in the early stages of atherosclerotic lesion development. Later, macrophages are likely to be the main source of LTC4. Indeed, the modulation of ABCC1 expression in human aortic endothelial cells affects vascular function [40,41,42]. ABCC1 is also involved in the export of sphingosine-1-phosphate (S1P), a lipid mediator that is involved in inflammation, angiogenesis, apoptosis and macrophage function, and regulation of endothelial barrier integrity through modulation of the endothelial cytoskeleton [43,44,45,46,47,48,49,50,51,52]. The transporters belonging to this group showed a multidirectional pattern of differential expression. Moreover, ABCC10 showed the most significant increase in expression in atherosclerotic samples, whereas ABCC4 showed no difference, and ABCC1 showed differences only for carotid artery samples. These data suggest that the role of these members of the ABC transporters in atherogenesis needs to be better investigated.The data obtained in the present study allow increased attention to several ABC transporters that have demonstrated differential expression and may potentially be involved in atherogenesis, or their differences may be related to the provision of impaired transport processes that are seen in atherosclerosis.Directions for further research may be to assess not only the differential expression of ABC transporter genes but also the protein, given the available literature suggesting that these may differ. In this regard, evaluation of gene expression and protein analysis will improve the interpretation of the data.Another important area for ABC transporter studies is the assessment of changes in protein expression and functional activity with age. There is evidence of age-related changes in ABC transporter gene expression, which may be of clinical significance. For example, in brain microvessels, the expression and functional activity of ABCB1 significantly decrease with age [53,54,55,56,57].The expression level of the ABCA1 gene can also be correlated with ageing and is reduced by the DNA methylation process of ABCA1 [58,59,60]. Similar data correlating age and expression are available for other transporters, such as ABCA7 [61]. However, analysis of the correlation between ABCC3 expression and age found no significant change [62]. In addition, there may be differences in different organs. For example, there are no data in the literature on age-related changes and the effects of physiological aging on breast cancer resistance protein (BCRP/ABCG2) expression and/or function in endothelial cells, but a decrease in expression at the protein level has been described in liver during aging, whereas no significant differences were observed in the level of ABCG2 gene expression during aging [63].These data are of potential interest from the perspective of the role of age-related changes in the expression and functional activity of ABC transporters in the pathogenesis of atherosclerosis.It should be noted that this study has a number of limitations due to the fact that the dataset contains a small number of patients; there are no data on the stage of progression of atherosclerotic lesions; there is a lack of sufficient information on the intake of drugs by patients whose data are available for analysis that can affect lipid metabolism and ABC transporters.An important limitation of the present study is that only ABC transporter gene expression data were evaluated, and protein-level expression data were not analyzed. Together, these data may significantly expand our understanding of the role of ABC transporters in the pathogenesis of atherosclerosis.However, the data obtained in this study will help to better plan an experimental study to assess the differential expression of ABC transporters in atherosclerosis.Thus, the bioinformatic analysis showed that atherosclerotic lesions are characterized by differential expression of ABC transporter genes. This can lead to disruption of transport processes for many groups of endogenous and exogenous substances, modulating the natural course of atherosclerosis and affecting treatment. Most of the ABC transporters differentially expressed in atherosclerosis have been shown to be related to lipid transport, ion channel function, and export of endogenous and exogenous substances, which are part of the mechanism of multidrug resistance. The obtained differences in the expression of ABC transporters in atherosclerotic specimens of different localization in the vascular bed may be related to both the peculiarities of atherosclerosis progression of different localization and the stage of atherosclerotic plaque development.In this regard, further studies on the role of ABC transporters in the pathogenesis of atherosclerosis, taking into account the data obtained in the present study, may be of clinical interest.Conceptualization, S.K.; methodology, S.K.; software, S.K.; validation, S.K. and A.K.; formal analysis, S.K. and A.K.; investigation, S.K.; resources, S.K.; data curation, S.K. and A.K.; writing—original draft preparation, S.K.; writing—review and editing, S.K. and A.K.; visualization, S.K.; supervision, S.K.; project administration, S.K. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.The data presented in this study are available on request from the corresponding author.The authors declare no conflict of interest.Gene expression patterns of the ABCA subfamily in the comparison groups: atherosclerotic—femoral artery (A–F); atherosclerotic—carotid artery (A–C); atherosclerotic—infrapopliteal artery (A–I); control—femoral artery (C–F); control—carotid artery (C–C); control—infrapopliteal artery (C–I). The red line shows the differences in gene expression in the group between all sets with atherosclerosis and all control sets. The data distribution is visualized in the form of box diagrams. Statistically significant differences (p values adjusted using the algorithm of Benjamini–Hochberg (adj. P. Val)) shown with asterisks: * p < 0.05, ** p < 0.001.Gene expression patterns of the ABCB subfamily in the comparison groups: atherosclerotic—femoral artery (A–F); atherosclerotic—carotid artery (A–C); atherosclerotic—infra-popliteal artery (A–I); control—femoral artery (C–F); control—carotid artery (C–C); control—infra-popliteal artery (C–I). The red line shows the differences in gene expression in the group between all sets with atherosclerosis and all control sets. The data distribution is visualized in the form of box diagrams. Statistically significant differences (p values adjusted using the algorithm of Benjamini–Hochberg (adj. P. Val)) shown with asterisks: * p < 0.05, ** p < 0.001.Gene expression patterns of the ABCC subfamily in the comparison groups: atherosclerotic—femoral artery (A–F); atherosclerotic—carotid artery (A–C); atherosclerotic—infra-popliteal artery (A–I); control—femoral artery (C–F); control—carotid artery (C–C); control—infra-popliteal artery (C–I). The red line shows the differences in gene expression in the group between all sets with atherosclerosis and all control sets. The data distribution is visualized in the form of box diagrams. Statistically significant differences (p values adjusted using the algorithm of Benjamini– Hochberg (adj. P. Val)) shown with asterisks: * p < 0.05, ** p < 0.001.Gene expression patterns of the ABCD and ABCG subfamily in the comparison groups: atherosclerotic—femoral artery (A–F); atherosclerotic—carotid artery (A–C); atherosclerotic—infra-popliteal artery (A–I); control—femoral artery (C–F); control—carotid artery (C–C); control—infra-popliteal artery (C–I). The red line shows the differences in gene expression in the group between all sets with atherosclerosis and all control sets. The data distribution is visualized in the form of box diagrams. Statistically significant differences (p values adjusted using the algorithm of Benjamini–Hochberg (adj. P. Val)) shown with asterisks: * p < 0.05, ** p < 0.001.The first 10 upregulated ABC genes, ranked by p values adjusted using the algorithm of Benjamini–Hochberg (adj. P. Val).The first downregulated ABC genes, ranked by p values adjusted using the algorithm of Benjamini–Hochberg (adj. P. Val).Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Arrhythmogenic cardiomyopathy is a familial heart muscle disease characterized by structural, electrical, and pathological abnormalities. Recognition of left ventricular (LV) involvement in arrhythmogenic right ventricular cardiomyopathy (ARVC) has led to the newer term of arrhythmogenic cardiomyopathy (ACM). We report on a family with autosomal dominant desmoplakin (DSP) related ACM to illustrate the broad clinical spectrum of disease. The importance of evaluation of relatives with cardiac magnetic resonance imaging and consideration of genetic testing in the absence of Task Force diagnostic criteria is discussed. The practical and ethical issues of access to the Guthrie collection for deoxyribonucleic acid (DNA) testing are considered.The proband died aged 19 years whilst trekking in India. After two autopsies the cause of death was determined as sudden arrhythmic death syndrome (SADS). “There was no blood or tissue specimen available to be used as source of deoxyribonucleic acid (DNA) for comprehensive genetic analysis in the proband. We obtained access to a historic DNA sample retained from the neonatal Guthrie screening card; this allowed targeted DNA analysis when a variant was identified in another relative.” The pedigree is shown in Figure 1. The family was referred for screening.A summary of the results of investigations in family members is shown in Table 1.III:B Sister of proband, an athlete. Initial investigations included 12 lead electrocardiograph (ECG), exercise tolerance test (ETT), and 2-D transthoracic echocardiogram, all of which were normal. She presented six months after the initial evaluation with chest pain after a flu-like illness. During this hospital admission, high-sensitivity Troponin I (hsTnI) measured greater than 5000 pg/nl and non-sustained ventricular tachycardia was recorded. Cardiac magnetic resonance imaging (MRI) showed mid-wall late gadolinium enhancement in the left ventricular inferior septum. No abnormalities of right ventricular size, function, or tissue characterization were detected. Twelve lead ECG showed interventricular conduction delay, prominent U waves, and loss of QRS voltage in the limb leads, when compared to the index evaluation. Holter monitoring showed 663 ventricular ectopics in a 24 h period, which were multifocal in nature. Repeat cardiac MRI 18 months later showed global myocardial edema and left and right ventricular epicardial late gadolinium enhancement in addition to the previously observed mid-myocardial enhancement (Table 1). Endomyocardial biopsy was performed to exclude an alternative cause for the presentation with presumed myocarditis. Microscopy showed a focal increase in intramyocardial interstitial collagen with small foci of replacement fibrosis (Figure 2). A varying degree of myocyte hypertrophy with patchy vacuolar change and myocytolysis was observed. There was no evidence of active myocarditis at multiple histological levels. Overall, the histological features were compatible with a cardiomyopathy that is difficult to classify on the basis of this biopsy alone. The case was reviewed by an expert cardiovascular pathologist in a tertiary referral centre and the possibility of arrhythmogenic cardiomyopathy (ACM) was raised, although it is recognised that the histological features did not meet Task Force criteria.Given the evolution of clinical findings in this family member and the history of sudden arrhythmic death (although not confirmed ACM or ARVC/D) in a first degree relative, a diagnosis of ACM was suspected. According to the revised Task Force Criteria for the Diagnosis of ARVC/D, the non-sustained ventricular arrhythmia (arrhythmia-1 minor) and the sudden death of a family member under the age of 35 years with suspected ARVC/D (family history-1 minor) suggest a possible diagnosis of ARVC/D. After genetic testing, a shared decision was made between the clinical team and patient for implantation of a subcutaneous implantable cardioverter defibrillator.II:B Mother of proband. The mother was asymptomatic at the initial clinical evaluation and the 12 lead ECG and echocardiogram were normal by Task Force criteria. The ECG showed low voltage QRS complexes in the limb leads. Cardiac MRI revealed septal mid-wall and inferolateral epicardial late gadolinium enhancement (Table 1). Prior to genetic evaluation of the family, II:B met one minor criteria for a diagnosis of ARVC/D by the revised Task Force criterion.II: C. Maternal aunt. A normal 12 lead ECG and echocardiogram were recorded at the initial evaluation. Cardiac MRI also showed mid-wall septal late gadolinium enhancement (Table 1).III:C Maternal cousin. At the time of the initial evaluation, the proband’s cousin was asymptomatic. A normal 12 lead ECG and echocardiogram were recorded. Cardiac MRI also showed septal mid-wall late gadolinium enhancement (Table 1).I: A. The proband’s maternal grandmother had New York Heart Association (NYHA) functional class II heart failure and palpitations at the index evaluation. She had an abnormal 12 lead ECG with interventricular conduction delay in the inferior leads with T wave inversion extending from V1-V6 in the precordial chest leads. The echocardiogram showed left ventricular systolic dysfunction. Cardiac MRI showed fibrofatty infiltration of the right ventricle with epicardial and mid wall late gadolinium enhancement in the basal inferolateral wall.Familial evaluation with the above investigations resulted in the identification of four further individuals as being affected by ACM. Of the individuals evaluated, only one fulfilled the Task Force criteria for a definite diagnosis of ACM or ARVC/D. The revised Task Force criteria for a diagnosis of ARVC/D for each family member are shown in Table 1 [1].Despite not meeting the Task Force criteria for a diagnosis of ACM, after detailed clinical evaluation, there was a high clinical suspicion of ACM in III:B. Next generation sequencing was undertaken for an ACM (nine gene) panel identifying two sequence variants. The variants were classified according to the American College of Medical Genetic classification [2]. A heterozygous desmoplakin (DSP) variant (c.1288G>T; p.(Glu430*) was predicted to result in the formation of a premature translational stop signal, resulting in an absent or disrupted protein product and therefore was considered pathogenic. ‘The DSP variant is predicted to undergo nonsense-mediated decay and therefore PVS1 was applied, along with PM2 as it is not recorded on gnomAD.’ The variant is not present in population databases (no frequency on the Exome aggregation Consortium, ExAC) and has previously been described in individuals with ARVC [3,4].In addition, a desmocollin-2 (DSC2) variant was identified (c.2335G>T; p.(Gly779Arg), which was considered to be a variant of uncertain clinical significance. The missense variant was predicted to result in the substitution of a glycine by an arginine residue at codon 779, which is conserved through evolution and in silico analysis indicated that the substitution may not be tolerated by the protein. This variant has been recorded in very low frequency (0.009%) on the ExAC database and reported previously as a variant of uncertain clinical significance on ClinVar (NCBI, Bethesda, MD, USA).Following the identification of the genetic findings in patient III:B, DNA was obtained from the Guthrie card of the proband (III:A). Targeted Sanger DNA sequencing confirmed the presence of the pathogenic variant in DSP but not the variant of unknown significance in DSC2. Cascade predictive genetic testing was performed, looking for the presence of the pathogenic variant in DSP, and was found in the proband’s mother (II:B), maternal grandmother (I:A), maternal aunt (II:C) and maternal cousin (III:C).In this case example, the proband’s sister presented after reassuring clinical screening with chest pain, elevated hsTnI, and non-sustained ventricular arrhythmia. The clinical evolution and history of sudden death in the family mounted sufficient clinical suspicion to initiate genetic evaluation, despite not meeting the Task Force criteria for a diagnosis of ACM. This allowed cascade predictive genetic testing in family members for the detection of gene carriers and appropriate evaluation of risk and clinical follow-up. In this case, clinical evaluation of family members with ECG, holter monitoring, and echocardiogram may not have detected a phenotype in three out of four individuals. The clinical diagnosis of ACM in relatives is often difficult because of the low penetrance of mutations and variable expressivity. The proband in this case example presented in the second decade, illustrating the age-dependent penetrance of ACM [5,6].The presentation of patient III:B with acute chest pain and elevated troponin levels is in keeping with the previously described ‘hot-phase’ of the condition. In a recent retrospective analysis of 23 patients with ACM meeting the clinical definition of a ‘hot-phase’ nearly 40% of patients were diagnosed with ACM at the time of their first presentation. In 60% of the patients, an alternative diagnosis was made at the index presentation (myocarditis, acute myocardial infarction, acute pericarditis, and unstable angina). This highlights the importance of distinguishing between acute myocarditis and the hot phase of ACM and reinforces the importance of a detailed family history in such patients.Historical nomenclature and diagnostic criteria do not account for left ventricular involvement in this condition or for cardiac MRI tissue characterisation findings [7]. It is increasingly recognised that patients with DSP-associated cardiomyopathy frequently have left ventricular involvement and that the subepicardial pattern of late gadolinium enhancement is common [8,9]. This may account for the poor sensitivity of the revised Task Force criteria when diagnosing patients with ACM associated with desmoplakin. The recently published Padua criteria propose the inclusion of left ventricular late gadolinium enhancement and low QRS voltage to diagnose the previously overlooked LV phenotype [10,11]. The Padua criteria are shown in Table 1.In this case, use of the stored blood spot (Guthrie) card to obtain DNA from the deceased for testing for the variants found in the affected sister provided insight and understanding into the otherwise unexplained death and important answers for the family. This blood spot test obtained on day five of a newborn’s life is used to screen for serious health conditions that can be reversed with early treatment, for example, phenylketonuria. The cause of sudden cardiac death in a young person can be challenging to identify. Often a drawn blood sample or tissue storage at post-mortem are not available for the deceased. The dried blood spot provides the opportunity for DNA extraction and advances in genomic testing technology allow genetic testing and confirmation of a familial variant when an alternative stored sample is otherwise unavailable. A recent study of 22 patients with a post-mortem diagnosis of ARVC found clinically relevant variants in 63% of families on whole exome sequencing of dried blood spots [12]. Of the variants detected, four were in ARVC-associated genes and a further six were associated with arrhythmia genes. This has important implications for preventing sudden cardiac death in surviving family members. In Scotland, the Guthrie collection system began in 1965 and there are now over 3 million samples stored. Only from 2003 were parents asked for consent for storage of their children’s samples. Whilst in the presented case, the Guthrie card was relevant for an individual and their family, obviously, this unique dataset raises potential opportunities for genetic-based and wider health research. It is also particularly relevant for families with a historical diagnosis of cardiomyopathy where genetic testing did not take place or in those affected by sudden cardiac death. Navigating the future use of the Guthrie cards is currently under consideration by the Scottish Government and a document published in 2014 exists to determine the legal status, the role of consent and governance, and importance of transparency in processes involving the collection [13]. Public engagement with this process has highlighted important areas for future consideration including the possibility that information obtained from the Guthrie card could disadvantage individuals, or the potential for the neonatal screening panel to be extended to screen for variations causing a wide variety of diseases.Individuals with DSP-related ACM display a range of clinical phenotypes and thorough cardiac investigation should be undertaken, especially where there is a family history of sudden cardiac death. Advances in genomic testing technology allows the confirmation of genetic variants on stored blood spot samples and in the case of sudden cardiac death provides confirmation of the diagnosis and closure for the family.Data acquisition, J.S., J.A., D.O. and S.W.; processing and interpretation, J.S., S.W. and C.C.; writing original draft preparation, J.S.; clinical evaluation, J.A., D.O., R.M. and C.C.; study concept, R.M. and C.C.; critical revision of manuscript, R.M.; data curation, C.C.; supervision, C.C. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Informed consent was obtained from all subjects involved in the study.The data that support the findings of this study are available from corresponding authors upon request.The authors declare no conflict of interest.Family pedigree. Squares and circles indicate male and female family members, respectively. Symbols with a single slash mark are deceased family members. Arrows indicate proband. Open symbols indicate unaffected family members, and symbols with a cross indicate family members who did not have a clinical evaluation. The presence (+) or absence (−) of a desmoplakin (DSP) variant is indicated for family members with DNA samples available for testing.(A) Ultra low power image (H&E). In this image, four fragments of endomyocardium are included for assessment. Note the presence of adipose tissue in some of the fragments. Other fragments appear to show some fibrous replacement of the cardiac myocytes. (B) Medium power (×10 H&E). This medium power image shows myocardium with areas of interstitial fibrosis. There is mature adipose tissue and a suggestion of associated fibrous tissue at the top of the section, but this was not demonstrated on tinctorial staining. There is myocyte nuclear hypertrophy and focal areas of myocytolysis were seen. No increase in the cellularity of the interstitium was seen. No granulomas were identified. (C) This medium power (×10, H&E) image of another fragment from the biopsy set shows myocardium with areas of replacement fibrosis. Tinctorial staining for excess iron and amyloid deposition were negative (not shown). (D) In this medium power image (×10 MSB), the arrow demonstrates an area of fibrosis. No definite established fibrosis can be seen in association with the adipose tissue in this section.Summary of investigations and the revised Task Force criteria.BSA, body surface area; CMR, cardiac magnetic resonance; ECG, electrocardiograph; F, female; LGE, late gadolinium enhancement; LV, left ventricle; LVEF, left ventricular ejection fraction; LVIDd, left ventricular internal diastolic diameter; LVEDV, left ventricular end diastolic volume; LBBB, left bundle branch block; M, male; NYHA, New York Hear Association; PLAX, parasternal long axis view; RV, right ventricle; RVEF, right ventricular ejection fraction; RVEDV, right ventricular end diastolic volume; RVOT, right ventricular outflow tract; RWMA, regional wall motion abnormality; RVSD, right ventricular systolic dysfunction; ACM, arrhythmogenic cardiomyopathy; PVC, right (R) and left (L) premature ventricular contractions. * Prior to genetic evaluation in patient III:B.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+MicroRNAs (miRNAs) are single-stranded small non-coding RNA (18–25 nucleotides) that until a few years ago were considered junk RNA. In the last twenty years, they have acquired more importance thanks to the understanding of their influence on gene expression and their role as negative regulators at post-transcriptional level, influencing the stability of messenger RNA (mRNA). Approximately 5% of the genome encodes miRNAs which are responsible for regulating numerous signaling pathways, cellular processes and cell-to-cell communication. In the cardiovascular system, miRNAs control the functions of various cells, such as cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts, playing a role in physiological and pathological processes and seeming also related to variations in contractility and hereditary cardiomyopathies. They provide a new perspective on the pathophysiology of disorders such as hypertrophy, fibrosis, arrhythmia, inflammation and atherosclerosis. MiRNAs are differentially expressed in diseased tissue and can be released into the circulation and then detected. MiRNAs have become interesting for the development of new diagnostic and therapeutic tools for various diseases, including heart disease. In this review, the concept of miRNAs and their role in cardiomyopathies will be introduced, focusing on their potential as therapeutic and diagnostic targets (as biomarkers).With age, the heart undergoes enormous stress and pathological stimuli that lead to cardiac remodeling and consequent cardiovascular disease [1,2]. During the last twenty years, non-coding microRNAs (miRNAs) have been identified as fundamental negative gene regulators. Among the functions regulated by these RNAs, there are also physiological and pathological aspects of the heart [3,4]. For a long time considered as junk RNA, today they are considered extremely important. MiRNAs control physiological functions such as the proliferation and differentiation of stem and progenitor cells, the function of cardiac myocytes, pacemaker cells, endothelial cells and smooth muscle cells [5]. These small sequences play a fundamental role in regulating cardiomyocyte contractility, maintaining heart rhythm, plaque formation, lipid metabolism and angiogenesis [6]. Even the stress response of the heart muscle is controlled by a precise spatiotemporal gene regulation of miRNAs [7,8].MiRNAs play a fundamental role in the pathophysiology of myocardial remodeling, causing damage to cardiomyocytes, cardiac hypertrophy, cardiac fibrosis and abnormal inflammatory response, binding to multiple targets. The balancing and minute regulation of different miRNAs are key to guiding cellular events towards functional recovery, and any variation can lead to detrimental effects on cardiac function following various insults. The discovery of miRNAs, therefore, changed our understanding of the regulation of gene expression.As the expression pattern of miRNAs varies according to the type of heart disease, it is believed that dysregulation of miRNA expression can be detected in the blood of patients with cardiomyopathies, making them optimal candidates as non-invasive biomarkers for diagnosis, prognosis and therapeutic response [9,10,11]. The characteristic that makes miRNAs possible biomarkers and even potential therapeutic targets are their stability and presence in circulating biofluids [12]. Instead, their specificity concerning different cardiac diseases needs to be studied more.In this review, we will analyze the main characteristics of miRNAs and their role in various cardiomyopathies such as hypertrophic cardiomyopathy, arrhythmogenic cardiomyopathy and dilated cardiomyopathy. It will be also mentioned the possible role that miRNAs could have as biomarkers of pathology, as well as an overview of future therapeutic approaches of miRNAs for cardiomyopathies.From an early age, we have always been taught the “Dogma of Biology”, which states that DNA is transcribed into RNA and then translated into protein. This suggested that only coding RNA could determine gene expression. Only 1% of the human genome encodes genes which will then be translated into proteins [13]. The remaining 99% of the DNA was considered junk and therefore not useful for gene regulation. In recent years, the importance of non-coding RNAs (ncRNAs) as regulators of gene expression has finally been understood [14]. Although ncRNAs cannot encode proteins, an important role of ncRNAs in the regulation of gene expression has been observed, both in physiological and pathological conditions (Table 1) [15].In this review, we will focus on a specific class of ncRNA, namely, microRNAs (miRNAs), which correspond to ~5% of the human genome [16,17]. MiRNAs are a class of single-stranded, non-coding RNA, approximately 18–25 nucleotides in length, which negatively regulate gene expression at the post-transcriptional level. They are highly conserved ncRNA and can be identified in animals, plants and viruses [18], and are thought to be a vital evolutionary component of gene regulation [19,20].MiRNAs influence protein production by binding to messenger RNA (mRNA) via imperfect base pairing and complementary sequences in their target mRNA, resulting in translational repression or transcript degradation [21,22]. The mRNA and miRNA binding occurs through a sequence of 2–8 nucleotides [7] of the 5′ end of the miRNA with the 3′ untranslated region (3′UTR) of the target mRNAs [23,24,25].It has been estimated that miRNAs control the activity of 30–50% of protein-coding genes [26]. Unlike transcriptional regulators, which have an activation and deactivation function in controlling gene expression, the various miRNA profiles appear to adapt the level of protein expression to changes in environmental conditions [6]. Due to the imperfect binding between the two strands, a single miRNA can influence the expression of many target genes in a cell and a target mRNA can be regulated by multiple miRNAs, as mRNAs host binding sequences for multiple miRNAs (Figure 1) [25,26]. MiRNAs are essential in various biological processes, including cell differentiation and proliferation, cell death and metabolism [27,28,29,30]. Dysregulation of miRNAs often disrupts critical cellular processes, leading to the onset and progression of various human diseases including heart diseases [31]. Furthermore, miRNAs can enter the circulation through microvesicles (exosomes) in an extremely stable form and can therefore be quantified through plasma sampling and then used as biomarkers [32,33].MiRNAs are an evolutionarily conserved integral part of the cellular genome. Depending on the genomic location of the miRNA coding sequences, miRNAs can be transcribed as independent transcription units starting from intergenic regions or in the introns and exons of the protein-coding genes. Intergenic miRNAs are transcribed under the control of distinct promoters, while the transcription of intronic and exonic miRNAs is mainly controlled by their host gene promoters. The genes encoding miRNAs can be transcribed singly or in polycistronic clusters forming a long transcript which is then cleaved into the different miRNAs [22,39]. In the latter case, more than one miRNA is transcribed together by the same transcription factor, generating a transcript with miRNAs belonging also to different families. Therefore, miRNAs from the same cluster can target multiple mRNAs [40]. Alternatively, miRNAs can be transcribed from introns of protein-coding genes, from introns or exons of non-coding genes, or even from 3′UTR of protein-coding genes [41].The biosynthesis of miRNAs occurs in several enzymatic steps, both in the nucleus and in the cytoplasm (Figure 2) [23,42]. The miRNAs are transcribed into the nucleus by RNA polymerase II (RNA Pol II) into primary miRNAs (pri-miRNA), a long strand of hundreds to thousands of nucleotides that overlaps on itself with an imperfect pairing in a stem-loop structure with two flanking single-stranded regions at the end [43,44,45].At this point, it activates an enzyme called Drosha (protein complex containing the RNase III endonuclease) that is associated with a nuclear protein called DGCR8. Drosha (with DGCR8) cleaves the 5′-end and 3′-end of the pri-miRNA and stabilizes the molecule, giving rise to precursor miRNAs (pre-miRNA) of ~70 nucleotides with a hairpin structure [22,39,44]. Then, the process continues in the cytoplasm.Exportin 5 (EXP5) and RAN-GTP (GTP-dependent protein) form a transport machinery that recognizes a short sequence of 2–3 nucleotides at the end of the pre-miRNAs to transport them from the nucleus to the cytoplasm [22,46]. EXP5 also protects pre-miRNAs from the degradation process which avoids the accumulation of pre-miRNAs in the nucleus [47].A final trimming step is performed in the cytoplasm by RNase III endonuclease called Dicer. Dicer interacts with the 5′-end and 3′-end of the hairpin and creates double-stranded miRNAs (miRNA*duplex) of approximately 22 nucleotides [48,49]. A double- strand presents an imperfect complementarity which will facilitate its division [50]. The miRNA*duplex splits to form two single strands: the guide strand, which acts as a functional unit, and the passenger strand [51,52]. The choice of the guide strand depends on the thermodynamic stability of the 5′-end of the miRNA*duplex [53]. In general, the strand with lower stability preferentially combines with Argonaute protein (AGO). This causes some miRNAs to have both of their strands loaded into the RNA-induced silencing complex (RISC) with the same proportion, while for others only one strand will dominate [53].The guide strand combines with AGO in the RISC complex to become a mature and functional miRNA [54,55]. The passenger strand is often degraded or incorporated into microvesicles and released in the bloodstream [12,56]. Both mature miRNAs and pre-miRNAs can be found in microvesicles [12].Thanks to the link with RISC, a mature miRNA is formed, ready to inhibit target mRNAs at the post-transcriptional level [22,57]. The RISC binding sites are complementary sequences to the 3′-untranslated region (3′UTR) of the mRNAs [58]. Therefore, this complex can direct mRNA towards miRNA through sequence complementarity. The miRNA sequence, incorporated in the RISC, binds to the mRNA by a seed sequence (first 2–8 nucleotides of the 5′ end of a miRNA).When the miRNA sequence matches its target perfectly, AGO cleaves the mRNA resulting in direct degradation. More frequently, when complementarity is lacking, mRNAs translation is inhibited, blocking protein synthesis [59]. Therefore, AGO does not induce target cleavage, but represses translation by three different mechanisms: translation initiation block, elongation block, or deadenylation [60]. However, these processes will not be discussed in this review.Thanks to the imperfect base-pairing with complementary sequences between miRNA and mRNA, a single miRNA can regulate multiple mRNAs targets which are involved in different biological processes and, conversely, a single mRNA can be regulated by several miRNAs.Generally, gene suppression is partial, and one mRNA can have multiple binding sites for different miRNAs.Not surprisingly, the binding of a single miRNA may not be sufficient to block the translation and several miRNAs are needed to regulate a single mRNA [18].Mature miRNAs are indicated with the prefix “miR-” followed by an identification number that reflects their order of discovery. In case of miRNAs with sequences that differ only in one or two nucleotides, an additional letter or number is added to the name, e.g., “miR-120a” and “miR-120b” or “miR-232a-1” and “miR323a-2” [10,39].Little is known about the half-life and degradation of mature miRNAs, which are stable in cells and have also been shown to play a central role in cell-to-cell communication [12,43]. Most miRNAs are located at the intracellular level, but some of them are released into the blood in association with proteins (e.g., Ago2, nucleophosmin 1 and HDL). They can be packaged in microvesicles, exosomes or apoptotic bodies in circulation, allowing resistance even to changes in temperature, pH and multiple freeze/thaw cycles.They are present in all blood compartments, including plasma, platelets, erythrocytes and nucleated blood cells [57]. MiRNAs bind RNA-binding proteins or high-density lipoproteins, forming miRNA–protein complexes which give them protection from circulating RNases [12].As previously mentioned, miRNAs are relatively stable within biofluids and tissues, allowing us to measure them reliably [61,62]. We indicated that mature miRNAs, pre-miRNAs and stand passengers can be found in the blood due to their inclusion in microvesicles. Therefore, it is possible to measure these molecules not only in the tissues, but also in the blood of patients with CMP. Several techniques are available to identify and quantify miRNAs, but the gold standard is the real-time PCR, which remains the most reliable technique. This technique can be used only with predefined primers to amplify and measure individual miRNAs in a sample. Alternatively, miRNA arrays could be used, which consists in miRNA hybridization on specific primers. MiRNA arrays allow obtaining a less accurate quantification than the previous one; however, they allow evaluating a greater number of miRNAs at the same time at lower costs.Both of these techniques can be used only and exclusively for already known miRNAs, as they are based on predefined primer sequences. The identification of uncharacterized miRNAs can be evaluated with non-coding RNA sequencing technologies, essential for the identification of miRNAs with unknown sequences. In this way, it is possible to have a quantitative analysis of a complete miRNA transcriptome [63].In recent years, advances in molecular technology, such as the Next-Generation Sequencing (NGS), have enabled the discovery of new disease sequences with higher yields and lower costs than conventional technologies [64]. NGS has become a fundamental tool for the clinical work-up of cardiomyopathies.Therefore, these techniques can be used to evaluated miRNAs, which can play the role of pathological biomarkers. Not only is their expression important, but also the mutations in seed regions or the deletion of some miRNAs themselves can cause a pathology [65]. This is possible because an aberrant expression of miRNA profile during disease has been demonstrated. Not surprisingly, variations in miRNA expression have been found in cardiomyopathies, and specific plasma miRNAs have been identified in correlation with these illnesses. Different miRNA expression profiles have been studied in pathological and normal conditions and the result highlights that dysregulation of miRNA levels can detect a pathology.The identification of miRNAs with the technique explained can allow evaluating the differential expression between healthy and diseased subjects [66,67]. In various studies, it has been shown that miRNA expression pattern was different and specific for different heart diseases; this led to the hypothesis that miRNAs could have etiological implications in the development of the disease. Therefore, their profile expression could be used as potential biomarkers for diagnosis, prognosis and response to therapy.MiRNAs are present in target cells and tissues, in biological fluids, including plasma and serum, stored within microvesicles. The microvesicles confer protection from the cleavage of endonucleases and stability in biofluids and which makes them easily detectable in circulation [68,69,70].With NGS technologies it is possible to obtain a rapid and accurate measurement of miRNAs which can be also measured with real-time PCR (even in small volumes of biofluids) [71,72]. The stability and diversity of miRNAs in combination with the available technologies make them new and promising diagnostic biomarkers [73,74]. However, miRNAs also have some limitations [75]. Unfortunately, miRNA detection is time-consuming and rapid diagnosis is not possible; there are no standardized laboratory protocols for the extraction, dosage and normalization of the analysis, so for now they may not be usable [76]. Furthermore, different miRNAs can regulate more genes and, at the same time, several genes can be regulated by more miRNAs, so there is a low correlation between miRNA and specific pathologies.In genetic diseases, pathogenesis mechanisms are often unknown or difficult to interpret. Mutations in a specific gene can be associated with different phenotypes. In this perspective, miRNAs and other ncRNA represent a further mechanism to be investigated in homogeneous groups of patients to better understand the mechanisms associated with particular pathologies.MicroRNAs are a group of short, non-coding and endogenous RNAs that regulate gene expression through the sequence-specific recognition of their target transcripts [18]. They are involved in various cellular processes such as apoptosis, proliferation or differentiation. Aberrant levels of miRNA are implicated in numerous pathophysiological conditions, including cardiomyopathies. The overexpression or underexpression of certain miRNAs also plays a crucial role in cardiomyopathies such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) or arrhythmogenic cardiomyopathy (AC), demonstrating that several cardiomyopathies can have a specific miRNA signature [12].MiRNAs are involved in cardiac biogenesis. Blocking specific genes essential for miRNA biogenesis, such as DROSHA, DGCR8, AGO or Dicer, in the heart tissue of murine embryos, results in an interruption of gestation due to severe developmental defects of the heart and blood vessels. However, the deletion of individual miRNAs is not lethal [6]. Thanks to several studies, we now know that heart muscle phenotypes are tightly regulated by multiple miRNA species [77]. Cardiomyopathies are a very heterogeneous group of diseases that affect the heart muscle. They are generally characterized by changes in the size of the heart chambers, ventricular wall thickness or contraction abnormalities.The main cardiomyopathies affecting the population are HCM (1:500), DCM (1:2.500) and ACM (1: 5.000) [78,79] which we will deal with in this review. Table 2 shows the miRNAs studied in this review and their associated cardiomyopathies.HCM is the most common primary cardiomyopathy (1:500) with heterogeneous clinical and genetic characteristics, the leading cause of sudden cardiac death (SCD) in adolescents and athletes [97,98,99]. It is an autosomal dominant disease with incomplete penetrance.HCM is a highly complex and heterogeneous disease as regards not only the number of associated mutations, but also the severity of the phenotype and the frequency of complications, such as heart failure and SCD [66,100]. HCM is also characterized by the varying degree of left ventricle hypertrophy (LVH) [101], symptoms and risk of SCD [102] or heart failure [103,104]. The disease is characterized by the so-called “myocardial disorder” which involved the hypertrophic non-dilated left ventricle. The pathological mutations generally affect genes that encode proteins belonging to the contractile components of the heart. Thus, mutations can occur in one or more of the eight genes encoding sarcomere proteins [105,106]. Seventy percent of HCM-causing mutations affect cardiac myosin binding protein C (MYBPC3) genes with nonsense mutations, as well as cardiac myosin heavy chain (MYH7) with missense mutations. A recent study showed that sarcomeric variants are mostly associated with female gender and young age, presenting asymmetric septal hypertrophy, a family history of HCM and SCD [107]. This pathology presents morphological and pathological heterogeneity, penetrance and age dependence with the consequence of different clinical outcomes, with conditions ranging from asymptomatic patients to cardiac arrhythmias and SCD [108].Furthermore, it has been observed that genetic and environmental modifiers may also be involved in the pathogenesis of the disease. Other genes that can cause HCM include TNNT2, TNNI3, TPM1, MYL2, MYL3 and ACTC1 even if with lower frequency (1–5% each) [109,110,111,112]. In recent years, other genes besides sarcomeric ones have also been identified. These include CSRP3, PLN, CRYAB, TNNC1, MYOZ2, ACTN2, ANKRD1 FLNC [64,113,114,115,116,117,118] and FHL1 [114,119].Interesting results have also been obtained with the study of miRNAs, but this will be discussed later. Given the variety of genes involved, there is an equivalent high phenotypic variety of recognized HCM. Unfortunately, the genotype–phenotype relationship may not be obvious. A high percentage of patients are asymptomatic or mildly asymptomatic, and the diagnosis is made during family screening or by accidental observation in mid or late adulthood where functional heart weakness has already progressed. Not surprisingly, members of the same family with the same mutation can have very different phenotypes [106]. Often, the phenotype is mild, but the arrhythmic prognosis could be particularly poor. Fortunately, the identification of causal mutations in a proband with HCM facilitates pre-symptomatic diagnosis of family members and clinical monitoring [97]. To explain the phenotypic variability, gene and environmental modulators were investigated. Interesting results have been obtained from miRNA studies which could therefore become potential biomarkers and therapeutic agents. If confirmed, the results obtained by the miRNAs would allow to have an early diagnosis, to evaluate the prognosis and future therapies. In this regard, the transcriptional profile modified by miRNA expression and other post-transcriptional modifications appears to be crucial for understanding the onset of HCM.Currently, therapies for HCM are purely symptomatic. Beta-blockers, calcium channel blockers and disopyramide are generally given to patients [120]. Severe patients require surgical myectomy and alcohol ablation to relieve left ventricular obstruction [121].The discovery of miRNAs and their role at the post-transcriptional level has made it possible to identify pathological regulatory mechanisms that have been little known until now. To date, an ever-growing list of HCM-associated miRNAs varied in animal models and human samples has been created and the miRNA profile may allow understanding of the pathophysiology of the disease [122]. For this reason, some miRNAs have been reported as useful biomarkers of diseases and future therapeutic agents [82]. In this section, the best-known miRNAs involved in HCM will be evaluated (Table 2).In one study, 370 miRNAs were analyzed in pathological HCM tissue, with and without mutation in the MYH7 gene. Two miRNAs—miR-590-5p and miR-92a—were overexpressed in HCM patients compared to healthy controls, and their expression was different between HCM-mutated and HCM-unmutated tissues [80]. In addition, the same group studied miRNA profile expression in plasma samples from HCM patients, where 10 miRNAs were analyzed in 24 patients and compared with healthy controls. Only miR-483-5p was upregulated in HCM patients compared to healthy ones [81].Increased levels of miR-29a have been found in the blood of patients with HCM carrying mutations in the MYH7 gene [82]. The same miRNA appears to be upregulated also in fibroblasts during the process of cardiac fibrosis. Therefore, miR-29a could be used as a biomarker of both fibrosis and hypertrophy [82]. Instead, miR-155 appears to be downregulated in HCM patients with MYBPC3 mutations [84].Duisters et al. have shown that in patients with LVH there is a correlation between the downregulation of miR-133 and the upregulation of CTGF and the effects of their interaction on collagen synthesis [83]; miR-133 is also downregulated in myocardiocytes during cardiac hypertrophy. RhoA (a GTP-GDP exchange protein), CDC42 (a kinase that transduces the signal involved in the development of hypertrophy) and WHSC2 (a nuclear factor involved in cardiogenesis) have been identified as possible target modulating negatively by miR-133 to counteract hypertrophy [83].MiR-1 is downregulated in myocardiocytes in the process of cardiac hypertrophy. Under physiological conditions, this miRNA negatively modulates the Insulin-like Growth Factor-1 (IGF-1) pathway. In this way, it can block a series of processes including myocardial hypertrophy. Therefore, its downregulation can lead to cardiac hypertrophy [85].C. Kuster and colleagues studied miRNA profile expression in six patients with a loss-of-function mutation in MYBPC3 [86]. Among the 699 miRNAs analyzed, 13 of them formed a unique miRNA signature for HCM: 10 were upregulated (miR-181-a2, miR-184, miR-497, miR-204, miR-222, miR-96, miR-34b, miR-383, miR-708 and miR-371-3p) and three were downregulated (miR-10b, miR-10a and miR-10b). Studies in silico demonstrated that a large number of differentially expressed miRNA-regulated genes were associated with the cardiac hypertrophic signaling pathway in which a large number of predicted mRNA targets were involved in β-adrenergic signaling [86]. In particular, they found that miR-204, incorporated in the TRPM3 gene (Transient receptor potential cation channel subfamily M member 3), appears to be upregulated in HCM patients carrying a mutation in the MYBPC3 gene, one of the genes most frequently mutated in HCM [86]. TRPM3 encodes a cation-selective channel involved in calcium entry and is found to be upregulated with consequent alteration of calcium homeostasis in HCM [123]. This finding suggests that TRPM3 may be involved in the pathogenesis of the disease caused by MYBPC3 mutations.MiR-139-5p is also one of the most downregulated miRNAs in the hearts of HCM patients [87,124].Finally, Sun et al. screened miR-20, which was one of the highly expressed miRNAs in HCM [88]. To do this, they constructed the cardiomyocyte hypertrophy model in vitro to validate whether miR-20 was associated with cardiomyocyte hypertrophy. In the study, a total of 1451 miRNAs were identified in both groups, 195 of which were upregulated and 172 downregulated in the HCM group compared to the control group; among these, miR-20a-5p was 2.26 times higher in the HCM group than in the control group. Over-expressed miR-20 could induce cardiomyocyte hypertrophy by suppressing MFN2 expression. Among the target genes of miR-20 are MFN2, PTEN, SMAD4 and DUSP1, which are associated with cardiac remodeling [88]. This study identified 367 miRNAs differentially expressed between HCM cardiac samples and healthy control samples, meaning that some miRNAs may be indispensable in the development of HCM. It has been reported that miR-1 [125], miR-22 [126], miR-29a [82], miR-106a [127], miR-133 [128], miR-181a [129] and miR-195 [122] took part in the development of myocardial hypertrophy and showed significant differences between an expressed model of hypertrophic and physiological myocardium.Dilated cardiomyopathy (DCM) is a major cause of SCD and heart failure with an unknown etiology. It has a prevalence of 1:2500, can occur at any age and is usually identified when associated with severe symptoms [130]. Unfortunately, it is the leading indication for heart transplantation in children and adults worldwide [98]. DCM is a heart muscle disease characterized by ventricular dilation and systolic dysfunction with cardiac fibrosis in the absence of abnormal load conditions or coronary artery disease [130,131]. It progressively leads to heart failure and a decline in left ventricular (LV) contractile function.DCM is a complex disease with a common phenotype, but the pathological mechanisms are heterogeneous and still poorly understood. Early diagnosis is essential for the clinical management of the patient. Clinical diagnosis is currently made using imaging methods and genetic tests. However, the assessment of the disease still remains challenging; therefore, new non-invasive indicators are needed. The European Society of Cardiology has proposed a genetic and non-genetic classification of DCM, including etiologies such as peripartum, cancer therapies, drug or alcohol abuse, and myocarditis [132]. However, overlapping phenotypes often make the differential diagnosis unclear.Currently, myocardial biopsies are used as a diagnostic tool. However, due to the invasiveness of the procedure, the use of the method is limited to a few patients [133]. The most used imaging techniques for the diagnosis of DCM are transthoracic echocardiography and magnetic resonance imaging as they are non-invasive and of wide applicability [134,135].Approximately 20–35% of patients with DCM exhibit a family inheritance with incomplete penetrance associated with at least 40 genes [98]. Most DCM-associated mutations have been found in genes encoding proteins related to the cytoskeleton, sarcomere, nuclear envelope, ion channels and unclassified proteins [130,136]. Identifying the genetic factors that lead to the clinical manifestation of diseases is the key to understanding the triggering mechanism that initiates the disorder [137]. In recent years, great efforts have been made to explore the molecular mechanisms underlying DCM which, however, still remain little known. Currently, attention is focusing on the myocardial gene expression of miRNA and their role in DCM [138,139]. Research suggests that miRNAs signature may constitute a novel source of non-invasive biomarkers for a wide range of cardiovascular diseases. Specifically, several studies have reported the potential role of miRNAs as clinical markers among the etiologies of DCM. However, this field has not yet been explored in detail.Currently, several manuscripts have evaluated the relationship between miRNA and DCM profiles, without a detailed definition of the etiological mechanism. Among these is the manuscript of Tao et al., in which the interaction between long non-coding RNA (lncRNA), miRNA and competing for endogenous RNA (ceRNA) in patients with DCM were studied [140]. In this study, a miRNA array was initially performed to determine the differentially expressed miRNAs in samples from DCM patients and healthy controls. The results obtained were confirmed by RT-PCR. Cardiac tissue from patients with DCM and the mouse model of the pathology were used as samples. The results showed that miR-144-3p and miR-451a are downregulated compared to healthy controls, while miR-21-5p is upregulated [140]. Based on the ceRNA theory, a triple global network was developed using data from the NCBI-GEO (National Center for Biotechnology Information Gene Expression Omnibus) and the results obtained from the miRNA array. The results showed that two lncRNAs (NONHSAT001691 and NONHSAT006358) targeted at miR-144/451, both highly related to DCM. Therefore, clusterization and the use of an adequate random walk with restart algorithm for the analysis of the ceRNA network have identified four lncRNAs (NONHSAT026953/NONHSAT006250/NONHSAT133928/NONHSAT041662) that interact with miR-21 and are significantly related to DCM. This study could provide a new strategy for diagnosing DCM or other diseases. Furthermore, lncRNA–miRNA pairs can be considered potential diagnostic biomarkers or therapeutic targets for DCM [140].In another study, 3100 miRNAs were evaluated in the plasma of four DCM patients. Forty-seven miRNAs were differentially expressed compared to three healthy subjects. Among these 47 miRNAs, miR-3135b, miR-3908 and miR-5571-5p were chosen because their levels were significantly increased in the plasma samples. The results obtained from this first comparison were then confirmed by a larger cohort, demonstrating that indeed the upregulation of miR-3135b, miR-3908 and miR-5571-5p had a discriminatory power to distinguish DCM patients from controls utilizing analysis of the ROC curve [89].Highlighting the regulatory role that miRNAs can play, it has been understood that miR-148a is downregulated in DCM, while it is upregulated in concentric hypertrophy in human cardiac biopsies [90]. These results were confirmed in transgenic mouse models for DCM and concentric hypertrophy. The knockdown of miR-148a, obtained through the use of antagomiRNAs in WT mice, led to chamber dilation, increase in the volume of the left ventricle, cardiac wall thinning and reduction of the ejection fraction. Instead, the upregulation of AAV-mediated miR-148a protects against systolic dysfunction caused by pressure overload [90].Yu et al., in 2011, evaluated the plasma levels of circulating miR-185 in patients with DCM [91]. These patients were compared with healthy ones. The analysis showed that miR-185 expression was significantly higher in DCM patients. DCM patients were placed in two distinct subsets, “high-group” and “low-group”, clustered by miR-185 plasma levels. Essays were performed at DCM diagnosis time and 1-year follow-up. During this year, DCM cases had received standard therapy. At follow-up, circulatory levels of miR-185 were stable. Patients in the “high-group” showed evident improvements in left ventricular size and systolic function with a significant decrease in blood pressure and cardiovascular mortality. This suggests that higher levels of circulating miR-185 determine the better clinical outcomes in DCM patients, proposing miR-185 as a new prognostic biomarker for DCM [91].Interestingly, mutations in enzymes that participate in miRNA biogenesis can also induce DCM. Studies conducted on subjects with DCM have found altered levels of Dicer expression. Dicer targeted cardiac deletion was performed in a mouse model. The result was progressive DCM, heart failure and early postnatal lethality [141]. This led to a reduction in mature miRNA levels and an increase in pre-miRNA levels. Dicer expression was reduced in human patients with DCM or heart failure. However, therapeutic implantation of a left ventricular assist device resulted in an increased Dicer expression and improved cardiac function [141].Despite all these data, further studies will have to be carried out to demonstrate a unique relationship between miRNA and cardiomyopathies in general; therefore, they can be used as early biomarkers of disease or for possible gene therapies.Arrhythmogenic cardiomyopathy (ACM) is a relatively rare genetic disease of the heart muscle with a frequency of 1:5000 [142,143,144,145], characterized by palpitations, syncope and/or cardiac arrest secondary to ventricular tachycardia (VT) or fibrillation; ventricular dysfunction and heart failure and high risk of SCD may also develop in some patients [146]. Structural changes include dilation of the right ventricle, aneurysms, abnormalities of regional wall movement, fibrosis, adipose infiltration and impaired ventricular function.Histologically, cardiomyocyte death, inflammation and progressive substitution of adipose or fibro-adipose ventricular cardiomyocytes are observed [147,148,149]. Consequently, there is progressive atrophy of the ventricular myocardium which interferes with the conduction of the electrical impulse [98,150]. The disease progresses outwardly, first involving the sub-epicardial tissue and extending towards the endocardium, eventually resulting in a thinned and trans-mural lesion [150]. Inflammatory infiltrates are typical of the ACM and are often observed in both ventricular walls [151]. Generally, males are more frequently affected by ACM than females; therefore, it is thought that there is a correlation between the disease and sex hormones, especially on the severity of ACM [152]. ACM is a disease that exhibits variable expression and age-related reduced penetrance. Clinical symptoms typically present in the third to fourth decade of life, with arrhythmic manifestations that generally precede structural features. Sometimes ACM affects adolescents and rarely children [153]. The early onset of ACM can occur during adolescence and early adulthood and in many cases, they present with nonspecific symptoms, such as syncope and palpitations [154]. It particularly affects athletes, as intense exercise worsens the phenotype of the disease [154].It is a cardiomyopathy caused by heterozygous mutations in genes coding mainly for proteins of the desmosomal protein complex, the adhesive junctions that connect cardiomyocytes, identified in almost 50% of subjects and with low and age-dependent penetrance [155]. However, the molecular mechanisms that lead to the destruction, remodeling and arrhythmic predisposition of the myocardium remain poorly understood. ACM inheritance is autosomal dominant with incomplete penetrance being the most common mode of transmission [156], although recessive forms are also known, namely, Naxos and Carvajal syndromes, and are associated with a cutaneous phenotype [157].The most frequently mutated genes are desmosomal and cardiomyocyte junction genes [157], but mutations in non-desmosomal genes are also known [156]. Desmosomes influence intracellular transduction signals via the Wnt pathway, which is impaired in patients with ACM [92]. Cardiomyocytes form electrical and structural connections due to desmosomes, adherent junctions and gap junctions, located at the intercalated disc [158,159]. The most affected genes are on JUP and DSP [160,161,162], and truncating and missense mutations in the desmosome genes PKP2 (encoding plakofilin 2), DSG2 (encoding desmoglein 2) and DSC2 (encoding desmocollin 2) in patients with ACM [163,164,165]. About half of ACM patients have mutations in one or more of these desmosomal genes [166,167]. PKP2 is the most commonly affected gene in adult cohorts [153,163], while some studies have suggested that the pediatric age group has more frequent mutations in the DSP [153,168].Mutations have also recently been identified in the adherent junction genes CDH2 (encoding cadherin 2) [169,170] and CTNNA3 (encoding catenin-α3) [171] in patients with ACM.Less frequent, there are also mutations in non-desmosomal genes. It is included genes that are involved in cytoskeletal architecture, calcium manipulation, sodium transport and cytokine signaling [172,173]. After desmosomal genes, genes encoding cytoskeletal proteins constitute the second largest category of ACM-associated mutations. These defects alter the architecture of cardiomyocytes [174,175]. The genes affected are DES (coding for desmin), LMNA (coding for lamina A), TMEM43 (coding for transmembrane protein 43), TTN (coding for titin) and FLNC (coding for filamin C).These pathways include canonical and non-canonical WNT signaling, the Hippo-Yes-associated protein (YAP) pathway and transforming growth factor-β signaling. Despite the discovery of multiple pathogenic genes, a large percentage of patients (35–50%) have no identifiable disease-associated variants. It suggests a more heterogeneous and complex etiology, with both polygenic and environmental factors contributing to the phenotypic expression [175,176]. For patients with an identifiable genetic cause, the exact biological mechanisms underlying this diverse and pleiotropic disease remain poorly characterized. Increasingly, asymptomatic relatives with variable penetration disease can be detected by cascade family screening [176].Diagnosing ACM can be challenging and requires a high degree of clinical suspicion as well as supporting diagnostic tests. Primarily, it is based on a scoring system of criteria that include structural and electrocardiographic changes, tissue characterization, previous arrhythmic events and family history. In selected cases, a genetic test is recommended [168]. Criteria for the clinical diagnosis of ACM were defined by the International Task Force (ITF) to inform the diagnostic process and improve consistency between research studies [168]. These criteria consider cardiac morphology and function, tissue characterization, electrical rhythm conduction and family history, including identification of pathogenic mutations.The clinical heterogeneity of the ACM and its incomplete penetrance suggests that there are also other mechanisms, such as the involvement of more than one pathogenic allele and epigenetics [177,178]. These two factors contribute together with known gene mutations to the severity of the disease. Therefore, how the synergy of genetic, epigenetic and environmental factors acts to modify the phenotype and the onset of the disease is crucial for understanding the pathophysiology of the disease [179]. Unfortunately, few studies have evaluated the miRNAs circulating in the ACM.Most of the miRNA identification studies were performed on serum or plasma samples, but also myocardial tissue samples from subjects with ACM. As previously reported, circulating miRNAs are extremely stable, often found in association with exosomes or proteins and represent potentially informative biomarkers [180]. The underexpression of specific miRNAs on tissues and the overexpression in the circulation lead us to hypothesize that miRNAs may be released into the circulation by apoptotic or necrotic cardiomyocytes. Consequently, elevated levels of a specific miRNA can indicate disease progression, as its level increases as more cardiomyocytes die. This evidence suggests that circulating overexpressed miRNAs may be potential prognostic biomarkers. Conversely, miRNA overexpression in tissue could indicate cardiomyocyte malfunction.Zhang and colleagues studied miRNA profile expression in 24 histologically confirmed ACM patients compared with controls. They evaluated 1.078 miRNA levels by RT-PCR and identified 21miRNAs differentially expressed in ACM samples [93]. The data obtained were validated and the analysis of the ROC curve was performed to determine whether these miRNAs have diagnostic power. The conclusion was that the overexpression of miR-1251, miR-21-3p, miR-21-5p, miR-212-3p and miR-34a-5p and the downregulation of miR-135b, miR-138-5p, miR-193-3p, miR-302b-3p, miR-302c-3p, miR-491-3p, miR-575, miR-4254 and miR-4643, allowed discrimination between ACM and healthy controls. In silico analyzes suggested the correlation of two miRNAs (miR-21-5p and miR-135) with the Wnt and Hippo pathways, which have been associated with the pathogenesis of ACM [92,181]. MiR-21-5p and miR-135 may play an important role in the regulation of Wnt/β-catenin and Hippo signaling pathways resulting in the phenotypic manifestation of ACM [93].In 2017, Sommariva and colleagues identified miR-320a as a potential plasma biomarker of ACM. In particular, they correlated the ACM and low plasma levels of miR-320a [94]. In this study, 377 miRNAs present in the plasma of three ACM patients and three healthy controls were screened [94]. One-hundred-and-twenty-one miRNAs were detected in all plasma samples and five showed potential differential expression between ACM patients and controls. When these five miRNAs were evaluated in 36 ACM patients and 53 healthy controls, only miR-320a exhibited significantly lower expression. Plasma levels of miR-320a showed a 0.53-fold difference in expression between ACM and healthy control. Furthermore, the miRNA expression profile in ACM patients was also compared to patients with idiopathic ventricular tachycardia (IVT) and miR-320a showed 0.78-fold lower expression, suggesting not only miR-320a as a potential biomarker for patients with ACM, but also as a potential discriminatory biomarker for ACM vs. IVT. Plasma levels of miR-320a were not influenced by intense physical activity, so the authors hypothesized that the differential expression of miR-320a is independent of the increase in mechanical elongation and adaptive cardiac remodeling induced by the training [94]. Furthermore, miR-320a appeared to have mechanistic implications in the pathogenesis of ACM. The diagnostic value of miR-320a must be validated in larger cohorts of patients with ACM.A similar study was performed on 62 patients with ventricular arrhythmia (VA), where 28 had definite ACM, 11 had borderline ACM and 23 had IVT [95]. In this study, they observed plasma levels of miR-144-3p, miR-145-5p, miR-185-5p and miR-494 with significantly higher expression in ACM patients with VA than in healthy controls. Among these, miR-494 levels appeared to have a central prognostic value because it was linked to recurrent VA after ablation [96]. Furthermore, based on in vitro results, a possible correlation between high expression of miR-494 and the apoptotic process that occurs in ACM hearts, although the role of miR-494 in apoptosis is unclear [96].Depending on how miRNAs are dysregulated when the heart is under stress, their manipulation has great potential for developing new treatments to restore the normal phenotype.The central action of miRNAs is to suppress protein expression by binding and silencing specific target mRNAs, which reduce protein synthesis. The overexpression of a miRNA will suppress its direct targets, while the inhibition of endogenous miRNAs will decrease the expression and therefore gene inhibition is lacking. Therefore, miRNAs constitute extremely attractive targets for possible therapies [182,183].The strong impact of miRNAs on the cardiac phenotype is of particular interest in the possibility of targeting these molecules as therapeutic substrates.The most effective way to use miRNAs as drugs is to modulate intracellular levels of miRNAs by transfecting target cells with miRNA mimics or inhibitors (Table 3). However, these methods require the development of efficient methods for cell/site-specific release, which we have not been able to achieve for the moment. As reported in the introduction of this review, a single miRNA can affect several genes at the same time. This offers miRNAs both an advantage and a disadvantage over conventional drug therapies, which traditionally target a single target within a cellular pathway [184]. To date, few clinical trials are in Phase I that show significant clinical promise of miRNAs. Only one treatment for chronic hepatitis C has managed to move into phase II clinical trials.Currently, the activity of miRNAs can be modulated by different approaches based on the imitation of the functions of miRNAs or the silencing of their function due to the use of miRNA inhibitors (antagomiRNAs and miRNAsponges) (Figure 3) [7].If miRNAs levels are compromised in any way, exogenous miRNAs can be administered in vivo. MiRNAmimics are small chemically synthesized double-stranded RNAs that mimic endogenous miRNAs and cause gene silencing. One strand of this molecule is identical to the native form of the miRNA, the other is complementary. The double-stranded structure is needed so the RISC complex can accurately recognize miRNAmimics [185].AntagomiRNAs are synthetic single-stranded RNAs made up of nucleotides complementary to an endogenous miRNA so that they can be silenced. The inhibitors are complementary to the entire sequence or the seed sequence of the miRNA [185]. This method allows the repression of an over-regulated miRNA by blocking its inhibiting action [186].MiRNAsponges are another approach to reduce miRNA levels. MiRNAsponges are exogenous transcripts that contain complementary regions for multiple miRNAs that have the same target site. The release of miRNAsponges into a cell determines their binding to the target miRNAs and reduces the number of free and active miRNAs [185].To enter the cell, these external miRNAs have to pass the lipid bilayer of the cell membrane. The lipid bilayers allow small neutral and slightly hydrophobic molecules to passively diffuse through them, while preventing large, charged molecules, such as RNA, from passing through them [187]. To do this, an approach has been developed that exploits the use of chemically modified liposomes or cationic polymers with specific ligands for the receptors on target tissues to improve the ability of nanoparticles to bypass the plasma membrane and enter target cells [188]. These modifications allow cellular uptake of exogenous miRNAs via receptor-mediated endocytosis.Alternatively, other systems exploit recombinant viral systems such as lentiviruses, adenoviruses and adeno-associated viruses (AAVs) as vectors.Currently, viral vectors are best suited for delivery miRNAs in the myocardium [189]. Viruses have already been used for some time to target genetic material in a given cell. In recent studies, excellent results have been obtained at the cardiac level through the use of lentiviruses and AAVs.In one experiment, miR-378, a regulator of cardiac hypertrophy, was administered via AAV9 (a serotype with cardiac tropism) in vivo, improving cardiac function [190]. AAV9 administration has also been exploited to treat dilated cardiomyopathy (DCM) in mouse models. The resulting is overexpression of miR-669a, downregulated in DCM. This increase in miR-669a levels reduced cardiac fibrosis, hypertrophy and apoptosis of cardiomyocytes for up to 18 months [191].Currently, AAV cardiotropic viruses achieve efficient miRNA delivery in cardiomyocytes [190]. However, this mechanism can only be used to inhibit overexpressed miRNAs. The use of antagomiRNAs or miRNAsponges are not suitable methods for the overexpression of an endogenous miRNA. However, antagomiRNAs have been observed to have low efficiency in the heart and vascular system. Therefore, the use of exogenous miRNAs for cardiovascular applications will require solutions for local or specific cell type release.Another method to allow the entry of miRNAs into cardiomyocytes is ultrasound-mediated sonoporation [192]. It uses albumin-coated microbubbles, which carry genetic material to target sites. Microbubbles are gas-filled acoustic microspheres that explode with ultrasound and release their contents to the target site [193]. The ideal would be to develop new techniques that use electromechanical mapping [194] or the use of positron emission tomography to study blood flow to carry miRNAs into cardiomyocytes [195].Unlike conventional drugs, which are specific for a cellular target, a single miRNA does not act on just one target, but on multiple ones. The multiple mechanisms of action of miRNAs can cause numerous side effects if they are released into the bloodstream. Potentially, most miRNA-based therapies would act systemically, which could preclude widespread clinical use. The development of insertion method of highly site-specific miRNA could be a step forward for effective clinical use, leading to the development of miRNA-based therapies [196].In the future, it is hoped that exploiting miRNAs will provide an effective therapeutic tool in the field of vascular and cardiovascular biology. Monogenic therapy has had limited success, and a single miRNA has far greater therapeutic potential with its unique ability to alter complex genetic networks. Therefore, it is hoped that miRNAs will become a new line of treatment in multiple human diseases. However, at the moment, the limitations are greater than the potential therapeutic benefits.The discovery of miRNA changed our understanding of gene expression regulation. Indeed, miRNAs can regulate the expression of proteins at the post-transcriptional level and are involved in cardiovascular physiology, while their expression is altered in various cardiovascular diseases. Many different biological events interact to determine the cardiovascular phenotype and its response to injury or stress. Due to their multitarget ability, a multitude of miRNAs is involved in these processes. Studies on miRNAs in cardiomyopathy could represent an important advance both for their use as biomarkers and for their therapeutic potential [7]. Treatment strategies currently focus on the systemic delivery of exogenous miRNAs. Exogenous miRNAs act by miming the function of endogenous ones, while exogenous complementary sequences bind miRNAs and limit their role, but every miRNA act on multiple genes. Systemically delivered drugs reach virtually every body district, acting also in non-desired organs and potentially leading to side effects and iatrogenic pathology. Due to their lack of targeting, future efforts should be aimed at evaluating site-specific strategies. For the cardiovascular system, targeting could be achieved by the use of adeno-associated viruses as vectors for the release of miRNAs or antagomiRNAs linked to nanoparticles or miRNA mimics [197].In hereditary cardiomyopathies, miRNAs could provide an answer to the search for factors responsible for broadly variable expressivity and thus bridge the genotype–phenotype gap, improving the therapeutic strategy. Research has mainly focused on identifying the mechanisms within a single tissue or cell type. However, care must be taken because the regulation of miRNA protein expression is highly dependent on the context and cell type. Therefore, their ubiquitous expression makes the side effects of miRNA therapies unpredictable. Targeting individual miRNAs, therefore, requires a meticulous evaluation of systemic effects.Although several studies have identified several altered circulating miRNAs in the plasma or serum of patients with cardiomyopathy, these are differentially expressed across disease phenotypes and are potentially usable as a novel early non-invasive biomarkers. To achieve this, however, more in-depth studies and standard protocols on the mechanics of these non-coding RNAs and their validation in larger cohorts of patients are needed to use them as disease biomarkers. In the same way, it is essential to develop faster analytical technologies, to make the use of miRNAs as biomarkers effectively competitive with, respect to current techniques.This research received no external funding.The authors declare no conflict of interest.Graphical representation that a miRNA can control several genes (A) and that one gene can be controlled by different miRNAs (B).Graphical representation of miRNA biogenesis and how they inhibit mRNA translation at the post-transcriptional level.Different approaches to using exogenous miRNAs as gene therapies. (a) Endogenous miRNA (blue) binds to the seed sequence present in the 3′-UTR of the mRNA target; (b) representation of miRNA mimic (red), a synthetic double-stranded RNA molecule. The double-stranded structure mimics the endogenous miRNA*duplex that binds RISC to inhibit translation of the mRNA target. (c) AntagomiRNA (brown) synthetic miRNA complementary to the target miRNA that has to be inhibited. (d) miRNAsponge(orange) synthetic oligonucleotide containing several seed sequences for different multiple miRNAs.Brief description of the main ncRNAs.Expression of the different miRNAs in human cardiomyopathies and the main target genes according to Targetscan (http://www.targetscan.org/vert_71/ accessed on: 17 October 21).The potential advantages and disadvantages of using miRNAs as an alternative therapy to conventional drugs.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Understanding the function of a locus is an issue in molecular biology. Although numerous molecular data have been generated in the last decades, it remains difficult to grasp how these data are related at a locus. In this study, we describe an analytical workflow that can solve this problem using the knowledge available at the single-nucleotide polymorphism (SNP) level. The underlying algorithm uses SNPs as connectors to link biological entities and identify correlations between them through a joint bioinformatics/statistics approach. We demonstrate its application in finding the mechanism whereby a mutation causes a phenotype and in revealing the path whereby a gene is regulated and impacts a phenotype. We translate our workflow into publicly available shell scripts. Our approach provides a basic framework to solve the information overload problem in biology surrounding the annotation of a locus and is a step toward repurposing GWAS data for new applications.Over the past several years, high-throughput studies have generated omics data for various biological entities, including functional features and phenotypes. These data are usually generated, analyzed and published separately due to logistical/technical restraints. Genome browsers then add these data to their repertoire and make them available as annotation tracks. This, however, has created an information overload problem. For example, a biologist that wishes to know the function of a locus starts the task by looking at a stack of annotation tracks provided by a genome browser and investigates their connections visually. However, this is a tedious task and often not fulfilling. In this study, we describe a different approach that statistically evaluates the relationship between biological entities that converge at a locus and provides the user with a report for decision making.Our approach relies on summary association statistics from GWAS studies, which are becoming increasingly available for various functional features and phenotypes. GWAS studies uniformly report their association findings with regard to SNPs. Therefore, SNPs can be used as common identifiers to investigate correlation between biological entities. This is valuable because, by scanning various entities converging at a locus and identifying those that interact, we can understand the function of a locus, which could represent a rare variant (mutation), a gene or an unannotated genomic region. In the next sections, we describe our approach and demonstrate its application through known examples for a rare variant and a gene. We provide freely available shell scripts that carry out the tasks and generate the report.Advances in sequencing technology allow us to capture rare variants efficiently; however, often, it remains elusive among the identified variants which variant causes the phenotype. Furthermore, it is important to know the mechanism whereby a rare variant triggers a phenotypic change for therapeutic purposes.From the molecular perspective, a rare variant is expected to cause a phenotype by disrupting a functional feature, i.e.,Rare variant → Functional feature → PhenotypeTherefore, if we detect that the above condition is true for a variant;
+      (i)We have identified the variant that causes the phenotype(ii)We have identified the underlying mechanismWe have identified the variant that causes the phenotypeWe have identified the underlying mechanismHowever, how can we relate a rare variant to a functional feature and the functional feature to the phenotype? The solution is SNPs. Traditionally, SNPs have been used to study both the genetics of phenotypes and the genetics of functional features. Therefore, if we can find a set of SNPs in a region that meet the following criteria, we have fulfilled our aims ((i) and (ii)):(a)The location of the rare variant overlaps with the coordinate of the SNP set(b)The SNP set is associated with the functional feature and the phenotype(c)The functional feature causes the phenotypeThe location of the rare variant overlaps with the coordinate of the SNP setThe SNP set is associated with the functional feature and the phenotypeThe functional feature causes the phenotypeBased on these requirements, we devise the following analytical pipeline (Figure 1). We review each step and the reasoning behind it below.Haplotype mapping: The search starts by finding the haplotype block that the queried mutation resides within. After finding the haplotype block, our algorithm retrieves the list of SNPs within the block to identify corresponding functional features. A haplotype block is a genomic region whereby SNPs within it are in linkage disequilibrium (LD). Haplotype mapping has been traditionally used to identify genes for Mendelian diseases. A mutation that arises in a block is expected to cause the disease by impacting the function of the block. Therefore, haplotype mapping is a viable step towards narrowing the search space for functional features.We estimated the boundaries of human haplotypes using PLINK (v 1.9) [1], based on the definition of blocks suggested by Gabriel et al. [2] and using the European sample (n = 503) of the 1000 Genomes Project. The majority of the existing GWAS data come from studies conducted in European populations and choosing a different population could cause downstream issues such as population stratification; besides, the average size of haplotype blocks in the European population is higher than in the African and Asian populations, which is beneficial in genetic studies. Nonetheless, our pipeline is flexible and if a researcher wishes to use a different definition of a block or use data from another population, this can be done by simply replacing the input file for haplotype blocks with another file.It is also important to note that not all of the human genome is within haplotype blocks; therefore, in situations where a variant cannot be assigned to a haplotype block, our algorithm searches for nearby SNPs that are within 5 Kbp of the mutation (locus), and if the search result is null, it increments its search window by 10 Kbp and stops until it reaches the search window of 45 Kbp (the largest size of an average haplotype block across human populations) [2].Finding functional features: The list of SNPs obtained from the previous step then is used to identify the functional features (probes) associated (p < 5 × 10−8) with them. We use the SMR software (version 1.03) [3] for this step because it stores quantitative trait locus (QTL) data for functional features in binary format; moreover, it provides flexible options to query the data. QTL data can be obtained from previous studies; we also provide a collection of QTL data for different types of functional features (see the Data/Code Availability section).Statistical analysis: The aim of this stage is to test whether any of the functional features obtained from the previous step are associated with the studied phenotype. For this purpose, we use Mendelian randomization, which can infer causality between a functional feature and a trait. QTLs included in the instrument for the MR must be non-pleiotropic (p < 0.01), be associated with the functional feature (p < 5 × 10−8) and be in linkage equilibrium (r2 < 0.05). MR analysis was performed using the GSMR algorithm implemented in GCTA software (version 1.92) [4]. During this stage, if we find a significant association between a functional feature and a trait, we can conclude that our queried variant/locus impacts the trait through the functional feature.MR analysis requires GWAS summary input files in GSMR format. For functional features, our workflow automatically generates such files from the SMR binary files; however, for a phenotype, this file must be obtained from a previous study. Of note, we provided a script that can generate the required input file for a trait by specifying its identifier from the OpenGWAS website (https://gwas.mrcieu.ac.uk/ (accessed on 6 December 2021)), which is a repertoire of GWAS summary datasets [5].Test of interaction: The purpose of this step is to obtain additional functional insight by testing whether the identified probes contribute to the trait independently or not. The underlying script uses MR to carry out pairwise interaction analysis between the probes; however, in addition to causality (SNPs → Probe A → Probe B), it also examines the presence of pleiotropy (Probe A ← SNPs → Probe B) between the probes by keeping the pleiotropic SNPs in the instrument. This helps to identify probes that are under the regulatory impact of the same set of SNPs.Summary report: If the algorithm finds a significant association, it reports the findings in a comma-delimited file that contains statistics that describe the nature of the association between the outcome and exposure and the magnitude of association. The columns in this file are described in Table 1. In Table S1, we provide the output for the search examples underlined in the Section 3. The estimated effect size (β) is interpreted in standard deviation (SD) units. Namely, for one SD change in the level of the exposure, the outcome changes by β (assuming that the data are unadjusted). The sign of β indicates the direction of association, i.e., a positive β indicates that a higher value (level) of the exposure is associated with a higher value of the outcome and a negative β indicates an inverse association.In the following section, we explain the performance of our approach through examples of well-studied loci.PCSK1 was one of the first genes linked to monogenic early-onset obesity. It encodes the enzyme proprotein convertase 1. Substrates of PCSK1 enzyme, such as POMC, insulin, NPY, ghrelin and GLP-1, are involved in the regulation of energy homeostasis and food behavior. Patients with mutations in PCSK1 develop a profound appetite that results in significant weight gain and eventually obesity early in life [6]. PCSK1 is located in chromosome 5q15 and rare variants within this locus are reported for obesity in the ClinVar database (Table S2). In this study, we tested whether our algorithm could link the rare variants to this gene and this gene to obesity. We passed the location of a rare variant within this locus and the name of the relevant phenotype (BMI) to the wrapper script and executed it as:Bash wrapper.sh chr5:95734724 BMI_PMID30239722The first argument represents the coordinate of the variant in the human reference genome (build GRCh37). The second argument indicates the phenotype name and its study identifier (represented by PMID). Our algorithm performed haplotype mapping and retrieved the list of biomarkers (functional features) tagged by the SNPs surrounding the rare variant (chr5:95734724). It then tested the association between the biomarkers and BMI using MR and reported the result. The report indicated that PCSK1.13388.57.3, which is a biomarker that measures the level of PCSK1 in the blood, is associated with obesity (B = −0.02, p = 4 × 10−19). This finding indicates that the rare variant is among the pQTLs for PCSK1 (Figure 2a) and, as such, contributes to obesity by lowering the level of this protein in the blood (Figure 2b).APOE is involved in the metabolism of lipids, and mutations in this gene cause lipid abnormalities. We tested whether our approach could link this gene to lipid phenotypes. For this purpose, we passed the genomic coordinates of the locus (chr19:45409039-45412650) and the name of the lipid phenotype to the wrapper script to initiate the search, e.g.,bash wrapper.sh chr19:45409039-45412650 LDL_PMID24097068.gzThe result (Figure 3) indicates that a higher protein level of APOE in the blood is associated with higher LDL (B = 0.8, p = 1.2 × 10−83) and total cholesterol (B = 0.5, p = 1.3 × 10−56) and lower HDL (B = −0.14, p = 9 × 10−23). The report generated by the interaction test indicates that the APOE protein level is under the impact of the cg13375295 site. Higher methylation at this site was associated (B = −3.5, p = 4.2 × 10−9) with a lower level of APOE in the blood (Figure 3).In this study, we devised a workflow to understand the function of a locus using knowledge available at the SNP level and we demonstrated its application through examples for the PCSK1 and APOE locus. The underlying algorithm that carries out the task is written in the shell scripting language. This allows the use of parallel computing and therefore the possibility to conduct screening at phenome-/genome-wide scales. Considering the volume of existing and upcoming functional data, parallel computing will become a necessity to integrate/relate various layers of omics data in a time-efficient manner.In this study, we used Mendelian randomization to quantify the association between two entities. MR allows the incorporation of summary association statistics from large GWAS consortia and therefore provides higher statistical power as compared to traditional association studies conducted in a sample of individuals. The MR design also provides a shield against the confounding effect of environmental factors because it uses a set of independent SNPs (an instrument) to gauge the relationship between two entities and alleles of independent SNPs are allocated to offspring randomly. Of note, the use of SNPs as an instrument also brings the caveat of weak instrument bias when testing the association between entities that are highly polygenic (e.g., complex traits); however, this is a lesser issue for a functional feature that is under the regulatory impact of fewer SNPs.Rare variant characterization is a pending problem in molecular biology. Overall, there are two approaches to infer the causal impact of a rare variant on a phenotype. Bioinformatics approaches use functional data to prioritize variants and identify those that carry more functional weight and, as such, are more likely to be the causal variant. Functional data that are used in this approach were previously generated by considering the genome itself, i.e., without taking the studied outcome (phenotype) into account. In contrast, statistics approaches directly test the effect of a rare variant on the phenotype. Hence, they require a sample of subjects with available genotype and phenotype data (in order to detect a significant association), which are not always readily available. Here, we present a workflow that exploits the benefits of both approaches while addressing the shortcomings. Our approach does not require a study sample; it takes functional data into account and carries out the analysis with regard to the studied phenotype. The underlying algorithm is provided as a series of shell scripts. This gives the user the flexibility to adjust them according to the research requirements. For example, by providing a list of phenotypes, it is possible to comprehensively investigate the impact of a rare variant on the entire phenome.By using SNPs as connectors, we demonstrate that it is possible to merge various omics data and repurpose them for new applications. Such an initiative also has the potential to unite findings from different fields of omics into a single network of knowledge. This would allow biologists to better investigate the mechanism that regulates a biological entity, and to track its impact on other components of the network.Our workflow relies on the power of GWAS data to provide molecular insights; however, downloading and reformatting these data could be cumbersome for a user. This is a limitation of our approach. One solution would be to provide an online platform where data are collected and centralized and a user can obtain the results by simply entering a search term. Unfortunately, we did not receive support to set up such a platform; however, this is a direction of work for future studies that are interested in this subject. Another limitation of our approach is that QTL data are mainly available at the transcriptome, proteome and methylome levels and, to a lesser extent, at other levels of functional annotation. Therefore, it would be valuable if the practice of reporting the GWAS summary statistics for various functional elements becomes frequent. Another issue in this field of research is inconsistencies in SNP density. New studies are using denser genotyping panels and improving their coverage through genotype imputation; however, this is not always the case in older studies. This impacts the power of analyses; therefore, the development of convenient tools that make the GWAS data uniform by imputing the missing SNPs is valuable.In summary, this study provides a solution to navigate through various layers of omics data in order to understand the function of a locus. We show its application in finding the mechanism whereby a mutation causes a disease and in revealing the path whereby a gene is being regulated and impacts phenotypes. We provide freely available shell scripts that perform the analyses.The following are available online at https://www.mdpi.com/article/10.3390/cardiogenetics11040024/s1, Table S1. The output of the analyses based on search examples presented in the manuscript; Table S2. List of genomic variants within 5q15 chromosome band from ClinVar database that are reported to cause obesity/proprotein convertase 1/3 deficiency.Conceptualization, M.N., R.M.; formal analysis, M.N.; investigation, S.R.; data resources, R.M. and R.D.; draft preparation, M.N.; editing, R.M. and S.R. All authors have read and agreed to the published version of the manuscript.Supported by the Canadian Institutes of Health Research # FDN-154308 (R.M.).Not applicable, this study was done using publicly available data.Not applicable.Data, instructions and shell scripts to carry out the analyses are available from: https://github.com/mnikpay/locus-annotator.git (accessed on 6 December 2021).This research was enabled in part by computational resources and support provided by the Compute Ontario and the Compute Canada.The authors declare no conflict of interest.The design of our workflow to understand the function of a locus and its impact on a phenotype. The search starts by finding the haplotype block that the queried locus resides within. Our algorithm then uses SNPs in the haplotype block to find functional features (probes) associated with them (p < 5 × 10−8). The next step is to test the association between the probes and the trait using Mendelian randomization (MR). Our algorithm also uses MR to test whether the identified probes act together (interaction) or independently. The final step is to generate a report that summarizes the nature and the magnitude of association between each probe and the trait as well as between the probes. In Figures 2 and 3, we provide examples and interpret the meaning of the statistics. A detailed description of each step is provided in the Section 2.Impact of rare variants within 5q15 on obesity. Rare variants within 5q15 chromosome band are reported for obesity in ClinVar database. In this region, PCSK1 deficiency is known to cause obesity. We investigate whether our workflow can link variants within this locus to PCSK1 and PCSK1 protein level to obesity. The haplotype mapping indicates that the reported variants are within the coordinate of pQTLs of PCSK1 (A). MR analysis indicates subjects that are genetically susceptible to lower levels of PCSK1 in blood tend to have higher risk of obesity (B). MR was done using non-pleiotropic SNPs (p > 0.01) that are independently (r2 < 0.05) and significantly associated (p < 5 × 10−8) with PCSK1 protein level. Each point on the MR plot represents a SNP; the x-value of a SNP is its beta effect size on PCSK1 protein level, and the horizontal error bar represents the standard error around the beta. The y-value of the SNP is its beta effect size on BMI and the vertical error bar represents the standard error around its beta. The dashed line represents the line of best fit (a line with the intercept of 0 and the slope of β from the MR test). pQTL summary statistics (beta and SE) were obtained from PMID: 29875488. For BMI, we obtained these data from PMID: 30239722.Impact of APOE blood level on lipid phenotype. In this example, we tested whether our workflow could detect the association of APOE protein level with lipid phenotypes. The results indicate that the APOE level in blood is under the regulatory impact of the cg13375295 site upstream of this gene. Furthermore, we detected that a higher level of APOE is associated with higher LDL and higher total cholesterol but lower HDL. Summary statistics provided in parentheses are from the Mendelian randomization analysis using non-pleiotropic SNPs (p > 0.01) that were independently (r2 < 0.05) and significantly associated (p < 5 × 10−8) with the exposure. Each point on the plots represents a SNP, the x-value of a SNP is its beta effect size on the exposure (cg13375295), and the horizontal error bar represents the standard error around the beta. The y-value of the SNP is its beta effect size on the outcome and the vertical error bar represents the standard error around its beta. The dashed line represents the line of best fit (a line with the intercept of 0 and the slope of β from the MR test). pQTL summary statistics (beta and SE) are from PMID: 29875488; mQTL summary statistics are from PMID: 30401456 and lipid summary statistics are from PMID: 24097068.Description of the columns provided in the report file.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Arrhythmogenic cardiomyopathy (ACM) is a genetically determined myocardial disease associated with sudden cardiac death (SCD). It is most frequently caused by mutations in genes encoding desmosomal proteins. However, there is growing evidence that ACM is not exclusively a desmosome disease but rather appears to be a disease of the connexoma. Fibroadipose replacement of the right ventricle (RV) had long been the hallmark of ACM, although biventricular involvement or predominant involvement of the left ventricle (LD-ACM) is increasingly found, raising the challenge of differential diagnosis with arrhythmogenic dilated cardiomyopathy (a-DCM). A-DCM, ACM, and LD-ACM are increasingly acknowledged as a single nosological entity, the hallmark of which is electrical instability. Our aim was to analyze the complex molecular mechanisms underlying arrhythmogenic cardiomyopathies, outlining the role of inflammation and autoimmunity in disease pathophysiology. Secondly, we present the clinical tools used in the clinical diagnosis of ACM. Focusing on the challenge of defining the risk of sudden death in this clinical setting, we present available risk stratification strategies. Lastly, we summarize the role of genetics and imaging in risk stratification, guiding through the appropriate patient selection for ICD implantation.Initially assumed as developmental anomaly, the term arrhythmogenic right-ventricular dysplasia (ARVD) was used to describe a condition characterized by replacement of the right-ventricular (RV) myocardium with fibrofatty tissue [1]. Arrhythmogenic right-ventricular cardiomyopathy (ARVC) has been included in the European Cardiomyopathies Working Group classification since 1994. On that iteration, morphological abnormalities were predominantly confined to the RV with little or no left-ventricle (LV) impairment [2]. Over the recent past, the term arrhythmogenic cardiomyopathy (ACM) has been increasingly used to describe a broader spectrum of primary myocardial diseases. This emerging term encompasses morphological involvement of the RV, the LV, or both. Importantly, the hallmark feature in ACM is prominent nonischemic fibrosis/scarring and ventricular arrhythmias of genetic or nongenetic etiology [3]; the adjective arrhythmogenic is disease-specific and denotes the distinctive propensity of the ACM to develop ventricular arrhythmias due to the underlying fibroadipose myocardial replacement [4].ACM is typically inherited as an autosomal dominant trait with incomplete penetrance and variable expressivity. Naxos disease and Carvajal syndrome are cardio-cutaneous syndromes in which ACM is inherited as a recessive trait and associated with palmoplantar keratosis and woolly hair [5]. About half of ACM cases are caused by mutations in five genes encoding desmosomal proteins, with plakophilin-2 (PKP2) being the most involved, accounting for approximately 43% of the cases [6]; desmoplakin (DSP), desmoglein-2 (DSG2), democollin-2 (DSC2), and plakoglobin (JUP) are the others [7]. More recently, mutations in fascia adherens genes cadherin-2 (CDH2) and catenin-α3 (CTNNA3) have been identified in ACM patients [8,9].A consistent proportion (2–6%) of mutation-positive ACM patients harbors a second pathogenic variant [10], mostly among ACM patients with DSG2 and DSC2 variants [11]. Furthermore, there is strong evidence that exercise contributes to the pathogenesis of ACM, promoting disease phenotypic expression, worsening structural damage, and increasing risk of arrhythmias and sudden cardiac death [12].The intent of this review is to shed light on the newer aspects of the etiopathogenesis, diagnosis, and risk stratification of ACM, underling the necessity to reappraise the currently available rigid classification system in the clinical setting.Cardiomyocyte loss, fibrosis, adipogenesis, inflammation, and arrhythmogenesis are classical features of ACM pathophysiology. The molecular genetics of ACM indicate that it is a desmosomal disease. Desmosomes and fascia adherens junctions (together forming the area composita, located at intercalated discs (ID) in the epithelium and muscle tissue) maintain mechanical adhesion, while gap junctions and ion channel complexes provide electrical continuity between cardiomyocytes [13]. Growing evidence seems to confirm that these subcomplexes form a single functional entity, the connexome [14]: a common protein interaction network controlling excitability, electrical coupling, and intercellular adhesion. Therefore, ACM seems to be not just a desmosomal disease, but rather a disease of the connexome, whose pathogenetic mechanism may include loss of mechanical integrity, altered signaling pathways, and disruption of ion channel complexes and gap junctions [15].Desmosomal and junctional gene mutations cause the disruption of the normal interactions in the area composita, altering the signaling function of the intercalated disc and, in particular, the following pathways: Wnt/β-catenin, Hippo/Yes-associated protein (YAP), and transforming growth factor-β (TGF-β), which have been implied in cardiomyocyte loss, adipogenesis, and fibrosis in ACM.Wnt ligand signaling through β1-catenin is known as canonical Wnt/β-catenin signaling [16]. β-catenin participates in intercellular adhesion and is also a transcriptional coactivator of the Wnt/β-catenin signaling pathway. This pathway is a key regulator of myogenesis versus adipogenesis and plays an essential role in heart development, as well in cardiac tissue homeostasis. Its abnormal regulation has been linked to a variety of cardiac disease conditions, including fibrosis and arrhythmias. Plakoglobin (PG), also known as γ-catenin, has some structural similarities to β-catenin, and it is an important component of the cardiac desmosome. Binding of Wnt ligands to their receptors allows the translocation of β1-catenin to the nucleus, where it coactivates target genes of the Tcf/lef transcription factor family [17]. On the other hand, in the absence of Wnt ligand–receptor, the intracellular β1-catenin is rapidly degraded by a cytoplasmic destruction complex. Nuclear PG localization, as a consequence of PG mutation, has been proposed to contribute to ACM pathogenesis by suppressing canonical Wnt signaling interfering with β1-catenin transcriptional activities, thereby enhancing adipogenesis driven by PPARγ and C/ERBα [16] (Figure 1).Mutation in catenin-α3 (CTNNA3) seems to affect the canonical Wnt/β-catenin signaling pathway in a similar manner [9]. The abnormal Wnt/β-catenin signaling pathway causes redirection of cells destined to become cardiomyocytes toward an alternative mesoderm fate [18], malformation of the outflow tract [19], and abnormal development of the cardiac conduction system [20].Moreover, Wnt ligands can activate signaling pathways that are independent of β1-catenin. This noncanonical Wnt/β-catenin signaling pathway, through its effector pathway ROCK (Rho GTPase/Rho-associated protein kinase), has been implicated in ACM-associated adipogenesis. Indeed, Rho has a key role in desmosomal architecture, and its disruption has been linked to PG nuclear localization and subsequent β1-catenin inhibition, as described in classical desmosomal mutations [21].Another intracellular signaling pathway implicated in ACM pathogenesis is the Hippo/YAP [22]. This pathway controls the activity of transcriptional coactivators that stimulate the expression of genes that promote proliferation; it is a key regulator of organ growth that has been linked to cardiomyocyte proliferation and heart size [23]. Intercalated disc abnormalities, such as impaired ID assembly, reduced ID stability, and abnormal regulation of ID gene expression by nuclear PG, seem a probable cause of the Hippo/YAP pathway deregulation [22]. Interestingly, a tight crosstalk between Wnt and Hippo/YAP pathways in controlling cardiomyocyte proliferation has been proven. Indeed, YAP interacts with β1-catenin and suppresses its nuclear translocation in presence of inhibitory Hippo kinase activity [24]. However, the Wnt-Hippo/YAP crosstalk seems more complex, happens at multiple other levels, and remains to be explored (Figure 2).In ACM, the Wnt/β-catenin signaling pathway could be suppressed, activated, or unchanged. Wnt/β-catenin signaling suppression in concomitance with Hippo pathway activation causes the inability of β-catenin to translocate into the nucleus. As a consequence, the expression of the effector of both Hippo and Wnt pathways is suppressed, and adipogenesis in enhanced [22]. Moreover, in the presence of Wnt/β-catenin signaling activation, the loss of PG leads to activation of Akt and subsequent inhibition of glycogen synthase kinase 3β (GSK3β), resulting in the stabilization of β-catenin and its translocation in the nucleus. Here, β-catenin interacts with Tcf/Lef, causing the enhanced expression of effectors such c-myc, c-fos, and cyclin D1 and promoting cardiac hypertrophy [25]. Lastly, the disruption of ID integrity can result in the increased presence of β-catenin without the involvement of Wnt/β-catenin signaling. However, the increased expression of transforming growth factor β-1 (TGFβ1), phospho-SMAD2 (pSMAD2), and Pai1 is consistent with activation of the TGFβ pathway responsible for progressive fibrosis in ACM hearts [26]. These findings suggest a central role of Wnt/β-catenin signaling in the disease pathogenesis. However, the direct causal relationship between mutant desmosomal proteins and perturbed Wnt signaling pathway remains poorly understood.Some variants of non-desmosomal genes have been linked to ACM: CDH2, desmin (DES), CTNNA3, phospholamban (PLN), sodium voltage-gated channel alpha subunit 5 (SC5A), cardiac ryanodine receptor (RYR2), filamin C (FLNC), lamin a/c (LMNA), titin (TTN), transmembrane protein 43 (TMEM43), transforming growth factor β-3 (TGFβ3), and p63 (TP63) [2]. However, albeit indirectly, some of these also play a role in cellular adhesion mechanisms [27], but their pathogenic role is controversial in ACM.SCN5A variants have been described in some ACM families [28]. SCN5A encodes NaV1.5, a pore-forming subunit of the voltage-gated cardiac sodium channel (VGSC). To date, over 300 pathogenetic variants of SCN5A have been associated with Brugada syndrome (BrS). Similarly, most of the other genes implicated in BrS pathogenesis encode cardiac ion channel proteins, suggesting that BrS is a pure channelopathy [29]. Leo-Macias et al. demonstrated that NaV1.5 and N-cadherin are implicated in adhesion and excitability molecular complexes in the intercalated disc, suggesting that NaV1.5 can contribute to intercellular adhesion strength [30]. Indeed, NaV1.5 appears to be sensitive to local mechanical forces with its proximity to mechanical junctions, conferring mechanical stability and limiting membrane deformation during the cardiac cycle [31]. This function of NaV1.5 as a modulator of cell adhesion and mechanical integrity is a likely explanation of how SCN5A can lead not only to rhythm disorders but also to structural abnormalities. Inversely, the close interconnection between mechanical and electrical subcomplexes would explain the early occurrence of major arrhythmic events in desmosomal mutation carriers, even before structural damage becomes overtly detectable.Intriguingly, the first non-ion channel-encoding gene to be implicated in BrS was PKP2, the most frequently causative gene for ACM. Cerrone et al. identified PKP2 variants in five of the 200 patients with BrS diagnosis, no mutations in BrS-related genes (SCN5A, CACNa1c, GPD1L, and MOG1), and no macroscopic signs of ACM [32]. This study experimentally demonstrated that siRNA-mediated loss of PKP2 expression in isolated cells affected the amplitude and kinetics of the sodium current (INa), exhibiting that PKP2 not only participates in intercellular coupling, but also interacts, directly or indirectly, with the VGSC complex [33].Similarly, the intensity of immunoreactive Nav1.5 has been shown to be reduced in heart sections obtained from ACM patients with conventional desmosomal mutations [34]. Therefore, a reduction in Nav1.5 abundance may be a component of the phenotype in subjects with ACM, and impaired INa could play a role in arrhythmia susceptibility in the “concealed” phase of ACM. As such, BrS and ACM could represent two clinical entities on the opposite ends of the same disease spectrum. Clinical studies have shown that BrS patients may show minor RV structural abnormalities [35], whereas desmosomal mutation carriers can develop arrhythmic events in the absence of overt structural disease [36] (Figure 3). Likewise, both PKP2 and SCN5A mutations have been associated with phenotypes on both ends of the disease spectrum, ranging from long QT syndrome and BrS to ACM [37].Mutations in Ca2+ cycling genes and impaired calcium homeostasis are described in arrhythmogenic diseases such as BrS, catecholaminergic polymorphic ventricular tachycardia (CPVT), and long and short QT syndromes, as well as in cardiomyopathies at elevated arrhythmic risk such as a-DCM and ACM [38]. Forms of ACM associated with RYR2 gene mutations were first described [39] and then reconsidered [40] due to a lack of sufficient abnormalities to maintain the initial ACM diagnosis. The RYR2 channel is responsible for the release of Ca2+ from the sarcoplasmic reticulum (SR) into the cytoplasm during the excitation–contraction process in cardiomyocytes. The gene mutations destabilize the tetrameric structure of the channel, leading to failure of Ca2+ retention in the SR. This results in enhanced spontaneous release of Ca2+, determining delayed after-depolarizations (DADs) and ventricular arrhythmia, as described in some ACM patients [41].Conventional mutation screening in desmosomal genes is performed as a gold standard to identify point mutations in ACM patients. However, as stated before, it only allows detecting mutations in about half of ACM probands. Therefore, it has been proposed to include copy number variations (CNVs) (large genomic rearrangements such as deletions, insertions, and duplications), which have been recently described in ACM but can also be omitted by conventional screening mutation, into routine genetic testing in ACM. In a study by Pilichou et al., conducted on an Italian cohort of ACM patients, genomic rearrangements were detected in about 7% of ACM probands negative for desmosomal gene point mutations, highlighting the potential role of CNVs in increasing the diagnostic yield of clinical genetic testing [42].Moreover, the contribution of whole-exome sequencing in ACM patients was highlighted in a recent study by Fedida et al. in which putative pathogenic variants were screened in 96 candidate genes associated with other cardiomyopathies and channelopathies in 22 ACM patients with negative routine genetic testing for desmosomal gene mutation. All suspected deletions were verified by multiplex ligation-dependent probe amplification (MLPA) and performed in 50 additional gene-negative probands. About 6% of large PKP2 deletions, undetectable by routine sequencing, were detected as the cause of ACM [43]. However, due to the difficulties in interpreting the many variants of unknown significance (VUS), the classification of genetic variants remains challenging, and the WES remains confined to research rather than diagnostic purposes.Nevertheless, these two studies highlight the possible role, in the future, of CNVs and next-generation sequencing analysis in expanding the diagnostic yield of routine genetic testing in ACM.ACM includes different clinical phenotypes, and Sen-Chowdry et al. for the first time described three patterns of disease expression: the classic ARVC, which primarily affects the RV, left-dominant arrhythmogenic cardiomyopathy (LD-ACM), primarily involving the LV, and the biventricular forms [44]. They defined the clinical and genetic profile of LD-ACM and suggested that idiopathic myocardial fibrosis (IMF) might be another clinical manifestation of arrhythmogenic cardiomyopathy [45]. Distinctive ECG features of LV involvement in ACM include (1) T-wave inversion in the inferolateral leads with low QRS voltages (<0.5 mV) in limb leads, and (2) monomorphic ventricular tachycardia with a right branch block (RBBB) morphology, denoting its origin from the LV. The typical LV imaging phenotype is characterized by a ventricular remodeling pattern consisting of mild LV dysfunction with no or mild LV dilatation, in association with subepicardial/mid-myocardial (nonischemic) late gadolinium enhancement (LGE) affecting the LV (predominantly the inferolateral wall regions) at tissue characterization in cardiac magnetic resonance (CMR). The degree of systolic LV dysfunction appears related to the global extent of LGE which, in the advanced disease stage, affects multiple septal and LV free-wall segments [46].In order to better define ACM, a recently published International Expert Consensus Document proposed an upgrade of the 2010 Task Force diagnostic criteria for the diagnosis of ACM phenotypic variants. The novelty of the new proposed diagnostic criteria (so-called Padua criteria) consists of the introduction of tissue characterization by CMR for detection of fibrofatty myocardial replacement of both ventricles and the addition of new ECG criteria specific to the LV involvement. The accuracy of diagnostic criteria for left-sided ACM varies according to the disease variant, whether biventricular or left-ventricular dominant. In the context of biventricular ACM, the disease specificity of the left-sided abnormalities is ensured by the concomitant fulfilment of International Task Force (ITF) criteria for the RV phenotype. On the other hand, in patients with no (or minor) clinical RV abnormalities (not fulfilling the updated 2010 ITF criteria), the diagnosis of LD-ACM cannot be achieved only considering the LV phenotypic criteria. In fact, morpho-functional and structural LV abnormalities of ACM do not provide a sufficient disease specificity because of the overlap with the phenotypic features of other heart muscle diseases such as DCM, myocarditis, and cardiac sarcoidosis. Hence, diagnosis of LD-ACM requires, in addition to consistent LV phenotypic features, the demonstration of a positive genotyping for ACM-causing gene mutation [4] (Figure 4).A subset of patients with familial DCM present with disproportionate arrhythmogenic risk considering the degree of the morphological anomalies and systolic dysfunction, with consequently increased risk of sudden cardiac death [47,48]. Current guidelines recommend ICD implantation for primary prevention in DCM patients whose ejection fraction remains <35% despite optimal medical therapy [49]. In a general nonischemic DCM population, the DANISH trial demonstrated a reduction in sudden death mortality; however, this did not translate into a substantial total mortality benefit [50]. This suggested that the majority of deaths in this population were a consequence of heart failure rather than arrhythmic in origin. Nonetheless, considering the high risk of SCD and phenotypic overlap with DCM, accurate recognition of LD-ACM which would derive maximum benefit from ICD implantation could be a crucial step in risk stratification. Applying genetic knowledge to clinical practice could impact clinical care; however, genetic screening is not routinely performed for DCM, except in individuals with concomitant conduction system disorders where genetic testing for mutations in the LMNA and SCN5A genes is recommended [51].Furthermore, while ACM is more often caused by desmosomal variants, interestingly, genetic defects in non-desmosomal genes are thought to be more frequently involved in LD-ACM. Mutations in LMNA and SCN5A have been described in ACM, as already mentioned, but are mainly responsible for a-DCM and LD-ACM. Mutations in LMNA, which encodes the nuclear proteins lamin A and C, have been identified in over 5% of familial DCM cases. Mutations in the LMNA lead to a more aggressive course as they are associated with conduction disturbances and ventricular arrhythmias [52] and the burden of ventricular arrhythmias is disproportionate to the underlying structural disease, overlapping LD-ACM. Frameshift mutations of SCN5A, classically causative of BrS, also cause a-DCM with atrial or ventricular arrhythmias that exceed the degree of LV dysfunction [28]. SCN5A is the exemplary gene of overlap across channelopathies, DCM, and ACM.Moreover, filamins anchor membrane proteins to the cytoskeleton in cardiac and skeletal muscles; filamin C binds to several proteins of the Z-disc of the sarcomere [53]. Truncating mutations have been reported in families with an overlapping a-DCM and LD-ACM phenotype; an early onset, an aggressive ventricular dysfunction with a high burden of atrial and ventricular arrhythmias, and an increased incidence of SCD were common. CMR demonstrated extensive fibrosis, mainly of the LV, and, in a percentage of patients the LGE was subepicardial and circumferential [54]. Moreover, a recent study showed fibrofatty infiltration of the LV in addition to interstitial fibrosis of the RV among patients with truncating FLNC variants [55]. Interestingly, immunostaining assays of the LV showed decreased desmoplakin staining of cellular junctions, supporting the concept of the LD-ACM and a-DCM overlap.Bermúdez-Jiménez et al. for the first time described the largest LD-ACM family with p.Glu401Asp mutation in the DES gene [56]. Desmin, encoded by the gene DES, is a structural intermediate filament present in the cytoskeleton of the leiomyocytes, rhabdomyocytes, and cardiomyocytes. More than 70% of the described pathogenic DES mutations are associated with cardiac involvement and can be related to DCM most commonly, but also to restrictive, hypertrophic, and ACM. This usually implies specific conduction system disturbance and skeletal myopathy [57]. Instead, they investigated the pathogenicity of this novel DES mutation as a cause of biventricular inherited ACM with dominant primary LV affection, without conduction system abnormality or signs of skeletal muscular involvement. A nearly exclusive LV affection was seemingly determined, with hypokinesia localized on the mid-apical inferolateral wall of the LV, mildly depressed LV ejection fraction, and no ventricular dilation; CMR revealed extensive late gadolinium enhancement with a typical circumferential subepicardial pattern in most of the cases. The p.Glu401Asp mutation is located in segment 2B of the central rod domain and could eventually produce a critical break in the intra- and interhelical ionic bridges between desmin dimers, leading to a loss of structural integrity and, consequently, of cellular adhesion [56].Moreover, a PLN mutation (R14del) has been associated with DCM, as well as with a-DCM/LD-ACM [58]. Lastly, a high-penetrance mutation in TMEM43 (S358L), encoding a nuclear envelope protein (LUMA), was identified among Canadian population with a reported association with LDAC and high risk for SCD [59].Among desmosomal genes, DSP mutations are mainly responsible for LD-ACM, and they are identified in about 3% of patients diagnosed with DCM [60]. Multiple case series identified DSP mutations in LD-ACM [44,61,62]. Smith et al. [63] described desmoplakin cardiomyopathy as a distinct nosological entity marked by a high proclivity for LV fibrosis and arrhythmias and associated with intermittent myocardial inflammatory episodes that appear clinically similar to myocarditis or sarcoidosis. They found that DSP cardiomyopathy involves the LV in almost all cases and often without any apparent RV involvement in contrast to PKP2 cardiomyopathy, which always involved the RV predominantly and most often in isolation. Interestingly, in contrast to the DSP group in their study, none of the PKP2 patients had documented episodes of acute myocardial injury. Desmoplakin interacts with intermediate filaments, binds them to desmosomal plaques [64], and seems to be capable of sensing exposure to external mechanical stresses and reacting to them [65]. Truncating DSP mutations are evenly distributed throughout the DSP coding sequence without any clear correlation between specific truncating mutations and clinical presentation; DSP loss of function leads to significant LV dysfunction. Conversely, specific missense mutations may contribute to the specific disease phenotype (e.g., mutations in the desmin versus plakophilin/plakoglobin-binding domains), less frequently leading to LV dysfunction. Furthermore, risk stratification variables that perform well for PKP2-associated ACM and DCM appear to exhibit poor accuracy for diagnosis and risk assessment for DSP cardiomyopathy. In particular, Castelletti et al. found that the standard DCM LVEF threshold of <35% was an insensitive marker for future severe ventricular arrhythmias in DSP cardiomyopathy, with many events occurring in an LVEF range of 35–55% and occasionally at an EF >55%. Even more, criteria predictive of events in “classic” ARVC, including RV systolic dysfunction, anterior T-wave inversion, and male gender would play no role in either clinical recognition or risk stratification in DSP cardiomyopathy [66]. Heliö et al. identified the DSP c.6310delA, p.(Thr2104Glnfs*12) in 10 Finnish index patients with established diagnosis of DCM; the major findings were ventricular arrhythmias and dilatation of the LV, with three of the patients dying because of arrhythmogenic events rather than ventricular dysfunction, confirming the significance of DSP gene as a cause of arrhythmogenic cardiomyopathy [67]. A genotype-specific management approach might be useful for DSP cardiomyopathy.Therefore, the distinction between LD-ACM and a-DCM in practical terms may be challenging, particularly in cases which have mild phenotypic expression or are detected early in disease course due to better cascade family screening [68].In conclusion, genetic and clinical overlaps between a-DCM and LD-ACM seem obvious, and, on these bases, an expert panel of the Heart Rhythm Society (HRS) has proposed to include a-DCM, ACM, and LD-ACM in a common nosological entity whose hallmark is electrical instability [3].Advances in our understanding of the complex pathophysiological pathways implicated in the development of cardiomyopathic phenotype obviate the need to reappraise classification systems and defined diagnostic–therapeutic pathways. Novel, flexible approaches incorporating genetic, functional, and ultrastructural characteristics beyond simple morphological features should be explored, reflecting the complexity often met in clinical practice.Pathological findings in ACM patients and laboratory models show that inflammatory infiltration can be present in two-thirds of cases [69,70]. On this basis, focus has recently shifted toward the potential role of inflammatory insults in myocardial necrosis and in the subsequent fibrotic replacement [6,68,71], raising the key question of whether inflammation is the primary cause of myocardial damage or a consequence of it.Myocardial necrosis can be acute, accompanied by substantial troponin elevation in the absence of coronary artery obstructions. In such cases, it can often be preceded by episodes of ventricular arrhythmia (clinically perceived as chest pain and palpitations) in the absence of typical pathological and ECG findings of ACM [72,73], suggesting an initial “hot phase” [74] of the disease predating phenotypical expression in its typical form. A viral origin of myocardial inflammation has been sought and, in some cases, found [75,76], but data are insufficient to establish an unequivocal cause–effect connection. On the other hand, a possible genetic predisposition to myocarditis has been demonstrated. Introducing the concept of vulnerable myocardium, Campuzano et al. analyzed sera of three patients suddenly dying because of myocarditis and found various types of mutations typically associated with ACM, even if signs of the disease were absent at autopsies [77]. Furthermore, a large clinical and genetical study of patients’ relatives unveiled further carriers of desmosomal mutations. Interesting elements were also underlined in a study conducted on cardiac sarcoidosis and giant cell myocarditis [78]; histopathological analyses of these patients showed an altered distribution of plakoglobin, desmoplakin, and plakophilin-2 not only in the damaged area but also in apparently unaffected regions. The local expression of cytokines was analyzed, and a high level of IL-17 and TNFα was found in both tissues and blood. Similar alterations were found in the serum and myocardium of ACM patients, raising the hypothesis that a local production of inflammation mediators could play a role in the pathogenesis and evolution of both inflammatory cardiac disease and ACM.A study on a murine model with mutated or knockout DSG2 demonstrated that, when myocardial necrosis is established, the immune system reacts to eliminate dead cells and repair the damage. In particular, in the early, acute stage when macroscopical phenotypic features are not yet detectable, but the risk of sudden cardiac death may already be increased, local and blood-derived macrophages are activated by neutrophils to a much higher degree compared to the chronic stage. Normally, resident macrophages (CD11b+ and CD206+) can be found in healthy hearts and participate in electrical impulse conduction, but those implicated in scar development express different markers (MMP12 an SPP1) which correspond to proteins involved in proinflammatory processes. On this basis, it is reasonable to assume that they are not equally able to participate in electrical propagation, enhancing the proarrhythmogenic feature of this phase [79]. On the other hand, B lymphocytes (CD45+) and T cells (CD3 and CD4 mainly) are more involved in the acute phase; whether their stable persistence in the chronic phase suggests a modulating effect remains to be proven.In a murine model study with DSG2 mutations, similar inflammatory cells and cytokines were involved [80]. In addition, this study showed that inhibition of GSK3β (a kinase that promotes inflammation via the NF-κB pathway) was able to reduce inflammatory cells and molecules leading to clinical improvement, reflected by less ventricular necrosis and fibrosis, better systolic function, and reduced arrhythmic episodes. The authors underlined that the presence of mutations typically associated with ACM (PKP2 in particular) could alone activate the NF-κB pathway, recalling in a certain way the concept of genetically vulnerable myocardium.Recently, an autoimmune hypothesis emerged, and three types of autoantibodies were identified, as outlined below.Anti-heart (AHA, with alpha and beta myosin heavy chain being the principal antigens identified) and anti-intercalated disc (AIDA) antibodies were found in 45% of patients with sporadic ACM and 85% of familial cases [81]. Clinically, patients AHA-positive presented with more severe LV involvement and with higher incidence of chest pain, arrythmias, ICD implantation, thicker septum and LV posterior wall, and lower LVEF; AIDA presence was associated with biventricular manifestations. Follow-up of antibody-positive relatives with no disease features was suggested as a means to elucidate whether detection of AHA or AIDA constitutes an early sign of disease.Anti-desmoglein 2 autoantibodies (anti DSG2) were studied by Chatterjee et al. [82] in a small group of genetically diagnosed patients plus sera of boxer dogs affected by ACM. AntiDSG2 antibodies were present in all subjects with definite disease, exhibiting high sensitivity and specificity for its detection. Moreover, the intensity of the immune reaction correlated closely with disease severity, as expressed by ventricular ectopic burden.Neither of the abovementioned studies could reach a conclusion on whether autoantibodies were the triggers inducing myocardial damage in genetically predisposed patients or if the presence of altered junctional proteins induced the production of autoantibodies perpetuating myocardial damage.We would like to report another potential category: antimitochondrial autoantibodies (AMA) are notoriously linked to primary biliary cirrhosis and other autoimmune diseases, in which the cardiac involvement mostly progresses in dilated cardiomyopathy [83]. A Japanese case report described an AMA-positive myocarditis with arrhythmias, severe HF, and a fibrofatty replacement of myocardium at autopsy (a genetic study was not performed), revealing a hypothetical unexplored scenario [84].In another case series [85], two brothers with a truncated variant of desmoplakin were diagnosed with ACM after multiple episodes of myocarditis following sessions of intense physical exercise. In both, autoantibodies against troponin and myosin were detectable, suggesting that exercise induced a sort of “mechanical unstable” myocardium necrosis by exposing epitopes which triggered autoimmune reaction. As for the interaction between immune system and exercise, recent evidence is intriguing; a more general analysis conducted by Campbell and Turner showed that single episodes of intense exercise recruit T cells, particularly CD8+, and redistribute them in areas with ongoing damage (necrotic or neoplastic tissues for example), thereby enhancing the immune response. On the other hand, regular intensity physical activity seems to also play a role in B-cell and antibody production [86]. Moreover, there are also data [87] highlighting how, in a patient with known ACM, adrenergic stimulation due to physical activity can indirectly activate intracellular Ca2+-mediated pathways, enhancing myocardial necrosis and a subsequent inflammatory response, in addition to impaired mechano-transduction due to an altered desmosomal structure (Figure 5). The sum of these elements could create a molecular microenvironment able to induce cardiomyocyte trans-differentiation, resulting in fibrotic and fibrofatty replacement. In Figure 3, we summarize the interplay among the many possible starters and perpetuators of the damage in ACM patients.Novel cardiac imaging techniques such as SPECT [88], CMR [89,90], or PET CT scanning [91] could be used to effectively diagnose early stages of ACM, allowing early detection of myocardial inflammation, especially when the LV is involved, triggering further genetic screening of patients presenting with acute inflammation.From a therapeutic perspective [92] immune modulation, could be a promising treatment option aimed at ameliorating disease progression and improving patient outcomes.Clinical diagnosis of ACM is often difficult because of the nonspecific nature of the disease features and the broad spectrum of phenotypic expressions. Indeed, in the early phases, the so-called “concealed” forms prevail. They are characterized by propensity toward ventricular arrhythmias in the presence of well-preserved ventricular morphology and function. As the disease progresses, the morpho-functional abnormalities, caused by myocyte loss, inflammation, and fibrofatty scar replacement (starting from the epi- or midmyocardium and extending to become transmural), become more evident [93].Considering that most ACM patients are asymptomatic and sudden death often represents the first manifestation of the disease, diagnosis can ultimately be challenging. A scoring system to establish the diagnosis was developed on the basis of the fulfillment of major and minor criteria encompassing morphologic, electrocardiographic, clinical, and genetic components according to the statement initially proposed by the international Task Force in 1994 and revised in 2010 in an attempt to achieve better diagnostic specificity. ACM diagnosis can be established as “definite”, “borderline”, or “possible” on the basis of a qualitative score including both minor (one point each) and major criteria (two points each) [94,95]. Definite ACM is diagnosed when one of the following combinations is satisfied: (i) two major criteria, (ii) one major and two minor criteria, or (iii) four minor criteria (score ≥ 4). “Borderline” is considered in the presence of at least (i) one major and one minor criteria, or (ii) three minor criteria. Lastly, the diagnosis is considered “possible” when either one major or two minor criteria are satisfied (see Table 1).Electrocardiogram (EKG) represents the first instrumental exam, often crucial to address diagnosis in ACM patients. Fibroadipose replacement interferes with the physiological conduction of the electric impulse through the ventricular myocardium, triggering electrocardiographic abnormalities in up to 90% of patients [2]. As such, a correct setting of the electrocardiograph is crucial for a correct interpretation. In particular, sensitivity can be improved by changing the 40 Hz low-pass filters (which may not detect either “fragmented” or “epsilon” waves) to 100 or 250 Hz [96]. In addition, double amplification and an increased scrolling speed (50 mm/s) might be useful [97]. EKG changes, particularly in precordial leads, depend on both the extent and the location of the disease. EKG criteria from the 2010 Task Force include T wave inversion in the precordial leads, presence of “epsilon” waves (late potentials between the end of QRS complex and the beginning of T wave), and an extension of the terminal part of QRS (nadir-end interval of the S wave >55 ms). These changes may not be manifest in at least 30% of patients. However, other nonspecific abnormalities may occur, such as ST segment changes or QRS “fragmentation” [98,99]. Late potentials by signal-averaged EKG (SAECG) represented an electrical diagnostic marker of ACM, classified in the 2010 International Task Force guidelines as a minor diagnostic criterion [46]. However, due to the technological advances by contrast enhancement CMR and the availability of molecular genetic testing for preclinical diagnosis based on demonstration of causative gene defects, use of the SAECG technique has been questioned because of its low diagnostic accuracy compared to these modern diagnostic tests [100]. In addition, data on the efficacy of late potentials in predicting clinical arrhythmic outcome are conflicting and insufficient to recommend their use for risk stratification [101]. Actually, the majority of cardiomyopathy centers no longer routinely employ late potentials in the evaluation of patients with ACM.T-wave inversion in right precordial leads (up to V3) is present in most adult ACM patients; it correlates with the extent of RV dilation and, over the years, it may extend to left leads [102]. However, other conditions associated with T-wave inversion, either physiological or pathological, must be excluded. This finding in the right leads, indeed, may manifest in young Afro-Caribbean athletes, in ischemic heart disease, in acute pulmonary embolism, and in RBBB. T-wave inversion in the inferior leads is instead associated with the “left dominant” form. Alterations in the QRS (epsilon waves, enlarged S wave, QRS fragmentation, atypical RBBB) usually reflect the extent of the scar and slower conduction in the RV. In particular, the epsilon waves correspond to a slowed perivalvular epicardial activation, whilst S wave widening is the expression of a slowed perivalvular endocardial activation and of the right infundibulum.Echocardiography is the first-line imaging modality in ACM, as well as the most commonly used tool in the follow-up of patients with ACM. Evaluation of the RV by echocardiography can be challenging due to its retrosternal position and complex geometry. Moreover, the assessment of wall motion abnormalities is highly subjective even for experienced operators. Echocardiography allows for quantitative evaluation of RV dilation and dysfunction, as well as changes in segmental kinetics (segmental akinesia, dyskinesia, or dyssynchrony). Both minor and major ITFC criteria require the presence of wall motion abnormalities, differing with regard to the extent of RV dilatation and severity of dysfunction. However, these criteria, although specific, are hampered by poor sensitivity, particularly in the early stages of the disease. Some structural alterations, such as accentuated RV apical trabeculation and thickening of the moderator band, are also poorly specific. Echocardiographic techniques, such as 3D ultrasound and speckle-tracking have proven useful to increase the sensitivity of disease detection, mostly in the early stages [103]. A recent study recognized echocardiographic strain as a useful tool to predict structural disease progression in ACM, suggesting that baseline RV free-wall strain and rate of deformation could both be useful in identifying patients at risk of disease progression, who may require closer follow-up and treatment [104]. Previous studies using serial echocardiograms [105,106] demonstrated disease progression expressed as RVOT dilatation and RV-FAC reduction in two-thirds of patients during a mean follow-up of 6.4 years. Worsening of the peak RV free-wall longitudinal systolic strain and strain rate has also been associated with an increase in the size of the RVOT, as obtained from PSAX and PLAX views. Nonetheless, echocardiography is considered less sensitive than CMR in determining structural disease progression, given the improved capacity of the latter to identify segmental RV dilatation or focal wall motion abnormalities [107].CMR represents the gold standard as compared to echocardiography, as it allows a more precise estimate of morphology, volumes, thicknesses, mass, and segmental and global kinetics of the ventricles. The presence of regional akinesia or dyskinesia and accurate RV volume assessment may lead to the diagnosis of ARVD or biventricular ACM [95]. Extensive, sometimes circumferential late enhancement has been recognized as a phenotypical feature of LD-ACM patients, and it may be the only imaging finding in carriers of FLNC truncating mutations. Thus, LGE can help differentiate patients with arrhythmic DCM and suggest potential high-risk genotypes [45,55]. CMR is able to identify the presence of both fibrous and adipose tissue [108], which, in addition to corroborating the ACM diagnosis, represents a useful tool for the prognostic stratification [3]. The usefulness of a combined evaluation of the movement of the regional wall and the characterization of tissues by CMR in ACM diagnosis has been recently reported. Highest precision (98%) is obtained when wall motion abnormalities are accompanied by pre/post-contrast alterations on tissue characterization [109]. Identification of LV involvement on CMR is associated with a negative prognostic significance. In such cases, the subepicardial/intramyocardial distribution of fibroadipose replacement may explain why early disease stages are visualized as normal in size, function, and segmentary kinetics on echocardiography [46,110]. LV systolic dysfunction is accompanied by increased LGE extent, affecting more LV segments, with a more transmural involvement [111]. Compared to the LV, assessment of late enhancement on the thin walls of the RV can be limited by spatial resolution restrictions [112,113].CMR findings such as RV dilatation, severe RV dysfunction, and segmental wall motion akinesia or aneurysms are highly specific for gene carriers. On the other hand, specificity has been reported as low as 56% for abnormal trabeculae and 44% for mild localized RV dilation and/or regional wall movement abnormalities. Interobserver variability in detecting features of ACM has been described, being more substantial among inexperienced operators [114,115,116]. Due to this reason, CMR should ideally be performed in a center with high expertise in evaluating abnormalities suggestive of ACM.In recent years, myocardial wall motion abnormalities in ACM have been assessed by CMR strain analysis using feature tracking to measure regional and global ventricular dysfunction in order to identify the patients affected in the early stages of the disease [117,118], identify those at risk of progression [105], and stratify the prognosis [119].Multidetector computed tomography (MDCT) can identify morphological features of ACM, such as increased RV chamber size, RV trabeculation, and intramyocardial fat. Modern MDCT scanners enable fast acquisition and high isotropic spatial resolution (0.5 mm), enabling precise ventricular volume measurements [120]. An advantage of 4D CT imaging is its ability to reveal the complex anatomy of RV, alleviating the risk of image overlap and limited views of standard 2D angiography. In addition to these functional parameters, a high spatial resolution, combined with the high native contrast of adipose tissue, allows a precise representation of fat infiltration within the thin wall of the RV, either with or without contrast injection [121]. CT-detected fibrosis correlated closely with epicardial and endocardial low-voltage areas on electro-anatomical mapping [122]. Fusion imaging using CT could assist ablative therapy of the culprit lesions. MDCT is widely available, fast, and affordable and can be considered a viable option in patients for whom MRI is challenging or contraindicated (e.g., those with severe arrhythmia, claustrophobia, implantable cardioverter defibrillators, or with suspicion of focal ARVC/D) [2].Once the diagnosis is established, the most challenging aspect in ACM patient management is to stratify arrhythmic risk [102]. Implantable cardioverter defibrillator (ICD) implantation is a lifelong preventive measure for SCD with an established efficacy and safety in high-risk patients [123]; however, it is also associated with both short- and long-term complications (malfunction, device infection, inappropriate shocks, and others) [124].Many single-center reports and several small multi-center registries have reported a number of clinical predictors of adverse events and death. Most of these studies focused on the outcomes of patients who already had ICDs implanted [125,126,127,128,129]. These studies have used ICD therapy for fast VT or VF as a surrogate for aborted SCD, according to the assumption that these ventricular arrhythmias would have led to death if not terminated by the device, although ICD shocks have been found to be an imperfect substitute for SCD [130]. The main clinical variables identified as independent predictors of poor outcome were history of sustained VT or VF, non-sustained VT syncope, male gender, young age at the time of diagnosis, proband status, T-wave inversion extent at 12 lead ECG, a premature ventricular complex (PVC) burden >1000/24 h, VT inducibility at EPS study, and RV and/or LV dysfunction. Only a few studies have examined risk stratification in patients with no ICD implanted. Among these, a recent study by Brun et al. concluded that sustained or non-sustained VT and LV dysfunction are risk factors for arrhythmic events [131].The International Task Force of experts from Europe and North America have produced a consensus document (ITFC) on the treatment of ACM [132], suggesting the use of a simple algorithm to stratify the risk of SCD in patient with definite ACM. According to the presence of past major arrhythmic events and defined “major” and “minor” risk factors, three categories of risk for SCD were defined: a high-risk category, with an estimated annual event rate >10%, a low-risk category, characterized by an estimated rate of arrhythmic event <1%/year, and an intermediate risk category between those two.Patients with a previous cardiac arrest due to VT or VF (secondary prevention) are considered in the high-risk category, as are those with severe RV and/or LV dysfunction. This group has a class I indication for ICD implantation. The intermediate risk group includes patients with ≥1 “major” risk factor (syncope, NSVT, and moderate RV and/or LV dysfunction), in whom it is reasonable to implant an ICD (class IIa). In the same risk group, if only minor risk factors are present (young age at the time of diagnosis, male gender, proband status, compound and digenetic heterozygosity of desmosomal gene mutations, extent of T-wave inversion, and VT inducibility at EPS), ICD implantation can be considered (class IIb) on an individual basis, especially if there are multiple minor risk factors [128]. This decision will need to take into account the overall patient clinical profile, age, device complication rates, and patient preferences. Lastly, the low-risk category includes healthy gene carriers and patients who meet diagnostic criteria for ACM but without risk factors, in whom ICD implantation is not indicated (class III).The Heart Rhythm Society (HRS) criteria proposed by Towbin et al. in 2019 [3] included emerging risk factors for SCD, such as the presence of highly arrhythmogenic mutations and extent of LV involvement [54,133,134,135]. These criteria have not been developed specifically for ACM, but rather encompass a broad spectrum of arrhythmogenic cardiomyopathy. In line with ITFC criteria, there is a class I indication for ICD implantation for secondary prevention of major arrhythmic events and a class IIa indication for patients who have experienced hemodynamically tolerated sustained VT or a recent syncope. If none of the above criteria are met, the presence of major risk factors (non-sustained ventricular tachycardia (NSVT), inducibility at EPS, and ejection fraction of LV <49%) and minor risk factors (male sex, >1000 PVC in 24 h, RV dysfunction, proband status, and two or more desmosomal mutations) should be investigated. ICD implantation should be considered in patients with three major, two major and two minor, or one major and four minor risk factors (Figure 6).In some aspects, ITFC and HRS criteria share similarities. Both strongly indicate ICD implantation in secondary prevention of major arrhythmic events, and both provide major and minor risk factors with subtle differences. In HRS criteria, LV dysfunction has a greater weight in risk stratification than RV dysfunction, which is considered only a minor risk factor. Moreover, inducibility of VT on EPS is included among the major risk factors in HRS criteria, although its role in predicting SCD is not currently well established [136].In 2019, a new risk score model was proposed by Cadrin-Tourigny et al. to generate individualized 1, 2, and 5 year risk estimates for sustained VA in patients with definite ARVC and no prior ventricular arrhythmias [137]. This was the result of an international collaboration of 18 centers from North America to Europe, with the largest cohort of ARVC patients assembled to date. The score is available online (www.arvcrisk.com, accessed on 5 May 2021) as a “risk calculator” which uses seven easily available clinical parameters: male sex, age, recent syncope, NSVT, PVC count on 24 h Holter monitoring, leads with T-wave inversion, and RV ejection fraction. Compared to the ITFC algorithm, the 5 year ARVC risk score model led to a 20.6% reduction in ICD implantations while providing the same level of protection from ventricular arrhythmias.However, this risk score model is not without issues to be considered. Firstly, patients were predominantly Caucasian, and pathogenic variants, when available, were primarily identified in the PKP2 gene (76%); DSP and DSG2 mutations were identified only in 7% and 5% of cases, respectively. Hence, the effectiveness of this risk score to predict events in patients of other ethnic background or genotypes is not adequately explored. Secondly, a threshold of 5 year ARVC risk score to indicate ICD implantation has not yet been determined.Therefore, to date, there is no universally accepted risk stratification algorithm for ACM. Recently, an attempt to compare risk stratification strategies was made by Aquaro et al. [138]. In this study, the predictive performance of the three available prediction models (ITFC, HRS, and 5 year ARVC risk score) was validated in a cohort of 140 patients with definite ARVC. A 5 year score with a threshold of >10% appeared to be more effective for predicting arrhythmic events than the ITFC and HRS criteria. With this threshold, the 5 year ARVC risk score was able to predict 95% of events, which was 14% and 50% more than the ITFC and HRS criteria, respectively, but at the cost of ICD implantation in 81% of patients.However, it remains unknown whether this score is accurate for LD-ACM forms which do not fulfill ITFC as definite ACM. Interestingly, prevalence of DSP and DSG2 mutations in the validation cohort was higher than that of the original risk score study, also confirming its effectiveness in this subset of patients. The performance superiority of the 5 year ARVC risk score could be attributed to the fact that it was directly generated from ARVC patient data, while the other two models were based on expert consensus.Arrhythmogenic cardiomyopathy is a field of increased research interest owing to its complex pathophysiology, variable expression, and close association with risk of sudden death. Novel developments have allowed for improved understanding of all the above aspects, but major benefits in clinical outcomes are yet to be seen. Further efforts to develop a classification system are needed, focusing on more accurate distinction of phenotypes, pathophysiological substrates, and natural history of the entities comprising ACM, aimed at allowing for more clarity in clinical patient management.All authors contributed equally to conceptualization, methodology, resources, writing—original draft preparation. M.T.F. & G.P contributed to writing—review and editing. G.P. contributed to visualization. All authors read and agreed to the published version of the manuscript.This research received no external funding.The authors declare no conflict of interest.Interferences in Wnt/β-catenin pathway contribute to ACM pathogenesis. Binding of Wnt ligands to their receptors allows the translocation of β1-catenin to the nucleus, where it coactivates target genes of the Tcf/lef transcription factor family. On the other hand, in the absence of Wnt ligand–receptor, the intracellular β1-catenin is rapidly degraded by a cytoplasmic destruction complex. Nuclear PG localization has been proposed to contribute to ACM pathogenesis by suppressing canonical Wnt signaling interfering with β1-catenin transcriptional activities, thereby enhancing adipogenesis driven by PPARγ.Hippo/YAP pathway is implicated in ACM pathogenesis. The Hippo/YAP pathway is a key regulator of organ growth that has been linked to cardiomyocyte proliferation and heart size. A tight crosstalk between Wnt and Hippo/YAP pathways in controlling cardiomyocyte proliferation has been proven. Phosphorylated YAP binds to β-catenin and suppresses its nuclear translocation.Proposed mechanisms of arrhythmogenic cardiomyopathy (ACM) and Brugada syndrome (BrS). Desmosomal mutations trigger fibrofatty tissue replacement via modulation of the transforming growth factor (TGF)β1/p38 mitogen-activated protein kinase (MAPK)113, Hippo97, and Wnt/βcatenin signaling pathways. In addition, adhesion defects induce apoptosis in cardiomyocytes; together, these processes contribute to ventricular remodeling, which initially affects the right-ventricular outflow tract (RVOT). Changes in cardiac tissue prompt monomorphic ventricular arrhythmias via a reentry mechanism. By contrast, defects in Nav1.5 or associated proteins (such as PKP2) promote loss of function of the channel, inducing depolarization–repolarization defects. These abnormalities might participate in RVOT remodeling by affecting the function of cell adhesion molecules (such as cadherin 2). According to repolarization and depolarization hypotheses, ST segment elevation on the right precordial leads of the electrocardiogram is a consequence of electrical defects in the RVOT, whereas structural remodeling in the RVOT contributes to those abnormalities in BrS. Remodeling of the RVOT is a mechanism common to the pathogenesis of both ACM and BrS. Modified from Moncayo-Arlandi J, Brugada R. Unmasking the molecular link between arrhythmogenic cardiomyopathy and Brugada syndrome. Nat Rev Cardiol 14, 744–756 (2017).Diagnosis of phenotypic variants of ACM in patients fulfilling the Padua criteria. Demonstration of morpho-functional and/or structural ventricular abnormalities is required for diagnosis of each phenotypic variant of ACM. Although right-ventricular dominant (ARVC) and biventricular disease variants can be diagnosed in those patients fulfilling RV and LV phenotypic criteria, the diagnosis of left-ventricular dominant (ALVC) disease, without clinically demonstrable RV abnormalities, needs demonstration of an ACM-causing gene mutation, in association with a consistent LV phenotype. Adopted from Corrado D, Perazzolo Marra M, et al. Diagnosis of arrhythmogenic cardiomy left side dilated hearts opathy: The Padua criteria. Int. J. Cardiol. 2020.Immunity involvement in ACM. The comparison of many studies and case reports suggests that, regardless of the genesis, myocarditis seems to be at least a phase (if not the core) of ARVC pathophysiology, creating a vicious cycle which leads to arrhythmias and heart failure.Implantable cardioverter defibrillator (ICD) recommendations. ACM, arrhythmogenic cardiomyopathy; ARVC, arrhythmogenic right-ventricular cardiomyopathy; COR, class of recommendation; EPS, electrophysiology studies; FLNC, filamin-C; LOE, level of evidence; LVEF, left-ventricular ejection fraction; NSVT, non-sustained ventricular tachycardia; NYHA, New York Heart Association; PVC, premature ventricular contraction; VF, ventricular fibrillation; VT, ventricular tachycardia. Colors correspond to COR (green—class I; yellow—class IIa; orange—class IIb). Adopted from Towbin, J.A.; McKenna, W.J.; Abrams, D.J.; Ackerman, M.J.; Calkins, H.; Darrieux, F.C.C.; Daubert, J.P.; de Chillou, C.; DePasquale, E.C.; Desai, M.Y.; et al. 2019 HRS expert consensus statement on evaluation, risk stratification, and management of arrhythmogenic cardiomyopathy. Heart Rhythm 2019, 16, e301–e372.Diagnostic criteria for diagnosis of arrhythmogenic cardiomyopathy.Regional RV akinesia, dyskinesia, or bulgingplus one of the following:Global RV dilatation(increase in RVEDV according to the imaging test specific monograms for age and gender)Global RV systolic dysfunction(reduced RVEF according to the imaging test specific monograms for age, sex, and BSA)Regional RV akinesia, dyskinesia, or aneurysm of RV free wallGlobal LV systolic dysfunction (depression in LVEF according to the imaging test monograms for age, sex, and BSA or reduction in echocardiographic global longitudinal strain), with or without LV dilatation (increase in LVEDV according to the imaging test specific monograms for age, sex, and BSA)Regional LV hypokinesia or akinesia of LV free wall, septum, or bothTransmural LGE (stria pattern) of ≥1 RV region(s) (inlet, outlet, and apex in 2 orthogonal views)LV LGE (stria pattern) of ≥1 bull’s eye segment(s) (in 2 orthogonal views) of the free wall (subepicardial or midmyocardial), septum, or both (excluding septal junctional LGE)Fibrous replacement of the myocardium in ≥1 sample, with or without fatty tissueInverted T waves in right precordial leads (V1, V2, and V3) or beyond in individuals with complete pubertal development (in the absence of complete RBBB)Epsilon wave (reproducible low-amplitude signals between end of QRS complex to onset of the T wave)Terminal activation duration of QRS ≥55 ms (measured from the nadir of the S wave to the end of the QRS, including R’ in V1, V2, or V3) (in the absence of RBBB)Inverted T waves in left precordial leads (V4–V6) (in the absence of LBBB)Low QRS voltages (<0.5 mV peak to peak) in limb leads (in the absence of obesity, emphysema, or pericardial effusion)Frequent ventricular extrasystoles (>500 per 24 h), nonsustained or sustained ventricular tachycardia of LBBB morphologyFrequent ventricular extrasystoles (>500 per 24 h), nonsustained or sustained ventricular tachycardia of LBBB morphology with inferior axis (“RVOT” pattern)Frequent ventricular extrasystoles (>500 per 24 h), nonsustained or sustained ventricular tachycardia of RBBB morphology (excluding the “fascicular” pattern)ACM confirmed in a first-degree relative who meets diagnostic criteriaACM confirmed pathologically at autopsy or surgery in a first-degree relativeIdentification of a pathogenic or likely pathogenic AMC mutation in the patient under evaluationHistory of ACM in a first-degree relative in whom it is not possible or practical to determine whether the family member meets diagnostic criteriaPremature sudden death (<35 years of age) due to suspected ACM in a first-degree relativeACM confirmed pathologically or by diagnostic criteria in a second-degree relativeACM, arrhythmogenic cardiomyopathy; BSA, body surface area; CE-CMR, contrast enhancement cardiac magnetic resonance; CMR, cardiac magnetic resonance; ITF, International Task Force; LBBB, left bundle branch block; LGE, late gadolinium enhancement; LV, left ventricle; LVEDV, left-ventricular end-diastolic volume; LVEF, left-ventricular ejection fraction; RBBB, right bundle branch block; RV, right ventricle; RVEDV, right-ventricular end-diastolic volume; RVEF, right-ventricular ejection fraction; RVOT, right-ventricular outflow tract.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Genotyping divides transthyretin cardiac amyloidosis (ATTR-CA) in hereditary (ATTRv) and wild type (ATTRwt) forms. This study investigated the prevalence and clinical presentation of ATTRv in a contemporary cohort of consecutive ATTR-CA patients diagnosed at a tertiary Danish amyloidosis center. Age at diagnosis, clinical- and echocardiographic data, and transthyretin (TTR) genotype were recorded. Relatives of ATTRv patients underwent clinical phenotyping and predictive gene testing. Genetic testing in 102 patients identified four TTR variant carriers: p.Pro63Ser, p.Ala65Ser (n = 2) and p.Val142Ile. The mean age of ATTRv index patients was significantly lower compared to ATTRwt patients: 70.2 ± 1.2 versus 80.0 ± 6.2, p-value: 0.005. Evaluation of ATTRv families identified seven TTR variant carriers with a median age of 65 years (range 48–76) and three were diagnosed with ATTR-CA by DPD-scintigraphy. Family members with ATTR-CA were all asymptomatic and had normal levels of cardiac biomarkers. In conclusion, the prevalence of ATTRv in a contemporary Danish ATTR-CA cohort is 4%. ATTRv index patients were significantly younger age at diagnosis than ATTRwt patients. Non-p.Leu131Met TTR variants have reduced penetrance at the age of 65 years in which approximately half of variant carriers have asymptomatic ATTR-CA with normal LV systolic function and cardiac biomarker analyses.Transthyretin amyloidosis (ATTR) is caused by abnormal deposition of misfolded transthyretin protein that aggregates as amyloid fibrils in various organs [1]. Transthyretin is a plasma transport protein produced primarily in the liver. ATTR is divided into two subtypes; an acquired wild-type variant (ATTRwt) and a hereditary form (ATTRvt), the latter caused by genetic missense variants in the TTR gene encoding tranthyretin. Accumulation of transthyretin in the heart causes amyloid cardiomyopathy (ATTR-CA) which leads to progressive biventricular heart failure with restrictive pathophysiology [2,3,4,5]. In ATTRv, the variant transthyretin may also accumulate in other organs than the heart. Among patients with ATTRv, the nervous system is often involved with amyloid deposition in the peripheral nerves causing a phenotype with familial polyneuropathy while other TTR missense variants lead to a mixed cardiac and polyneuropathy phenotype [1]. Frequent clinical precursors of the ATTR-CA phenotype are carpal tunnel syndrome (CTS), spinal stenosis, or tendon ruptures caused by amyloid fibril accumulation in these connective tissues. These clinical precursors often precede a subsequent diagnosis of ATTR by several years [6].Data from the National Amyloidosis Center (NAC), United Kingdom (UK) using the NAC-score prognostic staging system has highlighted that the presence of a variant TTR genotype is associated with a higher disease burden and increased mortality than ATTRwt [7,8]. The different ATTRv genotypes also have prognostic impact in terms of the anticipated age-related penetrance and phenotypic disease expression, e.g., cardiac versus nervous system involvement [9]. Therefore, it is suggested that all patients with confirmed ATTR-CA undergo TTR genotyping to screen for ATTRv [1,10]. More than two decades ago the p.Leu131Met TTR founder variant associated with primary cardiac involvement was described in a large family of Danish ATTRv patients and has since been considered as the primary ATTRv variant in Denmark [9,11,12]. However, an increasing number of patients with ATTR are diagnosed due to an increased awareness of the disease and with the introduction of improved diagnostic methods that more easily raise the suspicion of ATTR-CA and subsequently confirm the diagnosis. In particular, the use of echocardiographic longitudinal strain analysis with identification of the relative apical sparring pattern in conditions with left ventricular (LV) hypertrophy (LVH) [13]. Furthermore, the increasing use of bone scintigraphy (99mTechnetium-3,3-diphosphono-1,2-propanodicarboxylic acid [99m-Tc-DPD]) has undoubtedly contributed to the increased number of patients diagnosed with ATTR-CA. This advancement in the screening process and the diagnostic algorithms used in routine clinical practice might change the conception of the prevalence and genetic spectrum of other TTR variants in Danish ATTR-CA patients [1,14]. The aims of this study were therefore to report the results of TTR genotyping in a contemporary cohort of consecutive ATTR-CA patients diagnosed at a tertiary referral amyloidosis center in Denmark; secondly, to describe the clinical characteristics among ATTRv versus ATTRwt patients at time of diagnosis; and, lastly, to report genotype-phenotype relations in identified ATTRv families.Consecutive patients who were given a definitive ATTR-CA diagnosis at the Department of Cardiology, Aarhus University Hospital in the period from January 2008 to December 2020 were included in a retrospective analysis. The study was carried out in accordance with the Declaration of Helsinki. All patients were investigated by 12-lead electrocardiogram (ECG), transthoracic echocardiography (TTE), and cardiac biomarker analysis with baseline determinations of plasma N-terminal pro-brain natriuretic peptide (NT-pro-BNP) and estimated glomerular filtration rate (eGFR). Echocardiographic parameters included measurements of LV end-diastolic diameter, LV ejection fraction, interventricular wall thickness, LV global longitudinal strain, left atrial size, and graduation of diastolic dysfunction according to standard protocols [15]. Relative apical sparring of longitudinal strain was calculated by the formula: (average apical LS/(average basal LS + mid-LS) with a cut-off value of 1.0 being suggestive for ATTR-CA [13]. Cardiac light chain amyloidosis (AL) was excluded in all patients as patients who had an abnormal plasma kappa/lambda free light chain ratio and presence of plasma- or urine monoclonal proteins. In such patients, transthyretin amyloid subtyping was made by immunostaining and protein mass spectrometry of endomyocardial biopsies [1]. In the remaining patients without suspicion of plasma cell disease, ATTR-CA was diagnosed by a positive 99m-Tc-DPD-scintigraphy (Perugini grade > 1) according to standard diagnostic algorithms [14]. Age and National Amyloidosis Center (NAC) stage at the time of diagnosis were recorded [7]. TTR genotyping was performed using next-generation sequencing methods.Next, relatives of ATTRv index patients were offered predictive genetic testing for the TTR variant by direct Sanger sequencing, and variant carriers underwent clinical phenotyping with ECG, TTE, and cardiac biomarker analysis as described previously [3]. A 99m-Tc-DPD scintigraphy was performed if ATTR-CA was suspected in variant carriers. If possible, the age and cause of death of deceased family members were recorded by interviews of family members, and if possible, DNA extracted from formalin-fixed paraffin-embedded non-cardiac tissue was tested for the identified TTR variant by direct Sanger sequencing. All alive ATTRv carriers gave written informed consent to publication of their genetic findings.Normal distributed continuous variables are expressed as mean with standard deviations (SD), and otherwise as medians with interquartile ranges. Differences between group means were compared with the Wilcoxon rank-sum test.During a 13-year period, 102 patients were diagnosed with ATTR-CA and underwent TTR genotyping (Table 1). A preliminary report from the entire ATTR-FAP and ATTR-CA cohort in the period from 2001 to 2019 has been published previously as a conference abstract [16]. In the last three years of the period (2018–2020), a 4- to 6-fold increase in ATTR-CA diagnostic activity was observed (Figure 1). All genotyped ATTR-CA patients except one were Caucasians and 93% were males. Half of the patients were diagnosed by 99m-Tc-DPD scintigraphy. The remaining patients had abnormal plasma kappa/lambda free light chain ratio or monoclonal plasma or urine protein. In these patients, transthyretin amyloid deposits were detected by use of immunohistochemistry or amyloid subtyping protein mass spectrometry of endomyocardial biopsies (Table 1). Two-thirds of patients had paroxysmal or persistent atrial fibrillation or -flutter; 25% had atrioventricular conduction disease treated with pacemaker device, and 28% had obstructive coronary artery disease with a >90% coronary artery stenosis or previous revascularization. Previous CTS surgery had been performed in 39% of the patients. None of the patients had obvious symptoms or signs of sensory-motor polyneuropathy or autonomic dysfunction.Sequencing of the coding regions of the TTR gene identified four (4%) patients, all with Danish (Caucasian) ethnicity, who carried the TTR variants: c.187C>T/p.Pro63Ser; c.193G>T/p.Ala65Ser (n = 2); and c.424G>A/p.Val142Ile. ATTRv index patients were characterized by a significantly younger mean age at diagnosis (70.2 ± 1.2, [range 69.0–71.3] versus 80.0 ± 6.2 [range 65.7–93.4]; p-value 0.005), than ATTRwt patients (Figure 2). All other clinical characteristics of ATTRv patients were comparable to ATTRwt patients (Table 1).The index patient (II-2, age 71, Figure 3, Table 2) was diagnosed with ATTR-CA after twelve months of progressive exertional limitation and dyspnea (NYHA IIB-IIIA) with subsequent development of edemas and orthopnea. Two years previously, he was diagnosed with chronic atrial fibrillation suspected attributed to hypertensive heart disease. Examinations showed severe left ventricular hypertrophy (LVH) with severely reduced LV function (LVEF 19%; LV-GLS-5.5%) and a longitudinal strain plot showing a relative apical sparring pattern. The patient complained of sensory disturbances in the radial fingers suggestive of CTS. A DPD-scintigraphy confirmed ATTR-CA being in NAC-stage 2 (NT-proBNP 3091 ng/L; eGFR 75 mL/min). A cardiac resynchronization pacemaker with a cardioverter defibrillator (CRTD) was implanted due to slow atrioventricular conduction and non-sustained ventricular tachycardia in chronic atrial fibrillation. Three years later, at the age of 74, the patient was still alive but had progressed to NAC-stage 3. There was no family history of heart failure, and the parents died 72- and 86-years old, respectively. The identified p.Pro63Ser TTR variant was considered of unknown significance since it had not been reported before in ATTR-CA patients and was absent in control exomes (https://gnomad.broadinstitute.org/, accessed on 31 October 2021).To assess the pathogenicity of the p.Pro63Ser variant family, screening was initiated. The index patient had two siblings both carrying the TTR variant. The younger brother (II-3), who was 72-years old had a pacemaker implanted due to total atrioventricular block but had no symptoms of heart failure. Thorough investigations with TTE, cardiac magnetic resonance imaging (CMR), and DPD-scintigraphy ruled out ATTR-CA, and a possible explanation for atrioventricular block and septal midwall late gadolinium enhancement on cardiac magnetic resonance imaging was a previous titer-verified Borrelia Burgdorferi infection (Table 2). The 76-year-old sister (II-1) had mild left ventricular hypertrophy on both TTE. A subsequent 99m-Tc-DPD scintigraphy showed no cardiac DPD uptake. Based on these findings, evidence for a pathogenic impact of the p.Pro63Ser variant in development of ATTR-CA could not be established.The p.Ala65Ser variant was identified in two non-related families. The index patient (II-1, age 69, Figure 3, Table 2) in family 2 was previously described [17]. In brief, the index patient was diagnosed with ATTR-CA in NAC-stage 1 (NT-proBNP 2191 ng/l; eGFR 46 mL/min) after admission for a syncopal episode. CTS surgery was performed seven years previously. A TTE showed severe LVH and endomyocardial biopsies stained positive for transthyretin. Continuous ECG monitoring revealed short runs of non-sustained ventricular tachycardia and episodes of atrioventricular block, and a dual-chamber implantable cardioverter was implanted. The patient died of septicemia after seven years of follow-up. The parents with unknown genetic status died of non-cardiac causes at the age of 83 and 92 years, respectively. The brother (II-2) and the youngest son (III-3) were variant carriers. Both were asymptomatic and had normal biomarker analyses. They were both diagnosed with ATTR-CA-based cardiac DPD uptake (Perugini grade 2–3).In family 3, the index patient was diagnosed with advanced ATTR-CA NAC-stage 3 (NT-proBNP 4452 ng/L; eGFR 26 mL/min) at the age of 69. He had significant comorbidity with diabetes, arterial hypertension, obesity, prior mitral valve annuloplasty, and CTS. Coronary artery disease was ruled out, and despite treatment with CRTD, he died after 18 months of follow-up of biventricular heart failure. Predictive genetic testing of the brother (II-2, age 69), and the son (III-1, age 48 years) was positive. However, in contrast with the affected variant carriers in family 2, both had normal biomarker analyses and Perugini grade 0 on DPD scintigraphy. Post-mortem DNA analysis of the father (I-1) confirmed a carrier status of the p.Ala65Ser variant. The father died suddenly at the age of 72 years after in-hospital cardiac arrest in relation to colorectal surgery. Two years prior to this, the father had a coronary artery bypass surgery procedure performed after which there had been considerations about pacemaker implantation due to bradycardia. The family reported a history of congestive heart failure symptoms. No data were available about ECG or TTE findings or information about other relatives.This 71-year-old index Caucasian patient (II-1, Figure 3, Table 2) presented with heart failure symptoms and a history of CTS. TTE, CMR, and biomarkers were suggestive of ATTR-CA, NAC-stage 1 (NT-proBNP 1732 ng/l; eGFR 87 mL/min), which was confirmed by a positive DPD scan and an endomyocardial biopsy with amyloid subtyping. Three years after the diagnosis, at the age of 74, the patient had progressed to stage 2 NAC. Relatives were invited for screening and the 64-year-old brother (II-3) also carried the p.Val142Ile variant and was diagnosed with an early asymptomatic stage of ATTR-CA with only mildly elevated NT-proBNP on 354 ng/l. Postmortem genetic analysis of their father (I-1) was positive for the variant. The father had died at 73 years old of prostatic cancer without a prior heart failure diagnosis. One younger carrier (III-3) was unaffected at the age of 40.Transthyretin cardiac amyloidosis is increasingly diagnosed worldwide. The increased disease awareness and availability of diagnostic DPD scintigraphy have identified the presence of ATTR-CA in approximately 13% of patients with heart failure with preserved ejection fraction and in 16% of patients with aortic valve stenosis [2,18,19,20]. Expert consensus recommends that all ATTR-CA patients should undergo TTR genotyping to rule out ATTRv [1]. In our cohort of genotyped Caucasian ATTR-CA patients with Danish ancestry, the overall frequency of identified TTR variants was less than 5%. Furthermore, we observed that the Danish ATTRv patients were amongst the youngest patients in our cohort having a mean age of 70 years, and all had a prior history of CTS. The previously reported and well-characterized Danish p.Leu131Met variant has a complete penetrance in the age span between 40 and 60 years [11]. Therefore, ATTRv patients in Denmark are typically characterized by being 10–30 years younger at the time of diagnosis compared to ATTRwt patients having a mean age of 80 years. Thus, our data indicate that genotyping in ATTR-CA patients with Danish ethnicity could be focused on those diagnosed before the age of 75 or those with a positive family history suspicious for ATTR-CA or CTS. As the ATTRv phenotype overlaps with hypertrophic cardiomyopathy, it is also recommendable to include the TTR gene in hypertrophic cardiomyopathy gene panels [21,22].The observed prevalence of ATTRv in Denmark corresponds well with the DISCOVERY study including 1007 ATTR-CA patients (83% US patients) with an overall prevalence of ATTRv of 7.4% [23]. The vast majority of ATTRv patients were African Americans (89%) and 92% were carriers of the p.Val142Ile founder variant [23]. The National Amyloidosis Center, UK, reported a much higher prevalence of ATTRv of 46.7% in their cohort with the vast majority being the p.Val142Ile variant [24]. This discrepancy in ATTRv frequencies may reflect selection bias where ATTRv patients are overrepresented at tertiary amyloidosis centers. Furthermore, the UK NAC cohort has a more mixed ethnicity in contrast with the Danish cohort, which likely explains the difference in TTR variant frequencies [24]. Moreover, our study was limited by the relatively small sample size compared to the UK NAC and DISCOVERY cohorts.The p.Val142Ile variant is very rare in Caucasians with a minor allele frequency of 0.003098% in control exomes compared to 1.62% in Africans/African American and 0.05926% in Latinos/Hispanics [25]. In UK NAC ATTRv patients, 42% had the p.Val142Ile variant and all were of African or Caribbean ethnicity [24]. In DISCOVERY, they reported four non-African Americans with the p.Val142Ile variant [23]. Surprisingly, we report a Danish family in which p.Val142Ile co-segregated with a late-onset ATTR-CA phenotype in two affected brothers with no known African ancestry. Likewise, the p.Val142Ile variant has been reported sporadically before in two English families and three Italian patients [26,27,28]. This could indicate a common worldwide founder p.Val142Ile allele originating in Africa, or that codon 142 is a mutational hotspot. A case-control study in African Americans showed that the p.Val142Ile allele is clearly associated with the risk of developing congestive heart failure, and the phenotype in the Danish p.Val142Ile carriers resemble the phenotype in African Americans and African Caribbean patients with respect to disease onset and lack of neurological involvement [29,30].Studies of TTR variant pathogenicity in terms of age-related clinical penetrance and variable phenotypic disease expression are often limited by small family sizes. However, we sought to investigate the pathogenicity of identified variants by in-depth genetic analysis of ATTRv families and clinical phenotyping of seven asymptomatic variant carriers.The ATTRv index patients with p.Ala65Ser and p.Pro63Ser variants all had CTS and developed ATTR-CA phenotypes comparable to p.Val142Ile carriers with a mean age of 70 years at diagnosis. With the inclusion of our data, the p.Ala65Ser variant has now been reported in four unrelated ATTR-CA patients [31,32]. Family studies of the variants were limited by small family sizes. Currently, our data from family 1 do not indicate any pathogenic impact of the p.Pro63Ser variant. Opposite to the p.Leu131Met variant that expresses complete age-related penetrance between the age of 40 to 50 years, the p.Ala65Ser and p.Val142Ile variant had reduced penetrance of the ATTR-CA phenotype as only three of five non-index carriers had signs of silent ATTR-CA without symptoms or elevated biomarkers [33]. Predictive genetic testing should be offered to relatives at risk of disease development to identify ATTRv patients as early as possible. Asymptomatic ATTRv patients could be future candidates for early disease-modifying treatment which is rapidly developing. Clinical trials have assessed the effect of different treatment strategies; transthyretin stabilizers that counteract tissue amyloid disposition, e.g., tafamidis, reduction in hepatic transthyretin secretion by antisense oligonucleotides (inotersen), and small interfering ribonucleic acids (patisiran/vutisiran) [34,35,36].The study has several limitations. The study included consecutive patients diagnosed with ATTR-CA a tertiary referral center over a 13-year period, but data was compiled retrospectively without a strict imaging protocol including cardiac magnetic resonance imaging. Furthermore, the study was a single-center study with a relatively small and unequal group sample size compared to other studies, which could introduce a lack of statistical power and risk of type 1 statistical errors.The prevalence of ATTRv in a contemporary cohort of Danish ATTR-CA patients was 4%, and symptomatic ATTRv index patients were characterized by a cardiac ATTR phenotype preceded by CTS and an age of approximately 70 years at the time of diagnosis. The prevalence of ATTRv may exhibit geographical variations, however, in Danish patients, genetic screening for ATTRv could be reserved to ATTR-CA patients with an age below 75 to 80 years at the time of diagnosis. Predictive genetic testing of relatives and clinical screening of variant carriers with 99m-Tc-DPD scintigraphy identifies individuals at risk of disease development and diagnoses ATTRv in early and asymptomatic stages.The authors were involved in the following different phases of the study; the conception and design (T.B.R. and S.H.P.), data acquisition (T.B.R., B.T.L., R.H.S., T.S.C., H.V., A.J.T., H.M. and S.H.P.), or analysis and interpretation of the data (T.B.R., B.T.L., A.M.D. and S.H.P.); the drafting of the paper (T.B.R., B.T.L. and S.H.P.), revising it critically for intellectual content (all authors); and the final approval of the version to be published (all authors). All authors agree to be accountable for all aspects of the work. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The study was carried out in accordance with Declaration of Helsinki. Ethical review and approval were waived for this study, due to the circumstance that genetic testing was carried out based on a clinical indication and follow clinical guidelines.All participants gave written informed consent to database inclusion, TTR genotyping and if relevant subsequent publication of data.The data presented in this study are available on request from the corresponding author. The data are not publicly available due to their containing information that could compromise the privacy of research participants.The authors declare no conflict of interest.Numbers of ATTR-CA patients diagnosed annually in the period from 2008 to 2020.Age distribution of ATTR-CA patients according to TTR genotype.Pedigrees of families with identified TTR variants. Squares indicate males and circles females. Black symbol: affected; non-filled symbol: unaffected; grey symbol: history suggestive of ATTR; crossed symbol: deceased individual. Age refers to age at death (crossed symbols), at diagnosis (black symbols), or at last evaluation (non-filled symbols). Arrows indicate index patients. Genotype-positive individuals are marked with (+) and genotype-negative individuals with (−).Baseline characteristics of 102 patients diagnosed with ATTR-CA.* Median with IQR; LV: left ventricular; NT-proBNP: N-terminal pro-brain natriuretic peptide; eGFR: estimated glomerular filtration rate; NAC-stage: National Amyloidosis Center stage [7]; TTR: transthyretin gene.Clinical phenotypes in TTR variant carriers.* At diagnosis or at last screening for ATTR-CA; ECG: 12-lead electrocardiogram; TTE: transthoracic echocardiography; DPD: 99mTechnetium labelled 3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) scintigraphy; NT-proBNP: N-terminal pro-brain natriuretic peptide; eGFR: estimated glomerular filtration ratio (CKD-EPI formula); CTS: carpal tunnel syndrome; IVS: interventricular septum; LVEDD: left ventricular end-diastolic diameter; LVEF: left ventricular ejection fraction; GLS: global longitudinal strain; RALS, relative apical longitudinal strain; LAE, left atrial enlargement (>34 mL/m2 body surface area); DD: diastolic dysfunction grade (1–4) [15]. Data from index patients are shown in bold letters.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Cardiovascular epigenomics is a relatively young field of research, yet it is providing novel insights into gene regulation in the atherosclerotic arterial wall. That information is already pointing to new avenues for atherosclerosis (AS) prevention and therapy. In parallel, advances in nanoparticle (NP) technology allow effective targeting of drugs and bioactive molecules to the vascular wall. The partnership of NP technology and epigenetics in AS is just beginning and promises to produce novel exciting candidate treatments. Here, we briefly discuss the most relevant recent advances in the two fields. We focus on AS and DNA methylation, as the DNA methylome of that condition is better understood in comparison with the rest of the cardiovascular disease field. In particular, we review the most recent advances in NP-based delivery systems and their use for DNA methylome modification in inflammation. We also address the promises of DNA methyltransferase inhibitors for prevention and therapy. Furthermore, we emphasize the unique challenges in designing therapies that target the cardiovascular epigenome. Lastly, we touch the issue of human exposure to industrial NPs and its impact on the epigenome as a reminder of the undesired effects that any NP-based therapy must avoid to be apt for secondary prevention of AS.Drug loading to nanoparticles (NPs) allows targeting to specific cells and tissues and generally increases drug potency. The latter is achieved by combinations of improved biodistribution, solubility, site-release characteristics, circulation half-life, bioavailability, and immunogenicity (for a recent general review of the topic, see [1]). An additional often-mentioned phenomenon that enhances NP effectiveness is enhanced permeability and retention (EPR). EPR refers to the tendency of NPs to accumulate at sites of poor endothelial barrier, as consequence of either abnormal angiogenesis or endothelial damage. Although a controversial concept, EPR has been documented in cancer. Conversely, the relevance of EPR in cardiovascular disease is not well understood [2].In recent years, NP technology and epigenetics have joined forces to pursue novel therapeutic strategies for human disease. Epigenetic marks are covalent chemical modifications of histones and DNA that are generally reversible [3]. The best understood epigenetic modification of DNA is methylation, the focus of this article. DNA methylation (DNAm) is catalysed by the DNA methyltransferase (DNMT) enzyme family [4]. DNMT target mainly cytosine in a 5′-CG-3′ context in mammals, resulting in the formation of 5-methyldeoxycytosine (5mC). In turn, loss of DNAm can occur passively—when DNMT activity is surpassed by the cell proliferation rate—or by active mechanisms that involve 5mC oxidation and DNA repair to reinstate the original cytosine. DNA methylation regulates gene expression in a context-dependent manner. For example, gene promoter DNA methylation generally leads to transcriptional silencing. Cellular DNAm profiles are tightly regulated during differentiation and participate in establishing tissue-specific gene expression, but stochastic changes in DNAm due to epigenetic drift also occur [5,6,7].Here, we briefly review the most recent (published in 2019–present) advances in the quest of NP-based strategies to modify the DNA methylome of atherosclerosis (AS). We keep a narrow focus on AS, as the DNA methylome of that condition is better described in comparison with the rest of the cardiovascular field. The literature was chosen among entries obtained by PubMed email alert with the ‘nanoparticle atherosclerosis’ and ‘nanoparticle’ search terms.Rudolph Virchow’s seminal work proposed that AS is an inflammatory disease of the artery almost two centuries ago [8]. AS is promoted by endothelial dysfunction and accumulation of lipoproteins, some of which undergo oxidation, into the vascular wall. In particular, small apolipoprotein B-containing low-density lipoprotein particles have been identified as highly atherogenic [9]. These early events are followed by infiltration of monocyte-derived macrophages, an inflammatory response aimed at scavenging excess vascular wall oxidized lipoproteins [10]. Lipid-loaded macrophages are referred to as foam cells. While the conditions that promote AS persist, the vascular inflammatory response fails to resolve. The resulting chronic inflammation is accompanied by discontinuous growth of the atheroma, a phenomenon that spans decades in humans [11]. The expansion of the atheroma is driven by sustained macrophage and other immune cell recruitment and migration of smooth muscle cells (SMC) from the underlying media. SMC are critical players in AS: they differentiate from contractile, quiescent cells specialized in maintaining blood pressure homeostasis, to synthetic cells capable of migration, proliferation, and extracellular matrix synthesis [12]. Additionally, cell lineage tracking indicates that the majority of foam cells in the atheroma derive from SMC [13]. These events lead to the formation of a lipid-rich fibrocellular lesion (atheroma). Single-cell phenotype analysis revealed the presence of at least 14 cell types in the atheroma, including the endothelium, both anti-inflammatory and proinflammatory macrophages, a variety of myeloid and lymphoid cells, SMC, and significant cellular interconversion—i.e., endothelial to mesenchymal transition or vice versa, and SMC displaying macrophage markers [14]. The atheroma eventually undergoes rupture in a minority of atheroma-bearing individuals, causing thrombosis that leads to the three main clinical complications of AS: myocardial infarction, stroke, and peripheral vascular disease [15]. Risk factors for AS include diabetes, hyperlipidaemia, hypertension age, male sex, and smoking, in addition to genetic predisposition and the accumulation of somatic mutations during haematopoiesis [16,17,18].DNAm is altered in AS and in virtually any disease. Disease-specific DNAm profiles are imposed by a combination of dietary, lifestyle-related, and environmental factors and stochastic mechanisms [9]. The epigenetics of AS is a relatively young but rapidly expanding field, thanks to increasingly affordable sequencing and high-coverage microarray platforms. The latter include the Illumina Infinium HumanMethylation27 BeadChip, HumanMethylation450 BeadChip Kit, and, more recently, the Infinium MethylationEPIC Kit microarrays (containing ~27,000, 450,000, and 850,000 CpG probes, respectively) [19,20]. The rapid pace of research is already pointing to plausible NP-based strategies for the delivery of drugs that target the vascular epigenome. From a general viewpoint, such novel therapies for AS promise to lower the residual cardiovascular risk—i.e., the risk that persists after normalizing blood lipid levels with drugs such as statins or fibrates [21]. Indeed, it has been suggested that AS could be an orphan disease if adult human blood lipid levels were the same as in newborns or non-primate mammals [22]. From a molecular viewpoint, epigenetic studies may identify AS-associated DNAm profiles, either inherited or acquired following exposure to risk factors. Observations from multiple independent studies suggest that DNA hypermethylation is a landmark and, importantly, a driver of AS. The comparison of human DNA methylomes of atherosclerotic and healthy vascular samples matched for artery type and donor revealed widespread DNA hypermethylation across the genome [23]. DNA hypermethylation is likely to be established at very early stages of AS or even before any detectable histological evidence of atheroma [24]. Furthermore, DNA hypermethylation increases with AS progression [25]. Accordingly, ten–eleven translocation 2 (TET2), a DNA dioxygenase that favours DNA demethylation, inhibits AS [26]. Notably, TET2 promoter hypomethylation coincides with global loss of DNA methylation and decrease of inflammatory markers in post-rupture human atheroma [27]. Mechanistically, systemic administration of DNAm inhibitors decreases the size of aortic atheromas in mice models of AS and hyperlipidaemia [28,29,30]. From a molecular perspective, DNAm inhibitors increase expression of antiatherogenic genes such as phosphatase and tensin homolog, liver X receptor, and peroxisome proliferator-activated receptor gamma and decrease proinflammatory cytokines. In the latter animal studies, the duration of the protocol was too short to thoroughly assess any adverse effects of systemic DNAm inhibitor administration.In the following paragraphs, we summarize candidate NP delivery systems able to target the DNA methylome of AS. Furthermore, although no NP-based strategy to modify the epigenome of AS has been reported to date apart from curcumin-loaded NPs (see below), we briefly review advances in the use of NPs in the AS-related field of general inflammation.A synopsis of the three main features of DNA methylome-targeting NPs discussed in this section—cargo molecules, core NP materials, and specific cell type-targeting molecules is presented in Table 1.The above-mentioned experimental advances in the description of the AS DNA methylome hint at possible NP-based epigenome targeting strategies. One interesting target is type 1 DNMT (DNMT1). Among other convincing evidence, mouse studies have detected epigenetic deregulation of the DNMT1-peroxisome proliferator-activated receptor (PPAR) gamma pathway in AS [28]. Vascular DNMT1 inhibition may be achieved by loading DNMT inhibitors (DNMTi) to NP that are functionalized with available endothelium or macrophage surface ligands [21]. A battery of non-nucleoside DNMTi exist, such as RG108, SGI-1027, hydralazine, (-)-Epigallocatechin-3-gallate, and others (see, for example [49]).In addition to biochemical inhibition by means of DNMTi, DNMT1 silencing by small interfering RNAs (siRNAs) is an obvious approach. Although to our knowledge DNMT1 siRNAs encapsulated in NPs have not been employed in vascular biology, the feasibility of that approach is clearly illustrated by the effectiveness of the chemically close microRNA (miRNA)-loaded NPs. In a notable example, gold NPs functionalized with a nuclear localization signal were loaded with a siRNA specific for a miR-211 and AS1411, an anti-cancer drug, effectively inhibited the nuclear factor kappa B-DNMT1 signalling pathway, and promoted DNA hypomethylation in an animal model of leukaemia [50]. Another study aimed to alter macrophage cholesterol efflux by activating reverse cholesterol transport and subsequently inhibit atheroma lipid accumulation and growth in a mouse model. NPs based on the polysaccharide chitosan loaded with miR-206 or miR-223 promoted the expression of ATP binding cassette subfamily A member 1, a crucial cholesterol efflux pump [51].Moreover, statins are universally known as effective blood cholesterol-lowering drugs but have been attributed an additional activity as epigenetic modifiers. A number of studies documented inhibition of DNMT activity and both inhibition and stimulation of histone deacetylases by statins (listed in [52]). Histone deacetylases participate in transcriptional control and regulation of metabolism [3]. Although these observations could explain the anti-cancer activity of statins, a recent survey disputes those conclusions [52]. Despite the current controversy, statin-loaded NPs, mostly liposome and lipid-based NPs, have been used to control inflammation in the atheroma microenvironment (see below) [31,53].Finally, a recent review of plant-derived molecules that represent candidate NP cargo for cardiovascular disease treatment reveals a multitude of molecules that are mostly untested as DNAm modifiers and therefore may provide additional epigenetic weapons to treat AS [54]. Among plant-derived epigenetically active molecules, curcumin attracted considerable attention. An ingredient of Asian cuisine, curcumin has been attributed a range of beneficial effects on human physiology and has been proposed as a potential therapeutic tool for a variety of chronic degenerative diseases [55]. It has been reported that curcumin inhibits DNMT activity [38]. Yet, poor solubility in water and low bioavailability significantly hinder the use of curcumin in experimental medicine. NP technology might bring a solution to that problem, as curcumin loaded to NPs decreased the size and improved markers of stability of the atheroma in a mouse model [39]. Time will tell whether curcumin delivers results as a candidate cardiovascular drug [56].Conventional lipid-based and polymer-based NPs have been used to successfully mitigate AS in animal models [57,58]. A relatively wide panel of molecules that specifically target macrophages and other functionally relevant cell types are available and represent critical components of NP delivery systems. Recent advances in this direction include the administration of the statin simvastatin in liposome NPs, resulting in decreased AS and augmented smooth muscle cell apoptosis in the aorta of a hyperlipidaemic rat model [31]. A significant caveat of that otherwise encouraging study is the potentially negative impact of increased apoptosis on atheroma stability (see concluding section), which will need thorough assessment. Another liposome-based delivery system exploited the liver X receptor (LXR) agonist GW3965 [32]. LXR agonists deplete the atheroma of lipids by promoting inverse cholesterol transport but, if administered systemically, increase hepatic lipid deposition. The authors demonstrate that GW3965 delivery within liposomes effectively avoids those secondary effects and still decreases AS in a hyperlipidaemic mouse model, thus confirming previous reports [33]. In this case, liposomes were targeted to the atheroma by functionalization with Lyp-1, a cyclic peptide that promotes p32 receptor-mediated internalization by foam cells. Indeed, peptide-mediated NP targeting has been successfully used in a variety of recent studies. cRGD, a peptidic ligand for integrins present on macrophage and platelet surface, was used to direct anti-inflammatory interleukin-10-loaded pluronic NPs to murine atheroma, resulting in AS mitigation [37]. Another effective targeting peptide is PP1, a ligand for the macrophage scavenger receptor AI [47]. Notably, PP1 binds to both human and murine receptors and thus may help to simplify the transition from animal to human studies. The choice of targeting peptides is not limited to specific receptor ligands. p5RHH undergoes receptor-independent internalization and can be assembled with the mRNA of choice to modify gene expression in the vascular wall [46]. Although not yet tested in vivo, a promising atheroma-targeting peptide is S2P, ligand for the endothelial scavenger receptor STAB2 (aka stabilin-2) [48]. The authors successfully synthesized S2P-conjugated poly(lactic-co-glycolic acid) (PLGA) NPs loaded with the platelet-derived growth factor receptor inhibitor imatinib. Incidentally, PLGA is an interesting topic, as it is a very popular ingredient for NP synthesis and has been recently used to deliver statins to the atheroma [59,60]. Yet, at least one study documented a proatherogenic increase in foam cell formation by PLGA NPs in a hyperlipidaemic mouse model, pointing to the need to answer important mechanistic and safety questions before any transition to clinical work can be made [61]. Antibody-mediating receptor targeting has also been successfully used in the case of lipid-based NPs loaded with anti-inflammatory fatty acids and conjugated with antibodies that recognize the endothelial adhesion molecule PECAM-1 [41]. Another example of polypeptide-based delivery system is phospholipid bilayers assembled around an apolipoprotein A-I (ApoA-I) scaffold. The ApoA-I–lipid complex is readily internalized by scavenger receptor BI and is versatile in terms of both lipid composition and nature of hydrophobic load. An obvious advantage of this system is that the NP building blocks have targeting activity per se, besides its exquisitely physiological nature. A recent review covers the applications of ApoA-I-based particles in AS imaging and therapy [42].Furthermore, non-polypeptidic targeting systems have been developed, particularly carbohydrate-based. Mannose receptors are expressed on macrophage, particularly of the M2 type. A novel mannose-based ligand was engineered that mediated highly efficient endocytosis of NPs in cultured macrophages [45]. Notably, a two-edge mannose-decorated dendrimeric NP delivery system has been developed to simultaneously target the scavenger receptor AI by RNA interference and promote cholesterol efflux with a liver X receptor ligand [62]. Predictably, those NPs significantly decreased AS and atheroma cholesterol load in hyperlipidaemic mice. Another example of carbohydrate-based targeting molecule is hyaluronic acid (HA), a ligand for the CD44 receptor. In one study, HA was both the self-assembling building block and targeting ligand for statin-loaded NPs that significantly decreased atheroma inflammation in mice [43]. A recent review summarizes the virtues of HA as a widely used, well-tolerated NP ingredient [44].Exciting, cutting-edge technology-based additions to the arsenal of promising delivery systems are macrophage or platelet plasma membrane-coated NPs. The underlying rationale is the natural affinity of those cell types for components of the atheroma. In one elegant study, oxidation-sensitive chitosan oligosaccharide NPs were coated with purified macrophage plasma membranes [34]. The NPs in question combine strong recruitment to the vascular wall due to macrophage mimicry, with specific cargo offload in the reactive oxygen species-rich atheroma, resulting in significant reduction of mouse aortic AS. As for platelet plasma membranes, they were used to coat photosensitizer-loaded upconversion NP cores [35]. This delivery system couples NPs that produce visible light upon near-infrared irradiation and are therefore suitable for deep tissue applications, with the photosensitizer chlorin e6 that generates highly reactive singlet oxygen and cell death upon visible light irradiation. The resulting NPs yielded AS mitigation in animal models, although the caveat of excessive atheroma cell death should be kept in mind (see concluding paragraph). For a recent review of the application of platelet plasma membrane-coated NPs in a range of diseases, see [36]. Interestingly, the studies mentioned in this paragraph creatively exploited reactive oxygen species in two different ways, i.e., as a pre-existing inducers of NP cargo offload in one case and as a consequence of NP cargo activity in the other.Another promising avenue is the use of recombinant lipoproteins as Trojan horses. In an elegant example, an inhibitor of the interaction between CD40 and its partner tumour necrosis factor receptor-associated factor 6 encapsulated in artificial high-density lipoprotein slowed AS progression without any detectable immune suppression, a known side effect of CD40 signalling [63].Finally, one interesting approach is the use of ß-cyclodextrin NPs. These are cargo-switching NPs that, when presented to the atheroma in a statin-loaded form, incorporate cholesterol and release statins to exert anti-atherogenic effects by compounded statin release and augmented cholesterol efflux [64]. As DNMTi such as SGI-1027 are hydrophobic, ß-cyclodextrin NPs could be engineered to achieve a double beneficial effect in the atheroma microenvironment by promoting DNA hypomethylation and cholesterol efflux.Again thanks to Virchow’s early work, inflammation is a recognized critical player in cancer; therefore, antitumoral NP-based therapy may identify potentially useful targets in the context of cardiovascular disease [65]. The transcription factor nuclear factor kappa B (NFκB) is a relevant target in AS, as it is a pivotal player in the proinflammatory response to lipoproteins [66]. NFκB together with the micro-RNA miR-221 and DNMT1 participate in the aetiology of human acute myeloid leukaemia (AML). A recent study documents the effectiveness of gold NPs coated with antagonists of that pathway, in inhibiting DNMT1 and thus targeting tumour suppression genes for promoter demethylation and transcriptional reactivation in cultured AML cells [50]. Another study focused on chronic lymphocytic leukaemia (CLL). MiR-29b expression is low in CLL, yet attempts to use it as a therapeutic tool have been frustrated by the many undesired outcomes in non-cancerous tissues. When lipid NPs targeted to specific B cells were used as miR-29b carriers, CLL cell proliferation and survival were reduced both in culture and in a mouse model [67]. Those changes were accompanied by a decrease in DNMT1 and DNA methyltransferase 3A expression, leading to re-expression of crucial tumour suppressor genes. DNMT are not likely direct targets; rather, miR-29b acts on the transcription factor SP1, which, in turn, activates NFκB signalling. Taken together, the two studies mentioned in this paragraph provide evidence that NFκB signalling is perhaps a target of NP-based epigenome modification in inflammatory diseases beyond cancer.Although the above-summarised studies are clearly promising, the documented impact of industrial NPs on human health represents a significant caveat that must guide any design of NP-based therapies. The issue is relevant in the context of AS, as the likely use of any effective antiatherogenic NP-based treatment is in relatively lengthy secondary prevention. Humans are exposed to nanomaterials contained in a variety of food, cosmetic, and personal hygiene products. Convincing evidence has linked environmental nanomaterials to cardiovascular disease and other pathologies [68]. One recently documented example is titanium dioxide nanomaterial (TiN). Maternally administered TiN aerosol increases reactive oxygen species and DNAm in mouse foetal hearts [69]. Intriguingly, adult progeny heart DNA was hypomethylated, thus pointing to a marked dynamism of the epigenome in this model. Similarly complex data were obtained in mice exposed to TiN at different ages, demonstrating demethylation in young but not old animals [70]. Additionally, TiN and a range of industrial NPs elicited modest although significant demethylation of significant portions of the genome and DNMT downregulation in a panel of lymphocytic and epithelial cell line surrogates of the physiological airway system [71].Another relevant finding is myocardial inflammation triggered by carbon NPs in a zebrafish model [72]. Although the work emphasizes environmental exposure to those specific NPs, it suggests caution when designing therapeutic strategies based on carriers chemically related to carbon NPs.Although the mentioned exposure models pose some unanswered questions and at least two surveys of a panel of metal NPs produced inconclusive data [73,74], the concerns that therapy-oriented NPs might elicit undesired outcomes need to be addressed.The idea of improving cardiovascular disease outcome by modifying the epigenome is an exciting one. Basic research is fast deepening our knowledge of the vascular wall epigenome in health and disease, thus pointing to therapeutic targets, whether genome-wide or in specific loci. In parallel, NP technology lures with ever-improving strategies for drug/bioactive molecule delivery. On the one hand, advances in the categorization and purification of proteins, peptides, and biologically active molecules will improve NP administration–recognition–release mechanisms. On the other hand, it is likely that novel NPs with geometries beyond the more traditional sphere—cubic, rod, sheet, columnar, tubules, dendrimer, and polymorphous structure (polymeric nanogels)—with unforeseen drug delivery capabilities will be tested [75]. Yet, a number of hurdles are visible on the horizon [76]. One is the choice of DNMTi. Non-nucleoside analogue DNMTi offer the advantage of acting on DNMT directly without the need to be incorporated into DNA [49]. Thus, those molecules should efficiently demethylate DNA in non-proliferating cells such as the endothelium and macrophage, without any gross alteration of genome integrity that could result in apoptosis and atheroma rupture. This last aspect illustrates the profoundly different challenges in NP-based cancer and AS therapy: in the case of cancer, cell death in the tumour mass is a desirable outcome, while in AS effective cellular phenotype control must be achieved without disrupting the integrity of the atheroma to avoid its rupture and downstream clinical complications (Figure 1). The future will tell whether non-nucleoside analogue DNMTi will dissipate the doubts raised about their effectiveness and reliability [52]. Furthermore, even if NP-based therapy passes the animal model stage, a variety of issues related to tolerability and long-term effects in humans will have to be answered to. Physical and chemical properties of NPs will have to be finely tuned to assure that off-target effects, thrombosis, blood flow, and immune and allergic responses, to mention just few parameters, are within acceptable range. The potential pitfalls of NP-based modification of the epigenome have been reviewed in detail [77]. A further issue is NP access to the atheroma. Luminal endothelial damage and the presence of microvasculature with poorly developed endothelial lining are landmarks of AS that, in principle, favour NP diffusion to the atheroma by EPR. Recent work using HA NPs showed that this scenario may be too simplistic, as endothelial continuity is unexpectedly recovered in advanced AS of hyperlipidaemic mice, thus decreasing the number of NPs reaching the atheroma [78]. The authors of the study point out that, in contrast to mice, human atheroma microvessels are well-connected to the adventitial vasculature, which should result in overall better NP accessibility to the atheroma. Further exciting work is necessary to appreciate the clinical implications of these findings.Conceptualization, S.Z.; literature search, A.C.M.-S., L.S.-S., and G.L.; writing—original draft preparation, S.Z.; writing—review and editing, A.C.M.-S., L.S.-S., and G.L. All authors have read and agreed to the published version of the manuscript.This work was supported by the Mexican National Council for Science and Technology (CONACyT) “Atención a Problemas Nacionales” Programme (grant no. PDCPN-2015-01-584 to S.Z.). CONACyT supported A.C.M.-S. with a Ph.D. Fellowship.Not applicable.Not applicable.Not applicable.S.Z. is currently applying for a relevant patent. All other authors report no conflict of interest.Challenges in NP-based AS therapy. Left, proposed therapeutic strategy consisting of lowering DNA methylation levels close to the ones observed in the unaffected portion of the artery. DNA hypomethylation is accompanied by activation of anti-inflammatory, antiatherogenic gene transcription. Right, cancer therapy-inspired strategy to erode atheroma mass by inducing cell death. The generation of reactive oxygen species (ROS) is presented as an example. Although providing extremely useful insights into mechanism of atherosclerosis, a possible pitfall of strategies aimed at inducing cell death is the risk of atheroma rupture. LXR, liver X receptor; PPARG, peroxisome proliferator-activated receptor gamma; PTEN, phosphatase and tensin homolog.Synopsis of candidate and tested NP delivery systems for DNA methylome targeting in AS.Abbreviations: Apo-AI, apolipoprotein AI; DNMTi, DNMT inhibitor; PECAM-1, Endothelial Cell Adhesion Molecule Plate 1; S2P, stabilin-2 peptide; siRNA, small interfering RNA. Other peptide acronyms were not described in the original publications.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Background: Variants in the desmoplakin (DSP) gene have been recognized in association with the pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC) for nearly 20 years. More recently, genetic variation in DSP has also been associated with left-dominant arrhythmogenic cardiomyopathy. Data regarding the cardiac phenotypes associated with genetic variation in DSP have been largely accumulated from phenotype-first studies of ARVC. Methods: We aimed to evaluate the clinical manifestations of cardiac disease associated with variants in DSP through a genotype-first approach employed in the University of Pennsylvania Center for Inherited Cardiovascular Disease registry. We performed a retrospective study of 19 individuals with “pathogenic” or “likely pathogenic” variants in DSP identified by clinical genetic testing. Demographics and clinical characteristics were collected. Results: Among individuals with disease-causing variants in DSP, nearly 40% had left ventricular enlargement at initial assessment. Malignant arrhythmias were prevalent in this cohort (42%) with a high proportion of individuals undergoing primary and secondary prevention implantable cardioverter defibrillator implantation (68%) and ablation of ventricular arrhythmias (16%). Probands also experienced end-stage heart failure requiring heart transplantation (11%). Conclusions: Our data suggest DSP cardiomyopathy may manifest with a high burden of heart failure and arrhythmic events, highlighting its importance in the pathogenesis of dilated and arrhythmogenic cardiomyopathies. Targeted strategies for diagnosis and risk stratification for DSP cardiomyopathy should be investigated.The expanding use of clinical genetic testing in patients with heart failure is facilitating new insights into the causes of dilated cardiomyopathy (DCM). DCM has been defined by the presence of: (a) fractional shortening less than 25% (>2 SD) and/or ejection fraction less than 45% (>2 SD) and (b) left ventricular end diastolic diameter greater than 117% (>2 SD of the predicted value of 112% corrected for age and body surface area), in the absence of another myocardial, valvular, or systemic etiology of cardiomyopathy [1]. Familial DCM, a subtype of DCM, is suspected when (a) two or more affected relatives with DCM meet the previously mentioned criteria or (b) a relative of a DCM patient experienced unexplained sudden death before the age of 35 years [2]. The prevalence of familial DCM is estimated to be 30–50% [1,2]. Forty percent of familial DCM has an identifiable genetic cause and over 60 genes associated with familial DCM have been reported [1].Natural history studies have demonstrated that pathogenic variants in DCM-associated genes, such as lamin A/C (LMNA), filamin C (FLNC), cardiac sodium channel NAv1.5 (SCN5A), and RNA binding motif protein 20 (RBM20), lead to a malignant arrhythmogenic phenotype, which can be unrelated to the degree of left ventricular (LV) dysfunction [1]. Arrhythmogenic DCM, which has been found in one-third of DCM patients [3], has emerged as an overlap phenotype with arrhythmogenic cardiomyopathy (ACM) and arrhythmogenic left/left-dominant arrhythmogenic cardiomyopathy (ALVC) [4]. The lack of specific and widely accepted diagnostic criteria for ACM and ALVC has limited the recognition and categorization of these entities.The desmosome and its components regulate cell–cell communication. Desmoplakin (DSP), a desmosomal protein essential to cardiac force transmission, functions as a linker protein and connects the desmosome to a network of cytoskeletal proteins, including intermediate filament proteins [5,6,7,8]. Disruption in these desmosomal structural components can result in myocardial structural and electrical alterations. Recently, the ACM/ALVC phenotype has been identified in association with genetic variation in desmin (DES), the gene that encodes a multipurpose intermediate filament protein [9,10].Genetic variation in DSP has been causally implicated in arrhythmogenic right ventricular cardiomyopathy (ARVC), with more recent suspicion in the pathogenesis of ALVC. A majority of the investigation of DSP-associated disease has been performed in cohorts of individuals with ARVC [11,12,13,14,15,16,17,18]. Emerging data from ACM cohorts have suggested features of DSP-associated cardiac disease that may be distinct from DCM, ARVC, and ALVC (Figure 1). We hypothesize that the predominantly right ventricular phenotype-first approach utilized to date to investigate DSP-associated cardiac disease has limited the identification of important clinical features and outcomes. In this study, we aimed to conduct a genotype-first approach to investigate the clinical manifestations of genetic variation in DSP.Individuals evaluated at the University of Pennsylvania Center for Inherited Cardiovascular Disease, a tertiary referral center for patients with suspected hereditary cardiomyopathies and otherwise unexplained heart muscle diseases, from 2011–2019 were eligible for inclusion. Individuals with “pathogenic” or “likely pathogenic” variants in DSP identified by Clinical Laboratory Improvement Amendments-certified clinical genetic testing and consistent with published standards by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology were included [19]. Cascade screening of first-degree relatives for all individuals with cardiomyopathy was routinely recommended as per current practice guidelines [20]. Probands and first-degree family members were classified as such. Clinical genetic testing results were independently confirmed by licensed genetic counselors (J.C., L.H.-A.). Entry into the cohort was assigned by date of initial evaluation by a cardiologist in the University of Pennsylvania Health System. The Institutional Review Board of the University of Pennsylvania approved this study (protocol number 843087). Due to the retrospective nature of the study and waiver granted by the Institutional Review Board, no informed consent from the subjects was required.Data obtained by review of medical records included clinical history, pedigree analysis, electrocardiography (ECG), transthoracic echocardiography (TTE), ambulatory electrocardiographic monitoring (MCOT), cardiac magnetic resonance imaging (CMR), treatment, and follow-up testing as clinically indicated. Baseline demographic data included age at diagnosis, sex, first-degree familial history of DCM or sudden cardiac death, and symptoms at initial visit. Testing was performed according to standard clinical protocols. Echocardiographic chamber quantification was performed in alignment with the 2015 American Society of Echocardiography/European Association of Cardiovascular Imaging recommendations on cardiac chamber quantification for adults [21]. Clinical outcomes were adjudicated by medical record review.Continuous variables were described with medians and interquartile ranges (IQR) and categorical variables as n (%) with group comparisons performed with Wilcoxon rank sum testing. Kaplan–Meier curves were produced to analyze event-free probability from date of initial evaluation to a composite event (ventricular arrhythmia ablation, heart transplantation) with right censoring at date of death or date of last follow-up and were compared using the log-rank test. A 2-sided p value < 0.05 was considered statistically significant. Analyses were performed with Stata 15.1/IC (College Station, TX, USA).Between 2011 and 2019, a total of 19 patients, 11 probands and 8 relatives, from 14 different families were identified (Table 1). Data on variant type and pathogenicity are presented in Table 2. Of 14 unique variants found, 7 had not been previously cited in ClinVar, the United States National Institutes of Health public archive of human genetic variants (as of 20 March 2021) [22]. Age at diagnosis was not significantly different between relatives (median 35.5 years, IQR 31–48) and probands (42 years, IQR 30–48; p = 0.53). Five probands (26%) had a family history of sudden cardiac death, and five pro-bands had a family history of DCM. At initial evaluation, most probands and relatives were asymptomatic or mildly symptomatic by New York Heart Association (NYHA) class. Palpitations were the most common symptom reported by both probands (27%) and relatives (63%) at initial evaluation.Eight of eleven probands (73%) and six of eight relatives (75%) had abnormal 12-lead ECGs on initial electrocardiographic screening. Over half of the probands (55%) and 25% of relatives had T wave inversions in leads II, III, and aVF. T wave inversions in the lateral precordial leads (V4, V5, V6) were more frequent among probands compared with T wave inversions in leads V1, V2, and V3 (p < 0.01).All 11 probands and 6 relatives (75%) experienced either or both atrial and ventricular arrhythmias. Probands had a significantly higher burden of ventricular ectopy as compared to relatives (median 554.1 beats per hour (IQR 194.3–1024.3) vs. 5.9 (IQR 1.5–103.4); p = 0.02). Nonsustained ventricular tachycardia (VT) was common in both groups but occurred significantly more frequently among probands compared to relatives (p = 0.02). Five probands (45%) and one relative (13%) experienced sustained VT. Atrial fibrillation was rare and occurred in one proband (9%) and one relative (13%).Nine probands (81%) had implantable cardioverter defibrillators (ICD), six of which were implanted for primary prevention. Median LVEF at the time of ICD implantation in the probands was 36% (IQR 28–47). Three probands received appropriate ICD shocks over a median 62.8 months of follow-up (IQR 32.8–173.5) and none received an inappropriate shock. Five probands (45%) underwent catheter-directed ablation of either premature ventricular contractions (PVC) or VT.Four relatives (50%) had an ICD, three of which were for primary prevention. Median LVEF at the time of ICD implantation in the 4 relatives was 32% (IQR 28–45). No relatives received appropriate ICD shocks, and one received an inappropriate shock for sinus tachycardia over a median 20.9 months of follow-up (IQR 13.7–37.0). For the three individuals with documented LVEF at the time of secondary prevention ICD implantation, LVEFs were 49%, 55%, and 60%.Median LVEF at the time of ICD implantation was significantly higher for those who underwent secondary prevention ICD versus primary prevention (55% vs. 30%, p = 0.01). Of the seven individuals who met 2010 ARVC Task Force criteria for “definite” ARVC [23], three had no phenotypic evidence of RV disease and four had evidence of biventricular cardiomyopathy (Table 3).Left ventricular ejection fraction (LVEF) was higher for relatives (46%, IQR 25–63) at first contact compared to probands (30%, IQR 25–45), though this difference was not statistically significant (p = 0.59). Four probands (36%) and three relatives (38%) had left ventricular enlargement by TTE at the time of first contact. Left ventricular end diastolic diameter (LVEDD) was larger for probands (57 mm, IQR 50–58) at first contact compared to relatives (48.5 mm, IQR 45–57), though this difference was not statistically significant (p = 0.13). LVEF and LVEDD did not significantly change for either probands or relatives over a median follow-up of 36.3 months (IQR 9.9–72.5). At the time of most recent follow-up, most probands and relatives had normal RV size (82% and 63%, respectively) and normal RV function (82% and 75%, respectively) on TTE.Thirteen patients underwent CMR either at our or an outside institution. Twelve patients (92%) had LV late gadolinium enhancement (LGE), while only one proband and one relative (unrelated) had RV LGE (Table 4). Among probands, LV LGE occurred most commonly in the subepicardial and mid-myocardial layers (Figure 2).Myocardial histopathology was available from 5 of 19 patients: right ventricle (Patient 1), left ventricle (Patients 15 and 19), and native explanted heart (Patients 7 and 9). All samples demonstrated myocyte hypertrophy and mild to moderate endocardial and interstitial fibrosis. No sample demonstrated granulomas, giant cells, significant inflammatory infiltrates, amyloid, or iron deposition. Periodic acid–Schiff staining with and without diastase on Patient 15′s sample revealed presence of intracellular glycogen; however, this was not demonstrated on other LV samples. Histopathology findings are summarized in Table 5.Over a median follow-up of 36.3 months (IQR 9.9–72.5), one relative required extracorporeal membrane oxygenation for cardiogenic shock and eventually died. Two probands underwent heart transplantation for NYHA IV/Stage D heart failure and refractory ventricular arrhythmias (Table 6). Kaplan–Meier curves showing a comparison of the incidence of the composite outcome of PVC ablation, VT ablation, and heart transplantation for probands and relatives are shown in Figure 3.Current knowledge regarding clinical phenotypes and outcomes of desmoplakin cardiomyopathies is largely based on published reports of small populations [5], including (1) large cohorts of individuals with ARVC [11,12,13,15,17,18,24], (2) other cardio-myopathy cohorts with fewer than 10 probands [25,26,27,28], and (3) single family/single DSP variant cohorts [29,30]. Our study is one of the larger single-center descriptions of the clinical characteristics of DSP cardiomyopathy and aligns with recent work describing the distinctive presentation and course of this disease and the importance of molecular diagnosis for appropriate detection and risk stratification [8,31].In this cohort of 19 individuals with pathogenic or likely pathogenic non-missense DSP variants, we found a high prevalence of left ventricular pathology by TTE and CMR. Probands and relatives exhibited LV systolic dysfunction relatively early, in the third to fourth decades of life, and similar to the average age at diagnoses of other genetic ACM, such as LMNA-associated DCM. Right ventricular dilation and dysfunction by TTE and/or CMR were rare in this cohort, supporting emerging knowledge that there may be a subset of individuals with DSP cardiomyopathy who do not manifest with the classical ARVC phenotype. The possibility of a distinct pathogenesis of DSP-associated disease, compared to other desmosomal cardiomyopathies, has been recently suggested [32,33]. Only 7 of 19 individuals met the 2010 Task Force criteria for a “definite” diagnosis of ARVC, despite each individual having one major criterion fulfilled at baseline with the presence of a pathogenic/likely pathogenic DSP variant. Our findings align with a recent multicenter study from Smith et al. that similarly highlighted the poor sensitivity of the 2010 Task Force criteria in identifying individuals with DSP cardiomyopathy [8]. The high prevalence of LV LGE on CMR in our cohort indicates that LV scarring and fibrosis, particularly in the subepicardial and mid-myocardial layers, might warrant consideration as a characteristic injury pattern in DSP cardiomyopathy. The etiology of this scarring and fibrosis remains unclear; however, autoimmunity and myocarditis have been proposed [34,35]. Further efforts to link biomarkers, histopathology, and imaging longitudinally in individuals with DSP cardiomyopathy will be critical to our understanding of the etiology and sequelae of the desmosomal cardiomyopathies.We describe a high prevalence of atrial and ventricular arrhythmias and sudden cardiac arrest in this cohort. In addition, we report substantial utilization of therapies related to the treatment of malignant ventricular arrhythmias and heart failure including, secondary prevention ICD implantation, appropriate ICD shocks, ventricular arrhythmia ablation, and heart transplantation. Our observations support recent work from Wang et al., derived from a single center prospective registry of ARVC [31], which demonstrated that individuals with disease-causing variants in DSP were at high risk for sustained ventricular arrhythmia and heart failure. Importantly, the three individuals in our cohort who underwent secondary prevention ICD implantation had LVEFs above the guideline recommended primary prevention ICD implantation threshold of <35%. Although a small sample, this raises the question of using LVEF thresholds established for DCM in DSP cardiomyopathy, similar to the exception recommended for LMNA cardiomyopathy [4]. Our findings also add further evidence that left-sided and biventricular involvement, and not just right ventricular disease, should be suspected in DSP cardiomyopathies.Limitations of our study include its single center retrospective nature with a small sample size, resulting in limited direct genotype-phenotype associations, possible referral bias, and possible lead time bias with differences between probands and relatives. Complete clinical data, including dermatologic phenotyping, was not available for all individuals. We acknowledge that the TNNT2 variant found in Patient ID 7 has been reported in association with DCM and may have influenced this individual’s phenotype. In frame deletions in DMD are associated with the X-linked recessive Becker muscular dystrophy, and this female patient (Patient ID 13) did not exhibit signs or symptoms of skeletal myopathy. Digenic heterozygosity in desmosomal genes has been reported as a potential contributor to the development of an ARVC phenotype [36,37,38,39]; however, the characteristics of individuals with digenic heterozygosity in DSP and either TNNT2 or DMD has not been previously reported.Variants in DSP are estimated to occur in 3% of European individuals with DCM; although they have been historically implicated in ARVC, we demonstrate that disease-causing non-missense DSP variants manifest with a high burden of LV injury, LV dysfunction, heart failure, and arrhythmic events. We highlight the distinct features of DSP cardiomyopathy and association of DSP in the pathogenesis of DCM and ALVC, supporting the need to incorporate a genotype-first approach and molecular diagnostics into risk assessment and clinical care.Conceptualization, N.R. and A.T.O.; data acquisition, N.R., A.d.F., J.L.C., L.H.-A., L.V., J.K. and A.M.; investigation, N.R., A.d.F., J.L.C., L.H.-A. and A.T.O.; methodology, N.R. and A.T.O.; project administration, J.K.; formal analysis, N.R.; writing—original draft preparation, N.R.; writing—review and editing, N.R., A.d.F., J.L.C., L.H.-A., L.V., J.K., A.M. and A.T.O.; visualization, N.R.; supervision, A.T.O.; funding acquisition, N.R. and A.T.O. All authors have read and agreed to the published version of the manuscript. N.R. was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under award number KL2TR001879. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. A.T.O. was funded by the Winkelman Family Fund for Cardiac Innovation.The study was carried out in accordance with Declaration of Helsinki and approved by the Institutional Review Board of the University of Pennsylvania (protocol number 843087). Due to the retrospective nature of the study and waiver granted by the Institutional Review Board, no informed consent from the subjects was required.The data presented in this study are available on reasonable request from the corresponding author and are not publicly available due to the potential to compromise the privacy of research participants.The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.Characteristics of the clinical presentations of dilated cardiomyopathy (DCM), arrhythmogenic cardiomyopathy (ACM), and desmoplakin cardiomyopathy. Dilated cardiomyopathy is typically characterized by ventricular systolic dilation and dysfunction in the absence an ischemic, valvular, hypertensive, or other systemic insult. Arrhythmogenic cardiomyopathy is distinguished by a clinical presentation with documented or symptomatic arrhythmia or conduction disease. This presentation can occur concomitantly with ventricular dilation and/or dysfunction. Desmoplakin cardiomyopathy presents with features of both DCM and ACM along with unique features such as episodic and recurrent myocardial injury. Abbreviations: DSP = desmoplakin; LV = left ventricular; RV = right ventricular; PVCs = premature ventricular contractions.Electrocardiography and cardiac magnetic resonance imaging of two patients with pathogenic truncating variants in the desmoplakin (DSP) gene. 12-lead (A) and extended outpatient electrocardiographic monitoring (C) of Patient ID 13 (c. 888C > G, p. Tyr296*) demonstrated frequent multifocal premature ventricular complexes. Paper speed and amplification: 25 mm/s and 1 mV/10 mm. Cardiac magnetic resonance imaging (B) with contrast of Patient 15 (c. 478C > T, p. Arg160*) demonstrated subepicardial late gadolinium enhancement in the left ventricular anterolateral wall at the mid-cavity (arrow).Kaplan–Meier estimates of event-free survival for the composite outcome of ventricular arrhythmia ablation or heart transplantation with right censoring at date of death or date of last follow-up. Tick marks indicate censored individuals. Over the observed time of follow-up, no relative underwent ventricular arrhythmia ablation or heart transplantation.Demographics and clinical characteristics of 19 probands and relatives with pathogenic or likely pathogenic variants in the desmoplakin gene (DSP). Abbreviations: NYHA = New York Heart Association; SCD = sudden cardiac death; DCM = dilated cardiomyopathy.Pathogenic and likely pathogenic variants in the desmoplakin gene (DSP) in 19 patients. * Patient ID 7 also carried a pathogenic truncating variant (c.629_631delAGA, p.Lys210del) in TNNT2 (cardiac troponin T). Patient ID 13 also carried an in-frame deletion (exons 49–51) in DMD (dystrophin). Classifications were based on interpretations from Clinical Laboratory Improvement Amendments-certified laboratories and confirmed by institutional genetic counselors. Abbreviations: ID = identifier; ACMG = American College of Medical Genetics and Genomics.Electrocardiographic and arrhythmia characteristics of 19 probands and relatives with pathogenic or likely pathogenic variants in the desmoplakin gene (DSP). Abbreviations: MCOT = mobile cardiac outpatient telemetry; VE = ventricular ectopy; NSVT = nonsustained ventricular tachycardia; VT = ventricular tachycardia; ICD = implantable cardioverter defibrillator; LVEF = left ventricular ejection fraction; PVC = premature ventricular contractions.Cardiovascular imaging characteristics of 19 probands and relatives with pathogenic or likely pathogenic variants in the desmoplakin gene (DSP). Abbreviations: TTE = two-dimensional transthoracic echocardiography; LVEDD = left ventricular end diastolic diameter; RV = right ventricular; LV = left ventricular; LGE = late gadolinium enhancement; CMR = cardiac magnetic resonance imaging.Myocardial histopathology findings of five patients with pathogenic or likely pathogenic variants in the desmoplakin gene (DSP). Abbreviations: ID = identifier; RV = right ventricle; LV = left ventricle; PAS = Periodic acid–Schiff.Clinical outcomes of 19 probands and relatives with pathogenic or likely pathogenic variants in the desmoplakin gene (DSP). Abbreviations: ECMO = extracorporeal membrane oxygenation.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Currently, there is an unmet therapeutic need for the medical management of cardiac arrest, as is evident from the high mortality rate associated with this condition. These dire outcomes can be attributed to the severe nature and poor prognosis of this disorder. However, the current treatment modalities, while helping to augment survival, are limited and do not offer adequate improvements to outcomes. Treatment modalities are particularly lacking when considering the underlying pathophysiology of the metabolic phase of cardiac arrest. In this study, we explore the three phases of cardiac arrest and assess the factors related to positive clinical outcomes and survival for these events. Furthermore, we evaluate the present guidelines for resuscitation and recovery, the issues related to ischemia and tissue reperfusion, and the benefit of oxygen-delivery therapeutic methods including blood transfusion therapy and synthetic hemoglobins (HBOCs). The current therapy protocols are limited specifically by the lack of an efficient method of oxygen delivery to address the metabolic phase of cardiac arrest. In this article, we investigate the next generation of HBOCs and review their properties that make them attractive for their potential application in the treatment of cardiac arrest. These products may be a viable solution to address complications associated with ischemia, reperfusion injury, and organ damage.During recent decades, modern medicine has benefited from many advancements addressing common and familiar ailments, allowing for improved mortality and morbidity for a large variety of pathologies. One area which has seen relatively little improvement is the treatment of cardiac arrest. Many studies have been conducted and published on this topic to increase our understanding of the underlying pathology, mechanisms, and sequence of events involved in this condition, and these have guided the search for new therapeutic approaches. However, the mortality in patients suffering from cardiac arrest still remains very high. Ischemic heart disease is a major cause of death worldwide and is categorized as either out-of-hospital cardiac arrest (OOHCA) or in-hospital cardiac arrest (IHCA). In the USA, approximately 360,000 people suffer from OOHCAs and this pathology has a staggeringly low survival rate, estimated to be 10% to time of discharge from the hospital [1]. Meanwhile, recent studies from the literature estimate that between 370,000 to 750,000 IHCAs occur annually in the US, with recent meta-analyses showing a pooled survival rate of 15% with minimal change over time. This event also confers significant morbidity afterwards, with an estimated 1-year survival rate of 13.4% [2,3].This review article aims to explore cardiac arrest as a currently significant and prominent pathology that also has significant mortality. First, we investigate predictors of survival and also define the three phases of cardiac arrest: the electrical (first) phase, the circulatory (second) phase, and the metabolic (third) phase. We then delve into the third phase of cardiac arrest, focusing on ischemia, complications of this pathology, and reperfusion injury. We then explore the current guidelines addressing oxygen delivery, which theoretically should address the underlying pathology of the third phase of cardiac arrest. Finally, we first investigate hemoglobin-based oxygen carriers (HBOCs) broadly and then perform an in-depth analysis of the novel generation. It is possible that this class of products may offer a benefit to the mortality associated with cardiac arrest. This review does explain the breadth of etiologies and pathophysiology associated with cardiac arrest but ultimately focuses on cardiac arrest in the setting of arrhythmias that are considered shockable rather than non-shockable rhythms. This review was based on a comprehensive PubMed search of the topic of cardiac arrest, blood transfusions, and hemoglobin-based oxygen carriers. Additional details regarding this search are described in Supplementary File 1.The term cardiac arrest is a broad term with a variety of potential etiologies ranging from underlying heart disease and arrhythmia to sepsis and trauma [4,5,6]. Arrhythmic causes of cardiac arrest are the most common, the most classically described, and the target of modern-day therapeutics for cardiac arrest, and as such they are the focus of this review article. These arrhythmic causes are subdivided based on their therapeutic approach, with “shockable” rhythms including ventricular fibrillation and pulseless ventricular tachycardia, while “non-shockable” rhythms include pulseless electrical activity and asystole [4,7]. The therapeutic approach applied for arrhythmic cardiac arrest is described in the form of an algorithm in the advanced cardiovascular life support (ACLS) guidelines. Generally, shockable rhythms are managed through a combination of cardiopulmonary resuscitation (CPR) and defibrillation with medications, while non-shockable rhythms are managed primarily with CPR in addition to various medications [8].The predictors of survival differ between IHCA and OOHCA. Considering OOHCA, the elements of the chain of survival first described in 1991 are paramount. The chain of survival requires the timely functioning of bystanders, dispatchers, first responders, paramedics, and, finally, hospital care [9]. Ultimately, survival has been linked to appropriate timely response by the earliest members of the chain of survival, which includes prompt recognition, initiation of CPR, and, ideally, minimizing time to defibrillation, if applicable [4]. It has been shown that survival to hospital discharge is improved for OOHCA that is witnessed by a bystander/EMS (Emergency Medical Service), in patients who received bystander CPR, in patients with shockable rhythms, and in patients who were able to achieve in-field return of spontaneous circulation (ROSC) [10]. Furthermore, it has been shown that patients who suffer from OOHCA have improved outcomes when transported to medical centers that are specialized and capable of managing cardiac resuscitation with available services such as cardiac catheterization [11]. When considering IHCA, the chain of survival is not as prominent in ensuring improved survival, as patients theoretically are already at the highest level of care required to manage this condition. Instead, survival is influenced by patient factors, with reduced survival in older-aged patients, in black patients, and in those with various clinical diagnoses including sepsis, renal failure, metastatic cancer, stroke, and house-bound lifestyle [3]. More generally, improved outcomes have been linked with shorter durations of CPR and more rapid attainment of ROSC [12]. The benefit that has been shown with the application of thrombolytics in cardiac arrest suggests that myocardial blood-flow occlusion is a major component of negative outcomes [13]. Studies have also shown dysfunction of the hypothalamic–pituitary–adrenal axis after cardiac arrest, with decreased levels of cortisol, which suggests that a decrease in the body’s response to stress likely plays a role in negative outcomes as well [14]. Furthermore, there are a select few accessory therapies that should be applied in specific scenarios to help improve mortality. One such therapy is therapeutic hypothermia, which is also known as targeted temperature management. When applied following cardiac arrest, this intervention has been shown to confer a 30% improvement in mortality [15].The classically described therapeutic approach to cardiac arrest resuscitation utilizes a rhythm-based approach but does not account for time elapsed after the onset of cardiac arrest. Conversely, cardiac arrest can be viewed using the three-phase time-dependent model first described in 2002 [16]. This three-phase model remains the basis for the current treatment paradigm and each stage features different degrees of ischemia, risks of reperfusion injury, and therapeutic approaches, which target the unique underlying pathophysiology.The electrical phase begins with the onset of cardiac arrest and lasts for about 5 min. The therapeutic gold standard for this phase is early and rapid defibrillation. Patients who are able to receive appropriate early treatment have survival rates exceeding 60% [16].The circulatory phase of cardiac arrest is classically defined as existing between 5 and 10 min after the initiation of arrhythmic cardiac arrest. Improvements to mortality within this phase are most seen with maximization of blood flow to the myocardium. As such, the greatest benefit arises from chest compressions with intermittent defibrillation, if applicable. However, the emphasis on chest compressions that is required to improve mortality is not commonly employed, as it becomes very difficult to pinpoint and distinguish the phases of cardiac arrest in real-life clinical situations [16].The metabolic phase of cardiac arrest typically begins 10 min after the initiation of cardiac arrest and is responsible for the largest proportion of deaths among the phases. The failure of treatment modalities during the metabolic phase suggests that both defibrillation and CPR are not adequate as resuscitation measures [16]. The underlying pathophysiology of the metabolic phase of cardiac arrest involves a lack of adenosine triphosphate (ATP) generation due to ischemia, which disrupts the intracellular redox balance and causes the generation of reactive oxygen species. The decline in ATP levels cause failure of cellular machinery responsible for maintaining ion gradients, which causes secondary swelling of the mitochondria [17]. Meanwhile, the body suffers from a state of global ischemia, which causes a variety of pathologic processes including the release of endotoxins and cytokines. These factors suppress myocardial contractility and can be lethal to cardiomyocytes [18].While the exact cytokine profile that categorizes the metabolic phase has not been established, it is likely very similar to the cytokine profile that is seen in the systemic ischemia/reperfusion response that arises with ROSC following a cardiac arrest. The systemic ischemia/reperfusion cytokine profile is similar to that of sepsis and is characterized by polymorphonuclear leukocyte activation, activation of inducible nitric oxide synthase, and the production of cytokines such as tumor necrosis factor alpha and interleukin-6, both vitally important for generalized immune activation. This response produces a pathological vasodilatory response while causing cardiodepression, which impedes resuscitative efforts [19]. From this comes the benefit of the application of epinephrine during cardiac arrest, which acts on α1-adrenergic receptors to cause vasocontriction in non-vital peripheral organs and increase coronary and cerebral blood flow [20]. Furthermore, in the metabolic phase, the health-care provider is placed in a complicated scenario, where successful CPR can re-introduce the metabolically deranged body to near-normal levels of oxygenation, which induces reperfusion injury as discussed below.Ischemia is a state of oxygen deprivation which occurs as a consequence of cardiac arrest when the heart is unable to appropriately circulate oxygenated blood. Ischemia in cardiac arrest is not only found in systemic tissues but is a major effector, as it occurs in the myocardium. The global ischemia characteristic to cardiac arrest is evidenced by elevated lactate levels, a hallmark of this pathology. Furthermore, the persistence of an elevated lactate level may be due to complications following cardiac arrest, including systemic inflammatory response, ongoing hypoxia, and cardiogenic shock due to myocardial dysfunction [21]. This widespread ischemia is known to produce a large quantity of reactive oxygen species (ROS), which are potentially toxic to most biological tissues, causing enzyme inactivation, protein oxidation, and mitochondrial respiratory chain inhibition [22].The generation of this various detrimental cellular free radical generations are believed to be the culprit for the consequences of hyperoxia—the alveolar oxygen concentration exceeds that of normal breathing conditions. Supplemental oxygen is currently ubiquitous in the management of cardiac arrest, but more recent studies have shown that targeting oxyhemoglobin saturations of 94–98% can prove more beneficial to overall survival [22]. The underlying pathophysiology behind the detrimental effects of oxygen supplementation in cardiac arrest extends beyond simply the generation of ROS and is not completely understood. It has been suggested that elevated levels of inspired oxygen in ischemic heart disease reduce coronary blood flow while increasing coronary vessel resistance [23].Under physiologic conditions, reactive oxygen species (ROS) are tightly regulated within cardiac myocytes and act as molecules that regulate gene transcription and alter enzymatic activity [24]. Reactive oxygen species are produced within cardiac myocytes by NADPH oxidases (nicotinamide adenine dinucleotide phosphate oxidase), xanthine oxidase, and nitric oxide synthase within the mitochondria as byproducts of cellular respiration. The balance within myocytes is maintained similarly to cells throughout the body by a careful equilibrium of ROS-producing pathways and neutralizing enzymes, including superoxide dismutase, catalase, and glutathione peroxidase [25]. Notably, during periods of ischemia, elevated ROS production from mitochondrial dysfunction opens various mitochondrial channels to cause ROS-induced ROS release [24]. The increase in ROS goes on to cause significant oxidative stress to myocytes and impair ATP generation [24,26]. This explains the myocardial component of reperfusion injury that typically occurs following restoration of blood flow after cardiac arrest. Reperfusion injury can be viewed within the myocardium specifically, but it can also be viewed systemically as a similar mechanism occurs in all tissues deprived of oxygen, causing increased production of ROS and resultant cellular dysfunction. Reperfusion injury systemically results in reperfusion syndrome, which can involve platelet aggregation, complement activation, capillary leak, tissue edema, and impaired microcirculation to vital organs [27]. The ROS generated due to ischemia are the basis of the ischemia/reperfusion injury, involving the release of cytokines like interleukin-6 and tumor necrosis factor alpha [19].Therapeutic hypothermia is a treatment option for select patients following cardiac arrest that targets the generation of ROS to help provide benefits [28]. Specifically, hypothermia is therapeutically indicated for unconscious survivors of cardiac arrest, following ventricular fibrillation, and its application outside of these parameters and for pathologies other than cardiac arrest is not well-described [29]. This therapy involves cooling comatose survivors to target temperatures of 33–36 °C (91.5–96.8° F), with cooler target temperatures being indicated in patients with evidence of severe brain injury [28]. Therapeutic hypothermia is employed primarily for its neuroprotective effect. In neurons, the depletion of ATP in the setting of ischemia causes a buildup of cellular calcium, causing depolarization and release of neurotransmitters such as dopamine and glutamate, which go on to enact downstream effectors to induce cellular damage [30]. Since the release of these neurotransmitters from neurons is temperature-dependent, therapeutic hypothermia helps to reduce this process and preserves neurons [30]. This therapy also helps to improve neurologic outcomes by reducing neuron metabolism and ROS generation [31]. However, it is important to note that therapeutic hypothermia is known to be arrhythmogenic and malignant arrhythmia is an unfortunate complication of this therapy [32]. Hypothermia prolongs the action potential duration and increases the heterogeneity of ventricular repolarization to predispose the myocardium to arrhythmia [33]. This has been explained by potassium shifts that occur during hypothermia resulting in systemic hypokalemia, which is linked to the development of polymorphic ventricular tachycardia [32]. It is interesting to contrast these studies with those investigating hypothermia as an adjunct to cardioplegia during cardiac surgery. In this setting, a decrementing increase in sodium influx helps to impair the severity of calcium influx following restoration of calcium levels and reduce hypercontractility and cellular injury [34]. During surgery, hypothermia functions to significantly reduce metabolism and oxygen consumption and attenuate the injury incurred by ischemia [35].Other therapies that target the generation of ROS have been studied but are not applied clinically today. It has been shown that the administration of polyethylene glycol-superoxide dismutase 24 h prior to global ischemia events helped to reduce reperfusion injury [36]. Many of the studies and potential therapies associated with ROS level modification are currently impractical in the clinical setting and their continued research is invasive and costly.Preconditioning is a phenomenon associated with myocardial ROS that has been described within the myocardium in which brief episodes of ischemia activate cardioprotective mechanisms, which help to minimize the effect of ischemia and ROS on the myocardium [37]. This phenomenon is believed to occur due to the production of ROS, which then modify gene expression to improve cellular tolerance to impending ischemia [24]. This phenomenon can be observed naturally in patients with intermittent arrhythmias and coronary artery disease but is also studied for application in cardiac surgery [37,38].The clinical complications that follow cardiac arrest are varied and contribute to the high mortality rate associated with this condition. There are a variety of complications that stem from the ischemia and metabolic dysfunction that arise within the myocardium, and they share a similar pathophysiology to the third phase of cardiac arrest. These complications include ventricular free-wall rupture, papillary muscle rupture, and aneurysm formation, which arise along different timeframes following myocardial infarction and result in increased morbidity and mortality [39]. An often overlooked complication of cardiac arrest, which is defined partially by the complications arising from the metabolic derangements of the third phase of cardiac arrest, is post-cardiac arrest syndrome. This syndrome is complex and unique to each patient, as it results from the myocardial ischemia and metabolic derangements, as described above, superimposed upon the underlying etiology for the cardiac arrest and underlying patient co-morbidities. Classically, this syndrome is defined by post-cardiac arrest brain injury, post-cardiac arrest myocardial dysfunction, the systemic ischemia/reperfusion response, and the persistence of the precipitating pathology [40]. The management of post-cardiac arrest syndrome is typically approached similarly to septic shock, with emphasis on hemodynamic care in the form of fluid administration and inotropes in addition to therapeutic hypothermia for select patients [41]. These complications all arise due to the metabolic derangements that result from the decline in oxygen delivery. As previously stated, no therapeutic options that are currently employed address this pathophysiology directly.The myocardial complications of cardiac arrest are plentiful and, in combination with the large-scale systemic dysfunction that arises due to reperfusion injury, cause significant damage to numerous tissues and organs throughout the body. However, it is important to consider a more basic complication of cardiac arrest: the cessation or reduction of blood flow to vital organs. It is known that improved vital organ perfusion during resuscitation and following cardiac arrest is associated with improved outcomes [42]. Critical organs including the brain and myocardium are particularly susceptible to ischemia, and perfusion maintenance is vital to sustain their high metabolic activity [43].The majority of the therapies utilized for cardiac arrest aim to maintain and restore vital organ perfusion. Optimal closed-chest CPR is known to generate circulation equivalent to 25–33% of normal cardiac output and as such is a major weak point in the resuscitation algorithm [44]. Percutaneous left ventricular assist devices (LVADs), such as Impella (Abiomed, Danvers, MA, USA) devices, have been applied in the acute setting to maximize cardiac output and improve vital organ perfusion. This therapy also has the benefit of continuing to augment cardiac output following ROSC and improving overall functionality after cardiac arrest [42]. These therapies have promise but are accessible only to select advanced medical centers. Meanwhile, therapy involving vasopressor medications, such as epinephrine, has been applied for cardiac arrest for the last 60 years. This therapy relies on its ability to combat the pathological vasodilation seen in the third phase of cardiac arrest and the vasoconstriction it causes diverts blood flow to maintain perfusion of vital organs [45]. While this therapy is known to result in improvements in outcomes, the high mortality associated with cardiac arrest shows that further therapies are required. The development of therapies that enhance oxygen delivery to vital organs is vital to the improvement of cardiac arrest resuscitation.Thus far, this article has primarily discussed the metabolic complications associated with cardiac arrest in reference to an arrhythmic etiology. However, when considering the broad range of therapies offered for cardiac arrest, it is important to discuss blood and intravenous (IV) fluid administration, which is most commonly seen in cardiac arrest resulting from blunt trauma. This scenario often presents clinically with severe blood loss and hemorrhagic shock. In this setting, it has been shown that IV fluid administration decreases mortality by helping to prevent vascular collapse [46]. However, it is important to recognize that this therapy offers no benefit beyond volume replacement, while the use of blood products helps to restore intravascular volume while also repleting oxygen delivery capacity. This therapy comes with the risk of infection and adverse immunological reactions, while survival rates in patients treated with blood transfusion are not significantly improved [46]. Retrospective studies have shown that traumatic cardiac arrest patients treated with packed red blood cells (PRBCs) had improved likelihood of attaining ROSC (44.6% compared to 28% in control groups). However, there was minimal improvement between studied groups in survival until discharge (3.4% compared to 2.4% in control) [47]. With this in mind, it is clear that administration of PRBCs provides a benefit to patients, likely due to their capacity to support both circulation and oxygen delivery. It is also interesting to recognize that this therapeutic option theoretically has the potential to help target the underlying pathophysiology of the metabolic phase of cardiac arrest. It is unclear why an improvement in the rate of ROSC does not translate into improved survival, but an investigation into more efficient oxygen delivery methods is warranted to address this disparity.It is important to recognize the shortcomings of red blood cell therapy when considering other oxygen delivery solutions for their potential application for cardiac arrest. During PRBC storage, potassium leakage through the red blood cell membrane generates a hyperkalemic supernatant solution, which explains the association between rapid blood transfusion and hyperkalemia in emergency settings [48]. Furthermore, it has been shown that cardiac arrest is normally associated with a hyperkalemic state, with serum potassium levels of 5.63 ± 2.39 mmol/L [49]. As such, this population has a significant baseline risk for hyperkalemia and treatment with PRBCs exacerbates this issue, with measured potassium levels of 8.23 ± 1.99 mmol/L [49]. It has been suggested that washing of PRBC units prior to administration can help to prevent this complication, but this practice is not feasible in the emergency setting [48]. Currently, the electrolyte imbalances resulting from blood transfusion therapy generate the potential to exacerbate cardiac arrest and are major limitations to the application of this treatment.The use of hemoglobin-based oxygen carriers (HBOCs) is a novel method for systemic oxygen-delivery therapy that is actively being researched. This therapy, named “artificial blood”, aims to emulate the function of hemoglobin in circulating red blood cells by delivering oxygen. HBOCs are classically solutions that are composed of acellular hemoglobin and deliver oxygen to tissues along the normal diffusion gradient [50]. This therapy option offers several advantages over PRBC transfusions including long shelf-life, the option for non-refrigerated storage conditions, the prevention of pathogenic agent transfer during blood transfusion, and the elimination of the need to cross-match blood types [51]. HBOCs can help to resolve the storage issues related to the use of PRBCs, including the potassium leakage that develops during storage discussed above. HBOCs also eliminate any immune reactions associated with transfusion procedures and can potentially also be used on patients who reject blood transfusion for religious reasons [50]. Nephrotoxicity is also a potential complication of PRBC transfusion therapy due to the release of unmodified free hemoglobin as red blood cell lyse. HBOCs are engineered with several modifications to help address the shortcomings of PRBC therapy and some have minimal renal excretion [52].Earlier-generation HBOCs contained hemoglobins that were chemically modified using non-site-specific techniques, including conjugation and polymerization. These changes were made in an attempt to alter the physiological parameters of the hemoglobin molecules to increase their overall size and decrease their oxygen affinity and renal clearance [50,53]. It is important to note that, although the size of the hemoglobin molecules is increased, they are still significantly smaller compared to other blood components, such as red blood cells. As such, HBOCs have the potential to flow with plasma past areas of vessel occlusion, which normally prevents the passage of larger blood components, such as red blood cells.One of these earliest HBOCs was a cross-linked hemoglobin tetramer known as diaspirin cross-linked hemoglobin. However, this product was found to decrease cellular perfusion and, as a result, drastically decreased the survival rate of study subjects [50,54]. This study and others similar to it demonstrated a need to further refine HBOCs before trials can be carried out.Further chemical modifications, including conjugation to non-protein particles such as polyoxyethylene (POE) or polyethylene glycol (PEG), were introduced to diminish extravasation while retaining a low oxygen affinity [50,55]. Studies suggest that increasing the size of the HBOCs through polymerization or cross-linking diminishes many of the adverse effects associated with these products [50,55]. However, the methods by which these chemical modifications were performed were imprecise and resulted in non-homogeneous polymerization and conjugation products. This heterogeneity caused HBOCs produced by this methodology to have unforeseeable physiological and biochemical traits [50].The proposed chemical modifications to hemoglobin produced a multitude of changes to its properties, including the alteration of its oxygen equilibrium curve, a decrease in cooperativity, the loss of the Bohr effect, and a decreased ability to bind CO2. It was also noted that autoxidation of heme in most of these HBOCs occurred two to three times faster compared to the rate for unmodified hemoglobin, resulting in a decreased redox potential and the loss of heme [50]. Furthermore, the long half-lives of several HBOCs led to over-exposure of the products to the body’s degradative and oxidative processes, causing the release of more free-heme into circulation. Aside from PolyHeme (Northfield Laboratories Inc., Evanston, IL, USA) and pegylated human hemoglobin, it was found that most other HBOCs are highly susceptible to oxidative stress, leading to increased levels of ferryl hemoglobin in circulation [50]. On the other hand, these HBOCs did improve mitochondrial respiration significantly [50].Despite many of these advancements having optimistic outcomes in initial preclinical and clinical studies, a JAMA (Journal of American Medical Association, US) meta-analysis by Natanson completely dismissed earlier-generation HBOCs as unsafe and too dangerous to be used in clinical settings, suggesting that all of the current HBOCs increase the risk of myocardial infarction and mortality. These outcomes were believed to be caused by the extravasation of HBOCs, leading to the depletion of NO and heightened vascular tone that hindered blood flow. The analysis also detailed further side effects, including the prevention of platelet inactivation and release of pro-inflammatory substances [51]. This analysis stifled the development of many HBOCs and slowed research within the field.Currently, a novel generation of HBOCs (NG-HBOCs) is being developed to address the shortcomings brought forward by Natanson’s meta-analysis. Research has been focused on finding solutions to circumvent the mentioned side effects, particularly extravasation and NO depletion. NG-HBOCs employ novel methods for the synthesis of hemoglobin polymers, such as a zero-linked polymerization process that helps to prevent extravasation [56].Considering their functional similarity to PRBC transfusions as well as their many potential advantages, HBOCs are a logical candidate as a therapeutic option for cardiac arrest. Animal studies have been performed that reveal the potential benefits of these therapies. Manning et al. examined the effectiveness of HBOC-201 in promoting ROSC in cardiac arrest in a swine exsanguination model [57]. The addition of oxygenated HBOC-201 to selective aortic arch perfusion (SAAP) was evaluated and compared to oxygenated lactated Ringer’s (LR) solution to determine the impact of HBOC-mediated oxygen delivery on circulation and survival rates. Of the six swine that received the HBOC-201 therapy, all six achieved ROSC at 1.9 ± 0.3 min, while five reached survival at one hour [57]. In comparison, only two of the six swine in the lactated Ringer’s (LR) group reached ROSC with the addition of epinephrine, and none survived past the hour [57]. These results demonstrate the potential benefit of the early administration of HBOCs in the setting of cardiac arrest resuscitation. The benefit of the use of HBOCs over LR solution can be likely attributed to the ability of these solutions to effectively deliver oxygen to deprived tissues. The benefit seen in animal studies warrants continued research and development of these products.There are a small number of products that have been developed that are considered NG-HBOCs. This class of compounds employs new methods of synthesis and production, seeking to improve the biological efficacy and safety compared to previous generations. One NG-HBOC utilizes a zero-link polymerization process to produce ultra-high-molecular-weight hemoglobin polymers while having a viscosity similar to human plasma in solution [56,58]. Being larger than any vascular pore, NG-HBOCs with high molecular weights are incapable of extravasation and thus cannot deplete local NO to cause vasoconstriction, assisting in the maintenance of a suitable mean arterial pressure (MAP) [56,59,60]. One beneficial aspect of certain NG-HBOCs is their high oxygen affinity, ensuring that oxygen is offloaded primarily to severely hypoxic tissues [56,59,61]. Animal studies employing NG-HBOCs have been performed that have affirmed their ability to deliver oxygen and offload significant amounts of oxygen to hypoxic tissues when administered in low volumes, while not hindering the coagulation capacity of blood [54,59,62].Since most acellular hemoglobins are prone to oxidation and denaturation, it is of utmost importance to minimize oxidative changes when introducing an acellular hemoglobin into the circulatory system. Furthermore, in order for an HBOC to work effectively as an oxygen carrier, it must remain in the reduced state (heme-Fe2+) in the circulatory system. With regard to NG-HBOCs, it is known that ascorbic acid, an endogenous reducing agent present in human plasma, is effectively able to maintain these products in their reduced state [58]. Furthermore, the stability of NG-HBOCs helps to ensure that minimal loss of heme iron occurs following infusion [63]. Some NG-HBOCs are available in both powder and liquid forms and have been shown to be resistant to oxidative changes, such as through exposure to urea, a strong denaturant [56,58]. This fact suggests that NG-HBOCs have the structural integrity and the redox stability required to emulate normal hemoglobin function.Numerous animal studies have been performed employing NG-HBOCs. One study by Mito revealed that one NG-HBOC, when applied to an artificially induced stroke in an animal model, reduced the size of affected brain tissue by 40% [64]. Another experiment by Reynolds found that small-volume resuscitation of Long–Evans rats with an NG-HBOC improved survival rate [61]. A study by Ning revealed that poly-hemoglobin and purified stroma-free hemoglobin do not activate complement as complement system activation is attributed to the presence of cell membrane debris and endotoxins [65]. Therefore, it is logical that some NG-HBOCs should also not activate complement, as their structures are remarkably similar to the molecules examined by Ning. This would help to minimize reperfusion injury during the treatment of cardiac arrest.The characteristics and successes of NG-HBOCs warrant further investigation into the application of these compounds for cardiac arrest resuscitation. These products could help to address the pathophysiology of the third phase of cardiac arrest by promoting oxygen delivery to tissues. Further trials are required to assess the application of these products in large animal models prior to their potential application in human clinical trials.Existing therapeutic guidelines have proven inadequate in controlling the high mortality rate associated with cardiac arrest, and the development and improvement of these guidelines would greatly benefit medical care [1,2,3]. The current treatment paradigm involving defibrillation and chest compressions targets the pathophysiology of the first two phases of cardiac arrests but does not address the third phase, which results in metabolic derangement and organ injury [16]. Previously attempted but unsuccessful therapies addressing this third phase include blood transfusion therapy and first-generation HBOCs. Novel-generation HBOCs are being developed to address the shortcomings of first-generation HBOCs and have been shown in animal studies to be successful in resuscitation and reducing tissue injury [56,58,59,61,64]. Through a mechanism of oxygen delivery, it is possible that NG-HBOCs, if administered during the earlier stages of cardiac arrest, may offer a benefit to mortality.The following are available online at: www.mdpi.com/article/10.3390/cardiogenetics12010004/s1, Supplementary File 1: Methods.Conceptualization, B.M.W. and H.W.; investigation, B.M.W., B.P.-G., W.Y. and U.Z.; writing-original draft preparation, B.M.W.; writing-review and editing, B.M.W., B.P.-G., W.Y., U.Z., N.S., K.S. and H.W.; supervision H.W. All authors have read and agreed to the published version of the manuscript.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Not Applicable.Not Applicable.Not Applicable.We disclose that two of the authors are shareholders of OXYVITA Inc. (Hanna Wollocko, Brian Wollocko), the proprietor and manufacturer of a novel hemoglobin-based oxygen carrier. The shareholder status of the authors did not influence the review process or discussion. This work was compiled in association with Touro College of Osteopathic Medicine. There are no other reported conflicts of interest.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Hypertension and atherosclerosis are debilitating diseases that affect millions each year. Long-term consequences include but are not limited to stroke, myocardial infarction, and kidney failure. Platelet-activating factor (PAF) is a proinflammatory mediator synthesized from a subclass of phosphatidylcholines that increases platelet activation, leukocyte adhesion, infiltration of macrophages, and intracellular lipid accumulation, thereby contributing to atherosclerosis. Magnesium, a key micronutrient and free radical scavenger, is a water-soluble mineral that regulates peripheral vasodilation and calcium, phosphate, and hydroxyapatite homeostasis. Magnesium’s antihypertensive ability stems from its role as a natural calcium antagonist and promoter of vasodilatory mediators, such as nitric oxide. Platelet-activating factor and magnesium share an inverse relationship, and elevated magnesium levels have been shown to have protective effects against plaque formation as well as antihypertensive and antiarrhythmic effects, all of which allow for healthier aging. The purpose of this literature review is to investigate the role of platelet-activating factor and magnesium in the pathophysiology of hypertension, atherosclerosis, cardiovascular disease, stroke, and aging. Since the pathophysiology of the platelet-activating factor biomolecule is underexplored, further research studies are warranted in order to navigate the putative signaling pathways involved in the cardioprotective effects of dietary magnesium as a natural anti-PAF agent.The homeostatic function of peripheral, coronary, and cerebral vasculature is dependent on the molecular functions of numerous chemical mediators. Magnesium, being one of these mediators, is a micronutrient that has been shown to help reduce the risk of developing cardiovascular complications resulting from hypertension [1]. This mineral has also been implicated in decreasing levels of platelet activating factor (PAF), a proinflammatory mediator involved in the development of thrombogenic plaques [2,3].In this review, we will discuss hypertension, followed by the role of PAF in atherogenesis as it relates to causing hypertension. Finally, magnesium will be introduced along with its beneficial role in reducing hypertension and antagonism of PAF. Ultimately, this study will explore the roles of PAF and magnesium in cardiovascular disease pertaining to atherosclerosis, hypertension, and other relevant cardiac pathology.Hypertension is a public health menace with an estimated prevalence rate of 45.4% in adults in the United States [4]. Defined as an increase in pressure exerted by blood in the arteries, a common metric used to assess hypertension is blood pressure [5]. The current guidelines by the American College of Cardiology categorize the levels of blood pressures into four different stages. These criteria are organized in Table 1. However, in order to stage a diagnosis of hypertension, an average of two or more readings must be performed [6].The exact cause of hypertension in approximately 95% of cases is unknown. This is termed as ‘essential hypertension’ [7]. Both genetic and environmental factors underlie essential hypertension [7]. On the other hand, if the underlying cause of hypertension is known, such as reduction in blood flow, kidney dysfunctions, or endocrine pathologies, it is termed as “secondary hypertension” [8]. A plethora of lifestyle factors such as smoking, psychological stressors, low vegetable/fruit consumption, excess alcohol, and obesity increase the chances of hypertension; with obesity and excess alcohol being at the forefront [9]. Furthermore, several genetic and epigenetic variations affecting the Renin-Angiotensin-Aldosterone System (RAAS) and endothelial elasticity can directly affect renal plasma flow, fluid/electrolyte balance, and systemic/peripheral vascular resistance, thereby predisposing individuals to hypertension [3,4,5,6,7,8].Hypertensive patients are at higher risk for atherothrombotic events such as ischemic and hemorrhagic stroke, retinopathies, and myocardial infarctions [10]. Hypertension is typically preceded by prothrombotic events such as vascular dysfunction, imbalance of procoagulants, and fibrinolytic activity, which ultimately affects platelet function [11]. Platelets play a critical role in maintaining vascular homeostasis and as such their maladaptive function underlies atherosclerosis [12]. Even though the sequence of events leading to atherogenic episodes is understood, the precise mechanism that activates platelets remains largely unknown [13,14,15]. Platelets when activated by Platelet-Activating Factor (PAF) or environmental stressors are prone to producing platelet derived microparticles (PMPs). These PMPs have been associated with numerous cardiovascular risk factors, one of them being hypertension [14]. PAF, a phospholipid mediator, may have a key role in linking maladaptive platelet function to atherosclerosis and subsequent hypertension. One of the common pathways that has been linked to atherogenesis is the Nuclear Factor Kappa-B (NF-kB) pathway (Figure 1) [15]. NF-kB is an important nuclear transcription factor involved in cytokine activation through regulation of inflammation [16]. Cytokines released by cells or oxidative stress can activate NF-kB, which upregulates genes such as VCAM1, a cell adhesion molecule, and tumor necrosis factor (TNF-α) in the blood vessels [17,18]. Constitutive expression of the NF-kB signal in blood vessels and smooth muscle cells leads to a chronic increase in oxidative stress, which further promotes cytokine activation. This causes a systemic inflammatory response, ultimately resulting in atherogenesis [15]. The evidence for the role of NF-kB pathway in facilitating atherogenesis has been found by the detection of NF-kB associated proteins in atherosclerotic vessels; it is not present in healthy blood vessels that do not exhibit evidence of atherogenesis [18,19].PAF is considered to play an important role in atherosclerotic plaque formation by supplementing the inflammatory processes that take place within the NF-kB pathway [21]. Clinical studies have shown an observable correlation between patients with severe atherosclerosis and elevated PAF levels in coronary artery circulation [22]. PAF is released by endothelial cells in response to thrombin, vasoactive mediators, and pro-inflammatory cytokines [9]. It is a known vasoactive mediator that causes increased leukocyte adhesion and infiltration of macrophages, resulting in cytokine release, inflammation, and intracellular lipid accumulation [3]. Macrophages are specifically activated by PAF, leading to an increase in intracellular calcium levels. This calcium triggers further downstream effects by first leading to macrophage adhesion to LDL [23], which results in the oxidation of LDL by macrophages. This is an important step in the atherogenic mechanism [24]. The oxidized LDL are taken up by macrophages and generate foam cells along blood vessel walls, which are seen in atherosclerotic plaques, indicated by Figure 2. In addition, macrophages can also release PAF, which further facilitates plaque formation [25]. PAF also increases oxidative stress in the blood vessels through indirect generation of reactive oxygen species and increasing the vascular permeability of arteries [26]. Furthermore, PAF directly activates platelets, causing them to aggregate and adhere to the injured endothelium, thereby initiating the plaque-forming cascade in blood vessels (Figure 3) [16]. PAF’s role in hypertension and arrhythmias is of concern. A study performed in rats has shown that low endogenous levels of PAF correlated with peripheral vasodilation, thereby highlighting a potential protective effect on peripheral vascular resistance and hypertension [27]. Furthermore, PAF levels were elevated in ischemic myocardium, particularly in conjunction with arrhythmias [28]. Hence, evidence suggests a potential role of PAF in mediating fatal arrhythmias such as ventricular fibrillation that are a known complication of ischemic myocardial injury [28].Role of PAF in atherosclerosis [29].Relationship between PAF and atherosclerosis [30].PAF’s (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) unique structure offers further insights into its role in atherosclerosis. PAF is synthesized from a subclass of phosphatidylcholines, which contain an ether bond instead of an ester bond with the backbone of glycerol [20]. It is a minor component of low-density lipoprotein (LDL), which, along with platelets, is another key pro-atherogenic mediator [27]. Moreover, the precursors of PAF can also undergo oxidative attack since they contain polyunsaturated fatty acids. Such oxidative damage could result in further numerous PAF-like molecules [31]. The PAF receptor is present on various cell types including immune, endothelium, smooth muscle, platelets, and cardiomyocytes [19,32]. It belongs to a G-protein linked receptor superfamily, which transduces a variety of functions via multiple heterotrimeric G proteins [19,32]. Protein kinase C activation, tyrosine phosphorylation, and proto-oncogene expression are among the diverse effects observed upon PAF receptor activation [33]. The inflammatory cascade also involves the stimulation of phospholipase A2 and subsequent production of arachidonic acid, which is a key precursor for several inflammatory mediators [33].PAF release has been noted to increase in the heart post-ischemia, indicating that myocardial tissue can produce and release PAF in the absence of perfusion. In addition, reperfusion of cardiac tissue post-ischemia has been followed by an increase in PAF release, which contributes to inflammation [34]. Sources of the rise in PAF levels include platelets, polymorphonuclear leukocytes, monocytes, mast cells, macrophages, and even cardiac myocytes [34]. PAF reciprocally promotes recruitment of these polymorphonuclear leukocytes, monocytes, and eosinophils to release pro-inflammatory cytokines, causing endothelial damage and inflammation [35]. Reactive oxygen species then oxidize low density lipoprotein, which contributes to atherosclerotic plaque formation. Subsequent recruitment of Th-1 cells leads to further inflammation, and disruption of the atherosclerotic plaque, causing acute cardiovascular disease [35].Generally, PAF has a depressive effect on the cardiovascular system’s function. It causes a decrease in venous return by inducing systemic venous vasodilation and increasing vascular permeability [34]. In addition, PAF is strongly associated with coronary vasoconstriction, believed to be mediated by serotonin, thromboxane, and leukotriene, which reduces coronary artery perfusion and oxygen supply [34]. PAF is also believed to have a minor impact on cardiac conduction, causing cardiac arrhythmias [34]. Studies conducted with both isolated hearts and cultured myocyte samples have shown that, when exposed to PAF, there is a decrease in contractile force, beat amplitude, and velocities of contraction and relaxation [34].As in the myocardium, PAF levels have been noticed to increase in cerebrovascular tissues post-ischemia. After an ischemic event, cerebral tissue undergoes stroke due to hypoxia, and PAF is believed to be responsible for the vasoconstriction of vessels that are supplying the ischemic regions of the brain [36]. Some studies have shown that the administration of a PAF receptor antagonist, such as indomethacin, decreases the PAF-mediated ischemia and hypoxia seen in stroke-affected cerebral tissues [36]. One study, conducted by K. Satoh et. al. in 1992, looked at PAF blood levels in stroke patients by performing a radioimmunoassay. When compared to the controls, post-stroke patients were measured to have an increase in PAF levels in the blood [37]. Hypersensitivity reactions by the immune system can target the cardiovascular system, and PAF is the major factor mediating these reactions. PAF, along with cyclooxygenase and leukotrienes, is responsible for the coronary vasoconstriction, arrhythmias, and decreased cardiac contractility seen in hypersensitivity reactions [34]. In addition, administration of PAF receptor-specific antagonists has been shown to decrease the cardiovascular effects of these hypersensitivity reactions. PAF has been found to be elevated in blood and tissues of animals deficient in magnesium [38]. A study using proton nuclear magnetic resonance spectroscopy on single vascular smooth muscle cells, excised canine and rat aortic, coronary, and cerebral arterial vessels illustrated that low levels of magnesium led to rapid PAF synthesis [39]. Magnesium is also independently implicated in causing maladaptive changes that lead to atherosclerosis. Deficiency of magnesium leads to a loss of its regulatory effects on sphingolipid pathways in cardiac and vascular smooth muscle cells, resulting in elevated ceramide levels from sphingolipid metabolism [40]. Elevated ceramide concentrations may lead to erosion of existing atherosclerotic plaques, thereby inducing thrombosis and plaque remodeling [39]. Deficiency of magnesium worsens lipid metabolism, resulting in a build-up of cholesterol, triglycerides, VLDL, and LDL [41,42]. These lipids pose a great danger as they can aggregate within the vasculature and cause atherosclerosis. They are also accompanied by enhanced oxidation of very-low-density lipoproteins and low-density lipoproteins (VLDL)/(LDL) and lipid peroxidation in cardiac myocytes. This can increase reactive oxygen species to levels that are known to perpetuate the inflammation preceding plaque formation [32,38]. Magnesium also helps maintain the elasticity of vessels by antagonizing calcium deposition [41]. Magnesium binds phosphate in the gastrointestinal tract, effectively inhibiting plaque formation as both calcium and phosphate are needed [43]. It also inhibits the maturation of hydroxyapatite, which is the most abundant crystal present in atherosclerotic plaques [43]. The antihypertensive effect of magnesium is supported by studies showing that decreased magnesium levels caused activation of the RAAS pathway, increased angiotensin II induced plasma aldosterone concentration, production of thromboxane, and vasoconstrictor prostaglandins [44]. Magnesium also increases prostacyclin release in cultured cells as well as healthy individuals [45]. Normally, the endothelium regulates its vasomotor tone by synthesizing prostacyclin [45]. Magnesium promotes vasodilatory effects of blood vessels through increasing prostacyclin release, thus possessing potential antihypertensive effects [45].Long term and significant magnesium deficiency were associated with overactive RAAS, hypertension, and oxidative stress that induced damage to the endothelium [46]. Figure 4 demonstrates the far-reaching, deleterious effects of magnesium deficiency.Figure 5 demonstrates the multitude of effects that magnesium has on cellular transporters and channels. The regulation of cardiac conduction and contractility is highly dependent on maintaining sufficient magnesium ion levels. Magnesium can regulate K+ and Na+ ion transport within cardiac myocytes by acting on the ATPase to hydrolyze ATP in order to promote Na+/K+ ATPase pump activity [42]. Deficiency of magnesium has been implicated in, but not limited to, coronary artery disease, cardiac arrhythmia, and heart failure. Magnesium deficiency can lead to cardiac thickening and calcifications, specifically causing left ventricular hypertrophy and coronary artery calcification [42]. Magnesium deficiency, also known as hypomagnesemia, typically occurs with hypokalemia, resulting in significant cardiac arrhythmias. One meta-analysis found that patients with acute myocardial infarctions, after treatment with intravenous magnesium, experienced a 49% reduction in ventricular tachycardia and ventricular fibrillation [42].Magnesium also plays an important role in the prevention of heart failure, by ensuring proper cardiac function and blood pressure. Magnesium promotes ATP synthesis and regulates intracellular Ca2+ levels to promote cardiac contraction, while decreasing aldosterone levels to decrease blood volume and blood pressure [42]. Patients with chronic hypomagnesemia are seen to suffer from heart failure due to the loss of these protective effects.As the world’s population continues to age, many people have shown signs of aging including metabolic decline, atherosclerosis, high blood pressure, cardiovascular diseases, and type 2 diabetes. These symptoms of aging have been associated both experimentally and clinically with the presence of Mg-deficient states [47].Magnesium deficiency has been found to accelerate the cellular aging process. Studies performed by Shah et al. suggested that short term magnesium deficiency resulted in an upregulation of p53 and neutral sphingomyelinase (N-SMAse) in heart cells and smooth muscle cells of the aorta [47]. N-SMAse upregulation leads to the synthesis and release of ceramide and possibly other sphingolipid products [47]. Ceramide synthesis has been shown to downregulate telomerase activity, which is required for maintaining telomere length, but further studies are warranted [47]. All together, these have pivotal roles in atherogenesis, hypertension, and heart failure, all of which are involved in the aging process (Figure 6) [47]. These results uphold that magnesium deficiency, unless corrected early, will contribute significantly to aging [47].Aging has also been associated with an increase in the levels of proinflammatory cytokines in tissues and cells [47]. Interestingly, recent findings in Mg-deficient animals, tissues, and different cell types have shown elevated levels of many of the proinflammatory cytokines such as IL-1, IL-6, TNF-alpha, among others [47]. TNF-alpha is known to be associated negatively on telomerase activity in some cell types [47]. Furthermore, magnesium acts as an antioxidant against free radical damage of the mitochondria. Chronic inflammation and oxidative stress are pathogenic factors in aging and age-related diseases (Figure 7) [49]. It is crucial to supplement magnesium especially in the elderly population. Studies have shown that simple increases in daily intake of magnesium can allow for healthier aging [47]. The recommended allowance of magnesium in both males and females is 400–420 mg/day and 310–320 mg/day, respectively [50]. An essential feature of heart failure associated with complex ventricular arrhythmias is hypomagnesemia, most likely related to increased urine magnesium excretion. By supplementing with magnesium, these arrhythmias can be alleviated or abolished [51]. Magnesium supplementation above 15 mmol per day is required to normalize high blood pressure in unmedicated hypertensive patients as well as lower high blood pressure in patients treated with anti-hypertensive medications [1]. Unfortunately, the average dietary intake of magnesium has decreased among both men and women living in North America [52]. Several studies have shown a relative decreased intake in magnesium content in people following Western diets [53]. In addition, areas with soft water have low magnesium content in drinking water [54]. Residents of areas with soft-water and magnesium poor-soil have a higher tendency to suffer from ischemic heart disease (IHD), coronary vasospasm, hypertension, and sudden cardiac death (SCD) [55]. Low levels of magnesium are also associated with prehypertension and hypertension in children [56]. In the general population, a meta-analysis of one million patients revealed reduced risk of heart failure, stroke, diabetes, and all-cause mortality in those receiving magnesium supplementation [57]. Intravenous magnesium can be therapeutically administered to reduce the risk of potentially fatal arrhythmias after a myocardial infarction [58,59]. Studies have also shown that complex ventricular arrhythmias in patients with heart failure can be abolished by magnesium supplementation [52]. Overall, these findings highlight the importance of magnesium in preventing and treating cardiovascular disease.Hypertension leads to changes in blood vessels that contribute to atherosclerosis and platelet activation. This in turn contributes to cardiovascular and cerebrovascular disease through oxidative stress. Oxidative stress via low magnesium and increased PAF has also been found to reduce telomerase activity and shorten telomeres, which directly contributes to aging and is correlated to increased myocardial infarction risk [48]. In this review we have discussed the role of PAF and magnesium in the pathophysiology of these conditions. Hypertension was defined within the context of clinical medicine and its pathophysiology was explained. Secondly, PAF’s structure and function were discussed. Thirdly, magnesium was introduced as a micronutrient that plays a key role as an antihypertensive. Through calcium deposition antagonism and release of vasodilatory prostacyclin, magnesium is theorized to reduce high blood pressure. Experimental evidence has shown that supplementing magnesium deficiency with dietary magnesium has significantly improved cardiovascular health [60]. In a study performed by Altura et al. on rabbits who were fed a cholesterol diet, oral supplementation with Mg salt magnesium aspartate hydrochloride lowered levels of cholesterol and triglycerides in normal (25–35%) and atherosclerotic (20–40%) animals and inhibited the atherosclerotic pathway [60].There is clinical evidence to support that hypomagnesemia contributes to vasospasm and ischemic injury through several mechanisms, including induction of mitochondrial dysfunction, activation of apoptosis, and facilitation of ceramide synthesis and release. Furthermore, platelet-activating factor (PAF) has been implicated in atherogenesis and inflammation, especially in relation to cardiovascular and cerebrovascular injury. The promotion of platelet aggregation, oxidative stress, and vascular permeability were some of the highlighted mechanisms that PAF augments in atherogenesis. Figure 8 below summarizes the interrelationship between magnesium and PAF in the context of atherogenesis and vascular injury.As discussed, PAF has been implicated in inflammatory processes in relation to the NF-kB pathway. In 2016, Altura et al. provided evidence for a novel hypothesis interrelating PAF with activation of the NF-kB pathway, proto-oncogenes c-fos and c-jun, and ceramide synthesis, in a low-Mg2+ environment, which in turn contributes to the elaboration of PAF [39]. However, the precise interrelation between free Mg2+ concentration and PAF in the context of vascular disease is not yet clear. Ultimately, the present review (summarized below in Figure 9) encourages further investigation into platelet-activating factor and the cardioprotective role of dietary magnesium supplements, particularly in patients with a history of coronary artery disease and arrhythmias. Further investigation into the crosstalk between magnesium and PAF in the prevention of atherosclerosis and hypertension is also warranted. Conceptualization, N.S., R.S. and S.S.; investigation, N.S., R.S., S.S. and K.J.; writing—original draft preparation, N.S.; writing—review and editing, N.S., R.S., S.S., K.J., J.D., Y.C. and C.S.; supervision, H.W. All authors have read and agreed to the published version of the manuscript.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Not applicable.Not applicable.Not applicable.We are very grateful to Zumit Shah, Laxit Shah, Sasha Taylor, Ved Bhavin Patel, Radha Sanjay Shah, and Kena Piyush Patel for their technical assistance and generous help in writing this manuscript.The authors declare no conflict of interest.NF-kB signaling [20].Cardiovascular implications of magnesium deficiency.Effect of magnesium (Mg2+) on potassium (K+) channels, sodium (Na+) channels, and sodium/potassium (Na+/K+) ATPases transporting ions through cardiac myocytes.Impact of magnesium deficient states on telomerase activity [48].Effect of magnesium deficient states on age related diseases [49].Interrelation between magnesium and platelet-activating factor (PAF).Effects of low magnesium and increased PAF on hypertension, atherogenesis, cardiovascular disease, stroke, and aging.Current blood pressure guidelines by American College of Cardiology (ACC).Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Genetic testing plays an increasing diagnostic and prognostic role in the management of patients with heritable thoracic aortic disease (HTAD). The identification of a specific variant can establish or confirm the diagnosis of syndromic HTAD, dictate extensive evaluation of the arterial tree in HTAD with known distal vasculature involvement and justify closer follow-up and earlier surgical intervention in HTAD with high risk of dissection of minimal or normal aortic size. Evolving phenotype–genotype correlations lead us towards more precise and individualized management and treatment of patients with HTAD. In this review, we present the latest evidence regarding the role of genetics in patients with HTAD.Over the last two decades, genetic developments have significantly improved our understanding of heritable thoracic aortic disease (HTAD). The identification of new syndromes [1] and novel candidate genes [2] has changed the paradigm in the diagnostic evaluation of these patients. Specific genotype–phenotype correlations continue to emerge, promising a more precise and effective approach in the treatment of HTAD [3,4,5]. The goal of this review is to inform clinicians about the value and effect of genetics in the diagnosis, management, surveillance, risk stratification and familial evaluation of patients with HTAD.The presence of syndromic systemic features and a positive family history of aortic aneurysm or dissection are the key elements that determine the classification and thus the management of patients with HTAD. Thoracic aortic disease at a younger age occurs more often in the context of a genetic syndrome.Syndromic HTAD (sHTAD) typically exhibits a multiorgan phenotype and is caused by genetic variants that are involved in the transforming growth factor-β (TGF-β) pathway and genes encoding extracellular matrix proteins [2]. Nonsyndromic HTAD (nsHTAD) is typically characterized by isolated thoracic aortic aneurysm or dissection, without any recognizable systemic features, and can be familial in up to 20–25% of cases. A genetic defect, mainly in genes of the contractile apparatus, may be identified in up to 20% of familial nsHTAD [6].Only 5% of patients present with alarming symptoms before an acute aortic event [7]. Most patients are usually diagnosed following a major complication, e.g., an aortic dissection, as part of familial evaluation, or based on characteristic physical findings suggestive of a specific syndrome.Physical examination and history are vital in the assessment of patients with HTAD. The physician should be able to recognize any systemic features such as specific facial characteristics, skin lesions or skeletal manifestations, which suggest the presence of sHTAD. A detailed personal history should be obtained including history of recurrent pneumothorax, history of eye operations or ocular conditions. Ophthalmology evaluation, including slit-lamp examination, should be offered in all patients with a suspicion of Marfan syndrome (MFS). The reduced penetrance or incomplete expression and the phenotypic overlap and variability of hereditary aortopathy consist of major challenges, which make essential a multidisciplinary diagnostic approach.Marfan syndrome (MFS; Online Mendelian Inheritance in Man, OMIM®. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, USA OMIM #154700, Orphanet rare disease nomenclature, French National Institute for Health and Medical Research, Paris, France ORPHA:558) is the most common syndromic aortopathy. It is characterized by aortic dilatation, ectopia lentis and skeletal abnormalities. MFS is associated with variants in the FBN1 gene, encoding the extracellular matrix protein called fibrillin-1 [8]. Other genes, typically not causing any ocular involvement, also can lead to a phenotype resembling MFS [2].The diagnosis of MFS is based on the revised 2010 Ghent criteria [8]. A pathogenic FBN1 variant, along with ectopia lentis or enlarged aortic root (Z score ≥2 that corresponds to a diameter ≥2 standard deviations above normal, according to established aortic nomograms [9]), establishes the diagnosis. In the absence of a pathogenic FBN1 variant, the diagnosis can be made in patients with aortic dilatation (Z score ≥2) and ectopia lentis or a systemic score ≥7 (encompassing systemic features suggestive of MFS) [8]. In patients with a family history of MFS, the diagnosis can be made in the presence of ≥1 of the following criteria: (a) ectopia lentis, (b) systemic score ≥7, (c) aortic root enlargement with Z-score ≥2 in patients >20 years and (d) Z score ≥3 in patients <20 years [8].Over half of MFS patients are diagnosed after adolescence (median age of diagnosis is 19 years) [10], and the first cardiovascular procedure occurs at average at the age of 36 years [11]. Aortic dilatation typically occurs at the level of the sinuses of Valsalva (SoV), but aortic dilatation or dissection can occur at every level of the aorta [12]. Most patients with MFS are diagnosed before severe cardiac complications occur. This is particularly relevant, as MFS patients appear to have better survival rates with appropriate medical management and when undergoing prophylactic elective surgery (complication rate of 1.5% vs. 11.7% of urgent procedures) [13]. Established clinical factors of high risk are: (a) aortic diameter at the SoV ≥5 cm, (b) rapid increase in aortic dilatation (≥3 mm per year), (c) family history of aortic dissection at a low aortic size, (d) progressive aortic regurgitation, (e) personal history of spontaneous vascular dissection and (f) desire for pregnancy [14,15]. Therapy is based on optimal blood pressure control, and medical management includes beta-blockers or angiotensin-1 antagonists (losartan) [14,16,17].Most FBN1 variants are missense, having in most cases a dominant-negative effect (DN-FBN1), resulting in a disorganized extracellular matrix incorporating both mutated and nonmutated fibrillin-1 proteins. Haploinsufficient FBN1 variants (HI-FBN1), leading to reduced production of normal fibrillin-1 protein, are also well documented in up to 35% of MFS patients [5]. It has been reported that patients with HI-FBN1 are at increased risk of aortic dissection and death, have more rapid aortic root and ascending aorta dilation rates, manifest more severe cardiovascular complications and respond better to losartan therapy than patients with DN-FBN1 variants [5,18,19,20,21]. In a large cohort of MFS patients, premature termination codon variants leading to haploinsufficiency were associated with an 83% lifelong aortic event risk during life, shorter life expectancy, severe scoliosis and relatively lower rates of ectopia lentis surgery [22].Specific DN-FBN1 variants have been linked with a similarly severe phenotype compared to HI-FBN1 variants. MFS patients with in-frame DN-FBN1 variants leading to a cysteine loss at the level of fibrillin-1 showed 73% lifelong risk of aortic dissection or surgery, high rates of severe scoliosis and ectopia lentis surgery. In-frame variants leading to a neutral cysteine effect were associated with an intermediate phenotype (61% of lifelong aortic event risk). In-frame variants leading to cysteine gain were associated with a better cardiovascular profile (29% of lifelong aortic event risk) and lower rates of severe scoliosis but high risk for ectopia lentis surgery [22]. Takeda et al. identified deleterious high-risk variants among DN-FBN1 Japanese patients, in-specific variants affecting or creating cysteine residues and in-frame deletion variants in exons 25–36 and 43–49 [19].Furthermore, despite a greater frequency of surgery and type B aortic dissections in MFS patients harboring HI-FBN1 variants, all type A dissections that occurred at an aortic root diameter <50 mm were DN-FBN1 variants in a cohort of 954 MFS patients followed for a mean 9.1 years [23].Loeys–Dietz syndrome (LDS, ORPHA:60030) is characterized by the combination of arterial tortuosity with ascending aortic aneurysm/dissection, also involving the distal aorta and branching arteries, hypertelorism and bifid uvula or cleft palate. It was first described in 2005 as a novel autosomal dominant syndromic aortopathy [1]. Since 2005, loss-of-function variants in six genes have been linked to LDS (TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2 and TGFB3), all of which are involved in the TGF-β signaling pathway [4].LDS has been originally associated with a very aggressive natural history, probably reflecting a selection bias in the first series of patients. Aortic dissection in young patients (mean age at death of 26 years), very high incidence of pregnancy-related complications and aortic dissections at only mildly increased or even much normal aortic dimensions have been reported [1]. Current American Heart Association/American College of Cardiology guidelines suggest an aggressive approach with prophylactic surgery in aortic size ≥42 mm in all patients with LDS [24]. Recent European Society of Cardiology (ESC) guidelines suggest surgery in patients with TGFBR1 or TGFBR2 pathogenic variants with maximal aortic sinus diameter ≥45 mm [15]. Although there is no scientific evidence published to date, drug treatment with beta-blockers and/or angiotensin blockade and optimal antihypertensive management is thought to improve prognosis in LDS patients in a similar fashion to MFS patients. Extensive and distal vascular involvement in LDS patients warrants regular and more extensive imaging of the arterial tree (from head to pelvis).Most recent data from the biggest cohort to date, consisting of 441 patients with TGFBR1 and TGFBR2 variants from 228 families, showed a relatively more favorable overall clinical profile than previously reported and provided very important genotype–phenotype information [4]. No differences in survival or prevalence of syndromic characteristics between TGFBR1 and TGFBR2 carriers were identified.The investigators identified a subgroup of female patients with a TGFBR2 variant, marked systemic features and low body surface area who exhibited aortic dissection in aortic sizes <45 mm. Hypertelorism, aortic tortuosity and wide scars were significantly associated with aortic dissection in this cohort and were present in all women with aortic dissection and minimal aortic enlargement or pregnancy-related dissections. In a considerable percentage of patients that had surgery for an aortic root aneurysm (10%), dissection of the ascending aorta occurred during follow-up. Therefore, in patients with TGFBR1 or TGBR2 variants, replacement of both the aortic root and the ascending aorta when an indication for surgery exists should be considered.Variants in SMAD3 cause a type of LDS also known as aneurysm–osteoarthritis syndrome, which is characterized by arterial tortuosity, aneurysms and dissection, as well as early-onset osteoarthritis [25,26,27]. Aside from osteoarthritis-related symptoms, which often may be missed if there is no high clinical suspicion [6], patients with SMAD3 variants present with fewer and milder syndromic features compared to the other types of LDS or MFS. This possibly leads to a belated diagnosis, with the majority of SMAD3 variant carriers presenting with type A dissections.In contrast to TGFBR1 and TGFBR2, patients with SMAD3 variants show a relatively later onset of dilatation or dissection. Aortic events are extremely rare in children or adolescents [3,28]. A recent study indicated that missense variants in the region encoding the MAD homology 2 (MH2) domain may lead to a lower median age of the first aortic event compared with patients with haploinsufficient variants [3]. Aortic dissections typically occur with prior aortic root enlargement. In the largest series of patients with SMAD3 variants to date, dissections occurred at root diameter from 43 mm to 66 mm, with the majority of dissections or elective surgical repair happening in aortic root diameters of 50 mm or greater [3].These variants are found in 10–15% of LDS cases and typically show milder phenotypes and reduced penetrance [27,29]. Aortic dissection in lower-than-standard surgical thresholds of 50 mm has been reported in LDS patients with TGFB2 variants [30], indicating a possible intermediate risk. A more conservative approach regarding aortic surgery following standard aortic size thresholds is reasonable in patients with SMAD2 or TGFB3 variants until new data emerge [2].It is a rare autosomal dominant syndromic HTAD caused by genetic defects in the COL3A1 gene. Rarely, vEDS can be caused by specific arginine-to-cysteine substitution variants in the COL1A1 gene. The syndrome is characterized by arterial, uterine or bowel ruptures, skin translucency with visible veins and easy bruising and characteristic facial features (thin pinched nose, prominent eyes and lobeless ears, lack of subcutaneous fat). Diagnosis is established using the 2017 International Classification of the Ehlers–Danlos syndrome [31], which updated the earlier nosology of Villefranche [32]. Men seem to have a poorer prognosis than women (median survival age of 46 ± 1.8 years vs. 54 ± 2.5 years) [33]. Surveillance may include periodic arterial screening.Genetic testing is highly specific and sensitive for vEDS, revealing a genetic defect in 95% of cases [34]. Lethal arterial events in classic (nonvascular) Ehlers–Danlos syndrome (EDS) caused by COL5A1 or COL1A1 variants have also been reported [35]. Identification of a pathogenic variant establishes the diagnosis [32]. The clinical phenotype and prognosis of vEDS may be influenced by the type of COL3A1 variant. Patients heterozygous for “null” COL3A1 variants, leading to loss of the stable mRNA from one COL3A1 allele, show late-onset disease, reduced penetrance, solely vascular events and longest survival compared to missense and splicing variants [33,36,37,38]. Glycine substitutions, splice-site and in-frame insertions/deletions bear the poorer prognosis leading to earlier complications [39].Although aortic dissection can occur at normal aortic sizes in up to 33% of patients [40], aortic surgery is not usually recommended due to the high rate of intraoperative mortality caused by extreme fragility of the vessel walls. Surgery is usually performed urgently to treat potentially life-threatening complications. Endovascular repair with coil embolization has shown promising results in selected cases of ruptured pseudoaneurysms, visceral aneurysms and carotid-cavernous fistulas. A multicenter, randomized and blinded open trial study showed significantly lower arterial events (rupture or dissection) in vEDS patients receiving celiprolol, a β(1)-adrenoceptor antagonist with a β(2)-adrenoceptor agonist action, compared to controls [41]. Encouraging reports from animal models and an observational study in favor of celiprolol have been published since; however, no randomized prospective trials exist to date [38,42].Loss-of-function variants in the X-linked biglycan gene (BGN) have been described in five families with syndromic features overlapping with those of LDS and MFS patients. It is characterized by early-onset aneurysms of the aortic root or ascending aorta (as early as age 1) and aortic dissection (earliest at the age of 15 at an aortic size of 45 mm at the SoV) in male probands. Distal aneurysms in the brain have been detected in one patient. Female patients showed a relatively milder phenotype [43].Pathogenic variants in the X-linked filamin A (FLNA) gene, encoding an actin-binding protein that regulates the cytoskeleton and cell motility, cause the brain malformation periventricular heterotopia (PVNH; OMIM #300049, ORPHA:82004), which may also occur in association with EDS features [44]. Neurological symptoms include mainly seizures and dyslexia. Chen et al. reported on the largest series to date of 114 patients, with loss-of-function FLNA pathogenic variants and found aortic dilatation in 18.4% of the patients [45]. Aortic rupture occurred in a 41-year-old male patient at an aortic root size of 42 mm. Pulmonary artery dilatation and aneurysms of other vessels (in the subclavian, middle cerebral and internal carotid arteries, as well as in the abdominal aorta) were common.Loss-of-function variants in the LOX gene, encoding a lysyl oxidase involved in the remodeling of the extracellular matrix, have been shown to predispose to aortic root and fusiform aneurysms, involving both the aortic root and ascending aorta [46,47]. These patients exhibit some overlapping syndromic MFS features, without, however, fulfilling the Ghent criteria. No cases of aortic dissection in minimal aortic dimensions have been reported to date. Presence of a bicuspid aortic valve (BAV) has been found in up to 15% of LOX carriers [46].Recently, four case reports of aortic dilatation have been reported in patients with GCMS, including a 45-year-old female patient who presented with aortic dissection at an aortic size of 51 mm [48,49,50]. The syndrome is caused by variants in the SLC25A24 gene and is characterized by craniofacial dysostosis, hypertrichosis, underdeveloped genitalia, ocular and dental anomalies.Pathogenic variants in the SKI gene, coding a protein that regulates the TGF-β signaling pathway, cause a hereditary syndromic aortopathy, phenotypically overlapping with MFS and LDS, characterized by craniosynostosis, marfanoid habitus, intellectual disability, camptodactyly and typical facial dysmorphism [51]. Infantile hypotonia, early developmental delay and intellectual disability are distinct features of the syndrome. Aortic dilatation is generally restricted to the aortic root, and a more benign course than MFS and LDS has been described [52].Pathogenic variants in the SLC2A10 gene, coding the protein GLUT10 that regulates the TGF-β signaling pathway, lead to ATORS. It is characterized by widespread arterial involvement with elongation, tortuosity and aneurysms of the large and middle-sized arteries along with craniofacial, skin and ocular manifestations [53]. Cardiovascular findings also include aortic coarctation, abnormal implantation of the aortic branches, pulmonary stenosis and aortic stenosis. Patients with ATORS are at higher risk of ischemic events. Aggressive aortic root aneurysm formation even in infancy and aortic dimensions up to 60 mm has been reported; however, there have been no reports of aortic dissection to date [54].The LTBP3 gene encodes an extracellular matrix protein regulating the TGF-β signaling pathway in a latent state. Homozygous loss-of-function pathogenic variants in the LTBP3 gene have been associated with dental anomalies and short stature (DASS) syndrome (OMIM #601216). Guo et al. reported segregation of compound heterozygous or homozygous LTBP3 variants in two families with DASS and thoracic aortic disease [55]. The affected individuals also manifested arterial involvement, including abdominal aortic aneurysms and multiple visceral and peripheral arterial aneurysms as well as mitral valve prolapse. Zhu et al. recently reported another compound heterozygous patient with DASS and aortic dissection at the age of 42 and an aortic sinus size of 53 mm [56].Monoallelic LTBP3 variants appear to be involved in nsHTAD [55,56]. Guo et al. showed that relatives with heterozygous LTBP3 variants presented with late-onset aortic aneurysms and dissection without any systemic features. Additionally, heterozygous LTBP3 variants were identified in 9 out of 338 patients, with thoracic aortic dissection at <56 years of age and no family history or syndromic features [55]. Zhu et al., investigating a cohort of 266 Asian patients with thoracic aortic dissection and/or aneurysm, detected 4 patients with heterozygous LTBP3 variants who had experienced aortic dissections at 33–52 years of age.Pathogenic variants in the ACTA2 gene, encoding the vascular isoform of the smooth muscle cell contractile protein alpha-actin, lead to diverse forms of syndromic and nonsyndromic HTAD [6,57,58]. Heterozygous missense ACTA2 variants cause a form of HTAD commonly associated with persistent livedo reticularis (a purplish skin discoloration caused by constriction or occlusion of deep dermal capillaries), iris floccule [58], premature onset of coronary artery disease, premature ischemic strokes due to Moyamoya disease (MYMY5; OMIM #614042, ORPHA:2573) (stenosis or occlusion of the terminal portion of the internal carotid artery with the formation of an abnormal vascular network in the vicinity of the arterial occlusion) or fusiform cerebral aneurysms [59], and BAV. The penetrance of thoracic aortic disease in these patients was estimated to be 50–70% and did not appear to be age dependent. Type A dissections were more common than type B dissections [58,60].Pathogenic ACTA2 variants leading to arginine replacement by histidine, leucine or cysteine at position 179 (R179 variants) cause a distinct syndrome called multisystemic smooth muscle dysfunction syndrome (MSMDS, OMIM #613834, ORPHA:404463) characterized by congenital fixed pupils (mydriasis), large patent ductus arteriosus or aortopulmonary window, small vessel disease, urinary bladder dysfunction, intestinal malrotation, severe cerebrovascular disease and fully penetrant thoracic aortic disease by the age of 25 years [61,62,63,64,65].The p.R179 and p.R258 ACTA2 variants are characterized by a significantly increased risk for aortic events, very early presentation typically in childhood warranting early repair, whereas the p.R185Q and p.R118Q variants seem to bare a more benign course [60]. Larger cohorts are required before implementation of these genotype–phenotype correlations into clinical practice. Dissections have been reported at aortic aneurysm sizes of as low as 40 mm [64,66]; therefore, earlier surgical intervention is advised in patients with established ACTA2 variants.Biallelic variants in the IPO8 gene, encoding the nuclear import protein importin 8, lead to a form of sHTAD presenting with a LDS/SGS-like phenotype with early-onset aortic aneurysms (before 1 year of age in the youngest), marked arterial tortuosity, structural heart disease in involving atrial or ventricular septal defects and patent ductus arteriosus, facial and skeletal anomalies, developmental delay, umbilical and/or inguinal hernias, immune dysregulation and allergic diseases [67,68,69]. No aortic dissection has been reported in a total of 28 patients who have been identified so far, despite a severe aneurysm phenotype in most affected individuals.Turner syndrome (TS; OMIM #300082, ORPHA:881). TS is characterized by short stature, premature loss of ovarian function, webbing of the neck, lymphoedema, kidney and skeletal abnormalities in women and girls with complete or partial absence of the second X chromosome. Aortic coarctation (reported prevalence 7–18%) and BAV (reported prevalence 12–30%) are the most common congenital heart disease detected [70]. Aortic dilatation and dissection usually occur in TS patients with BAV or coarctation. However, the onset of aortic complications in TS occurs at a much younger age (20s and 30s) than nonsyndromic BAV or coarctation cases, aortic dilatation and dissection in the presence of apparently normal aortic valve has been reported, TS is an independent risk factor for aortic dilation and dissection and cystic medial necrosis has been found in a considerable portion of TS patients with aortic dissection during histology, indicating an inherent abnormality of the aortic tissue [70,71,72]. Aortic dissection in the absence of coarctation, BAV or hypertension has been reported in up to 11% of TS patients [73].Although metabolic storage diseases are not typically classified as syndromic HTAD, there is evidence of thoracic aortic disease in these conditions. El-Gharbawy et al. reported dilatation of the ascending aorta and/or aortic arch in five female patients (12.5% of the cohort) with late-onset Pompe disease (OMIM #232300, caused by variants in the GAA gene, leading to a deficiency in the acid α-glucosidase enzyme), including a 42-year-old patient with concomitant BAV who presented with aortic dissection of the ascending aorta at an aortic size >50 mm [74]. Aneurysms of the aortic root and ascending aorta, developing by the fifth decade of life, have been reported in 9.6% of male and 1.9% of female normotensive patients with Fabry disease (FD; OMIM #301500), an X-linked recessive disorder that is caused by deficiency of the lysosomal enzyme α-galactosidase A (GLA gene) [75,76,77]. Although vertebral artery and carotid artery dissection are relatively common and lead to stroke in FD, no cases of aortic dissection or rupture have been reported to date [76]. Aortic root dilatation (in 35–39% of the patients) has also been described in mucopolysaccharidoses, a group of 11 different lysosomal storage disorders, characterized by enzymatic deficiency leading to attenuated degradation and increased storage of glycosaminoglycans. No cases of aortic dissection have been published, and absolute aortic root dimensions greater than 45 mm are uncommon [78,79]. Hyperplasia and glycogen deposition vascular of smooth muscle cells, as indicated in animal models, may explain the presence of thoracic aortic disease in lysosomal storage disease [80].Bicuspid aortic valve (BAV; OMIM #109730, ORPHA:402075) is the most common congenital heart disease with an estimated prevalence of 0.5–0.8% [81]. Although BAV can be part of the phenotype in some cases of sHTAD such as MFS or LDS [82], there is also mounting evidence of familial clustering in up to 6–9% of first-degree relatives of nonsyndromic BAV [83,84]. An autosomal dominant inheritance pattern with variable expressivity and typically incomplete penetrance is recognized [82,84]. Up to 75% of patients with BAV might develop aortic dilatation [85], although this typically occurs later than other syndromic or nonsyndromic HTAD and at relatively slower growth rates (average of 0.19 cm/year) [86].Variants in the NOTCH1 gene have been described in approximately 1% of sporadic BAV cases and in up to 7% of familial BAV cases [82,87,88] and are typically associated with prominent valve calcification [89]. Recently, loss-of-function SMAD6 variants have been found in up to 11% of nsHTAD patients with BAV [90,91]. ROBO4 variants, encoding a factor known to contribute to endothelial performance, and TBX20 variants, a transcription factor involved in the regulation of heart development, were shown to contribute to aortic aneurysm formation in families with nonsyndromic BAV [92,93].Echocardiography screening of first-degree relatives of patients with BAV should be offered especially in boys, athletes and if hypertension is present [24]. Families with multiple affected relatives, a combination of other left-sided congenital abnormalities and a particularly malignant clinical profile should be offered genetic testing for at least ACTA2, SMAD6, TBX20, ROBO4 and NOTCH1 genes. Multiple gene panels should be considered in selected cases, taking into consideration the variable and incomplete penetrance of sHTAD that might lead to a mild phenotype with minimal or no systemic features in some patients. No specific genotype–phenotype correlations currently exist that could possibly guide surgical interventions or provide specific prognostic information. Recently, Pileggi et al. indicated that specific NOTCH1 variants could be associated with better prognosis and later-onset development of aortic stenosis [88].Based on the presence of familial disease or not, nsHTAD is further categorized into familial and sporadic nsHTAD. A positive family history of thoracic aortic disease is associated with an increased aortic growth rate, a bigger chance of gene identification and earlier phenotypic manifestation [94]. The genetic etiology of familial nsHTAD is highly heterogeneous and usually involves genes that regulate the smooth muscle cell contractile apparatus. The genetic substrate of sporadic nsHTAD is largely unknown and seems to differ from familial nsHTAD cases.To date, over 10 genes and 2 linked loci have been involved in the pathogenesis of nsHTAD, including genes involved in the (TGF-β) pathway and genes encoding extracellular matrix proteins that are typically associated with syndromic aortopathies [2,6,47]. Common single nucleotide polymorphisms at the 15q21.1 locus of the FBN1 gene have been shown to be associated with sporadic nsHTAD [95] without other systemic features of MFS. Arnaud et al. performed genetic screening in 226 consecutive nsHTAD, either sporadic in patients under 45 years of age or in documented familial cases, and identified an overall yield of pathogenic or likely pathogenic variants (SMAD3, FBN1, TGFBR1, TGFBR2, TGFB2, ACTA2, MYLK, FLNA, FBN2, LOX, MFAP5 genes) in 18% of the patients (11% in sporadic cases vs. 22% in familial cases), with almost two-thirds located in SMAD3 and FBN1 genes. Exclusively missense variants and no premature termination codon variants were identified in the FBN1 gene in this cohort. More careful clinical evaluation after the genetic result revealed clinical findings consistent with LDS in approximately half of the cases with SMAD3 variants and history of periventricular heterotopia in patients with the FLNA variant, reclassifying these cases as syndromic [6]. Weerakkody et al. investigated a cohort of 1025 unrelated HTAD cases, including many cases of sporadic HTAD, and reported a 4.9% yield of genetic testing for a 15-gene genetic panel. Patients with a family history of HTAD were four times more likely to carry a pathogenic or likely pathogenic variant than those without a family history (9.8 vs. 2.4%) [96]. Since clinical information (syndromic features or clinical diagnosis) was not available in a significant percentage of the cases, these cases cannot automatically be categorized as sporadic nsHTAD.Overall, pathogenic ACTA2 variants are the most frequently encountered, as they are detected in 1–21% of nsHTAD [57,97,98] and are associated with a malignant aortic phenotype. Pathogenic variants in the MYLK gene [99,100,101], with missense pathogenic variants showing an earlier onset aortic event, and variants in the MYH11 [102] and PRKG1 genes [103] have also been recognized as relatively rare but aggressive causes of thoracic aortic dissection (~1% prevalence of each), in nsHTAD which are not always preceded by obvious aortic dilatation. There is no evidence to date that defects in the other genes identified (LOX, MFAP5, FOXE3, MAT2A, SMAD2, SMAD4, NOTCH1, PLOD1, TGFB2, TGFBR2, FBN1, FBN2) are linked to a more severe phenotype or earlier presentation of HTAD [6,57,58].Genetic testing of patients with established or suspected HTAD is an essential part of their assessment and should follow clinical evaluation and proper genetic counseling (Figure 1). Specific gene testing may be considered when the phenotype indicates a distinct syndromic aortopathy to aid clinical decisions and offer prognostic and diagnostic information. In most cases, however, a multigene panel should be used, consisting of at least of the 11 genes (MFS–FBN1, LDS–TGFBR1, TGFBR2, SMAD3, TGFB2, vEDS–COL3A1, ACTA2, MYLK, LOX, PRKG1, MYH11) that have been identified to have a “definitive” or “strong” association with HTAD [104]. Specific criteria for genetic testing have been proposed by the Rare Disease Group of VASCERN, based on expert opinion (Table 1) adopted in the most recent European guidelines of the ESC and the European Society of Human Genetics [105]. Exome sequencing may be performed in cases with equivocal diagnosis, nonsyndromic HTAD or for research purposes [106]. Cascade genetic screening should be offered in all family members of patients with well-established pathogenic or likely pathogenic variants. Surgery should be offered in patients with a malignant genetic and/or clinical profile and mildly dilated aortas (Table 2).Genetic testing plays an increasing role in the management of patients with HTAD. The identification of a pathogenic variant can establish or confirm the diagnosis of syndromic HTAD, dictate extensive evaluation of the arterial tree in HTAD with known distal vasculature involvement and justify closer follow-up and earlier surgical intervention in HTAD with high risk of dissection of minimal or normal aortic size. Evolving phenotype–genotype correlations should soon allow for more precise and individualized management and treatment of patients with HTAD.Conceptualization, E.P. and A.A.; methodology, E.P., D.D. and A.A.; writing—original draft preparation, E.P.; writing—review and editing, E.P., D.D. and A.A.; supervision, E.P., D.D. and A.A.; All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.The authors declare no conflict of interest.Clinical and genetic evaluation of suspected heritable thoracic aortic disease. HTAD: Heritable thoracic aortic disease; TAD: thoracic aortic disease, BAV: bicuspid aortic valve. 1 This includes one of the following: (a) Aortic root diameter Z-score >3.5 (b) Aortic root diameter Z-score ≥3 in patients <18 years (c) Aortic root diameter Z-score 2.5–3.5 in patients 18–60 years (d) Aortic root diameter Z-score 2.5–3.5 without hypertension in patients >60 years. 2 1st- or 2nd-degree relative with aortic dissection or aneurysm aged <60 years or sudden cardiac death <45 years. 3 Ectopia lentis, hypertelorism, bifid uvula, premature and extensive osteoarthritis, club feet, cleft palate and other systemic features. 4 Familial cases, syndromic or extra phenotypic characteristics, young patients with severe disease. 5 In familial and/or syndromic cases consider exome sequencing, copy number variant testing if next-generation sequencing is negative and there is high clinical suspicion of HTAD or for research purposes.Criteria for genetic testing in patients with suspected heritable thoracic aortic disease.HTAD: Heritable thoracic aortic disease, a Based on expert opinion [105].Genotype–phenotype correlations to be considered before aortic surgery in patients with heritable thoracic aortic disease.HTAD: heritable thoracic aortic disease. a A lower threshold may be considered if there is a family history of aortic dissection, pregnancy or rapid aortic size increase on an individual basis; b High-risk factors for Marfan syndrome patients are: (a) aortic diameter at the sinuses of Valsalva ≥5 cm, (b) rapid increase in aortic dilatation (≥3 mm per year), (c) family history of aortic dissection at a low aortic size, (d) progressive aortic regurgitation, (e) personal history of spontaneous vascular dissection and (f) desire for pregnancy. c High-risk factors for Turner syndrome: (a) Bicuspid aortic valve, (b) elongation of the transverse aorta, (c) Aortic coarctation or (d) hypertension. d High-risk factors for bicuspid aortic valve: (a) Family history of dissection at a low diameter, (b) desire for pregnancy, (c) systemic hypertension, (d) increase >3 mm/year.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+These authors contributed equally to this work.In thelast few decades, the roles of cardio-oncology and cardiovascular geneticsgained more and more attention in research and daily clinical practice, shaping a new clinical approach and management of patients affected by cancer and cardiovascular disease. Genetic characterization of patients undergoing cancer treatment can support a better cardiovascular risk stratification beyond the typical risk factors, suchas contractile function and QT interval duration, uncovering a possible patient’s concealed predisposition to heart failure, life threatening arrhythmias and sudden death. Specifically, an integrated cardiogenetic approach in daily oncological clinical practice can ensure the best patient-centered healthcare model, suggesting, also the adequate cardiac monitoring timing and alternative cancer treatments, reducing drug-related complications. We report the case of a 14-month-old girl affected by neuroblastoma, treated by cisplatin, complicated by cardiac arrest. We described the genetic characterization of a Ryanodine receptor 2 (RYR2) gene mutation and subsequent pharmacogenomic approach to better shape the cancer treatment.In the last few decades, the roles of cardio-oncology and cardiovascular geneticsgainedmore and more attention in research and daily practice, shaping a new clinical approach and management of patients affected by cancer and cardiovascular disease. As it is common practice when something new is discovered, scientists and clinicians followsuper specialized pathways to investigate in detail each single brick of the new process, while the final aim is to manage each patient in their own fragmented complexity [1].In this regard, the genetic characterization of patients undergoing cancer treatment could allow a better cardiovascular risk stratification beyond the typical risk factors, such as contractile function and QT interval duration, uncovering a possible patient’s concealed predisposition to heart failure, life threatening arrhythmias and sudden death. An integrated cardiogenetic approach in daily oncological clinical practice will ensure the best patient-centered healthcare model, suggesting, also the adequate cardiac monitoring timing and alternative cancer treatment, reducing drug-related adverse events [2].Although the creation of dedicated registries shared between oncologists, cardiologists and geneticists will be the main tool to increase our knowledge about this issue, case reports represent an important causefor reflection, the first step to guarantee a comprehensive knowledge sharing on the worst case scenarios, as “mistakes are the portals of discovery” [3].Patient predisposition to heart failure and cardiac arrhythmias relies depend on the strong relationship between genetic features and the specific cancer treatment, the latter representing a genetic modifier; early recognition of these complex interactions will improve personalized treatments and will reduce side effect [4].We describe the case ofa 14-month-old girl affected by congenital sensorineural deafness, carrying a cochlear implant, who presented with an abdominal and mediastinal mass. Lymphnode biopsy allowed the diagnosis of stage IV, N-Myc-non-amplified (Figure 1) neuroblastoma [5,6]. Results of the preliminary resting electrocardiogram and echocardiogram were normal. After complete staging, she was enrolled in the SIOPEN/HR-NBL1 Protocol (Société International d’Oncologie Pédiatrique European Neuroblastoma/High Risk Neuroblastoma Protocol 1) by COJEC scheme (rapid, platinum-containing induction schedule: CBDCA, CDDP, CYC, VCR, VP16) including cisplatin [7]. During the 7th cycle with cisplatin, on the second day from therapy she had irritability and sudden cardiocirculatory arrest with evidence of ventricular fibrillation (VF). After one hour of continuous cardiopulmonary resuscitation associated to repetitive epinephrine iv and cardiac defibrillation, according to the current guidelines [8], she was returned to spontaneous circulation (ROSC). Then the child was transferred to the intensive care unit. First electrocardiogram (ECG) after cardiac arrest showed QTc prolongation (Figure 2), normalized at subsequent controls, likely due to acute metabolic acidosis and hyperkalemia, while magnesium concentrations were normal before cardiac arrest (2.4 mg/dL, normal range 1.6–2.6 mg/dL). Echocardiography showed normal biventricular function, without any evidence of cardiac structural disease.Nevertheless, in the presence of sensorineural deafness and evidence of long QT after cardiac arrest, in the absence of familial history of sudden death and deafness, we hypothesized the Jervell and Lange-Nielsen syndrome, an autosomal recessive variant of the familial long QT syndrome; consequently, genetic analysis was performed [9].On the contrary, the next generation sequencing (NGS) panel for bilateral sensorineural deafness was negative for potassium channel mutation, while it showedhomozygosity for a GJB2 frameshift pathogenic variant c.35delG, p.(Gly12Valfs*12), implicated in a recessive form of congenital sensorineural deafness [10].Therefore, we performed the NGS panel for suspected channelopathies showing a missense mutation in theRYR2 gene (RYR2: c.1927T > A; p.(Leu643Ile), classified as a variant of uncertain significance (VUS). The RYR2 gene provides for making the protein ryanodine receptor 2, involved in calcium transport handling within cardiomyocytes cells. The RYR2 gene is closely related to catecholaminergic polymorphic ventricular tachycardia (CPVT). According to present consensus, after parents screening, the mother resulted to be a carrier of the same mutation. A more detailed maternal clinical history revealed her positivity to palpitations and two syncopal events.An exercise testing displayed that the mother had frequent premature ventricular beats during effort, disappearing at rest. Although the reported RYR2 mutation was considered a variant of uncertain significance (VUS) after bioinformatics’ analysis, the particular familial segregation may support its plausible pathogenicity. Cancer restaging after cardiac arrest, by means of (MIBG) single photon emission computed tomography (SPECT, GE Healthcare, Boston, MA, USA) and computer tomography (CT) scan, showed an important mass reduction and minimal uptake, while brain CT showed a picture of widespread suffering, but the clinical and neurological conditions of the child were good. On the basis of the cardiogenetic results, nadolol treatment was started, according to the current indications [11]; because of poor prognosis and low body mass index, cardiac defibrillator implantation was temporary avoided, with a strong indication to continue ECG monitoring during the treatment. A brief period of maintenance therapy with temozolomide and topotecan was useless, burdened by disease progression.After multidisciplinary discussion, the need for treatment continuation was evident, avoiding the acknowledged cardiotoxicity; for this reason, the patient was administered a stepwise treatment by MIBG therapy, autologous peripheral blood stem cells (PBSC) infusion, followed by conditioning therapy with busulfan and melphalan and a second autologous PBSC infusion. Afterwards, she receiveda treatment with retinoic acid and effective immunotherapy based on Anti-GD2, a surface glycolipid, associated to improved survival and quality of life by reducing exposure to cytotoxic agents [12]. After twenty-eight months the patient is alive, without any evidence of further ventricular arrhythmias; unfortunately, cancer restaging by MIBG confirmed a tracer capturing mass. Currently, she is on etoposide maintenance treatment and continues cardiological follow up, according to the international consensus.The patient’s parents provided written informed consent to molecular testing and to the full content of this publication. This study was performed in accordance with the Declaration of Helsinki (1984) and its subsequent revisions. Genomic DNA was extracted from peripheral blood samples using the Bio Robot EZ1 (Quiagen, Solna, Sweden). DNA quantity and quality were measured by NanoDrop 2000 C Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The sample was then quantified with the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using the Quant-iT dsDNA BR Assay kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The sequencing of 75 genes responsible for several forms of cardiomyopathies and of 85 genes responsible for Hearing loss of the index patient was performed by using the SureSelectQXT Target Enrichment system (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer’s instructions. The libraries were then sequenced on the NextSeq500 Sequencing System (Illumina Inc., San Diego, CA, USA), using a Mid Output 300 cycles flow cell (Illumina Inc., San Diego, CA, USA).All putatively pathogenic variants were confirmed by Sanger sequencing and the segregation analysis was carried out. Sanger sequencing was performed using theBigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) on the 3130xl Genetic Analyzer Sequencer (Thermo Fisher Scientific, Waltham, MA, USA).The quality of the sequenced sequences was checked using the FastQC software [13] and then aligned against the hg19 human reference genome by the BWAmem v.0.7.17 [14]. Depth of coverage analysis was carried out through the TEQC R Package on the produced *.bam file, thus obtaining measures of sample and region-specific sequencing coverage [15]. The identification of single nucleotide variants and small insertions/deletions was performed using GATK ver. 3.7 [16]. Variants were functionally annotated through the Annovar Software v.2017Feb15 [17], with the NCBI Human RefSeq as annotation reference system [18]. Annotated variants were also checked for novelty in public collections, such as dbSNP ver. 151 [19], ExAC and gnomAD [20]. Furthermore, prediction of functional effect of nonsynonymous and splice site variants were retrieved by the dbNSFP v3.4 database [21]. Subsequently, the prioritization of the variants started excluding those described as benign and likely benign. Then the remaining variants which passed this filtering were classified on the basis of their clinical relevance as pathogenic, likely pathogenic or variant of uncertain significance by using the following criteria: (i) nonsense/frameshift variant in genes previously described as disease causing by haploinsufficiency or loss-of-function; (ii) missense variant located in a critical or functional domain; (iii) variant affecting canonical splicing sites (i.e., ±1 or ±2 positions); (iv) variant absent in allele frequency population databases; (v) variant reported in allele frequency population databases but with a minor allele frequency (MAF) significantly lower than expected for the disease; (vi) variant predicted and/or annotated as pathogenic/deleterious in ClinVar v.2018Oct12 and/or LOVD v.2.0. The putative pathogenic variants identified following this pipeline were confirmed by direct Sanger sequencing, and the segregation analysis was carried out. The Sanger sequencing was performed using theBigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) on the 3130xl Genetic Analyzer Sequencer (Applied Biosystems, Foster City, CA, USA). The clinical significance of the identified putative variants was interpreted according to the American College of Medical Genetics and Genomics Recommendations [22].The NGS analysis of the proband revealed that the patient was heterozygous for the RYR2 (NM_001035.3):c.1927T > A (p.Leu643Ile) (rs368918887) variant, and homozygous for the GJB2 (NM_004004) c.35delG, p.(Gly12Valfs*12) (rs80338939) variant. The detected variants were identified with a read depth of 604X and 1158X, respectively. After Sanger sequencing of the proband’s parents, the RYR2 variants resulted to be inherited from the mother, while both parents were heterozygous the GJB2 variant.The occurrence of a major adverse cardiac event (MACE) gives rise to many questions in the clinical practice. The cardio-oncology approach requires the assessment of the patient’s status and risk stratification before treatment for adequate risk assessment, patient’streatment and an appropriate follow up timing with close surveillance for side effects. In this case, preliminary cardiological assessment was normal, in particular cardiac contractility and ventricular repolarization on echocardiography and electrocardiogram. Serum electrolytes concentration was normal in the morning, before the cardiac arrest; in particular magnesium level, as it is an important RyR2 regulator, a potential mechanism by which Cisplatin may promote RyR2 hiperactivity duringhipomagnesemia. After cardiac arrest due to ventricular fibrillation, in the presence of long QT and congenital sensorineural deafness, the first hypothesis was represented by the Jarvell and Lange-Nielsen syndrome, characterized by an autosomal recessive inheritance pattern of potassium channel mutation, yet it seems questionable based on familial absence of deafness and sudden death. A first clinical approach based on epidemiological and probabilistic considerations can be helpful, leading to an initial intuitive orientation. However, in this case, the Ockham’s razor showed its fallacy [23].The NGS panel for genes responsible for hearing loss showed homozygosity for a pathogenic GJB2 gene variant; previous studies denied a major incidence of cardiac arrhythmias and electrocardiographic parameter abnormalities (atrioventricular conduction or ventricular repolarization) in patients affected by this condition [24].On the other hand, the presence of a VUS in the RYR2 gene, with autosomal dominant inheritance pattern, even in the presence of a weak familial segregation, represented by maternal history of palpitation, syncope and frequent premature ventricular beats during effort, brings a reflection on the strict role of intracellular calcium handling with implication on ventricular fibrillation induction.The RYR2 gene is implicated in the pathogenesis of catecholaminergic polymorphic ventricular tachycardia (CPVT), a rare inherited arrhythmia syndrome characterized by adrenergically driven ventricular arrhythmia predominantly caused by pathogenic variants in the cardiac ryanodine receptor (RyR2) [25]. RyR2 is responsible for sarcoplasmic reticulum calcium release, hence its dysfunction leads to an impairment of intracellular calcium handling [26]. On the other hand, although a direct interaction between RyR2 and cisplatin cannot be ruled out [27], it is described that cisplatin induces an irreversible inhibition of SERCa2, reducing calcium reuptake in sarcoplasmic reticulum [28]. Broadening the comprehension of the complex biological system interactions during chemotherapy, we need to consider patient psychological distress, leading to anxiety and increased adrenergic burden, well described by the recent paradigm of “emotional chemobrain” [29]. As described by Trafford et al., beta adrenergic stimulation induces calcium overload mainly by RyR2 phosporylation, increasing Ca2+ leaking and enhancing L-type Ca2+ current, with consequent increase in the intracellular calcium concentration. High Ca2+ concentration leads to both Ca2+-induced Ca2+ release phenomenon (CICR) by SR and Na-Ca pump exchange (NCX) activation, causing Na+ inward current and delayed after-depolarization (DAD) [30]. Summarizing, patient concealed predisposition to cardiac arrhythmias, represented by pathological calcium release due to the RYR2 mutation, is worsened by further calcium intracellular concentration, secondary to reduced calcium uptake by cisplatin-induced SERCa2 inhibition and stress-related beta-stimulation, impairs cytosolic Ca2+ waves propagation and full development [31]; this complex processleads to early and delayed after depolarizations (EADs and DADs), precipitating in ventricular fibrillation [32].According to the international cardiopulmonary resuscitation protocol [8], epinephrine is strongly indicated. In our case, although a prompt intervention was guaranteed, ROSC was obtained after one hour. Our plausible consideration is that epinephrine has a detrimental effect in the presence of the RYR2 mutation, leading to further calcium overload. The recent literature supports our hypothesis, confirming that epinephrine administration during cardiac arrest in children affected by CPVT is associated to significantly longer resuscitation efforts, more interventions and a longer time to return to spontaneous circulation [33]. Starting from this initial evidence, strengthened by biological plausibility, we suggest that a different protocol needs to be discussed and validated in pediatric patients affected by CPVT. In particular, vasopressine, recently ruled out in the setting of cardiac arrest by the 2017 AHA/ACC/HRS Guideline [34], with a current renewed adoption in pediatric intensive care [35], could be a valid alternative in this particular subgroup and represents an interesting hypothesis to investigate in further studies.After multidisciplinary discussion, even in the presence of an indication to ICD implantation in secondary prevention after the cardiac arrest, we decided not to implant for several reasons mainly represented by low body mass index with higher risk of complication [36], poor life expectancy and the presence of a reversible cause of ventricular fibrillation represented by cisplatin infusion. On the other hand, the evidence of disease progression needs to be addressed through a different available treatment, in particular by immunotherapy, dinutuximab, an anti-GD2 chimeric monoclonal antibody approved for high-risk neuroblastoma treatment [37]. In our case, according to the literature, this solution avoids the occurrence of any further arrhythmias, which is made evident by continuous ECG monitoring during hospitalization.Currently, pharmacogenomic focuses mostly on pharmacokinetics, in particular on drug metabolism modification induced by gene polymorphism, implicating different drug-response and side effect distribution among population; this approach is suffering from some limitations, due to cost-effectiveness and lack of reliable predictable models [38]. Future aims of pharmacogenomics will rely on a better characterization of the patient’s whole genome to build predictable models and reach a personalized medicine by using big data analysis platforms [39].In conclusion, existing evidence demonstrates that genetic factors have the potential to improve the discrimination between individuals at higher and lower risk of cardiac complications, such as heart failure and life-threatening cardiac arrhythmias. We underline the role of genetic analysis on this issue, allowing the best support to patient care and healthcare decision making, improving prevention of cardiotoxicity. In the future, this combined approach will allow a correct recognition of this demanding characterization, bringing together oncological and cardiological features of this complex scenario, leading to proper clinical management in both the acute and the chronic patient pharmacological treatment.Data acquisition: A.M., S.M., G.D.S. and M.V.; processing and interpretation, A.M., S.M., G.D.S. and D.R.P.; writing original draft preparation, A.M., S.M., G.D.S., M.V. and M.P.S.; clinical evaluation, A.M., S.M., G.D.S., A.S., M.V. and M.P.S.; study concept, S.M., G.D.S., S.L., D.R.P. and M.V.; critical revision of manuscript, A.M., S.M., G.D.S. and M.C. (Massimo Carella); data curation, S.M., G.D.S., P.P., S.C. and M.P.L.; supervision, G.D.S., D.R.P., M.C. (Marco Castori), M.V., M.P.S. and M.C. (Massimo Carella). All authors have read and agreed to the published version of the manuscript.This work was supported by Italian Ministry of Health for research grant RC 2001CA08.Not applicable.Informed consent, approved by Fondazione IRCCS Casa SollievodellaSofferenza Ethical Committee, was obtained by both parents for the genetic analysis and case history.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.The authors declare no conflict of interest.HR-NB: High Risk Neuroblastoma(a) Meta-(radioiodinated)iodobenzylguanidine showing abdominal mass uptake. (b) CT scan showing neuroblastoma.Electrocardiogram after cardiac resuscitation.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Rigorous peer-reviews are the basis of high-quality academic publishing. Thanks to the great efforts of our reviewers, Cardiogenetics was able to maintain its standards for the high quality of its published papers. Thanks to the contribution of our reviewers, in 2021, the median time to first decision was 20 days and the median time to publication was 85 days. The editors would like to extend their gratitude and recognition to the following reviewers for their precious time and dedication, regardless of whether the papers they reviewed were finally published:
+    Andreassi, Maria GraziaMonda, EmanueleAnghel, LarisaNanda, VivekAsimaki, AngelikiNaydenov, StefanBaturova, Maria A.Nishida, NaokiBellin, MilenaOlcum, MelisBerger, MartinPandian, Vishnuprabu DurairajCaiazza, MartinaPersampieri, SimoneCappelli, FrancescoPitto, LetiziaChavan, SameerPohl, JuliaChoudhuri, SubhadipPollari, FrancescoCimmino, GiovanniPrilepskii, Artur Y.Cismaru, GabrielRobles-Mezcua, AinhoaCosta, Marina C.Rodríguez, IsabelDariolli, RafaelSabater-Molina, MariaDegano, Irene R.Seaby, EleanorEsposito, AugustoSharma, Umesh C.Fukuda, KeiichiSievers, Laura KatharinaGalani, AlessandroSpinelli, LetiziaGlowniak, AndrzejVan Bommel, Rutger J.Gragnano, FeliceVan Damme, TimGuo, LilongVatta, MatteoHirono, KeiichiWalsh, RoddyKhan, MohsinWelch, CarrieKozieł, MonikaYadav, ShambhuKutikhin, Anton G.Yin, KanhuaLee, Jyh-YeuanZoppi, NicolettaLi, ChangZorio, EstherLi, YaweiZorzi, AlessandroMohieldin, Ashraf M. 
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+Long QT syndrome (LQTS) is an inherited arrhythmic disorder associated with sudden cardiac death (SCD). This study aimed to identify the clinical and molecular genetic risk factors that contribute to major arrhythmic events (MAEs) in patients with genetically confirmed childhood onset LQTS 1–3. This study was a retrospective double-center study. An MAE was defined as the occurrence of SCD, aborted SCD, appropriate implantable cardioverter defibrillator discharge, or sustained ventricular tachycardia. During a median follow-up of 4.6 years (range 0.1–24.3 years), MAEs occurred in 18 (17.8%) of 101 patients diagnosed with LQTS at a median of 7.7 years (range 0.0–18.0 years) despite the use of beta-blockers in 91.6% of patients at the last follow-up. A multivariate analysis identified a genetic diagnosis of LQTS2 and LQTS3 and variants within the KCNH2 S5-loop-S6 pore region as independent risk factors for MAEs, independent of the QTc value or a history of syncope detected from a univariate analysis. MAEs occur frequently in childhood onset LQTS despite beta-blocker treatment. A detailed molecular genetic diagnosis can contribute to the arrhythmia risk stratification and optimize the use of preventive measures in this vulnerable patient population.Long QT syndrome (LQTS) is an inherited arrhythmogenic disorder caused by variants in the genes encoding cardiac ion channels [1]. These ion channels generate the cardiac action potential by regulating the flux of ions across the membrane such as the influx of sodium and calcium or the outflow of potassium [2]. In LQTS, an increase in the sodium and calcium currents or a decrease in the potassium outflow can prolong the action potential duration, leading to the eponymous phenotype of a prolonged QT interval [3]. Seventeen distinct subtypes of LQTS were initially described with their associated diseases/genes; seven of those were recently confirmed in a re-evaluation study [4].The most commonly affected genes are KCNQ1 (LQTS1) and KCNH2 (LQTS2), encoding for potassium currents (IKs and IKr), and SCN5A (LQTS3), which regulates the sodium inflow (INa) [5]. A pathogenic variant in one of those three disease genes is identified in approximately 75% of all patients carrying a clinical diagnosis of LQTS, based on the calculated Schwartz score [6,7]. The clinical consequences of LQTS include severe cardiac arrhythmia manifesting as ventricular tachycardia (VT) (e.g., Torsades de pointes), which can either be self-terminating or develop into ventricular fibrillation leading to sudden cardiac death (SCD) [8]. The rate of these life-threatening arrhythmic events is described in the literature as varying between 2.4% and 12.1% of cases [9,10,11,12,13,14].The disease presentation and clinical course are variable amongst patients. Clinical symptoms such as syncope and the occurrence of sudden cardiac death can effectively be prevented by treatment with beta-blockers and by the implantation of implantable cardioverter defibrillators (ICDs) [15,16,17]. The identification of risk factors is crucial to identify patients at a high risk of life-threatening arrhythmias.Previous studies, mostly carried out on adults, have described certain risk factors such as carriers of certain genotypes, age, sex, the length of the prolonged QTc value, and the presence of syncope [9,11,18,19,20].More than 50% of patients with LQTS experienced their first cardiac event before the age of 15 years [20]; this subgroup of patients is of particular importance when it comes to individual risk stratification in LQTS.Given the scarce data available for the risk stratification of pediatric patients, the aims of this study were to identify and confirm the clinical and genetic risk factors in this vulnerable patient population.In this retrospective double-center study conducted at the German Heart Centers in Munich and Leipzig, the data were extracted from the medical records of 101 patients out of 82 families with genetically confirmed LQTS 1–3 (Munich: n = 69, Leipzig: n = 32). The patients were treated at the respective centers in the time between 1995 and 2019. All participants were 18 years or younger at the time of diagnosis. Those who were lost to follow-up were censored at the time of their last contact; part of this patient cohort was included in a recent publication [21].The data extracted included the demographics, family history, use of medication, need for hospitalization and interventions, history of syncope, and the occurrence of major arrhythmic events (MAE, defined as sudden cardiac death (SCD), aborted cardiac arrest (ACA), appropriate cardioverter defibrillator discharge, or sustained ventricular tachycardia (VT)). Twelve-lead electrocardiogram (ECG) measurements of the heart rate and QT interval corrected for the heart rate by the Bazett formula (QTc) were obtained for all study subjects. The QTc measured on the baseline ECGs at enrollment was used for the statistical analysis. The Schwartz score [7] was calculated for each patient in order to summarize and objectify the clinical outcomes. Transthoracic echocardiography (TTE) was used to exclude patients with structural heart defects.Details on the molecular genetic diagnosis were extracted from the reports of the respective accredited laboratories or from medical patient records. Only patients with variants in the three main causing genes KCNQ1, KCNH2 and SCN5A—classified as pathogenic or likely pathogenic variants according to criteria of the American College of Medical Genetics and Genomics (ACMG) [22]—were selected for the statistical analysis. These variants were characterized by their location and type. For the KCNQ1-encoded Kv7.1 channel, amino acids before position 122 were defined as the N-term, residues from 122 to 261 as the S1–S4 region, from 262 to 348 as the S5-loop-S6 region, and from 349 as the C-term. Amino acids located before 404 (N-term), from 404 to 547 (S1–S4 region), from 548 to 659 (S5-loop-S6 region) and from 660 onwards (C-term) were defined for the KCNH2-encoded Kv11.1 channel. For the SCN5A-encoded Nav1.5 channel, the N-term, the transmembrane region of each domain (D1–D4) with their S5-loop-S6 region (pore region), and the C-term were defined as follows: up to 131 as the N-term; 132 to 410 as D1 (pore region: 253–410); 718 to 938 as D2 (pore region: 838–938); 1207 to 1466 as D3 (pore region: 1334–1466); 1530 to 1771 as D4 (pore region: 1657–1771); and 1772 to 2016 as the C-term. The location of the variants was reviewed by using the UniProt dataset (https://www.uniprot.org/, last accessed on 26 December 2021). The type of variant was divided into missense, frameshift, nonsense (stop codon), splice site, nearsplice, in-frame deletion/duplication, and intragenic deletion.Univariate and multivariate analyses were performed to identify the clinical and molecular genetic risk factors for the occurrence of MAEs. The chi-squared test was used for the categorical variables; Mann–Whitney U and Kruskal–Wallis tests were used for the continuous variables given the small sample size. The cumulative probability of a first MAE was developed for the baseline covariates with the Kaplan–Meier estimator and compared with the log-rank test. To investigate the influence of the clinical and genetic factors as well as the time-dependent occurrence of syncope with the first occurrence of an MAE, the multivariable Cox proportional hazard regression model was used. Testing for multicollinearity was conducted to determine the degree of correlation between the variables. Multicollinearity was considered to be a Spearman Rho correlation coefficient of two variables greater than r > 0.8 [23]. The statistical software used for the analysis was SPSS version 28.0.0 (SPSS Inc.; IBM Company, Armonk, NY, USA) and a p-value of less than 0.05 (two-sided) was considered to be statistically significant.The data of 101 patients (54 females) diagnosed at a median (range) age of 7.7 years (0.0–18.0 years) were analyzed. The median (range) Schwartz score at the time of diagnosis was 4.0 (1.0–7.0). A total of 54 patients were affected by LQTS1 (53.5%), 40 by LQTS2 (39.6%), and 7 by LQTS3 (6.9%). During a median (range) follow-up time of 4.6 (0.1–24.3) years, MAEs occurred in 18 (17.8%) patients. Almost two-thirds of the patients (71.3%) received a beta-blocker therapy at the time of the study enrollment and almost all (91.6%) at the last follow-up. Metoprolol and propranolol were the most frequently used (47.4% and 39.5% of patients receiving beta-blockers, respectively). An ICD was implanted in 25 (24.8%) patients. The indication was a primary prevention in 16 (64.0%) patients and implantation occurred at a median (range) age of 12.8 years (1.8–30.0) in those subjects. Clinical risk factors prompting a primary prophylactic ICD implantation included syncope in 10 (62.5%) and VT in 6 (37.5%); all subjects with VT also suffered from syncope. The indication for a primary prophylactic ICD implantation was not clear in 6 out of 16 patients (37.5%). All patients received an appropriate beta-blocker therapy before the ICD implantation. Complications of primary prophylactic implanted devices occurred in 3 (18.8%) patients. A total of 9 patients received an ICD at a median (range) age of 14.3 years (4.9–23.7) for a secondary prevention with 1 of them (11.1%) experiencing an ICD complication. Complications included lead failure in 3 (12.0%) patients and an upper extremity deep vein thrombosis in 1 (4.0%) patient.The detailed clinical and molecular genetic information can be found in Table 1.There were no differences in sex, age at diagnosis, follow-up time, occurrence of syncope, and medication among the different LQTS types. Features varying upon the underlying affected genotype included the QTc value, rate of ICD implantation, and location of the pathogenic variant within the gene. The median (range) QTc was significantly longer in the LQTS2 and LQTS3 groups (LQTS2: 490 ms (400–630 ms); LQTS3: 480 ms (417–740 ms)) compared with the LQTS1 group (LQTS1: 460 ms (370–554 ms); p = 0.047, Kruskal–Wallis test for multiple independent non-parametric variables). There was a higher rate of ICD implantation in the LQTS2 group (45.0%) compared with the LQTS1 (9.3%) and LQTS3 (28.6%) groups (p < 0.001, Pearson chi-squared). In the LQTS1 group, the location of the pathogenic variant was most frequently found in the S1–S4 region and the C-term; it was in the S5-loop-S6 region in the LQTS2 group and in the C-term in the LQTS3 group (Table 1, Figure 1, and Supplementary Figure S1).A total of 18 patients (17.8%) experienced an MAE during the follow-up (no SCD, 8 ACA in 8 patients, appropriate ICD discharge in 6 patients, and sustained VT in 4 patients (Table 1)). Comparing patients with and without MAEs in the univariate analysis, patients with MAEs had a longer follow-up time, higher Schwartz score, higher QTc value, higher rate of syncope, and were more often treated with beta-blockers at the time of enrollment or with an ICD (Table 2, Figure 2).Patients with MAEs were more likely to carry a pathogenic variant in KCNH2 (13/40, 32.5%) or SCN5A (2/7 patients, 28.6%) than in KCNQ1 (3/54, 5.6%; p = 0.002) (Table 1 and Table 2, Figure 3a). The probability of a cumulative MAE-free survival was significantly lower if the patient carried the variant within the pore region (S5-loop-S6 region) of the KCNH2 gene (Figure 1 and Figure 3b).A multivariate analysis revealed that patients with a variant in the KCNH2 or SCN5A gene had a significantly higher risk of developing an MAE than the carriers of variants in the KCNQ1 gene, independent of the QTc value and the occurrence of syncope (Table 3). This was also confirmed if only the index patients were analyzed (Supplementary Table S1). In addition, a multivariate analysis identified carriers of a pathogenic variant within the KCNH2 pore region as an independent risk factor, increasing the risk of occurrence of an MAE four-fold (Table 4, Supplementary Table S2).This study with LQTS patients diagnosed during childhood shows the importance of molecular genetic findings for risk stratification. A molecular genetic diagnosis of LQTS2 and LQTS3 as well as variants in the KCNH2 S5-loop-S6 region were identified as independent risk factors for an MAE, irrespective of the QTc value and the occurrence of syncope.Overall, life-threatening arrhythmic events occurred in almost 18% of a population with childhood onset LQTS despite the use of beta-blockers. When only taking ACA and SCD into account, the observed frequency of 8% in this study was in accordance with previous studies that reported events in 2.4–12.1% of cases. These studies did not include appropriate ICD discharges and a sustained VT [9,10,11].The clinical risk factors associated with a higher risk of MAEs included a longer QTc duration and a history of syncope in the pediatric population of this study. The median QTc was 470 ms, which was about 10 ms higher than in a comparable study including only genotyped patients aged 0–18 [24]. Compared with the other studies, which also included non-genotyped patients, the QTc was lower (490–494 ms) [9,10]. In most studies, a high QTc was shown to be an independent risk factor of life-threatening events [9,19,20,24]. In line with the findings of other studies [10,24], an event-free survival was significantly lower in the pediatric patients of our study with QTc values greater than 500 ms. The finding of a history of syncope as a risk factor for the occurrence of MAEs was also reported by others [9,10,20].Patients diagnosed with LQTS2 and LQTS3 were more likely to experience MAEs compared with LQTS1 patients. The findings of the present study were in line with studies of young adult LQTS patients who showed a higher rate of cardiac events in the carriers of variants in the KCNH2 and the SCN5A genes [18]. Those findings were later confirmed in patients who had already been treated with beta-blockers [15], which was also the case in the present study. Previous studies focusing on children and adolescents with LQTS failed to identify the genotype as a risk factor for MAEs [9,10]. When adults were included, LQTS1 and LQTS2 were shown to be independent risk factors. However, there were differences in the study design in that a syncope was not counted as a separate risk factor but as a cardiac event [25].Consistent with reports from others [26], about two-thirds of the patient population in our study carried a missense variant. The location of the variants was within a pore region in about a third of the patients, similar to the findings from previous studies (17–30%) [27,28,29]. Although the type of variant did not influence the risk of an MAE, the location of variants in the S5-loop-S6 pore region of KCNH2 was identified as an independent risk factor for life-threatening arrhythmic events in the present study. This was in line with findings from others that showed an association between the variant location and the risk of life-threatening events in young adults [29]. The pore region often contains variants with dominant negative effects on IKr because the potassium conductance pathway is generated in this region and is, therefore, a critical zone [30].Due to the limited number of patients, a subgroup analysis on the genotype-related risks dependent on age and sex [9,12,31] was not performed. Given the retrospective character of the present study, there was a lack of information on the circumstances under which MAEs occurred. Therefore, a subanalysis on the triggering factors dependent on the underlying genotype could not be performed. Exact information about the methods used for the respective molecular genetic diagnosis was not available for all patients, specifically if information about the molecular genetic diagnosis was extracted from the medical records of the patients. Finally, given that this study included only genotyped patients with variants in the KCNQ1, KCNH2, and SCN5A genes, this risk stratification could not be extrapolated for all LQTS patients.Despite these limitations, the current study has contributed toward a better understanding of variant-specific risk stratification in LQTS in pediatric patients. The clinical phenotype of patients with LQTS in childhood and adolescence is heterogeneous and influenced by numerous factors. As variant-specific risk stratification has still not been explicitly studied in this age group, further studies are needed to identify and prove the predictors of severe arrhythmias leading to SCD in pediatric patients with LQTS.MAEs frequently occur in childhood onset LQTS despite the use of beta-blockers. The clinical factors associated with a higher risk of life-threatening events from a univariate analysis included a longer follow-up time, longer QTc intervals, and a history of syncope. From a multivariate analysis, a molecular genetic diagnosis of LQTS2 and LQTS3 as well as variants in the KCNH2 S5-loop-S6 pore region were identified as independent risk factors irrespective of the QTc and clinical symptoms. These findings underline the importance of genetic diagnostics in the risk stratification of pediatric LQTS patients.The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/cardiogenetics12010009/s1. Figure S1: Distribution of variants in KCNQ1 and SCN5A gene; Table S1: Cox regression model: risk factors for MAE in index patients; Table S2: Cox regression model: risk factors for MAE with KCNH2Pore in index patients; Table S3: Genetic characteristics in the KCNQ1 gene; Table S4: Genetic characteristics in the KCNH2 gene; Table S5: Genetic characteristics in the SCN5A gene.Conceptualization, C.M.W.; methodology, C.M.W. and T.B.; software, C.M.W. and T.B.; validation, C.M.W., D.S.W. and G.H.; formal analysis, T.B. and C.M.W.; investigation, T.B., F.M. and R.A.G.; resources, C.M.W., R.A.G. and G.H.; data curation, C.M.W.; writing—original draft preparation, T.B.; writing—review and editing, D.S.W., G.H., R.A.G. and C.M.W.; visualization, C.M.W. and T.B.; supervision, C.M.W. and D.S.W.; project administration, C.M.W. and G.H.; funding acquisition, C.M.W. All authors have read and agreed to the published version of the manuscript.This study was supported by the 2020 research funding award of the German Society for Pediatric Cardiology and Congenital Heart Defects (Forschungsförderung 2020, Deutsche Gesellschaft für Pädiatrische Kardiologie und Angeborene Herzfehler e.V., DGPK).The study was conducted according to the guidelines of the Declaration of Helsinki. The study was approved by the institution’s ethical committee (approval number 243/17S from 16 October 2017).All parents or legally authorized representatives of the pediatric patients gave written consent for the anonymous publication of their data.The data that support the findings of this study are available from the corresponding author upon reasonable request.The authors declare that there is no conflict of interest related to the content of this study.(a) Distribution of variants in the KCNH2 potassium channel, depending on major arrhythmic event (MAE). Black dot: occurrence of MAE; white dot: without occurrence of MAE. (b) Kaplan–Meier estimates of survival without major arrhythmic event (MAE) among the 40 LQTS2 patients, depending on location of variant. Pore: S5-loop-S6 region. Non-pore: N-term, S1–S4 region, or C-term.Kaplan–Meier estimates of survival without a major arrhythmic event (MAE) among the 101 patients with Long QT syndrome depending on (a) QTc value and (b) the occurrence of syncope.Kaplan–Meier estimates of survival without a major arrhythmic event (MAE) among the 101 patients with Long QT syndrome depending on (a) genetic locus of the variant and (b) location of the variant in the KCNH2 gene. KCNH2Pore: variant in the KCNH2 gene as well as in the S5-loop-S6 region (pore region). No KCNH2Pore: variant in the KCNH2 gene and not in the S5-loop-S6 region or variant in other genes (KCNQ1, SCN5A).Patient characteristics with clinical and genetic findings by genotype.Abbreviations: LQTS: Long QT syndrome; MAE: major arrhythmic event; SCD: sudden cardiac death; ACA: aborted cardiac arrest; ICD: implantable cardioverter defibrillator; VT: ventricular tachycardia; BW: body weight; n.a.: not available. a: chi-squared test; b: Kruskal–Wallis test; c: from first diagnosis; d: ICD implantation or beta-blocker therapy; e: corresponds to the pore region.Characteristics with clinical and genetic findings by MAE.Abbreviations: LQTS: Long QT syndrome; MAE: major arrhythmic event; SCD: sudden cardiac death; ACA: aborted cardiac arrest; ICD: implantable cardioverter defibrillator; BW: body weight; n.a.: not available. a: chi-squared test; b: Mann–Whitney U-test; c: from first diagnosis; d: ICD implantation or beta-blocker therapy; e: corresponds to the pore region.Cox regression model: risk factors for MAE.Abbreviations: LQTS: Long QT syndrome; MAE: major arrhythmic event; SCD: sudden cardiac death; FH: family history. a: patients with syncope before the occurrence of MAE vs. patients without syncope or patients with syncope after the occurrence of MAE; b: corresponds to the S5-loop-S6 region; c: patients with beta-blocker use before the occurrence of MAE vs. patients without beta-blocker use or with beta-blocker use after the occurrence of MAE.Cox regression model: risk factors for MAE with KCNH2Pore.Abbreviations: MAE: major arrhythmic event. a: patients with syncope before the occurrence of MAE vs. patients without syncope or patients with syncope after occurrence of MAE; b: patients with a variant in the KCNH2 gene as well as in the S5-loop-S6 region (pore region) vs. patients with a variant in the KCNH2 gene and not in the S5-loop-S6 region or with a variant in other genes (KCNQ1, SCN5A).Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+These authors contributed equally to this work.Fabry disease (FD) is a multiorgan disease, which can potentially affect any organ or tissue, with the heart, kidneys, and central nervous system representing the major disease targets. FD can be suspected based on the presence of specific red flags, and the subsequent evaluation of the α-Gal A activity and GLA sequencing, are required to confirm the diagnosis, to evaluate the presence of amenable GLA mutation, and to perform a cascade program screening in family members. An early diagnosis is required to start an etiological treatment and to prevent irreversible organ damage. Here, we describe a case of a 37-years-old patient, with a surgically repaired congenital heart defect in his childhood, who had a late diagnosis of FD based on the clinical history and targeted genetic evaluation. This case highlights the importance to perform a correct phenotyping and definite diagnosis of FD, to start an early and appropriate treatment in the index patient, and a cascade clinical and genetic screening to identify other family members at risk, which may benefit from specific treatment and/or a close follow-up.Fabry disease (FD) is a rare X-linked lysosomal storage disorder, caused by a mutation in GLA, that results in lower activity of α-galactosidase A (α-Gal A) enzyme and progressive accumulation of globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (lysoGb3), potentially affecting any organ or tissue [1]. Clinical manifestations are extremely heterogeneous, depending on the patient’s sex and type of GLA mutation, which influences the degree of α-Gal A deficiency [2,3]. In adulthood, the main involved organs are represented by kidneys, heart, and central nervous system, and this condition is suspected based on the presence of clinical markers, or red flags, which help to guide the subsequent investigations [4,5], such as the evaluation of the α-Gal A activity and GLA sequencing. Genetic analysis is mandatory to confirm the diagnosis, it is required to perform a cascade program screening in family members [6], and it is indicated to evaluate the presence of amenable GLA mutation [7].This case report exemplifies the importance of the clinical markers in performing diagnosis of FD, to start an early and appropriate treatment in the index patient and a cascade program screening to identify other family members at risk, which may benefit from specific treatment and/or a close follow-up.A 37-year-old man was referred to the Inherited and Rare Cardiovascular Diseases Clinic of the University of Campania “Luigi Vanvitelli”, Naples, Italy, for evaluation of left ventricular hypertrophy, in absence of hypertension or aortic valve stenosis, identified at echocardiography in a previous cardiological evaluation. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the University of Campania “Luigi Vanvitelli”. Informed consent was obtained from all subjects involved in the study.The patient has been followed since birth from the Division of Pediatric Cardiology and subsequently from the Division of Adults with Congenital Heart Defects of our Department following the neonatal diagnosis of a partial atrioventricular septal defect. At 6 months old, he underwent surgical repair (the approximation of the edges of the valve cleft with interrupted nonabsorbable sutures and the closure of the interatrial communication with bovine pericardial patch) and at 14 years old he underwent reoperation (left valve repair) for severe left atrioventricular valve regurgitation. At 21 and 22 years old, he experienced two transient ischemic attacks (TIAs) manifested with left arm weakness and speech difficulty. Thus, he underwent a comprehensive diagnostic work-up to identify the possible cause of the TIAs. In detail, he underwent complete laboratory investigations, including complete blood count, electrolytes, coagulation, renal function, glucose and homocysteine levels, screening for autoimmune diseases and thrombophilia, non-contrast brain computerized tomography (CT) and magnetic resonance imaging (MRI), carotid Doppler ultrasound, 12-lead electrocardiogram (ECG), repeated 24-h ECG monitoring, transthoracic and transesophageal echocardiography. However, no underlying cause of TIA was identified, thus the episodes were labeled as “idiopathic TIAs”. At 36-years-old, he experienced a third TIA. The echocardiographic evaluation showed the presence of a mild concentric left ventricular hypertrophy, which was considered unrelated to the congenital disease and/or other potential causes, and the patient was referred to our clinic for further evaluation.The patient was asymptomatic, the physical examination showed a systolic heart murmur, the ECG showed sinus rhythm, normal atrioventricular and interventricular conduction, and repolarization abnormalities in the inferior leads (Figure 1). The echocardiogram confirmed the presence of concentric left ventricular hypertrophy with the maximal wall thickness of 13 mm at the level of the anterior interventricular septum and showed papillary muscles hypertrophy, normal ejection fraction (EF, 65%), and mild reduction of global longitudinal strain (GLS, −18.2%) (Figure 1). Thus, he underwent a cardiac MRI that evidenced basal inferolateral late gadolinium enhancement (LGE) and reduced cardiac native T1 time (Figure 1).Based on the patient’s sex, the history of TIAs, and the presence of the mentioned cardiological abnormalities, FD was suspected. Alpha-Gal A activity was significantly reduced and the sequencing of GLA identified the presence of the pathogenic variant c.1066C>T (p.Arg356Trp) for FD.A multidisciplinary evaluation (i.e., genetic, neurologic, ophthalmologic, nephrological, dermatological, otolaryngological) failed to show other organ involvement. After a careful discussion about the risk/benefit balance of the available treatment options, the patient decided to refuse the intravenous enzymatic replacement therapy (ERT), and considering the presence of an amenable mutation, chaperone therapy with Migalastat was started. At 6-months of follow-up, echocardiographic parameters, including left ventricular mass, left ventricular EF, and GLS, remained stable.After the identification of the disease-causing mutation in GLA, family members were invited to join the cascade program screening (Figure 2). The pathogenic mutation was identified in the mother, both the two sisters and the two daughters of the proband.All the subjects underwent a comprehensive multidisciplinary evaluation and cardiological investigations, including ECG, echocardiography, and cardiac MRI. The mother (II-2) and the two daughters (IV-1 and IV-2) showed normal α-Gal A enzyme activity and no signs of organ involvement. The sister III-2 (33 years old) experienced a TIA when she was 13 years old and showed mild proteinuria, while the sister III-3 (37 years old) showed a significant elevation of lysoGb3 levels, in absence of any sign of organ involvement. No cardiac abnormalities were evidenced in these two patients (Figure 3).Thus, a careful discussion was performed with both the sisters. Sister III-2, in consideration of cerebrovascular and renal involvement, started enzyme replacement therapy, while for sister III-3, no specific therapy was recommended, and a close follow-up was initiated.FD is a multiorgan disease, which can potentially affect any organ or tissue, with the kidneys, heart, and central nervous system representing the major disease targets [1]. The classic form of the disease manifests during childhood, and gastrointestinal disorders, neuropathic pain, hypohidrosis, and angiokeratomas are the most common manifestations of the disease [1]. In adulthood can appear signs and symptoms of heart, kidneys, and cerebral involvement, which is responsible for the increased mortality and morbidity in these patients [8]. On the contrary, males with the non-classic form or later-onset FD and female patients generally show mild clinical presentations and tend to have single organ involvement.Several messages emerge from this case report:The diagnostic delay that generally characterizes rare diseases;The potential coexistence with other diseases (i.e., congenital heart defect) and the importance of identifying specific red flags that raise the possibility of the disease;The importance of the cascade program screening to identify family members at risk;The difficulty in the management of asymptomatic female patients or with mild clinical manifestations.The diagnostic delay that generally characterizes rare diseases;The potential coexistence with other diseases (i.e., congenital heart defect) and the importance of identifying specific red flags that raise the possibility of the disease;The importance of the cascade program screening to identify family members at risk;The difficulty in the management of asymptomatic female patients or with mild clinical manifestations.The high variability in clinical manifestations of FD, with different possible age and symptom onset, can lead to delayed diagnosis and treatment. Similar to patients with other rare diseases, also FD patients frequently had an initial misdiagnosis [9], and the diagnostic “odyssey” to which the patients are frequently subjected is negatively experienced. A recent study shows that the average diagnostic delay from symptom onset is 10.5 years in adults and 4 years in children [10]. The greater diagnostic delay in adults may probably be explained by the non-specific and milder clinical presentation than children. However, the early diagnosis is fundamental in FD since the organ damage, in particular, cardiac and renal injury is in large part irreversible [1,7].In the classic form of FD, with a clear cardiovascular and renal involvement, the diagnosis is generally easy. Patients are referred to nephrologists for proteinuria or to cardiologists for (generally concentric, non-obstructive) hypertrophic cardiomyopathy, and the coexistence of the two conditions is a well-recognized “alarm bell” to promote further investigation [4,11]. The presence of additional cardiac (including short PR interval, atrioventricular blocks, reduced GLS with involvement of the basal inferolateral wall, hypertrophy of papillary muscles, mid-layer inferolateral LGE, low T1 time) or non-cardiac “red flags” (hearing loss, angiokeratoma, cryptogenic TIA or stroke) may be of help to suspect the diagnosis [4,5,12,13,14]. In the present case, the proband was followed for a repaired congenital heart disorder. Clinical history after the second operation included multiple, cryptogenic strokes. The coexistence of mild, concentric hypertrophy, with no evident clinical cause, was the primary reason for referral to our center, and the prompt to look for additional markers (i.e., papillary muscle hypertrophy, reduced longitudinal strain in inferolateral walls, reduced T1).Genetic testing is an indispensable tool in patients with cardiomyopathies and inherited cardiac conditions [15,16,17,18,19,20,21,22,23,24] to confirm the diagnosis and to perform a cascade screening in family members. Thus, after the identification of a disease-causing mutation in the index patient, family members should be invited to join the cascade program screening. This program consent to early identify patients at risk and to start early and appropriate management. In the present case, though the cascade screening, it was possible to identify a symptomatic female patient (with proteinuria and history of TIA) who started ERT, and an asymptomatic female patient (with very low enzyme activity and high lysoGb3), which may potentially benefit from etiological therapy in the future.The etiological therapy shows the maximal benefit in terms of a decrease in incidence rates of adverse events in males; however, also female patients may benefit from a specific treatment [25,26]. In particular, a comprehensive systematic literature review showed that ERT in female patients was associated with significant reductions in plasma and urine GB3 accumulation, in those with elevated pre-treatment levels, and improvement of cardiac parameters and quality of life [25].Although there appears to be a common consensus on the initiation of therapy in symptomatic female patients [7], its role in asymptomatic or mildly symptomatic patients is less clear. Thus, the decision to proceed to etiological treatment in these patients should be evaluated after a case-by-case discussion, considering the risk/benefit balance of the treatment and the patient’s will.FD is a multiorgan disease, which can potentially affect any organ or tissue, with the heart, kidneys, and central nervous system representing the major disease target. Based on clinical markers which should guide the suspect, the clinical and genetic diagnosis in the index patient and family members allows starting appropriate treatment to prevent irreversible organ damage.G.L. designed the study and supervised the writing project of the manuscript. M.R. and E.M. prepared the manuscript and wrote the draft together. M.C. designed the pedigree and managed genetic analysis. M.R., E.M. and M.C. prepared the figures. G.P., M.L., A.C. (Annapaola Cirillo), A.F., F.V., A.P., G.D. (Gaetano Diana), F.A., A.C. (Arturo Cesaro), G.D. (Giovanni Duro), B.S., M.G.R. and P.C. actively participated in the discussion and suggestions for the manuscript. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the University of Campania “Luigi Vanvitelli”.Informed consent was obtained from all subjects involved in the study.The authors declare no conflict of interest.Electrocardiography (A), apical 4 chamber view showing left ventricular hypertrophy and global longitudinal strain at echocardiography (B) and native T1 mapping at cardiac magnetic resonance (C) of the proband (III-1).Pedigree of the members of the family studied and their phenotypic spectrum and genotype. The arrow indicates the proband (III-1). HCM, hypertrophic cardiomyopathy; N/A, not analyzed; TIAs, transient ischemic attacks.Global longitudinal strain at echocardiography and native T1 at cardiac magnetic resonance of the two sisters of the proband ((A): III-2; (B): III-3)) that showed no cardiac involvement.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Previously, Cardiogenetics [1] was published by PAGEPress from Volume 1 (2011) to Volume 10, Issue 1 (2020). From Volume 10, Issue 2, (2020) onwards, Cardiogenetics has been published by MDPI.Previous open access articles that were in Volume 1–Volume 10, Issue 2, and published by PAGEPress under a CC-BY (or CC-BY-NC-ND) licence are now hosted by MDPI on mdpi.com as a courtesy and upon agreement with PAGEPress.To standardize the metadata format of all of the previous articles, MDPI has republished 88 articles in Volume 1–Volume 10, Issue 1 with article numbers replacing page numbers (Table 1). MDPI has also corrected Issue Number errors that occurred while importing the batch data for 14 articles from Volumes 1–3 (Table 2).The author declares no conflict of interest.MDPI has changed the page numbers to articles numbers for 88 articles originally published in Volumes 1–10.MDPI has corrected the Issue Numbers for 14 articles originally published in Volumes 1–3.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+The deletion of the arginine 14 codon (R14del) in the phospholamban (PLN) gene is a rare cause of arrhythmogenic cardiomyopathy (ACM) and is associated with prevalent ventricular arrhythmias, heart failure, and sudden cardiac death. The pathophysiological mechanism which culminates in the ACM phenotype is multifactorial and mainly based on the alteration of the endoplasmic reticulum proteostasis, mitochondrial dysfunction and compromised Ca2+ cytosolic homeostasis. The symptoms of this condition are usually non-specific and consist of arrhythmia-related or heart failure-related manifestation; however, some peculiar diagnostic clues were detected, such as the T-wave inversion in the lateral leads, low QRS complexes voltages, mid-wall or epicardial fibrosis of the inferolateral wall of the left ventricle, and their presence should raise the suspicion of this condition. The risk stratification for sudden cardiac death is mandatory and several predictors were identified in recent years. However, the management of affected patients is often challenging due to the absence of specific prediction tools and therapies. This review aims to provide the current state of the art of PLN R14del cardiomyopathy, focusing on its pathophysiology, clinical manifestation, risk stratification for sudden cardiac death, and management.Arrhythmogenic cardiomyopathy (ACM) is a myocardial disease that affects the left ventricle (LV), right ventricle (RV), or both, whose most typical characteristics are the progressive fibrotic or fibrofatty myocardial replacement that predisposes to ventricular arrhythmias and can be responsible for global or regional ventricular dysfunction [1]. In the pre-genetic era, ACM was considered a myocardial disease that exclusively or predominantly involved the RV, the so-called arrhythmogenic RV dysplasia (ARVD) or cardiomyopathy (ARVC), whose clinical features were RV dysfunction and arrhythmias [2,3]. Therefore, the classical diagnostic criteria for ACM were focused on RV involvement [4]. Subsequently, autopsy investigation, cardiac magnetic resonance (CMR) and genotype-phenotype correlation studies showed that the LV was commonly involved by the fibrotic replacement, changing the paradigm of the disease [5,6].The current classification of ACM includes different clinical variants according to the prevalent ventricular involvement. The classical ARVC phenotype is characterized by isolated RV involvement. On the other hand, the LV phenotype, defined arrhythmogenic left ventricular cardiomyopathy (ALVC), is characterized by predominant LV involvement, and the biventricular phenotype is defined as a disease involving both the ventricles [7].The genetic basis of ACM is responsible for the clinical phenotype. The classic ARVC and the biventricular phenotype are mainly caused by pathogenetic variants involving desmosomal genes, such as PKP2, JUP, DSC2, DSG2, and DSP [8,9,10]. On the other hand, patients with ALVC phenotype carry non-desmosomal gene pathogenic variants, such as ion channel, sarcomere, cytoskeleton, mitochondrial, and sarcomeric genes [11,12,13]. Therefore, with the increased knowledge of the ALVC phenotype, specific diagnostic criteria for the left-side disease variants were proposed (the “Padua Criteria”) [14], based on the following phenotypic features: electrocardiographic (ECG) abnormalities, such as low QRS voltages and T-wave inversion in the lateral or inferolateral leads; ventricular arrhythmias with a QRS morphology which denotes its origin from the LV; normal or mild hypokinetic LV with no or mild dilation; extensive myocardial fibrosis evidenced by CMR as late gadolinium enhancement (LGE) with a non-ischemic pattern of distribution.However, in the absence of RV involvement, the diagnosis of ALVC cannot be formulated based on the phenotype criteria due to the extreme overlap with other inherited or acquired conditions, such as dilated cardiomyopathy (DCM), myocarditis, or cardiac sarcoidosis [15]. Thus, in the presence of a phenotype suggestive for ALVC, the demonstration of a pathogenic or likely-pathogenic variant of an ACM-related gene is required for the diagnosis [16].The identification of the genetic variant underlying the ACM phenotype is essential not only for the diagnosis but also for risk stratification and management. Indeed, several genetic variants were found to be associated with an increased risk of ventricular arrhythmias and sudden cardiac death (SCD) [17,18]. Among these, the pathogenic PLN R14del gene variant, commonly identified in patients fulfilling the diagnostic criteria for ALVC, is generally associated with early-onset arrhythmias and a worse prognosis [19]. Unfortunately, data on the natural history, risk prediction and management of ALVC caused by this pathogenic variant are scant.This review aims to provide the current state of the art of PLN (phospholamban) R14del cardiomyopathy, focusing on its pathophysiology, clinical manifestation, risk stratification for SCD, and management.Physiological cardiac muscle contraction is a finely tuned process mainly regulated by accurately synchronized Ca2+ fluxes in cardiomyocytes [20,21]. When an action potential depolarizes the cell, voltage-dependent L-type Ca2+ channels (LTCCs) open to generate an inward Ca2+ current, which leads to an additional release of Ca2+ from the sarcoplasmic reticulum (SR) through the activation of ryanodine receptor channels 2 (RYR2). The intracellular increase of the Ca2+ concentration is responsible for the myofilament activation and contraction. During diastole, Ca2+ is removed from the cytosol via the sarcolemmal Na+/Ca2+ exchanger (NCX1), which transfers Ca2+ in the extracellular space, and the SR Ca2+ ATPase (SERCA2a), which pumps the Ca2+ in the SR lumen [22]. SERCA2a-dependent diastolic Ca2+ uptake dominates over the extracellular extrusion via the NCX1.PLN finely regulates SERCA2a activity. PLN is a protein of 52 amino acids, localized into the SR membrane and involved in cardiomyocyte calcium handling. PLN activity is modified by its phosphorylation state [23,24]. Its phosphorylation by protein kinase A (PKA) at Ser-16 and/or by calmodulin-dependent kinase II (CaMKII) at Thr-17 releases its inhibitory effects on SERCA2a [24].From a theoretical point of view, the mutation of PLN and its subsequent dysfunction results in more significant inhibition of SERCA2a by non-phosphorylated PLN, thereby leading to an impairment of Ca2+ reuptake. Thus, the decrease in SR Ca2+ content is responsible for impaired systolic function, while the diastolic cytosolic overload is responsible for diastolic dysfunction and arrhythmias.However, the pathophysiological mechanisms which culminate in the ALVC phenotype are more complex and not fully understood. In recent years, several studies have been carried out to elucidate the underlying molecular features of the PLN R14del variant.This variant was identified for the first time in humans with hereditary cardiomyopathy by Haghighi et al. [19] and then studied in murine models. To understand the molecular mechanisms which link the PLN variant to ACM in human induced pluripotent stem cells (hiPSC-CMs), Feyen et al. [25] used single-cell RNA sequencing. They found the presence of elevated stress of the endoplasmic reticulum ER with an unfolded protein response (UPR), a signaling pathway with a critical role in the keeping of proteostasis in the ER [26], in the PLN R14del mutants compared with controls. These findings suggest that the PLN R14del variant is responsible for an altered ER proteostasis. This observation is of clinical interest since for long-term cell function preservation, the balance among protein production, folding and degradation is required. With the aging of the cells, this ability progressively reduces, and the aggregation of unfolded proteins is typical of different age-related diseases, such as Parkinson’s Diseases and Alzheimer’s Disease [27].The proteostasis involvement was also identified by Eijgenraam et al. [28], which postulated that this alteration, combined with the aggregation on PLN proteins, are among the first hallmarks of PLN R14del cardiomyopathy.Furthermore, Cuello et al. [29] reprogrammed dermal fibroblasts to hiPSC-CMs and established isogenic controls using CRISP/Cas9. Then, cardiomyocytes were differentiated. They found that cardiomyocytes that bring the PLN R14del variant showed a Ca2+ load-dependent irregular beating pattern, lower force and a prolonged Ca2+ transient decay time than controls. In addition, the ER, ribosomes, and mitochondria exhibited less protein content when analyzed using proteomic analysis. Finally, large lipid droplets in mitochondria and an ER dilation were observed using electron microscopy. This evidence suggests that the ER and mitochondrial impairment are a novel disease mechanism underlying the PLN R14del cardiomyopathy.In conclusion, the molecular mechanisms underlying the PLN R14del cardiomyopathy are complex and under investigation (Figure 1). Therefore, a better understanding of its pathophysiology is required to formulate a tailored therapy.PLN R14del cardiomyopathy presents overlapping clinical features between ALVC and DCM [30]. The identification of specific phenotypic features to distinguish patients with PLN R14del cardiomyopathy and their relatives from those with other forms of ACM or DCM has been investigated in different studies. Many signs of the disease can be identified in the pre-symptomatic phase, in particular repolarization abnormalities, frequent ventricular premature complexes (VPCs), and CMR LGE, as evidenced in the recently published iPHORECAST (PHOspholamban RElated CArdiomyopathy intervention Study) trial [31].The disease onset seems to be age-related, with a slightly higher prevalence in males [32]. The symptoms are usually non-specific and consist of arrhythmia-related (e.g., palpitation, syncope) or heart failure-related symptoms (e.g., dyspnoea, exercise limitation). Symptoms usually appear in the fifth decade of life [30]; however, cases of SCD have been described in patients younger than 30 years old [30,32,33].The ECG findings reflect the myocardial fibrosis substrate, as proved by histological examination studies, and typically consist of low QRS voltages with reduced R-wave amplitude [19,34]. These abnormalities have not been found in patients without the mutation [35]. Te Rijdt et al. [36] found a median R-wave amplitude of 5.3 mV, with more decreased QRS voltages in older mutation carriers. Negative T-waves are also common in PLN R14del cardiomyopathy. They were identified in the right precordial leads in 11% of carriers and in V4–V6 in 29% of them (80% of index patients) [36]. Moreover, 15% of patients experience ventricular tachycardia episodes.Van de Leur et al. [37] utilized deep neural networks (DNNs) to detect possible typical ECG abnormalities in PLN R14del cardiomyopathy, useful to identify pre-symptomatic mutation carriers. The elaborated algorithm was capable not only of confirming known features of the disease, such as negative T-waves and low QRS voltages, but also to define their characteristics. Low QRS voltages consisted more properly in R-wave attenuation with normal S-wave, localized in the right precordial leads V2 and V3, and in the lateral leads DI, aVL and V6. Moreover, T-waves attenuation/inversion was situated not only in V2, V3, and V6 but also in DI and aVL. Furthermore, it was also able to find a new distinctive element on surface ECG, the prolonged PR interval, that suggests a possible involvement of atrioventricular conduction.The most common CMR pattern of disease is the presence of epicardial or mid-wall fibrosis in the inferolateral LV wall, which usually corresponds with negative T waves in the LV inferolateral leads [33,38]. Functional and structural impairment of LV is common, usually represented by mild LV dilation and dysfunction, as confirmed either by echocardiography or CMR studies [33,38,39].A recent study showed an extensive LGE in the LV of the affected patients, even in those with preserved or mildly reduced LV ejection fraction (LVEF) (>45%) and was found to be independently associated with ventricular arrhythmias [36]. However, LGE was more significant in older and in those with reduced LVEF. RV was involved by LGE only in 5% of patients, and it was associated with reduced RV ejection fraction.PLN R14del variant carriers can experience early-onset ventricular arrhythmias, ranging from frequent PVCs to ventricular fibrillation and SCD, which in rare cases may be the first clinical presentation of the cardiomyopathy [30,32,33]. Due to the lack of specific recommendations for PLN R14del cardiomyopathy, the indication for the implantable cardioverter–defibrillator (ICD) implantation for SCD prevention follows the current ACM and DCM guidelines and consensus documents [7,40].However, while there is clear evidence that ICD implantation is recommended for secondary prevention in those patients who experienced sudden cardiac arrest or ventricular arrhythmias with hemodynamic instability, more difficult is the identification of subjects at high risk for SCD who require an ICD for primary prevention. In recent years, several studies investigated the SCD predictors in patients with PLN R14del cardiomyopathy.Firstly, van Rijsingen et al. [32] identified LVEF < 45%, and sustained and non-sustained ventricular tachycardia (SVT and NSVT) as independent risk factors for malignant ventricular arrhythmias.Subsequently, Te Rijdt et al. [36] investigated the extent and localization of myocardial fibrosis and its association with ECG features and ventricular arrhythmias in PLN R14del mutation carriers. They found that LGE in the LV, but not attenuated R-waves and inverted T-waves, was independently associated with ventricular arrhythmias. Of importance, 30% of patients with preserved LVEF showed a significant LGE in the LV. However, in this study, the occurrence of ventricular arrhythmias was determined on ambulatory 24 h ECG Holter or exercise ECG, which were not available for every patient, leading to a possible selection bias.Furthermore, the incremental value of the LV mechanical dispersion (LVMD) by echocardiographic deformation imaging for sustained ventricular arrhythmias prediction was recently investigated. Taha et al. [40] evaluated 243 PLN R14del mutation carriers, which were classified into three groups according to the “45/45” rule. Patients with overt LV dysfunction (LVEF < 45%) had the worst prognosis in terms of ventricular arrhythmic events and were considered to be at high risk, similar to a previous study [32]. In contrast, patients with normal LV function (LVEF > 45% and LVMD < 45 ms) showed a low risk of developing sustained ventricular arrhythmias, and those with mechanical LV dysfunction (LVEF > 45% and LVMD > 45 ms) exhibited an intermediate risk, falling into a “grey zone” where a multiparametric assessment for SCD risk prediction is required.Finally, a multiparametric algorithm to identify patients who may benefit from ICD implantation for primary prevention was developed [41] (https://plnriskcalculator.shinyapps.io/final_shiny/, accessed on 1 March 2022). The multivariable model, including LVEF, PVC count in 24 h, number of negative T-waves, and presence of low QRS voltages on ECG, showed an excellent discriminative ability (C-statistic 0.83 (95% CI 0.78–0.88)). However, the study suffers from some limitations, such as the endpoint used for the assessment of the arrhythmic outcome, the lack of an external validation cohort, and the insufficient amount of data on the presence and extent of LGE. In detail, the use of a combined endpoint, consisting of SVT, appropriated ICD intervention, and SCD, may overestimate the true risk of SCD. Indeed, ICD intervention is a poor surrogate of SCD since most ventricular tachycardia episodes treated by ICD are expected to be self-terminating. Moreover, the lack of data on LGE may affect the power of the prediction model and can be responsible for the identification of ECG abnormalities (i.e., low QRS voltages and T-waves inversion) as SCD predictors, in contrast with the previous study.In conclusion, two predictors (LVEF < 45% and extensive LGE in the LV) were found to be strongly associated with major arrhythmic events and, in their presence, an ICD implantation in primary prevention should be considered. However, in the remaining patients, risk stratification should be based on a multiparametric approach, including family and clinical history, ECG, ECG-Holter monitoring, echocardiography and CMR, and discussed case by case in the context of a multidisciplinary team of experts (Figure 2).As previously reported, the PLN R14del cardiomyopathy is associated with a high prevalence of ventricular arrhythmias, heart failure (HF), and SCD. Unfortunately, no specific treatments for this condition are currently available. For this reason, the primary efforts should be oriented toward the prevention and treatment of life-threatening arrhythmias and HF (Figure 3).The PLN R14del cardiomyopathy phenotype is typically associated with a reduction (<40%) or a mild reduction (LVEF 40–50%) of the LVEF, while the RV is rarely involved. Therefore, the treatment of HF in these patients is based on disease-modifying drugs, such as angiotensin-converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARBs), beta-blockers, mineralocorticoids antagonists (MRAs), angiotensin receptor–neprilysin inhibitor (ARNI), and sodium–glucose cotransporter 2 (SGLT2) inhibitor, and diuretics for the treatment of congestion, according to the current guidelines [42]. Furthermore, in patients with more severely reduced LVEF, cardiac resynchronization therapy should be considered. In addition, anticoagulant therapy is recommended if there are atrial fibrillation, intracavitary thrombosis and venous or systemic thromboembolism [7,43]. Moreover, it may be considered in individuals with LV or RV aneurysms.Next to its use in the setting of HF, beta-blockers are used for the management of arrhythmias. In particular, the use of beta-blockers is recommended in patients with ACM receiving inappropriate ICD interventions due to arrhythmias such as sinus tachycardia, supraventricular tachycardia, atrial fibrillation, or atrial flutter causing a high ventricular rate [44,45,46]. Moreover, in patients with ACM and ventricular arrhythmias, antiarrhythmic drugs such as amiodarone and sotalol may be used to control symptoms and reduce ICD shocks [47,48]. Finally, in patients with recurrent sustained monomorphic VT despite antiarrhythmic drug therapy (or intolerant to such therapy), catheter ablation can be opted to reduce arrhythmic events and ICD shocks [49].As stated before, no evidence-based treatment is available for pre-symptomatic carriers. The i-PHORECAST trial, aiming to address whether pre-emptive treatment of PLN R14del mutation carriers with eplerenone can prevent or delay the onset of cardiomyopathy, is still ongoing [31].PLN R14del cardiomyopathy is a rare cause of ACM and is associated with prevalent ventricular arrhythmias, HF, and SCD. In the spectrum of ACM, the identification of this condition is mandatory to approach a tailored risk stratification and management.However, several gaps in knowledge still exist in this field (e.g., pathophysiology is still poorly understood, no tailored therapy available, etc.). A better understating of the molecular mechanisms responsible for the cardiomyopathy phenotype is required to develop an aetiological therapy.Conceptualization, E.M. and G.L.; writing—original draft preparation, E.B., A.D.P., A.D.V., M.L.M., O.M. and A.V.; writing—review and editing, E.M., F.A., M.C., G.D., M.L., A.P., F.V. and G.L. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The authors declare no conflict of interest.Pathophysiology of PLN R14del Cardiomyopathy. ACM, arrhythmogenic cardiomyopathy; ER, endoplasmic reticulum; PLN, phospholamban; SERCA2a, sarcoplasmic reticulum Ca2+ ATPase.Risk stratification for sudden cardiac death in PLN R14del cardiomyopathy. CMR, cardiac magnetic resonance; ECG, electrocardiography; LGE, late gadolinium enhancement; LVEF, left ventricular ejection fraction; LVMD, left ventricular mechanical dispersion; NSVT, non-sustained ventricular tachycardia; PVC, premature ventricular contraction; SCD, sudden cardiac death; SVT, sustained ventricular tachycardia.Medical treatment of PLN R14del cardiomyopathy manifestations. ACE, angiotensin converting enzyme; ARNI, angiotensin receptor neprilysin inhibitor; CRT, cardiac resynchronization therapy; HF, heart failure; ICD, implantable cardioverter defibrillator; LVEF, left ventricular ejection fraction; MRA, mineralocorticoid receptor antagonist; OMT, optimal medical treatment; SCA, sudden cardiac arrest; SGLT2. sodium-glucose co-transporter-2; VA, ventricular arrhythmia; VT, ventricular tachycardia.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Cardiomyopathies (CMPs) are a heterogeneous group of diseases, frequently genetic, affecting the heart muscle. The symptoms range from asymptomatic to dyspnea, arrhythmias, syncope, and sudden cardiac death. This study is focused on MYH7 (beta-myosin heavy chain), as this gene is commonly mutated in cardiomyopathy patients. Due to the high combined prevalence of MYH7 variants and severe health outcomes, it is one of the most frequently tested genes in clinical settings. We analyzed the clinical presentation and natural history of 48 patients with MYH7-related cardiomyopathy belonging to a cohort from a tertiary center at Helsinki University Hospital, Finland. We made special reference to three age subgroups (0–1, 1–12, and >12 years). Our results characterize a clinically significant MYH7 cohort, emphasizing the high variability of the CMP phenotype depending on age. We observed a subgroup of infants (0–1 years) with MYH7 associated severe DCM phenotype. We further demonstrate that patients under the age of 12 years have a similar symptom burden compared to older patients.Cardiomyopathies (CMPs) are a heterogeneous group of diseases affecting the heart muscle characterized by alterations in the ventricle wall thickness, size of the cardiac chambers, or abnormal contraction in the absence of underlying heart disease. The symptoms range from asymptomatic to dizziness, palpitations, syncope, and sudden cardiac death (SCD) [1]. CMPs can be divided into specific morphological and functional phenotypes, including dilated, hypertrophic, restrictive, arrhythmogenic, and non-compaction patterns and they frequently have a genetic basis [2,3].This study focuses on the MYH7 gene (beta-myosin heavy chain), encoding myosin-7, which is a major contributor to several CMPs and is often associated with poor prognosis [4,5]. This large gene is located on chromosome 14 q11.2–q13 in humans (40 exons), and its product was the first sarcomeric protein linked with cardiomyopathy [6]. It is expressed predominantly in the human ventricle and skeletal muscle tissues. Mutations in MYH7 are associated with hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), left ventricular non-compaction (LVNC), restrictive cardiomyopathy (RCM), and multiple patients have shown overlapping phenotypes. In addition, MYH7 mutations can cause a combination of myopathy and cardiomyopathy, and pure skeletal myopathies. Due to MYH7 high combined prevalence and severe health outcomes, it is one of the most frequently tested genes in a clinical setting [4,5,7].This study analyzes the clinical presentation and natural history of patients with MYH7-related cardiomyopathy with special reference to patients under 12 years of age. For this purpose, we investigated a cohort of MYH7-related CMPs from a tertiary center in Helsinki University Hospital, Finland. We present here the clinical and genetic characteristics of the MYH7 cohort and demonstrate that patients under the age of 12 years have a similar symptom burden to older patients. Furthermore, we noticed a subgroup of infants with MYH7, which associates with a severe DCM phenotype.Our retrospective study aimed to include all identified MYH7 variants in the Helsinki University Hospital district in Finland between 2002 and 2021. Patients under 16 were followed and diagnosed at the children’s hospital and older patients at the adults’ cardiology department. Five patients declined their participation in the study. The final study population consisted of 21 index patients and 27 first-degree relatives. Of these 27 first-degree relatives, nine were not related to any other study patients.Cardiac imaging data were collected for each patient. All patients underwent echocardiography and resting and ambulatory electrocardiograph as a part of their clinical workup. Cardiac magnetic resonance imaging (CMR) was performed on selected patients to verify CMP diagnosis and to assess the severity of the disease [8]. Cardiac MRI was performed using a 1.5 Tesla clinical system (Ingenia, Philips Healthcare, Best, The Netherlands in pediatric patients and Siemens MAGNETOM Avanto/Avantofit, Siemens Healthcare in adult patients) and was in line with current ESC recommendations [9]. Imaging investigations were performed by a senior radiologist (CMR) or cardiologist (CMR, echocardiography) with a specialization in cardiac imaging settings. Cardiac echocardiography was performed using a GE Healthcare or Philips Healthcare ultrasound device. DCM was defined by reduced ventricular systolic function: fractional shortening <25%, ventricular ejection fraction (EF) <45%, and left ventricular end-diastolic diameter >27 mm/m2, or in children a Z score ≥+2. HCM was defined by otherwise unexplained septal hypertrophy, left ventricular free wall hypertrophy ≥15 mm, or in first-degree relatives of HCM ≥13 or in children Z score ≥+2 [9]. For LVNC, CMR was used in all patients to confirm the diagnosis of LVNC and to examine the disease’s severity. The following MRI criterion was used: the end-diastolic ratio of non-compacted to compacted layers above 2.3 in short axis view [10]. No patients were detected with MYH7 related arrhythmogenic right ventricular cardiomyopathy.Clinical data were collected from routine outpatient visits from all the patients, including heart failure symptoms according to the New York Heart Association (NYHA) or Ross functional classification [11,12], family history, resting and ambulatory electrocardiography (ECG), and transthoracic echocardiography (2D, Doppler, and color). Heart failure symptoms were defined as an NYHA or Ross functional class ≥2. Left ventricular outflow tract obstruction (LVOTO) was defined as an instantaneous peak Doppler left ventricular outflow tract pressure gradient ≥30 mmHg at rest [9]. Non-sustained ventricular tachycardia (NSVT) was defined as three or more consecutive ventricular beats at a rate greater than 120 beats/min lasting less than 30 s on ambulatory ECG recordings [9]. The patient was considered to have familial cardiomyopathy if at least one first-degree or two second-degree relatives were reported to have cardiomyopathy. Sudden cardiac death was defined as a sudden and unexpected death. This information was obtained from the patients during a routine clinical visit as a part of family history.Genetic testing was performed as part of usual care. Of the 48 patients, the NGS panel method was used for 34, and 12 were separately diagnosed for their gene defect. As part of usual clinical practice, targeted sequencing panels vary according to the phenotype presented by patients (e.g., HCM, DCM). The targeted panels used were the Cardiomyopathy Panel version 1.0–3.0 Panel (Blueprint Genetics, Helsinki, Finland), 155 cardiomyopathy-related genes; Hypertrophic Cardiomyopathy (HCM) Panel (Blueprint Genetics, Helsinki, Finland), 16 genes; Pan cardiomyopathy panel (Blueprint genetics, Helsinki, Finland), 103 genes; Dilated Cardiomyopathy (DCM) Panel Plus, (Blueprint genetics, Helsinki, Finland), 69 genes. Furthermore, whole-exome sequencing was performed for two patients. All the gene findings were verified with bi-directional Sanger sequencing. When an MYH7 proband was diagnosed, first-degree relatives were tested clinically independent of age by Sanger sequencing.All variants initially considered disease-causing were re-analyzed and classified for this study according to the American College of Medical Genetics and Genomics (ACMG) guidelines as pathogenic (P), likely pathogenic (LP), variant of unknown significance (VUS), likely benign (LB), or benign (B) [13]. Variants classified as P/LP were considered to be diagnostic. Pathogenic, likely pathogenic, and VUS variants were included in the study (Table 1). We excluded from the analyses one patient whose variant was classified as benign MYH7 c. 4472 C > G, p. (Ser1491Cys) and one patient who had wide homozygous regions in the exome. One patient also had another potential cardiomyopathy variant (DSG2 c. 2137 G > A, p. (Glu713Lys)), but the clinical phenotype did not support this variant as the patient’s underlying cause of HCM, so the patient was included in the study.The study outcomes and factors associated with more severe disease forms were sudden cardiac death (deceased), cardiac transplantation (TX), pre-transplantation examinations (preTX), LVOTO, or implantable cardiac defibrillator (ICD).All statistical analyses were performed using SPSS v. 25 (IBM Corporation). Continuous variables are described as mean ± standard deviation or median (range) where appropriate, with three group comparisons conducted using ANOVA or Wilcoxon Rank Sum. Categorical variables were compared using the Chi-Square test.The patients older than ten years of age or the parents of younger children gave written informed consent. The Ethics Committee of Helsinki and Uusimaa Region Hospital approved the study plan (ethical license HUS/2227/2018, HUS 291/13/03/03/2008, HUS/3225/2018).We identified 48 patients with MYH7 variants; 21 (43.8%) were index patients, 27 (56.3%) were first-degree relatives (nine were not related to any other study patients); 56.3% were men, and 43.8% were women. Table 2 shows the general characteristics of the study population divided by age (0–1, 1–12, and >12 years). Table 1 lists the variant and phenotype of each patient. A family history of the MYH7 variant or SCD was positive at the time of presentation in 27 (56.3%) and 5 (10.4%) patients, respectively. Four patients (8.3%) reported unexplained syncope, and five (10.4%) had neurological deficits at baseline. All the patients underwent resting and ambulatory ECGs as part of routine CMP protocol. Two patients had atrial fibrillation, six had left ventricular hypertrophy, and four had right bundle branch block. No signs of myocardial infarction or conduction disease, for example, a typical Brugada ECG pattern, were noted.Phenotype: Patient cardiomyopathy type differed significantly according to age. The median age of disease onset was 12.0 years (15.0 years in HCM and four months in DCM). In patients with an age of onset >12 years, HCM was the most frequent diagnosis present in 19/21 (90.4%) patients, DCM and LVNC were both present in 1/21 (4.8%) patients, and there were no variant carriers with a normal phenotype. In patients with an age of onset between 1 and 12 years, 9/15 (60%) showed HCM, no patients showed DCM or LVNC, and 6/15 (40%) were asymptomatic carriers. In patients with an age of onset younger than 1 year, 5/12 (41.6%) showed DCM, 3/12 (25%) showed HCM, 1/12 (8.3%) showed LVNC, and 3/12 (25%) were asymptomatic carriers.Imaging: CMR was performed for 26 (52.2%) patients. Late gadolinium enhancement (LGE) in the left ventricle was observed in 13/15 (86.7%) of HCM and in 2/4 (50.0%) of DCM. Median CMR global longitudinal strain (GLS) was −19.2% (−33–(−15)) in HCM patients and −20.6% (−24–(−10)) in DCM patients, p = 0.464. In echocardiography, 5/6 (83.3%) of DCM, 2/2 (100%) of LVNC patients, and 1/31 (3.2%) of HCM patients had reduced ejection fraction (EF) (<45%).Genetics: Following the American College of Medical Genetics and Genomics (ACMG) reclassification, 38 (79.2%) had a pathogenic/likely pathogenic (P/LP) variant, and 10 (20.8%) had a variant of uncertain significance (VUS). MYH7 variant c. 3158 G > A, p. (Arg1053Gln), which is classified as pathogenic, was the most common (26.3%), followed by MYH7 c. 1816 G > A, p. (Val606Met) (15.8%), and MYH7 c. 1357 C > T, p. (Arg453Cys) (7.9%), both also classified as pathogenic. Most of the disease-causing alterations were missense variants. There was one nonsense mutation and two in-frame variants (Table 1). Most of the MYH7 variants were heterozygous.Only one patient also had another potential variant (DSG2 c. 2137 G > A, p. (Glu713Lys)) in our genetic testing, but the clinical phenotype did not support this variant as pathogenic.The illustration of the MYH7 gene (Figure 1) presents the MYH7 variants in our cohort color-coded by cardiomyopathy type. The distribution pattern throughout the protein is similar to previous reports, with an enrichment in the head area. We highlighted the most relevant functional domains, such as the converter domain and actin-binding regions, to assess the association between the type of CMP and MYH7 variants/domains.The localization of mutations is depicted in relation to important functional domains of the protein. The broad domains of Myosin-7 were described as the head (aa 1–778), lever or neck (aa 779–838), and tail (aa 839–1935), or as subfragment-1 (aa 1–847), subfragment-2 (aa 848–1216), and light meromyosin (aa 1217–1935). They contain functional domains such as SH3-like domain, motor domain, actin-binding regions, converter, IQ domain, and the coiled-coil domain.Outcome: Factors associated with more severe disease forms were observed in 21/48 (43.8%) of the patients, including 11 patients who underwent ICD implantation (seven as primary and four as secondary prevention), five who underwent cardiac transplantation, two who died, and two who had LVOTO (Figure 2). The mean patient age at the primary outcome event was 26 years and the time from diagnosis to the primary outcome event was 8.0 years, respectively (Table 2). Factors associated with more severe disease forms were observed in 17/38 (45.0%) of P/LP patients and in 4/10 (40.0%) of VUS variants (p = 0.788).Pathogenic variants located in the converter domain of MYH7 were associated with particularly adverse outcomes. Amongst our studied patients, two variants were located in the converter domain: c. 2155 C > T, p. (Arg719Trp) and c. 2162 G > A, p. (Arg721Gln). Of these, one patient underwent a cardiac transplant, and one underwent an ICD implantation.Our study demonstrates that MYH7 patients under the age of 12 years have a similar symptom burden to older patients. Interestingly, we observed a subgroup of infants with MYH7 associated severe DCM phenotype. We also show clustering of mutations in the MYH7 gene map, highlighting that all pathogenic variants associated with DCM in the present cohort reside in the coiled-coil domain. The number of variants in our cohort is too small to predict domain-specific effects but can contribute to future protein structure-function analyses. For example, the age of onset for five out of six patients with DCM was under one year, and they also presented pathogenic variants in the coiled-coil domain. Future structural–functional analyses could determine why these variants are particularly damaging for MYH7 protein.Overall, over 800 MYH7 variants were classified as disease-causing, almost all of which are missense variants (HGMD®, available via http://www.hgmd.org (Accessed on 22 Decembar 2021)). The pathogenicity of truncating protein variants is uncertain. We mostly observed missense variants in our cohort, but one variant (MYH7 c. 335 G > A, p. (Trp112*)) caused premature truncation. This patient was diagnosed at the age of 61 with LVNC presenting with cardiac symptoms (e.g., palpitations and ventricular premature complexes, shortness of breath), and her LV EF was 40%, although her overall cardiac function was stable. The clinical significance of the MYH7 c. 335 G > A, p. (Trp112*) remains unknown. Usually, single truncations are not disease-causing but can associate with very severe disease when they are coincident with a disease-causing missense variant in the second allele.Our study’s most common MYH7 variant was c. 3158 G > A, p. (Arg1053Gln), which is the third most common pathogenic variant encountered in Finnish patients with HCM, explaining up to 7.9% of Finnish HCM cases in a recent study [14]. Because of its high prevalence in Finnish patients with HCM, this pathogenic variant is likely to be a founder variant as in many other genetic diseases in the Finnish population [15]. One patient with the p. (Arg1053Gln) variant underwent ICD implantation in our cohort.In order to further investigate the MYH7 gene and its related mutations, we drew a gene map highlighting the distribution of variants in relation to clinical phenotypes (Figure 1). The MYH7 protein is often divided into three different structural regions: head, neck, tail. The head (aa 1–778) of the protein corresponds in the gene from exon 3 to partway through exon 21, the neck (aa 779–838) extends from exon 21 to 25, and the tail (aa 839–1935) comprises exons 25–40. The functional sites for the ATP-binding domain are encoded between exons 5–12, the actin-binding domain from 13 to 16, the converter region from 18 to 19, and the light chain-binding region from 21 to 22. In our gene map, clustering of the mutations is apparent around some of these sites, although the number of pathogenic variants analyzed is relatively small. A similar finding was also reported in a previous study where it was also noted that variants in the head region were more likely to lead to more severe disease [16,17,18,19,20,21,22,23]. In accordance with this, we noticed that 11 out of 21 primary outcome events (52.4%) were among patients whose variants were in the head region.The head region contains functional sites for muscle contraction and thus plays a significant role in the functioning of the molecule. It is plausible that the mutation in this region could lead to a detrimental effect on the ability of myosin to fulfill its role in muscle contraction and therefore lead to more severe disease. An especially interesting functional site in the head region is the converter domain located between amino acids 709–777 [23]. This domain is essential for elastic distortion of the cross-bridge. Elastic distortion of a structural element is fundamental to the ability of myosin to generate motile forces. Recently, amino acids between 716 and 719 in this domain were linked to severe forms of CMP [23]. In our study, there were two patients with a variant (c. 2155 C > T, p. (Arg719Trp)) between amino acids 716–719. This variant was previously highlighted as entailing a high risk of early mortality. Both patients had HCM, and one of them also underwent an ICD implantation. This mutation affects the converter and light-chain-binding domain, making it more resistant to elastic distortion, thus affecting the ability of myosin to generate motile force [24]. The patient with another variant in the converter domain (c. 2162G > A, p. (Arg721Gln)) underwent a cardiac transplant operation at the age of 42. The variant was categorized as VUS.According to recent findings, the variants related to the DCM-phenotype were clustered in the tail and neck regions [22]. Variants in the tail region may sometimes predispose to a more severe form of cardiomyopathy. This could be due to amino acid substitution that disrupts the assembly or the structure of the thick filament component of the sarcomeres [22]. However, more studies are needed to draw a further conclusion from this finding.In our cohort, the phenotype of MYH7-related CMP was highly variable. DCM was more prevalent among patients under one year of age, and the prevalence of HCM increased with age. Although DCM is typically an adult-onset disease, the onset may already occur in infancy. Similar findings were also reported previously [25,26,27]. Our study describes high symptom burden and variable cardiac phenotype on MYH7 patients under 12 years, including severe hypertrophy, malignant ventricular arrhythmias, and cardiac transplantations. It highlights infants who might present a more severe form of CMP and have a poorer prognosis [28,29,30]. However, it is noteworthy that, in our study, 60% of the patients under one year of age responded well to heart failure medication, and their cardiac function recovered. One patient from this age group underwent cardiac transplantation, and one patient died.There were no significant differences in any primary outcome events stratified by age (0–1, 1–12, >12 yrs), suggesting that there might already be a considerable symptom burden among patients under 12 years of age with the MYH7 gene mutation. However, more research is needed to verify these results.The American College of Cardiology (ACC)/American Heart Association (AHA) recently modified the recommendation for family screening of first-degree relatives of affected probands with HCM in the absence of malignant family history. The age threshold for starting family screening [8,31,32,33] was eliminated. Our findings are in accordance with this recommendation. From the diagnostic point of view, echocardiography remains the cornerstone of cardiac assessment in CMPs. CMR is mainly recommended to describe the phenotype and diagnosis and is valuable for risk stratification for sudden cardiac death [34,35,36].Altogether, the prevalence of outcomes was high in our cohort, considering that many variants were classified as VUS (n = 10 (20.8%)). Historically, VUS was not considered when evaluating genotype-outcome association, as the pathogenicity of these variants is uncertain. However, a more significant variant burden was found in pediatric HCM patients, including pathogenic and VUS, a burden associated with worse outcomes [36,37]. These data suggest that some of the VUSs with a pathogenic pair are truly disease-causing while many are not. Thus, VUSs should not be used for risk stratification.We noticed a subgroup of infants with MYH7 associated severe DCM phenotype. We further demonstrate that patients under the age of 12 years have a similar symptom burden to older patients. These findings support the observations that clinical and genetic screening should also be considered for younger MYH7 family members to identify patients needing closer monitoring and interventions. However, more research is required to estimate causal relationships, considering our limited study sample.T.V.: data acquisition, processing, and interpretation; writing of the manuscript. T.O., T.H.: study concept and supervision, data interpretation, critical revision of the manuscript. L.M.: Data interpretation, critical revision of the manuscript. S.W.: data acquisition, critical revision of the manuscript. J.K.: genetic analysis, critical revision of the manuscript. A.H.: critical revision of the manuscript. C.V.: genetic analysis, data interpretation, critical revision of the manuscript. All authors have read and agreed to the published version of the manuscript.This work was financially supported by the State funding for university-level health research (TYH2020210, Y2020SK004, Y1016SK004), the Foundation for Pediatric Research Center, Aarne Koskelo Foundation, and the Finnish Foundation for Cardiovascular Research.Written informed consent was obtained from the patients (ethical license HUS/2227/2018, HUS 291/13/03/03/2008, HUS/3225/2018).Informed consent was obtained from all subjects involved in the study.The data that support the findings of this study are available from the corresponding authors upon reasonable request.We thank Anu Suomalainen for their valuable help in the interpretation of study data. We thank patients for participation in the study.Juha Koskenvuo is a full-time Executive Director, Lab and Medical, at Blueprint Genetics. Other authors declare no conflict of interest.Schematic representation of the Myosin-7 protein and the patient mutations analyzed in this study.(A) Patients by cardiomyopathy (CMP) types. (B) Age at diagnosis color-coded by CMP type. (C) The number of patients with endpoints. PreTX = pre-transplantations examination, lvoto = left ventricular outflow tract obstruction, ICD = implantable cardioverter defibrillator, TX = cardiac transplant.MYH7 variants observed in the study patients.HCM = hypertrophic cardiomyopathy, DCM = dilated cardiomyopathy, LVNC = left ventricular non-compaction cardiomyopathy, LVOTO = left ventricular outflow tract obstruction, ICD = implantable cardiac defibrillator, preTX = pre-transplantation examination, TX = cardiac transplantation.Clinical characteristics of patients with MYH7 related cardiomyopathy.Data expressed in mean ± standard deviation, unless otherwise indicated. FHx = family history, HCM = hypertrophic cardiomyopathy, DCM = dilated cardiomyopathy, LVNC = left ventricular non-compaction, FHx = family history, LVMWT = left ventricular maximal wall thickness, CI = 95th confidence interval, LVEDD = Left ventricular external end-diastolic diameter, EF = ejection fraction, CMR = cardiac magnetic imaging, LVOT = left ventricular outflow tract, ICD = implantable cardiac defibrillator.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+We describe a patient with sub-acute bacterial endocarditis, whose chief presenting feature was mild expressive dysphasia.A 68-year-old male presented to our hospital complaining of very mild word finding difficulties. He had instrumental dental cleaning four weeks previously. He had a background history of ischaemic heart disease, mitral valve repair (for mitral valve prolapse), hypertension and paroxysmal atrial fibrillation. On examination, he had a temperature of 37.9°, very subtle expressive dysphasia, left palmar erythema and a petechial rash on the left little finger pulps. He had raised inflammatory markers and blood cultures grew Enterococcus faecalis. His transthoracic echocardiogram revealed a 5×4 mm vegetation of the anterior mitral valve leaflet (Figures 1 and 2). His brain magnetic resonance imaging (MRI) showed acute bilateral embolic infarcts (Figures 3 and 4).
+
+Figure 1
+Parasternal long axis view-showing vegetation on anterior mitral valve leaflet.
+
+
+Parasternal long axis view-showing vegetation on anterior mitral valve leaflet.
+
+Figure 2
+Parasternal short axis view showing vegetation on anterior mitral valve leaflet.
+
+
+Parasternal short axis view showing vegetation on anterior mitral valve leaflet.
+
+Figure 3
+Magnetic resonance imaging head-DWI hyperintensity revealing a left temporo-parietal infarct.
+
+
+Magnetic resonance imaging head-DWI hyperintensity revealing a left temporo-parietal infarct.
+
+Figure 4
+Magnetic resonance imaging head-DWI hyperintensity in the right parietal cortex revealing an embolic infarct.
+
+
+Magnetic resonance imaging head-DWI hyperintensity in the right parietal cortex revealing an embolic infarct.The patient was treated with six weeks of intravenous antibiotics (amoxicillin, vancomycin and gentamicin) for infective endocarditis. He responded well with falling inflammatory markers and reduction in vegetation size.The epidemiology of infective endocarditis (IE) has changed substantially in the last few years in industrialised nations where the incidence increases with age (peak incidence 70–80 years of age).1 In 30% of patients, embolization to the brain, lung or spleen is the presenting feature.2 The incidence of IE after dental procedures is highly variable and can range from 10–100%.3 Good oral hygiene and regular dental review is of importance for the prevention of IE. Despite major advances in diagnostic and therapeutic procedures, the mortality rate (9.6–26%) of IE has not changed in the last 30 years.1

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+Isolated sphenoid pathology is uncommon. Nasal polyps that originate from the anterior wall of the sphenoid sinus and reach the nasopharynx are called sphenochoanal polyps. The atypical location of sphenochoanal polyps leads to misdiagnosis, and surgery risks injuring the surrounding structures, such as the optic nerve, carotid artery, and brain. For the differential diagnosis of sphenochoanal polyps, nasal endoscopy and computed tomography are very important. We present the clinical and radiological features of a sphenochoanal polyp and review the status of the optic nerve during endoscopic surgery for a sphenochoanal polyp.Nasal polyps are polypoidal masses arising mainly from the mucous membranes of the nose and paranasal sinuses. When they extend to the choana, they are called choanal polyps, which constitute 3–6% of all nasal polyps.1 Nasal choanal polyps occur in three different forms, which produce similar symptoms: sphenochoanal, antrochoanal, and ethmoido-choanal polyps. Sphenochoanal polyps are rare, while antrochoanal polyps are the most frequent and originate from the inflamed sinus mucosa.2The etiology of sphenochoanal polyps is not clear. They are most frequent in adolescents and young adults.3 They may occur with concomitant nasal polyps. Isolated sphenoid sinus pathology is relatively uncommon. Most nasal polyps produce the same symptoms: nasal obstruction, nasal discharge, and headache. Nasal polyps are seen in anterior rhinoscopy, while an endoscopic examination or computed tomography (CT) is needed to differentiate sphenochoanal polyps.4Nasal polyps can be removed via endoscopic sinus surgery, but the site of origin is very important, especially for polyps originating from the sphenoid sinus because the carotid artery and optic nerve are located near the sphenoid sinus. Any uncontrolled maneuver in endoscopic sinus surgery can damage these essential structures, so a preoperative diagnosis of the polyp origin is essential.A 16-year-old boy presented to our outpatient clinic with a 1-year history of bilateral nasal obstruction and discharge. He had no significant medical history or drug allergy. In the nasal examination, the nasal cavity was filled with a nasal polyp and there was right septal deviation. At nasal endoscopy, the origin of the nasal polyp was not clear; it seemed to originate posteriorly, occupying the left choana. Skin-prick testing was negative for the main commercial allergens. The total immunoglobulin E was within normal limits. Paranasal coronal images showed a homogeneously opacified maxillary sinus, totally opacified left sphenoid sinus, and obliterated choanal passage. The middle meatus was also opacified (Figure 1).
+
+Figure 1
+Paranasal sinus computed tomography shows homogeneously opacified maxillary sinus walls, a totally opacified left sphenoid sinus, and an obliterated choanal passage.
+
+
+Paranasal sinus computed tomography shows homogeneously opacified maxillary sinus walls, a totally opacified left sphenoid sinus, and an obliterated choanal passage.Endoscopic sinus surgery was performed under general anesthesia. After decongestion, the polyp was seen to originate from the sphenoethmoidal recess, not the maxillary sinus. Using nasal forceps, the nasal polyp was excised totally with its stalk (Figure 2). The sphenoidal ostium was naturally dilated (Figure 3). The polyp originated from the posterior wall of the sphenoid sinus. While attempting to excise the polyp stalk, dehiscence in the course of the optic nerve was observed. The operation finished after that.
+
+Figure 2
+The sphenochoanal polyp specimen after surgery. The stalk is seen at the bottom of the specimen.
+
+
+The sphenochoanal polyp specimen after surgery. The stalk is seen at the bottom of the specimen.
+
+Figure 3
+Endoscopic view of the dilated ostium of the sphenoid polyp caused by the sphenochoanal polyp. The posterior wall of the sphenoid sinus is seen after excising its stalk.
+
+
+Endoscopic view of the dilated ostium of the sphenoid polyp caused by the sphenochoanal polyp. The posterior wall of the sphenoid sinus is seen after excising its stalk.Postoperatively, saline was applied for one week and antibiotic was given to the patient. The nasal cavity was free of polyps and the sphenoid sinus was clear radiologically. Histopathologically, the specimen was an inflammatory polyp.Sphenochoanal polyps originate from the sphenoid sinus and are an uncommon form of choanal polyp. They are usually solitary. The etiology is not well understood, but allergy and chronic sinusitis might contribute.5 Intramural cysts can give rise to choanal polyps.2Most frequently, they originate from the maxillary sinus.6 However, nasal polyps can arise from the sphenoid or ethmoid sinus.Sphenochoanal polyps have to be differentiated from antrochoanal polyps to choose the correct surgery.The most common symptoms are nasal obstruction, snoring, and unilateral nasal discharge, although the polyps can sometimes be asymptomatic. Sphenochoanal and antrochoanal polyps can be misdiagnosed as hypertrophied inferior turbinate, adenoid vegetation, pansinusitis, and nasal polyps. The presentation of a sphenochoanal polyp is similar to that of the more common antrochoanal polyp. A sphenochoanal polyp can be diagnosed preoperatively using nasal endoscopy and paranasal sinus tomography.7Antrochoanal polyps are treated successfully with endoscopic sinus surgery, which allows complete removal of the polyp, including its site of origin, which minimizes the risk of recurrence.8,9 As choanal polyps arise from inflamed, edematous mucosa in the paranasal sinuses, at sinus surgery the origin of the polyp and diseased mucosa must be excised carefully to prevent recurrence. Our patient had an inflammatory polyp.A sphenochoanal polyp has intrasinusoidal, ostial, and extra-sinusoidal components. They originate from the sphenoid sinus wall, exiting the sinus via the sphenoid ostium, passing through the sphenoethmoidal recess, and reaching the choana. In this case, the nasal polyp was not diagnosed as a sphenochoanal polyp preoperatively as the polyp obscured the cavity and was not clear radiologically. When there is a lack of maxillary opacity with choanal polyps, one should think of the possibility of sphenochoanal polyp. Sphenochoanal polyps usually pass through the sphenoethmoidal recess directly and do not reach the middle meatus unless very large. Opacification in the maxillary sinus, such as in our case, might lead to a misdiagnosis. In addition, a large polyp can fill the middle meatus.There have been recent technical improvements in surgical procedures. Intraoperative CT surgery provides additional information about the surgical site in difficult anatomical situations. Intraoperative imaging provides near real-time imaging that has the potential to improve surgical outcomes and reduce operative morbidity. Complications can be avoided in nasal polyposis with CT-guided surgery. CT can also help to assess the origin of nasal polyps and surrounding essential structures, such as the carotid artery and optic nerve.10At surgery, the polyp was diagnosed as a sphenochoanal polyp. The polyp stalk was not from the maxillary sinus, but from the sphenoid sinus through an enlarged sphenoid sinus. Forceps are also used to excise the polyp instead of a microdebrider, as the latter might damage the optic nerve and carotid artery when used inside the sphenoid sinus. Uncontrolled pulling on the nasal polyp with forceps is dangerous due to the proximity of the carotid artery and optic nerve, which can be damaged during surgery.11,12 In our case, the sphenochoanal polyp was excised en bloc and there were no complications involving the optic nerve. The patient's ophthalmic examination and vision were normal postoperatively.Sphenochoanal polyps can be diagnosed at nasal endoscopy or with paranasal tomography. Isolated sphenoid sinus disease is under-reported due to a lack of recognition and experience. The symptoms are nonspecific. An adequate preoperative evaluation is necessary for a correct diagnosis. The stalk of the polyp should not be excised in an uncontrolled manner due to the proximity of the optic nerve and carotid artery.

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+Conflict of interest: the authors declare no potential conflicts of interests.A 72-year-old woman without any medical and psychiatric history, suffered from nausea, pain in the epigastria and constipation for over a year. She eventually lost 20 kilograms despite nightly drip-feeding. Extensive additional tests did not reveal any clues for her complaints. She remained convinced that her symptoms were a side-effect of anti-fungal medication she used. She was diagnosed with hypochondria. In the course of time her ideas about her somatic symptoms became delusional and she was diagnosed with a hypochondriacal delusion as part of melancholia, without depressed mood or loss of interest or pleasure as prominent features. It is important to recognize melancholia as soon as possible by continually evaluating other symptoms of depression. This may enable to avoid repetitive and exhaustive somatic examinations, which are not indicated, and to start effective treatment. In our patient electroconvulsive therapy resulted in a fast and complete recovery.Medically unexplained symptoms are frequent, 20–50% of symptoms presented to a general practitioner remain without a physical diagnosis.1 Most symptoms vanish over time but a small percentage persists without a somatic explanation despite extensive additional tests. Epidemiologic research shows that 2.5% of the general population suffers from chronic somatic unexplained symptoms and in the elderly this is 3.8%.2 A systematic review of the literature showed that approximately 50– 75% of patients with medically unexplained symptoms improve, whereas 10–30% of patients deteriorate.3 Especially in the elderly unexplained somatic symptoms are problematic since minor abnormalities in physical examination and work-up are frequently found without explaining the symptoms in a satisfying way. This may lead to underscoring of psychiatric diagnoses such as somatization disorder and hypochondriasis in elderly patients.In this report, we describe a woman with a hypochondriacal delusion who doesn’t meet the criteria for a severe mood disorder, somatization disorder or hypochondriasis according to the Diagnostic Statistical Manuel (DSM) IV-TR criteria.4 Eventually she was diagnosed with melancholia and recovered quickly with electroconvulsive therapy (ECT) (Figure 1). In addition we performed a literature search on ECT and hypochondriasis, hypochondriacal/ somatic delusion/psychosis and severe depression with hypochondriacal/somatic delusion/ psychosis.
+
+Figure 1
+Electroconvulsive therapy.
+
+
+Electroconvulsive therapy.A 72-year-old female without a somatic or psychiatric medical history was referred against her will to an outpatient psychiatric clinic by her gastroenterologist after a year of suffering nausea, pain in the epigastria and constipation. The patient was convinced she had a physical illness and no psychiatric illness. Her long lasting complaints had started 3 days after the start of oral nystatine prescribed for a Candida infection of the mouth, which she developed shortly after getting a complete set of dentures. Her appetite was diminished and she also complained of a bitter taste in her mouth. A thorough work up, including repeated physical examinations, gastroscopy, blood tests and an angiography of the abdominal arteries did not reveal a plausible cause for her complaints. Despite nightly drip-feeding the patient lost more than 20 kilograms during her frequent visits with the gastroenterologist.At first psychiatric examination she was preoccupied with her somatic symptoms that were, according to her, due to nystatine use. She was diagnosed with hypochondriasis that was considered to lead to lack of appetite and disturbed sleep, which were present as well. Since she had severe trouble sleeping the anti-depressant mirtazapine was started. Notably there were no complaints of depressed mood or anhedonia and there were no psychomotor symptoms; however she was very limited in her daily activities because of her physical complaints.She remained preoccupied with her physical condition and was convinced that there was a not yet identified somatic cause for her symptoms. A year after the use of nystatine she feared that her gastric mucosa would dry out completely and that she had a rotten tube in her throat. These ideas were diagnosed as delusional. She was referred to the psychiatrists as a patient with somatization and a hypochondriac delusion. Evaluation of her depressive symptoms revealed a MADRS score of 25/60 without the patient reporting depressed mood or anhedonia. She complained of lack of appetite, disturbed sleep, diminished interest and inner turmoil. There was no psychomotor disturbance. Assuming a depression with psychotic features she was treated in vain with amitriptyline during 4 weeks with adequate blood levels and then refused further treatment. A second opinion confirmed a probable diagnosis of melancholia, a mood disorder with a hypochondriacal delusion. ECT was started after extensive counseling with the patient and her husband. Unilateral brief pulse ECT was administered twice weekly using a Thymatron IV, starting with a 70% dose setting (75 mCoulombs) resulting in 54-second motoric seizure and recording 82 seconds on the EEG. After the first treatment her physical symptoms were markedly improved, her appetite and sleep pattern recovered in the following two weeks. After 13 sessions of unilateral ECT she recovered completely and maintenance pharmacotherapy was started with nortriptyline. The following 2.5 years she was in complete remission and functioning well. Than she experienced a relapse and again she quickly recovered with ECT. Four years after the initial complaints of gastrointestinal discomfort she was diagnosed with colon carcinoma.The DSM IV-TR defines strict criteria to identify medically unexplained symptoms as psychiatric somatoform disorders. Somatiza-tion disorder requires a combination of gastro-intestinal and pseudo neurological symptoms, sexual and pain complaints. Whereas in hypochondria the fear of being ill is central and patients realizes that their anxiety is exaggerated and that no major physical condition is present.The syndrome of monosymptomatic hypochondriacal psychosis (MHP) is a form of DSM-IV delusional disorder, somatic subtype, characterized by the delusional belief that one is afflicted with a medical disorder of defect. Such patients often present to dermatologists with delusions of parasitosis. The literature describes case studies and 1 double-blind cross over study indicating the benefit from several typical neuroleptics, especially pimozide for this disorder,5,6 atypical neuroleptics7–11 or anti-depressants.12 Our patient is crossing the borders of the DSM IV criteria. Although she didn’t fulfill the criteria for a delusional disorder because since she didn’t believe she had a specific serious illness or defect and moreover her daily functioning was restricted, she was considered as a delusional disorder of the somatic subtype. She didn’t meet criteria for any other somatoform disorder, as there were no other somatic complaints apart from nausea and weight loss. She was convinced that her complaints were side effects of nystatine, and a gastrointestinal problem could be found if more extensive diagnostic research was done.According to DSM IV criteria a depression can only be diagnosed in presence of five or more symptoms including depressed mood or loss of interest or pleasure. Since 1980 classifying according to DSM III increased the reliability of psychiatric diagnoses and stimulated scientific research. In clinic practice however, symptoms, which not fit a certain psychiatric category, are easily missed. The diagnosis depression is not considered when depressed mood or loss of interest or pleasure isn’t present. Recent research in four academic centers showed that the diagnosis depression with psychotic symptoms was missed in about 25% of the cases if the psychotic symptoms were more present than a disturbed mood.13In the European psychiatric textbooks from the first part of the 20th century it was stressed that melancholy, especially in the middle age and elderly, had a protean clinical expression and could mimic several psychiatric conditions like hypochondria, delirium, catatonia and anxiety disorder, depending on the symptoms that are most prominent.14 Severe depression with hypochondriacal delusion was described by Cotard in 1880 as du delire hypochondriaque and in 1957 by Schneider as depressio sine depressione. In the English literature the term masked depression was popular, however in most cases it appeared to concern the differential diagnostic considerations: somatoform or somatization disorder. There is limited research on depressive disorder with hypochondriacal delusions. As a subtype it would be more frequent in women15 and accompanied with a high risk for suicide.16 In a multivariate analysis of 187 patients with severe depression a subtype with hypochondriacal delusions could not be defined by specific characteristics, despite the number of variables that was analyzed.17 There are two case reports describing a patient with hypochondriacal delusion improving after ECT.18 There is a limited number of case reports on patients with hypochondriacal delusions treated with ECT.19,20 We believe that hypochondriacal delusions as part of a severe depression might be undiagnosed following the strict criteria of the DSM IV-TR, moreover these patients aren’t likely to consult a psychiatrist with their symptoms. Intensifying collaboration with our colleagues in internal medicine for example could make more cases of hypochondriacal delusion as part of a severe depression surface. Our patient was first diagnosed with hypochondria but over time she developed a hypochondrial delusion and was diagnosed with melancholia despite the absence of a prominent mood disturbance or loss of interest or pleasure. She was treated with electroconvulsive therapy and after one session her somatic complaints evaporated. Recognizing mood disorders without depressed mood or loss of interest or pleasure as prominent features will speed up adequate treatment and prevent unnecessary, costly test and delay.

Med-MDPI/clinpract/clinpract-02-01-e12.txt

@@ -0,0 +1,19 @@
+Aortic intramural hematoma (IMH) is related to but is pathologically distinct from aortic dissection. In this potentially lethal entity, there is hemorrhage into the aortic media in the absence of an intimal tear. With recent advances in imaging techniques, IMH is now increasingly recognized. The limited data available suggest that the clinical course of IMH mimics that of acute aortic dissection, and mortality rates are similar. Physicians need to be cognizant regarding this entity when they are evaluating chest pain. Here we report a case of IMH, in a 63-year-old female, which was managed conservatively.A 63-year-old Caucasian female presented to the emergency department with the chief complaint of intense pain between her shoulder blades, which began a few hours back. The patient reported that she was feeling well early in the day but began to experience a sharp, intense pain between her shoulder blades, which started suddenly while she was eating. She rated the pain as 8/10 and described the pain as a feeling of being hit with a baseball bat. The pain was unrelieved with rest and fluids. She denied any chest pain or shortness of breath, but reported some mild diaphoresis. She denied abdominal pain, but felt nauseated and vomited clear liquid mixed with food before coming to the hospital. Patient had no medical problems, was on no medications, and had not seen a physician in over five years. Patient had a 100-pack year smoking history and drinks 4–5 beers daily. She denied any illicit drug use.On physical exam, she was noted to be a thin, frail woman in mild distress secondary to her back pain. The patient was afebrile. Her pulse was regular with a rate of 94 beats per minute. Her blood pressure was 230/134 mmHg. Her oxygen saturation was 95% on room air. The patient's lungs were clear to auscultation bilaterally with normal respiratory effort. On cardiac examination, there was no jugular venous distension or parasternal heave. Auscultation revealed a normal S1 and S2 without murmur, rub, or gallop. Her abdomen was soft and nontender with normal bowel sounds and no bruit. The pain in her back was not reproducible with palpation and there were no signs of deformity or trauma. The peripheral arterial pulses were palpable and symmetric in all four extremities.Laboratory data showed mild leukocytosis, with a white blood cell count of 12.400 mm3 (normal range, 4.000–10.000) and hypokalemia, with a serum potassium of 3.3 mmol/L (normal range, 3.6–5.0). Amylase and lipase were within normal limits. An electrocardiogram showed normal sinus rhythm. The patient was diagnosed with hypertensive urgency and treated with aspirin, sublingual nitroglycerin, transdermal nitroglycerin, and sublingual clonidine. Her blood pressure decreased to 102/79 and her back pain abated. The patient was to be admitted to the hospital for continued control of her blood pressure and further evaluation. A computed tomography (CT) angiography of the chest with and without contrast was obtained (Figure 1) which was followed by magnetic resonance (MR) angiogram (Figure 2). CT angiogram showed diffuse abnormal wall thickening of the descending thoracic aorta, which is peripheral to the calcified intima and extends up to the origin of the celiac and superior mesenteric arteries (Figure 1). MR angiogram of thoracic aorta showed thickening of the wall of the descending thoracic aorta starting from just beyond the origin of the left subclavian artery, with no definite wall thickening of the ascending thoracic aorta or evidence of a pericardial effusion (Figure 2). Transesophageal echocardiogram revealed an abnormal descending aorta with a thickened wall, which was eccentric and had a uniform echo dense appearance consistent with intramural hematoma, which ended at the origin of the left subclavian artery. There was no evidence of any intimal tear (Figure 3). She was admitted to the intensive care unit, her blood pressure was controlled using intravenous labetalol. Since it was a Type-B intramural hematoma, it was decided to manage conservatively with close outpatient follow-up.
+
+Figure 1
+Computed tomography angiography of the chest showed diffuse abnormal wall thickening of the descending thoracic aorta.
+
+
+Computed tomography angiography of the chest showed diffuse abnormal wall thickening of the descending thoracic aorta.
+
+Figure 2
+Magnetic resonance angiogram of thoracic aorta showed thickening of the wall of the descending thoracic aorta.
+
+
+Magnetic resonance angiogram of thoracic aorta showed thickening of the wall of the descending thoracic aorta.
+
+Figure 3
+Transesophageal echocardiogram revealed an abnormal descending aorta with a thickened wall.
+
+
+Transesophageal echocardiogram revealed an abnormal descending aorta with a thickened wall.Acute aortic syndrome refers to the spectrum of aortic emergencies that include aortic dissection, intramural hematoma, penetrating atherosclerotic ulcer of the aorta, aortic aneurysm leak and rupture and traumatic aortic transection. A classic aortic dissection begins with a laceration of the aortic intima and inner layer of the aortic media forming an entrance tear that allows entering blood to split the aortic media.1Aortic intramural hematoma (IMH), first described in pathological literature in 1920, is related to but is pathologically distinct from aortic dissection. It is characterized by the absence of an entry tear, absence of an intimal flap, and absence of evidence of communication between the medial hemorrhage and the aortic lumen.2 In a review of 505 autopsy cases of aortic dissection from 1933 to 1954, Hirst et al. found a 4% incidence of IMH.3 These intramural hematomas like dissections, involve the ascending aorta, arch or both (Type A) or the descending aorta (Type B). Though deemed as pathologically distinct entities, intramural hematoma may lead into acute aortic dissection, aneurysm or aortic rupture.4Aortic intramural hematoma may be a primary event in hypertensive patients due to spontaneous bleeding from vasa vasorum into the media or may be caused by a penetrating atherosclerotic ulcer.4 Vaso vasorum vessels of vessels, forms network of vessels in adventitia, which also supply the outer media plays a significant role in pathogenesis of all aortic pathologies including intramural hematoma, aortic dissection and aortic aneurysm.4 Animal studies have shown that aortic distensibility decreases when vaso vasorum are removed from the vessel wall. Penetrating atherosclerotic ulcer develops, when an atheromatous plaque, ulcerates through the internal elastic lamina and exposes the media to high-pressure aortic blood flow leading to intra medial hematoma.5The risk factors for IMH are very much similar to cardiovascular diseases, with hypertension being the most common among them. Pregnancy and some congenital disorders such as Marfan's syndrome, Ehlers-Danlos syndrome, annuloaortic ectasia and bicuspid aortic valve are the other possible predisposing factors for IMH.6 Aortic intramural hematoma can occur as a primary event or as a result of blunt chest trauma, in which there is spontaneous rupture of the nutrient vasa vasorum, with circumferential or longitudinal spread of the hematoma over a variable distance along the media layer of the aorta. Cases of intramural hematoma following cardiac resuscitation have been reported. IMH is difficult to distinguish from classic dissection on purely clinical grounds. In clinical series 13 to 27% of patients with a diagnosis of aortic dissection in fact had IMH.7 Patients with IMH are typically elderly with history of hypertension. Unlike classic aortic dissection, ratio of men to women appears equal. Risk factors and clinical features at presentation at presentation are similar to aortic dissection. Clinically IMH most commonly occurs in the descending aorta and in older patients. Chest pain and back pain are the most frequent symptoms.Diagnostic studies should reveal fresh thrombus within the aortic wall, which should manifest in TEE as either crescentic or circular thickening of the aortic wall with maximal thickness greater than or equal to 7 mm without intimal flap or tear or any longitudinal flow in the false lumen.8 On unenhanced CT, intramural hematoma is hyper dense. MRI identifies slow flow in the false lumen in dissection and no flow in an intramural hematoma. Dynamic phase-contrast MRI is more sensitive than gradient refocused echo sequences in differentiating aortic dissection from intramural hematoma.9 Intramural hematoma may be distinguished from mural thrombus by the identification of the intima; mural thrombus lies on top of the intima, which is frequently calcified, where as intramural hematoma is sub intimal. Some patients with intramural hematoma have limited hemorrhage and responds well to medical therapy. Intramural hematoma leads to weakening of the aorta and may progress either to outward rupture of the aortic wall or to inward disruption of the intima which leads to communicating aortic dissection.6 Rate of conversion of IMH to dissection varies according to the site of IMH, with various studies reporting 3% to 14% conversion rates in IAH involving the descending aorta and in 11% to 88% of IMH involving the ascending aorta.9 Although a rare phenomenon, spontaneous resorption of IMH has also been reported.6Type A IMH may advance to complete dissection and can rupture through adventitia causing pericardial effusion, hemothorax, and mediastinal hemorrhage. Mortality from proximal lesions is greater than distal IMH and mortality is highest within the first 24 to 72 hours after hospital admission. The maximum thickness of the hematoma on the initial CT is the most significant factor for predicting the development of aortic dissection and aortic aneurysm. Patients with Type A IMH and ulcer like projections, as revealed by initial and short term follow up CT examinations, should be follow-up with subsequent CT examinations to monitor for the development of an aortic aneurysm, which is a relatively common chronic complication of IMH.10 If IMH develops at the convexity of the distal arch, supra-aortic branches prevent retrograde extension toward the ascending aorta. If an aortic IMH develops at the free lateral wall or at the concavity, it may affect the entire thoracic aorta, due to the lack of the natural barrier of the supra-aortic branches.Definite guidelines for the management of intramural hematoma are yet to be derived. Initial medical treatment, endovascular surgery or classic open surgery is the common treatment of IMH. A study by Ledbetter et al., on morbidity and mortality for 168 patients with IMH, 30-day mortality was 18% with surgical repair of proximal IMH, and 33% with surgery to distal IMH compared to 60% and 8% with medical treatment of type A and type B IMH, respectively.11 Most authorities suggest treatment of intramural hematoma similar to aortic dissection with early surgical intervention in Type A and medical management for Type B lesions.12 Surgery is usually indicated when there are signs of expansion of the IMH, rupture into the pleural or pericardial cavity, uncontrollable symptoms like chest pain, if the patient becomes hemodynamically unstable or dilated aorta more than 5 cm.6,13 Other possible alternative is endovascular repair if there are no other comorbidities like uncontrolled hypertension or persistent pain.6 Oral β-blocker therapy helps in control of heart rate and blood pressure and may improve long-term prognosis of IMH independent of anatomical location. Intramural hematomas have a more favorable outcome compared to aortic dissection, as the hematoma is non-communicating with the aortic lumen.

Med-MDPI/clinpract/clinpract-02-01-e16.txt

@@ -0,0 +1,25 @@
+Contributions: TK, diagnosis and therapy, manuscript writing; KI, manuscript writing.Conflict of interest: the authors declare no potential conflicts of interests.We report a case of a neuroendocrine carcinoma arising in a wound of the postoperative maxillary sinus that was difficult to distinguish from a postoperative maxillary cyst. The patient was a 65-year-old Japanese woman who complained of left exophthalmos with cheek swelling and eye movement disorders. In past history, she had, 40 years previously undergone operation on the bilateral maxillary sinus by Caldwell-Luc's method. In a preoperative computed tomography, a mass occupied the left maxillary sinus showing irregular densities with destruction of the posterior bone walls and invasion into the left orbital. Both TI and T2 weighted magnetic resonance imaging showed low intensities and unevenness in the mass. We performed a biopsy of the maxillary tumor according to Caldwell-Luc's method. Histological examination diagnosed neuroendocrine carcinoma. Radiation therapy (total 66Gy) resulted in partial response for this tumor. However, sinonasal neuroendocrine carcinoma has been identified as highly aggressive, with a high probability of recurrence and metastasis.Maxillary carcinoma has seldom been known to arise in the postoperative maxillary sinus. Primary neuroendocrine carcinomas of the paranasal sinuses are newly recognized, extremely uncommon, and aggressive tumors with the capacity to metastasize locally and distantly.1–3 We encountered neuroendocrine carcinoma arising in the wound of a postoperative maxillary sinus. In preoperative diagnosis, it was difficult to discriminate between maxillary carcinoma and postoperative maxillary cyst. Biopsy of the maxillary mass revealed neuroendocrine carcinomas. After biopsy, tumors became partial remission in response to radiation therapy. This case suggests that the possibility of a carcinoma should be kept in mind in cases of postoperative maxillary sinus. Below we describe our case, as well as some of the considerations suggested by the literature on this topic.A 65-year-old Japanese woman consulted our hospital with a 1-week history of left exophthalmos with cheek swelling and eye movement disorders. Forty years prior, she had undergone an operation on the bilateral maxillary sinus by Caldwell-Luc's method. In a pre-operative computed tomography (CT) scan, a mass occupied the left maxillary sinus showing irregular densities with destruction of the posterior bone wall and invasion into the left orbital (Figures 1 and 2). Both TI and T2 weighted magnetic resonance imaging (MRI) showed low intensities and unevenness in the mass (Figure 3). Thus, CT and MRI suggested a solid mass in the maxillary. At the first medical examination, we suspected carcinoma arising in the postoperative maxillary sinus in addition to postoperative maxillary cyst. We performed biopsy of the maxillary tumor according to Caldwell-Luc's method. On the histological examination, the carcinoma tissues showed nesting patterns with necrosis, proliferation of cells with round nuclei and numerous abnormal mitotic features (Figure 4). Immunohistochemical studies showed positive staining for keratin, CAM5.2, and CD56, but not LCA (leukocyte common antigen). From the above results, we diagnosed neuroendocrine carcinoma. After biopsy, radiation therapy (total 66Gy) resulted in partial remission (PR) for this tumor. We consulted her concerning radio-chemotherapy or chemotherapy after radiation therapy for the remains of the tumors, but she refused the above combined chemotherapy. Regression of the tumor after therapy has been continued 24 months.
+
+Figure 1
+In the preoperative computed tomography, a mass occupied the left maxillary sinus, showing irregular densities with destructions of the posterior bone wall (arrow).
+
+
+In the preoperative computed tomography, a mass occupied the left maxillary sinus, showing irregular densities with destructions of the posterior bone wall (arrow).
+
+Figure 2
+In the preoperative computed tomography, a mass occupied the left maxillary sinus, invading the orbita (arrow).
+
+
+In the preoperative computed tomography, a mass occupied the left maxillary sinus, invading the orbita (arrow).
+
+Figure 3
+T2 weighted magnetic resonance imaging showed low intensities with unevenness in the mass and destructions of the posterior bone wall (arrow).
+
+
+T2 weighted magnetic resonance imaging showed low intensities with unevenness in the mass and destructions of the posterior bone wall (arrow).
+
+Figure 4
+Histopathological examination (H&E staining, ×200) revealed nesting patterns with necrosis and the proliferation of cell round nuclei.
+
+
+Histopathological examination (H&E staining, ×200) revealed nesting patterns with necrosis and the proliferation of cell round nuclei.Forty years previously, the patient had undergone an operation on the bilateral maxillary sinus by Caldwell-Luc's method. The majority of histopathological classifications of maxillary carcinomas are squamous cell carcinoma. Squamous cell carcinoma can be seldom observed in wounds of the postoperative maxillary sinus because removal of the maxillary mucosa membranes by Caldwell-Luc's method eliminates the place where squamous cell carcinoma arises. Silva2 and Mendeloff4 suggested olfactory epithelium as the original cell of neuroendocrine carcinoma, Schall5 reported spheno-palatine nerve. A main aggressive finding in this case showed destructions of the posterior bone wall in the maxillary sinus. Therefore, the origin of this neuroendocrine carcinoma may be spheno-palatine nerve. Neuroendocrine carcinoma consists of carcinoid and small cell carcinoma. It is difficult to be discriminate between carcinoid and small cell carcinoma. Moreover, the neuroendocrine carcinoma of nasal-parasinus has not been classified. Therefore, Tojima6 described it as small cell neuroendocrine carcinoma. In general, the cyst showed high intensities by T2weighted MRI.7 In this case, T2 weighted MRI showed low intensities with unevenness in the maxillary mass. Therefore, it was difficult to preoperatively distinguish carcinoma from a postoperative maxillary cyst. Finally, we diagnosed neuroendocrine carcinoma by biopsy. Silva et al.2 the first proposed sinonasal neuroendocrine carcinoma as an entity. In this case, destruction of the posterior bone wall and aggressive invasion into the orbital were found on CT. Smith et al.3 reported that sinonasal neuroendocrine carcinoma was a rare, and aggressive neoplasma.6,8,9 Our hematoxylin and eosin staining showed nesting patterns with the cells having round nuclei. These findings have been reported as one of features of the neuroendocrine carcinoma. It has been known that it is difficult to discriminate between the neuroendocrine carcinoma and olfactory neuroblastoma because the olfactory neuroblastomas as well as neuroendocrine carcinomas have filaments, microtubules, and secretory granules on electron microscopy.10 Immunohi stological examinations are helpful for the diagnosis. Our case showed positive staining for keratin, CAM5.2, and CD56. Their antibodies can be used to detect neuroepithelia. CAM 5.2, epithelia membrane antigen, neuron specific enclose, synatophysin, and chromogranin were reported to be reactive for neuroendocrine epithelia.1,3,9 Keratin is negative for olfactory neuroblastoma.11 Our patient was treated by radiation therapy alone. She has been alive 24 months after therapy with regression of the tumor. Presently, radiation therapy has been considered to be the first choice for the therapy of neuroendocrine carcinoma.12,13 Some cases have undergone chemoradiotherapy.6,13,14,15 Morikawa13 reported that a patient treated with chemo-radiotherapy was alive 21 months after diagnosis without local recurrence and distant metastasis. Reversely, Tojima6 documented that a patient received chemo-radiotherapy died from bone metastasis 7 month after diagnosis. Takahashi14 performed chemo-radiotherapy in 2 cases. But they died 6 months (bone metastasis) and 15 months (liver and brain metastasis) after diagnosis. Georgiou15 described that despite radiotherapy and chemotherapy; the patient died 4 months after diagnosis due to widespread dissemination and bone marrow failure. From the above reports, there has been no evidence of any difference in effectiveness between radiation therapy alone and chemo-radiotherapy. Bailey16 and Cartrell17 reported that only radiation therapy contributed to remission. Sinonasal neuroendocrine carcinoma is identified as highly aggressive, with a high probability of recurrence and metastasis. Takahashi et al.14 reported that the 5-year mortality of nasal-paranasal neuroendocrine carcinoma (30 cases) was 69%. Perez-Ordonez et al.1 reported that, out of 6 patients, 4 were alive with disease recurrence and 2 had died of the disease. Slivia et al.2 reported that recurrences and metastasis in 70% of the case occurred later than the third year. Multiple recurrences were present in 54% of the cases. The metastases affected lymph nodes, brain and spine. Therefore, careful follow -up after treatment of this disease is indicated.We encountered a very rare case that a neuroendocrine carcinoma arising in a wound of the postoperative maxillary sinus was difficult to distinguish from a postoperative maxillary cyst. MRI and immunohistological examinations (e.g., Keratin, CAM5.2 and CD56) were helpful for diagnosis of this case. Radiation therapy has been considered the first choice for the therapy of neuroendocrine carcinoma. In our case, radiation therapy (total 66Gy) resulted in PR for this tumor. Regression of the tumor after therapy has been continued 24 months.

Med-MDPI/clinpract/clinpract-02-01-e18.txt

@@ -0,0 +1,146 @@
+Contributions: the authors contributed equally.The aim of the study was to determine whether intravenous gamma globulin (IVIG) treatment is effective in patients with West Nile Virus (WNV) neuroinvasive disease.We contacted hospital based infectious disease experts in Israeli hospitals to identify patients with WNV neuroinvasive disease who were treated with IVIG. The main outcome measure was neurological response after treatment. There were 12 patients who received IVIG and four improved within 48 h. Three patients died, 6 had partial recovery, and 3 recovered completely. Eleven of the 12 patients were infected with Israeli genotypes that are highly homologous to Europe/Africa viruses. The rapid response in some patients suggests that IVIG is effective, and might be used to treat patients with WNV neuroinvasive disease with IVIG.The West Nile virus (WNV) is an arthropod-borne flavivirus that is associated primarily with epidemics of flu-like febrile illness. Neurologic manifestations are uncommon with overlapping clinical syndromes including encephalitis, meningitis and acute flaccid paralysis.1 WNV has been endemic in Israel since the 1950s usually causing a mild, self-limiting disease but an isolated epidemic occurred in the year 2000 with 428 hospitalized cases of neurological disease and 42 deaths.2The observation of a patient with chronic lymphocytic leukemia and WNV encephalopathy who made a prompt and complete recovery after receiving high dose intravenous immunoglobulin (IVIG)3 lead to the finding that immunoglobulin preparations in Israel contain high titers of antibodies to WNV (1:1600).3 Since then, case studies have continued to suggest that IVIG therapy can be effective in some patients including immunosuppressed post-transplant patients.4,5 Recent reviews have called for randomized controlled trials6 and a multicenter randomized, placebo-controlled trial sponsored by the National Institute of Health is in progress.7 The results of such studies are urgently needed because of the high morbidity and mortality rates of neuroinvasive WNV. Nevertheless until results of randomized controlled trials are available, an accumulated series of cases can provide a higher degree of evidence than isolated case reports. In this report, we summarize the clinical course of all patients known to have received intravenous immunoglobulin for WNV neuroinvasive disease in Israel until the end of 2007. We also characterized the prevalent genotypes found in mosquitoes (highly correlated with human infection) according to time and place in order to determine if there is a relationship to the outcome according to the various genotypes.We contacted the Infectious Disease consultants of Israeli Hospitals to request details of the clinical course of patients with serious WNV neuroinvasive disease who received treatment with at least one day of high dose intravenous immunoglobulin. Details of 12 patients were compiled from case notes. Ten cases have been reported previously and were not included.Serological testing was performed in the Ministry of Health’s National Center for Zoonotic Viruses, at the Central Virology Laboratory by using an IgM-capture enzyme-linked immunosorbent assay (ELISA). Diagnosis of primary WNV infection was made on the basis of clinical symptoms and signs, together with laboratory confirmation of the presence of immunoglobulin M (IgM) antibodies, with or without IgG antibodies, and low IgG avidity. Diagnosis was also made on the basis of a significant rise in antibody level between paired samples (≥4 fold), and a specific reaction calculated from the reaction to viral antigen over the reaction to mock antigen (≥2 fold).8,9 All tests were developed in house and performed with a local WNV isolate (as antigen) either genotypically similar genotype to the New York 1999 strain or a genotype similar to both the New York 1999 and to the Romania 1997 strain.9For identification of the circulating WNV genotypes in the country, RNA was extracted from mosquito pools, as part of the yearly routine mosquito surveillance program. RNA was then amplified by Real-Time RT-PCR with specific primers of the ENV gene.10 Virus isolates were identified by standard RT-PCR methods,11 amplified, sequenced,12 and the gene compared with published sequences in the Gene bank.There were 12 patients who received IVIG; three patients died, 6 had partial recovery, and 3 recovered completely (Table 1). Eleven of the 12 patients were infected with Israeli genotypes that are highly homologous to Europe/Africa viruses. A standard dose of IVIG of 0.4g/kg/day was used, for a variable number of days (Table 1). The effects of therapy were often dramatic and occurred within 48 hours in 4 patients.
+
+Table 1
+Clinical characteristics, treatment and outcome.
+
+
+
+Case
+Gender/age
+Significantpast history
+Presentation
+Daysof Rx
+Daysto response
+Response/residualdisease
+Recovery
+
+
+
+
+1
+F/72
+Dementia-meningioma
+Stupor, seizures,weakness
+2
+-
+No
+Died
+
+
+2
+F/86
+Dementia
+Seizures, paralysis,loss of consciousness
+2
+2
+Complete
+Complete
+
+
+3
+F/37
+None
+LOC, paralysis, ventilated
+1
+3
+Awake/ Weakness
+Partial
+
+
+4
+M/74
+Diabetes mellitus
+Stupor, paralysis, ventilated
+5
+<5
+Awake/ Weakness
+Partial
+
+
+5
+M/76
+Diabetes mellitus
+Loss of consciousness, paralysis,ventilated
+5
+<20
+Awake/Tracheotomy
+Partial
+
+
+6
+F/65
+High grade NHL
+Loss of consciousness, paralysis,ventilated
+4
+-
+No
+Died
+
+
+7
+F/83
+Toxic Goiter
+Stupor, weakness
+1
+1
+Complete
+Complete
+
+
+8
+M/40
+Alcoholic
+Stupor, muscle weakness
+1
+2
+Complete
+Complete
+
+
+9
+F/41
+None
+Paralysis
+5
+1
+Weakness/Ataxia
+Partial
+
+
+10
+M/67
+Thymoma
+Stupor, muscle weakness, tremor
+1
+<3
+Awake/ weakness
+Partial
+
+
+11
+F/45
+Lung transplant- IPF
+Stupor, muscle weakness, ventilated
+3
+-
+None
+Died
+
+
+12
+F/87
+Dementia
+Stupor, weakness
+3
+<20
+Complete
+Complete
+
+
+
+
+
+Rx, treatment; CLL, chronic lymphocytic leukemia; NHL, Non-Hodgkin’s lymphoma; LOC, loss of consciousness; weakness means objective muscle weakness.
+
+
+Rx, treatment; CLL, chronic lymphocytic leukemia; NHL, Non-Hodgkin’s lymphoma; LOC, loss of consciousness; weakness means objective muscle weakness.The major finding of our study is the prompt response to treatment with IVIG observed in some patients with WNV neuroinvasive disease. Our findings are consistent with the response observed in 10 patients reported previously with Israeli genotypes that are highly homologous to American viruses3,5,13 and with cases reported outside of Israeli.4,14,15 Eleven of the 12 patients reported here had genotypes homologous to the Europe/Africa viruses. Furthermore although none of the 5 new cases ventilated had complete recovery, there were 3 of 5 such patients reported previously with complete recovery. This is in contrast to the prolonged recuperation and recovery,16 and lack of complete recovery in 21 patients who did not receive IVIG reported in a review of the literature.17 The use of immunoglobulin for treatment West Nile virus illness is biologically plausible. Animal data indicate an important role for humoral immunity in controlling West Nile virus infection, and treatment with antibodies is still used in certain viral illnesses such as disseminated Vaccinia after small pox vaccination.15,18 Recent studies in mice have shown that antibodies present in Israeli plasma are effective in reducing morbidity in mice.19 We conclude that it is warranted to treat WNV neuroinvasive disease with IVIG because of the rapid response to treatment observed in some patients, and the favorable outcome in patients requiring respiratory support. Efforts should be made to initiate randomized control trials.

Med-MDPI/clinpract/clinpract-02-01-e19.txt

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+Compartment syndrome is an orthopedic emergency that require early recognition and urgent intervention to avoid catastrophic complications. High index of suspicion is required for early diagnosis based on a constellation of signs and symptoms that include pain out of proportion and worsened by passive stretching, altered sensorium and palpable tenseness. Any event thus, that masks pain, may lead to delay the diagnosis of compartment syndrome. We report here a case of polytrauma where post-operative analgesia was administered using epidural catheter, which obscured pain and lead to delay in recognition of compartment syndrome. Authors wish to share a lesson, learned at the expense of tragedy.Compartment syndrome, first described by Volkman1 is an orthopedic emergency, which occurs when perfusion pressure of fascial compartment falls below the tissue pressure with resultant ischemia of muscles and nerves of the compartment.2 It is shown that muscles can tolerate only four hours of ischemia after which irreversible changes ensue.3 Early recognition and urgent intervention are of paramount importance to avoid complications like muscle necrosis, neurologic deficit, ischemic contracture and necrosis. High index of suspicion like pain out of proportion and worsened by passive stretching, is required to make the diagnosis. Any event thus, that masks pain, may lead to delay the diagnosis of compartment syndrome.3–5 Thus any clinical situation like intoxicated or unconscious patients or patients with concomitant nerve injury diagnosis of compartment syndrome is delayed. We report here a case of polytrauma where post-operative analgesia was administered using epidural catheter which obscured pain and lead to delay in recognition of compartment syndrome.A young 32-years-male patient victim of motor vehicle accident was admitted with the diagnosis of bilateral fracture femur and fracture of both bone right leg. After stabilizing the general condition and splintage of fractures, the patient was rushed to emergency operation theatre and fractures were stabilized. Epidural catheter was maintained for top up infusion of 3 mg Morphine in 10 mL normal saline every 12 hourly, to control pain. Patient was completely free of pain and passive stretching was not significant during the period of epidural analgesia. Epidural catheter was removed after 28 h after last dose at 24 h, when patient was asymptomatic. It is after four hours of removal, patient started complaining of progressive pain unrelieved by appropriate oral analgesic. Clinical examination revealed swollen compartment of leg with altered sensorium and significant pain on passive stretching. Extension of toe and dorsiflexion of ankle was remarkably absent. Dorsalis pedis was not palpable and posterior tibial artery was doubtful. Nail bed circulation was present. Compart ment pressure measurement further confirmed the diagnosis. Patient was immediately taken to operating room and four compartment fasciotomy was done. Dead muscle was noted in anterior and lateral compartment, which were excised. Pain significantly improved with return of distal pulses. Further debridement was performed on 3rd and 5th day. Fasciotomy wound was closed by split skin grafting by 10th day.Diagnosis of compartment syndrome is essentially clinical and high index of suspicion is the key. Most common cause of this entity is trauma, usually a fracture. About 4.3% of all patients of tibial shaft fracture, 3.1% of diaphyseal fracture of forearm and 0.25% of distal radial fracture develop compartment syndrome.6The underlying pathophysiology is ischemia - perfusion – ischemia cycle. In a clinical setting it is difficult to pin point the precise time when compartment pressure started increasing. Thus its measurement by either needle or catheter technique is recommended in high risk patients. Normal pressure in muscle compartment is below 10–12 mmHg. Various invasive7 and non-invasive methods2,6 are often employed for continuous measurement of compartment pressure.Patient controlled analgesia (PCA) gained increasing popularity8 for post-operative pain management after its first description in the year 1971.9 However, this technique has its own potential complications in polytraumatized victim with extremity injury, which were reported subsequently. Iaquinto et al.10 reported increased prevalence of neurological complications in patients who were managed with continuous epidural analgesia in comparison to those managed by oral analgesia. Strecker et al.11 also reported a case in which compartment syndrome remained undetected because early symptom of pain was masked by epidural analgesia. Price et al.3 further described compartment syndrome of thigh after corrective osteotomy associated with post-operative epidural analgesia. Harrington et al.4 reported delayed diagnosis of compartment syndrome in a case of Gustillo II tibial fracture who was on PCA. Richards et al.5 further reported four cases of in which the diagnosis of compartment syndrome was delayed due to PCA following intramedullary nailing and recommended avoiding PCA in favor of intermittent intramuscular morphine injections. Glynn et al.12 observed that epidurally administered local anesthetics causes sympathetic blockade and thereby tends to increases blood flow, which in turn contribute to rise in intra-compartmental pressure. In contrast, epidurally administered opioids do not abolish the normal vasoconstrictor response. Johnson et al.13 and Mar et al.,14 based on literature search concluded that there is no convincing report that PCA or regional analgesia delays diagnosis of compartment syndrome provided continuous monitoring of patients is done.Epidural infusion of morphine or local anesthetic is an excellent mode of analgesia; however, it has the potential to obscure cardinal clinical features of compartment syndrome especially in polytraumatized patients. In light of our experience and literature review, we recommend frequent clinical evaluation, extra vigilant monitoring of analgesic demand and invasive or non-invasive measures is warranted to detect early harbinger of the capricious entity called compartment syndrome.

Med-MDPI/clinpract/clinpract-02-01-e2.txt

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+Contributions: the authors contributed equally.Conflicts of interests: the authors declare no potential conflicts of interests.Bowel obstruction is a common surgical admission around the world. On the other hand, small intestinal tumors, such as midgut carcinoid, are uncommon neoplasms and an infrequent cause of intestinal obstruction leading to hospitalization. A foreign body is an extremely rare cause of intestinal obstruction and when ingested, foreign bodies most often lodge in the narrowest portion of the gastrointestinal tract. Narrowing of the small bowel due to a neoplasm can prohibit the passage of an accidentally ingested foreign object and produce an obstruction that neither the neoplasm nor the foreign body could have produced alone. We hereby report a case in which an accidentally ingested piece of foreign material leads to the finding of a small, early stage, asymptomatic, midgut carcinoid cancer in the proximal ileum that would have otherwise eluded detection for several years.Bowel obstruction is a common surgical admission around the world usually resulting from post surgical adhesions or abdominal hernias. Conversely, small intestinal tumors, such as midgut carcinoids, are uncommon neoplasms and an infrequent cause of intestinal obstruction. Although the small bowel is the most common site for these tumors, the overall incidence is still less than two per 100,000 of all medical or surgical admissions, however the actual reported incidence is closer to eight per 100,000 based on autopsy studies.1,2 A preexisting small bowel neoplasm can prohibit the passage of an accidentally ingested foreign object and produce an obstruction that neither the neoplasm nor the foreign body could have produced alone. We hereby report an interesting case in which a small piece of foreign material accidentally ingested by a patient lead to the detection of an asymptomatic midgut carcinoid in the proximal ileum that would have otherwise eluded detection for several years.A previously healthy, 39-year-old male presented to the emergency department (ED) with a less than 24 hour complaint of severe cramping abdominal pain and two episodes of emesis within the past five hours. He denied history of abdominal surgery, recent travel or changes in bowel habits. His past medical history is significant only for hypertension for which he was noncompliant and not taking any medication. Social history was relevant for cigarette smoking of one pack per day and for beer drinking several times per week but he denied ever being intoxicated. His last drinking event was a couple of cans of beer two days prior to his presentation. Review of systems was negative except for the gastrointestinal complaint resulting in his emergency room (ER) visit. The patient was employed as a mechanic and had previously been active until the onset of his current symptoms.At presentation, his pulse was 98 beats/min, his blood pressure was 186/88 and his temperature was 99.0 degrees Fahrenheit. Respiration rate was 16 times per minute and unlabored. Physical exam revealed a moderately obese Caucasian male in obvious discomfort. His chest was clear to auscultation, heart sounds were regular and without murmur, and he had no peripheral edema or cyanosis. The abdomen was mildly distended. The bowel sounds were hyperactive with some rushes, but not high-pitched. His bowel was diffusely tender to deep palpation, without focal tenderness; and no ventral or inguinal hernias were found. The patient displayed mild voluntary guarding however; there were no obvious rebound or other peritoneal signs.His laboratory analysis was remarkable only for a white blood count of 19.8×103 with left shift (28% bands). His chemistry panel was abnormal for a hypokalemia of 3.3 gm/dL.Plain films of the abdomen showed dilated small bowel loops with multiple air fluid levels, highly characteristic of a distal small bowel obstruction. A computed tomography (CT) scan of the abdomen with IV and PO contrast revealed dilated, contrast-filled, small bowel loops, a short segment of small bowel with thickened bowel wall, and a small metallic ring within the lumen (Figure 1). The colon was collapsed and mesenteric adenopathy was noted (Figure 2).
+
+Figure 1
+Metallic piece in the lumen of the thickened small bowel.
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+Metallic piece in the lumen of the thickened small bowel.
+
+Figure 2
+Mesenteric thickening and lymphadenoathy.
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+
+Mesenteric thickening and lymphadenoathy.The patient was resuscitated with 2 liters of intravenous Lactated Ringer's and then taken to the operating room (OR) for an exploratory laparotomy. Upon entering the abdomen, dilated loops of small bowel were immediately encountered that had a tapioca-like studding pattern, particularly around the proximal jejunum. An obvious stricture was noted in the distal jejunum/proximal ileum which displayed the characteristics of a transition point in which the distal bowel segment was decompressed, and the proximal segment was dilated and edematous. Palpationrevealed a patchy, white, firm, napkin-ring-like deformity in the bowel wall with thickening and contracture of the surrounding mesentery (Figure 3). A foreign body could be palpated within the lumen. Within the adjacent mesentery, a hard, subcentimeter nodule with surrounding desmoplastic reaction was visualized, this was consistent with a metastatic mesenteric lymph node. Several other lymph nodes were firm and easily palpable or visible within the remainder of the small bowel mesentery. Running the entire length of the bowel revealed two more palpable subserosal masses within the jejunum/ileum just distal to this site, each measuring less than one centimeter. A segmental en bloc small bowel resection to include all three lesions and the mesentery was performed. The total resected small bowel measured 29 cm in length. The adjacent remaining mesentery was then carefully explored and dissected for any additional metastatic lymph nodes, three were identified and removed. A cholecystectomy was also performed without difficulty.
+
+Figure 3
+Small bowel carcinoid tumor seen in operating room.
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+Small bowel carcinoid tumor seen in operating room.The specimen was immediately dissected in the pathology lab (Figure 4). Upon opening the bowel wall a dense, intraluminal mass, with an associated tight luminal obstruction was noted. Additionally, a tab from a beer can was seen immediately proximal to the intraluminal mass (Figure 5). This mass displayed the classic scirrhous desmoplastic reaction associated with carcinoid cancer.
+
+Figure 4
+Specimen opened to reveal the primary carcinoid tumor and the adjacent beer can lid induced mucosal damage.
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+Specimen opened to reveal the primary carcinoid tumor and the adjacent beer can lid induced mucosal damage.
+
+Figure 5
+Beer can lid retrieved from the small bowel lumen adjacent to the carcinoid tumor.
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+Beer can lid retrieved from the small bowel lumen adjacent to the carcinoid tumor.The final pathology revealedx a multifocal, low-grade neuroendocrine tumor (carcinoid) with multiple drop metastases throughout the segment of intestine and metastatic mesenteric lymph nodes. Two of the three additional lymph nodes submitted were positive for metastasis. Both the intraluminal mass and lymph nodes stained positive for chromogranin and synaptophysin, but were negative for Ki-67.The patient's recovery was uneventful with the return of normal bowel function on the fourth postoperative day. He was discharged home on the fifth postoperative day.Upon questioning at a postoperative visit, the patient recalled that two days prior to his admission he had dropped a beer tab into the beer can that he was drinking and he must have swallowed it without knowing. His 24 hour urine was collected and submitted for analysis of 5-HIAA levels, this was within normal limits.Carcinoids are rare neoplasms, first reported by Langhans in 1867 and first described by Lubarsch in 1888.3 The term carcinoid (karzinoide, meaning cancer-like in German) was originally given by Oberndorfer to this tumor for its' histological malignant appearance, but benign and indolent clinical course when compared to that of other cancers.4Patients with midgut carcinoid commonly present with vague and non-specific symptoms, which often results in clinical detection being delayed for years. As time progresses, the most common presenting symptom is intense, episodic, abdominal pain, which is typically indicative of advanced disease.2,4,5 This pain may be due to intermittent intussusceptions of an affected segment, mesenteric ischemia due to buckling of the fibrosed mesentery or arterial elastosis, or frank obstruction. Unlike other cancers, the size of the primary tumor does not necessary correlate with presence of metastatic disease (with the exception of appendiceal carcinoids).2–4 Often times, the diagnosis is made only after the development of carcinoid syndrome (flushing, wheezing, and diarrhea) due to the serotonin produced escaping hepatic clearance when cancer cells metastasize to the liver or retro-peritoneum strictures.Admission for intestinal obstruction can be due to a variety of causes and is a frequent event in hospitals around the world. In one series intestinal obstructions accounted for 3% of all surgical admissions and up to 20% of all acute abdominal conditions requiring admission by other observers.6,7 Post surgical adhesions are the most common etiology of acute mechanical obstruction of the small bowel (SBO) and account for 60–80% of all cases of intestinal obstruction in the developed world.6–8 Hernias are recognized as the second most common etiology of mechanical small bowel obstruction in developed countries.7Malignant small bowel obstruction is the third, and a much less frequent, cause of hospital admission in developed countries. In one series, it accounted for less than 5% of small intestinal obstructive cases, but can make up to 7.4% of all admissions for small bowel obstructions.7,9 Although most small bowel tumors found at autopsy are benign (75%), those that present with symptoms or are found during surgery are usually malignant.10 The most common tumor of the small bowel is an adenoma, which is generally asymptomatic. Gastrointestinal stromal tumors are the most common symptomatic benign tumors of the small bowel. Several theories attempt to explain the low incidence of tumorigenesis in the small bowel including rapid turnover of mucosal cells, rapid transit of luminal contents, low bacterial counts, and the alkalinity of chime.10,11 Population clusters do exist, but potential risk factors for the development of malignant disease of the small intestine are poorly defined.3,11Masses causing intraluminal obstruction or extrinsic compression are rarely obstructive in the small intestine due to the liquid nature of the small bowel enteric contents. Even a very small luminal channel is sufficient for the passage of all residuals of digestion, with the exception of an indigestible non-yielding foreign object. In this patient, a preexisting and previously undetected, asymptomatic small intestinal neoplasm caused the retention of a foreign body resulting in the complete obstruction of the small bowel. This resulted in an interesting and unique etiology of a small bowel obstruction. Such an unusual combination of factors resulting in a bowel obstruction has not widely been reported in literature due to the rarity of such a condition. A foreign object that is small enough to pass through the esophagus and pylorus is unlikely to cause obstruction anywhere else along the lower gastrointestinal tract. Therefore, when a foreign object is found to be obstructing at the mid portion of the small bowel, suspicions of a preexisting small bowel neoplasm should be raised and a timely exploratory surgery should be conducted.8