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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We therefore found the p.Gln172_Tyr176delinsHis variant as an excellent candidate for this cardiac disorder.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We then searched for additional individuals carrying bi-allelic variants in POPDC2 by screening this gene in 78 individuals that presented with a similar phenotype to family A (i.e., CCD with HCM) and a search using GeneMatcher and DECIPHER.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In total, we identified six affected individuals from four unrelated families of different ancestries (affected individuals 3–5 are siblings, Figures 1A and 1B; Tables 1 and S3) who harbor either homozygous or compound heterozygous rare POPDC2 variants.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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All six affected individuals were diagnosed with CCDs with or without HCM.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In summary (see Tables 1 and S3; Figure 1B), (1) AV conduction disease was present in all six individuals, mainly consisting of first-degree AV block or second-degree AV block type 1 (Wenkebach), (2) sinus node disease presenting as sinus bradycardia and sinus pauses was present in four out of six individuals, (3) cardiac arrest accompanied by sinus bradycardia or asystole was seen in two out of six individuals, (4) atrial arrhythmia (i.e., atrial flutter or fibrillation) was detected in three out of six individuals, (5) non-sustained ventricular tachycardia occurred in four out of six individuals, and (6) HCM was diagnosed in two out of six individuals (probands in families A and B with no other genetic variant causing HCM).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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A pacemaker was implanted in five out of six affected individuals with the age at implantation ranging from 15 to 23 years.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In two out of six affected individuals, an implantable cardioverter-defibrillator (ICD) was implanted to prevent lethal ventricular arrhythmia (ranging from 15 to 33 years), but no appropriate ICD shocks occurred.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Unlike the recessive syndrome associated with the other POPDC genes (i.e., POPDC1 and POPDC3-related limb-girdle muscular dystrophy), none of the affected individuals showed signs of muscular dystrophy.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Of note, individual 6 (II-1 in family D; Figure 1A) presented with acute myocarditis, for which he was admitted to the intensive care unit.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Detailed phenotypic descriptions of all affected individuals can be found in Note S1.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We noted an autosomal recessive mode of inheritance in families A and C wherein the identified variants in POPDC2 were inherited from each of the unaffected parents.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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While we expect a recessive inheritance, we could not assess the inheritance pattern in individuals 2 and 6 (from family B, II-4; and D, II-1; Figure 1A) as DNA of (one of) the parents was not available.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In total, we report five POPDC2 variants (RefSeq transcript: NM_001308333.2; protein ID: NP_001295262.1), of which two are missense variants (c.788G>A [p.Arg263His], c.787C>T [p.Arg263Cys]), one in-frame insertion deletion (c.516_527del [p.Gln172_Tyr176delinsHis]) and two are expected to result in protein truncation (c.563G>A [p.Trp188Ter], c.110_113del [p.Leu37SerfsTer20], Table S2).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The MAF of POPDC2 variants identified in affected individuals ranged from 1.4 × 10 to 1.3 × 10 in the gnomAD v4.0 (accessed March 2024; Table S2).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Furthermore, none of the variants were found homozygous in 730,947 exomes and 76,215 genomes from gnomAD, suggesting that homozygosity for these variants is not well tolerated.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In line with this, all variants are predicted damaging by multiple in silico prediction tools (Table S2).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Intriguingly, in families B and C, the same residue is affected: p.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Arg263His and p.Arg263Cys, respectively.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Of note, a homozygous variant (c.787G>A [p.Arg261Gln]) in POPDC3, which affects the paralogous residue of p.Arg263 in POPDC2 (families B and C), has been associated with muscular dystrophy without cardiac arrhythmia (MIM: 604577).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In family C, the three affected siblings homozygous for p.Arg263Cys inherited the variant from the heterozygous and clinically unaffected parents (II-1, II-2, and II-3 in Figure 1A).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We performed a look-up of the p.Arg263Cys variant in recent whole-genome sequencing dataset consisting of young affected individuals (n = 226) receiving a pacemaker because of AV block, and none of the individuals was homozygous for p.Arg263Cys, nor were there any individuals with bi-allelic variants in POPDC2 (Note S2).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In family D, we identified both p.Trp188Ter and p.Leu37Serfs20 variants that are expected to result in LOF due to premature truncation of the protein and/or nonsense-mediated decay (individual II-1 in Figure 1A).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The cardiac arrest accompanied by sinus bradycardia and first-degree AV block he displayed fitted the conduction disease seen in the other POPDC2-affected individuals.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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However, although the cardiac biopsy was negative for myocarditis, a potential causal role of myocarditis cannot be ruled out, as the disease can be focal and patchy, potentially reducing sensitivity due to sampling error.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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To explore the structural and functional consequences of the homozygous and compound heterozygous POPDC2 variants, we generated a predicted structural model of POPDC2, as no experimentally determined structures are currently available.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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SWISS-MODEL and AlphaFold Multimer were used since these protein-structure-prediction programs could generate dimeric models of POPDC2, and dimerization has been shown to be critical for the activity of POPDC1.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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As a complementary approach, AlphaMissense was used to predict relative pathogenicity scores of the identified single-amino-acid substitutions and deleted regions.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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AlphaFold Multimer generated a high-confidence dimer (Figure S2A) for full-length POPDC2 with a dimeric transmembrane domain composed of six transmembrane α helices (three α helices from each subunit) and two cAMP-binding Popeye domains that also contained an extensive dimer interface (Figure 2A).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In the AlphaFold model, Arg263 (altered in families B and C) was pointing into the cAMP-binding pocket of the Popeye domain from the adjacent subunit.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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SWISS-MODEL was the only program able to model a cAMP-bound state of the Popeye domain (Figure S2B).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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To generate a model of the cAMP-bound state of full-length POPDC2, we merged the AlphaFold Multimer and SWISS-MODEL predictions by swapping the Popeye domains (see section material and methods).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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This created a merged model of POPDC2 that included the transmembrane domain and the Popeye domains bound to cAMP (Figure 2A).Figure 2Functional characterization of the POPDC2 variants(A) Structural model of POPDC2 bound to cAMP generated using AlphaFold2 Multimer and SWISS-MODEL.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Dimer subunits are shown in green and cyan; cAMP molecules, green and cyan sticks.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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N′ and C′ indicate the N and C termini.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The positions of the transmembrane and Popeye domains are indicated by the labels.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The intrinsically disordered C termini (residues 275–364) are shown as dotted lines.(B) Zoom-in of the predicted cAMP-binding pocket of POPDC2.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Dimer subunits are shown in green and cyan; cAMP, green sticks; residues p.Gln172_Tyr176delinsHis, orange; p.Arg263, cyan sticks.(C) Structural models of the POPDC2 variants p.Leu37Serfs20 and p.Trp188Ter, which would both generate truncated proteins that would lack the ability to bind cAMP.(D) Homology model of POPDC2 by AlphaFold Multimer color coded by the average pathogenicity score for each residue as predicted by AlphaMissense.(E) Heatmap of predicted effects of amino-acid substitutions on POPDC2.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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AlphaMissense (AM) scores range from zero to one, with higher scores corresponding to increased pathogenicity.(F) Homology model of POPDC2 by AlphaFold Multimer color coded by the average pathogenicity score for each residue as predicted by AlphaMissense.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The position of non-disease-associated variants found in general population are shown as blue spheres, indicating predicted AlphaMissense pathogenicity scores <0.1.(G) Typical examples of TREK-1 currents upon 500-ms voltage-clamp steps to membrane potentials ranging from −100 to +50 mV from a holding potential of −80 mV in absence or presence of wild-type (WT) and mutant POPDC2.(H) Average current-voltage relationships of TREK-1 currents in absence or presence of WT and mutant POPDC2.(I) TREK-1 current amplitude at +50 mV in absence or presence of WT and mutant POPDC2.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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p < 0.05 with one-way ANOVA.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Error bars indicate the standard error of the mean (SEM).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Functional characterization of the POPDC2 variants (A) Structural model of POPDC2 bound to cAMP generated using AlphaFold2 Multimer and SWISS-MODEL.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
Dimer subunits are shown in green and cyan; cAMP molecules, green and cyan sticks.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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N′ and C′ indicate the N and C termini.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
The positions of the transmembrane and Popeye domains are indicated by the labels.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The intrinsically disordered C termini (residues 275–364) are shown as dotted lines. (
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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B) Zoom-in of the predicted cAMP-binding pocket of POPDC2.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Dimer subunits are shown in green and cyan; cAMP, green sticks; residues p.Gln172_Tyr176delinsHis, orange; p.Arg263, cyan sticks. (
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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C) Structural models of the POPDC2 variants p.Leu37Serfs20 and p.Trp188Ter, which would both generate truncated proteins that would lack the ability to bind cAMP. (
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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D) Homology model of POPDC2 by AlphaFold Multimer color coded by the average pathogenicity score for each residue as predicted by AlphaMissense. (
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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E) Heatmap of predicted effects of amino-acid substitutions on POPDC2.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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AlphaMissense (AM) scores range from zero to one, with higher scores corresponding to increased pathogenicity. (
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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F) Homology model of POPDC2 by AlphaFold Multimer color coded by the average pathogenicity score for each residue as predicted by AlphaMissense.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
The position of non-disease-associated variants found in general population are shown as blue spheres, indicating predicted AlphaMissense pathogenicity scores <0.1. (
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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G) Typical examples of TREK-1 currents upon 500-ms voltage-clamp steps to membrane potentials ranging from −100 to +50 mV from a holding potential of −80 mV in absence or presence of wild-type (WT) and mutant POPDC2. (
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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H) Average current-voltage relationships of TREK-1 currents in absence or presence of WT and mutant POPDC2. (
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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I) TREK-1 current amplitude at +50 mV in absence or presence of WT and mutant POPDC2.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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p < 0.05 with one-way ANOVA.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Error bars indicate the standard error of the mean (SEM).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Based on the structural model, all variants (p.Gln172_Tyr176delinsHis, p.Arg263His, p.Arg263Cys, and p.Trp188Ter/p.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Leu37Serfs20) are predicted to diminish the ability of POPDC2 to bind cAMP.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The first variant (p.Gln172_Tyr176delinsHis, family A) replaces five residues with a single histidine residue.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The deleted residues are predicted to form one beta strand that is part of a three-stranded beta sheet at the base of the cAMP-binding pocket (Figure 2B).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Replacement of the five residues with a single histidine residue would significantly alter the structural integrity of the cAMP-binding Popeye domain and directly affect the ability of POPDC2 to bind cAMP.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The second and third variants, p.Arg263His (found in family B) and p.Arg263Cys (found in family C), are also both predicted to interfere with proper cAMP binding, as the positively charged Arg residue is predicted to be near the negatively charged cyclic phosphate group of cAMP (Figure 2B).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Change of the Arg to either His or Cys would eliminate a key interaction predicted to stabilize cAMP binding and thus reduce or eliminate the ability of POPDC2 to bind cAMP.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In support of Arg263 being involved in a specific interaction to stabilize cAMP binding, alteration of Arg263 to any other residue is predicted to be pathogenic by AlphaMissense, with pathogenicity scores of 0.82 and 0.79 for the p.Arg263His and p.Arg263Cys variants, respectively.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The compound heterozygous variants are also predicted to eliminate cAMP binding by POPDC2 (Figure 2C).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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p.Leu37SerfsTer20 is a truncated protein that completely lacks the cAMP-binding Popeye domain and would also generate an incomplete transmembrane domain.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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p.Trp188Ter would retain the dimeric transmembrane domain, but truncate the Popeye domain, thus leaving an incomplete domain incapable of binding to cAMP (Figure 2C).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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In addition, both variants could be subject to nonsense-mediated decay, which would result in no protein product being made.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Notably, residues with high pathogenicity scores (>0.5) all cluster in the regions of the POPDC2 structure that are involved in either dimerization or cAMP binding (Figures 2D and 2E), which emphasizes the importance of cAMP binding and dimerization for POPDC2 function.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Consistently, other variants within POPDC2, found homozygously in gnomAD, and without a clear disease association (p.Arg17Val, p.Val29Ile, p.Arg98Cys, p.Val270Ile, and p.Ala321Thr), are all positioned away from the cAMP-binding pocket and are predicted to be benign by AlphaMissense with low pathogenicity scores <0.1 (Figures 2E and 2F).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Taken together, the disease-associated variants reported here are primarily located in regions of the POPDC2 structure that are critical for protein function.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We then aimed to functionally characterize the POPDC2 variants.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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TREK-1 is a recognized interacting protein of POPDC2, and co-expression of POPDC2 and TREK-1 has been shown to increase TREK-1 current in comparison to expression of TREK-1 alone.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Furthermore, cardiac-specific TREK-1-deficient mice display a sinus node phenotype characterized by bradycardia with frequent episodes of sinus pauses, partially resembling the phenotype in the affected individuals with bi-allelic POPDC2 variants presented here.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We therefore hypothesized that the effect of the p.Gln172_Tyr176delinsHis (family A) and p.Arg263His (family B) variants is mediated through modulation of the TREK-1 current.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We did not test the variants found in family C (p.Arg263Cys) and family D (p.Trp188Ter/p.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Leu37Serfs20) as they affect the same residue as the variant in family B (p.Arg263His) or are expected to result in premature truncation of the protein and possibly nonsense-mediated decay, respectively.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We co-transfected HEK293 cells with WT and mutant POPDC2 with TREK-1 containing plasmids.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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As expected, co-expression of WT POPDC2 with TREK-1 increased TREK-1 current density (Figures 2G–2I).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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However, when we co-expressed TREK-1 with a p.Gln172_Tyr176delinsHis or p.Arg263His POPDC2-containing plasmid, no increase in TREK-1 current density was observed, comparable to the co-expression of TREK-1 with an empty vector (Figures 2G–2I).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We then tested the effect of the POPDC2 variants on sodium current (INa) as it was recently hypothesized that POPDC2 may interact with NaV1.5.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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However, expression of WT, p.Gln172_Tyr176delinsHis, or p.Arg263His POPDC2 in an HEK293 cell line stably expressing human NaV1.5 channels (encoded by SCN5A) showed an effect neither on INa density nor on INa gating properties by WT or mutant POPDC2 (Figure S3).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Western blot analysis showed levels of expression of the mutant proteins that were comparable to the levels seen for WT POPDC2 (Figure S4).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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To evaluate whether the observed decrease in TREK-1 current density, associated with the POPDC2 variants, is responsible for the bradycardia observed in affected individuals, we conducted computer simulations using comprehensive mathematical models of both a human sinus nodal pacemaker cell and a human atrial cell.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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As shown in Figure 3A (blue line), the experimental data on the voltage dependence of the TREK-1 + POPDC2-WT current could be well fitted (r > 0.99) with the relationship ITREK-1 = −117.1 + 284.24 × exp(Vm/90.52), in which ITREK-1 and Vm denote TREK-1 current density (in pA/pF) and membrane potential (in mV), respectively.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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The mutant data could be well fitted by scaling down ITREK-1 to 59% of the TREK-1 + POPDC2-WT current over the entire volage range (Figure 3A, red line).
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We started our simulations with incorporating ITREK-1, which is not present in the original model cell, into the human sinus node model cell.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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Because data on the density of ITREK-1 in human sinus node cells are lacking, we set its density to 1.2 pA/pF (at +30 mV), as observed in mouse sinus node cells by Unudurthi et al. This, however, resulted in cessation of pacemaker activity in the human sinus node model cell.
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PMC12256823
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Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
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We then repeated our simulations with a 10 times lower density of ITREK-1.
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PMC12256823
|
Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
With this density, the cycle length of the simulated action potential amounted to 1,160 ms (Figure 3B, top panel, blue solid line), as compared to 813 ms in the original model (Figure 3B, top panel, gray dotted line).
|
PMC12256823
|
Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
Cycle length was reduced by 16% from 1,160 to 973 ms upon reduction of ITREK-1 density to 59%, thus simulating the effect of the variants in POPDC2 (Figure 3B, top panel, red solid line; Figure 3C, vertical arrow).
|
PMC12256823
|
Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
The decrease in cycle length was mainly due to an increase in diastolic depolarization rate (Figure 3B, top panel) as a result of the decrease in the small but effective ITREK-1 during this phase of the action potential (Figure 3B, bottom panel).
|
PMC12256823
|
Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
Qualitatively similar results were obtained with other ITREK-1 densities.
|
PMC12256823
|
Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
The observed effects on cycle length are summarized in Figure 3C.Figure 3Functional effects of the POPDC2 variants from in silico modeling(A) Fits to the experimental data on TREK-1 currents in absence or presence of wild-type (WT) and mutant POPDC2.
|
PMC12256823
|
Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
The fit to the mutant data were obtained by scaling the fit to the WT data by a factor of 0.59.(B) Membrane potential (top) and associated TREK-1 current (bottom) of a single human sinus nodal pacemaker cell as simulated using the comprehensive mathematical model developed by Fabbri et al. ITREK-1 was introduced into the original model cell using the fits of (A).
|
PMC12256823
|
Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
ITREK-1 magnitude was set to 0.12 pA/pF at a membrane potential of +30 mV(C) Cycle length of the simulated single human sinus nodal pacemaker cell as a function of ITREK-1 magnitude.(D) Membrane potential (top) and associated TREK-1 current (bottom) of a single human atrial cell as simulated using the comprehensive mathematical model developed by Maleckar et al. ITREK-1 was introduced into the original model cell using the fits of (A).
|
PMC12256823
|
Bi-allelic variants in POPDC2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy.
|
ITREK-1 magnitude was set to 4.0 pA/pF at a membrane potential of +30 mV. Action potentials were elicited at a rate of 1 Hz with a 1-ms, 20% suprathreshold stimulus current.(E) Diastolic potential of the simulated single human atrial cell as a function of ITREK-1 magnitude.(F) Threshold stimulus current of the simulated single human atrial cell as a function of ITREK-1 magnitude.
|
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