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In a first family (family#1), we identified 53 members of whom 17 present a syndromic cardiac disorder characterized by electrical disorders (sinus node dysfunction, atrial fibrillation...) and developmental defects (atrial septal defect, valvopathy...) following an autosomal dominant model. Despite a strong linkage pointing to the 4q25 region, exome sequencing failed to identify causal variant. Interestingly, 6 additional non-related families presenting the same phenotype have been also identified. Our aims are to identity the causal mutation and the molecular mechanism underlying this complex cardiac syndrome. Genetic study has been performed using whole-genome sequencing (WGS). Based on transgenic mouse strains, we assessed the impact of Family#1 mutation on the phenotype and on gene expression. Then, we generated human cardiomyocytes derived iPS cells (CM-iPS) isogenic models to evaluate the epigenome (CUT&RUN and ATAC-seq), transcriptome (RNA-seq) and topological associated domain (TAD) remodelling (Hi-C). By WGS we uncovered a deletion of 15 kb in a gene desert area on 4q25, segregating in all affected relatives of Family#1. The 6 other families present overlapping deletions. Mouse model recapitulates the cardiac phenotype and exhibit a dysregulation of Pitx2 expression in cardiac specific compartments. Based on human CM-iPS models, epigenetic data highlight among the 15 kb deletion a unique open region containing a CTCF binding site, crucial for delimiting TAD boundaries. Hi-C assay reveals the fusion of 2 TADs and highlights new interactions between PITX2 and atrial specific regulatory elements. We identified a deletion located within a gene desert area associated with a complex cardiac disorder. The CTCF binding site contained in the deletion seems key in the TAD border. The TAD remodelling leads to new (regulatory) interactions and expression dysregulation of PITX2. We describe a new molecular mechanism implying a yet unidentified non-coding regulatory element of PITX2 and responsible for a complex electrical and developmental cardiac syndrome. [ABSTRACT FROM AUTHOR]

Ventricular arrhythmic events are responsible for 50% of death in heart failure but no reliable predictive marker is known to discriminate patients at risk of fatal arrhythmia. Interestingly, familial predisposition has been reported suggesting a role of genetic factors. Identify genetic markers increasing the arrhythmic risk in heart failure population. We prospectively included heart failure patients with left ventricular ejection fraction (LVEF) under 35% and a cardioverter defibrillator in primary prevention in 22 French centres between 2009 and 2017. Patients were followed for 72 months and divided into two groups: cases with an arrhythmic event during follow-up and controls. A Genome Wide Association Study (GWAS) was done. Single Nucleotide Polymorphisms (SNPs) genotyping was performed on Affymetrix Axiom Precision Medicine Research Array plates. 332 cases and 567 controls were included (86% men, mean age at implantation 52 ± 11 years). 78% of patients had ischaemic cardiopathy, 20% had dilated cardiomyopathy. Mean LVEF was 27 ± 5%. No statistical difference was found between cases and controls on clinical parameters or electrocardiographic measures. No locus shows genome-wide significant association (P < 5.10 − 8) on the GWAS analysis. However, 16 signals with a p-value between 5.10 − 8 and 5.10 − 5 were investigated. eQTL and chromatin conformation point to 35 genes with cardiac expression previously associated with heart failure, cardiomyopathies, arrhythmias and inflammation. Variants identified point to regulatory regions of the genome and may then propose a molecular mechanism predisposing patients to arrhythmias (Fig. 1). No locus raises genome-wide significance, but several signals with a nominal p-value point to relevant genes and pathways. Replication of the GWAS is ongoing on a cohort of 156 new patients with a less severe cardiopathy implanted with a cardioverter defibrillator in secondary prevention. [ABSTRACT FROM AUTHOR]

Hereditary sinus node dysfunction (SND) with atrioventricular block (AVB) is a rare disease characterized by permanent heart rate decrease associated with paroxysmal dizziness and syncope. Pacemaker implantation remains the main therapy. To date, only few genes are known associated with this disease. Most of the cases remain genetically unsolved. The aim of the study is to identify a new gene responsible for familial sinus node and atrioventricular conduction dysfunction (SND +AVB). A genome-wide linkage analysis is performed to map the SND + AVB locus and is completed by performing Whole Genome Sequencing (WGS). Candidate variants (Single Nucleotide Variants (SNV) and Copy Number Variations (CNV) will be kept if rare and shared by affected members. We investigated a 4 generations pedigree with 25 family members, 13 of them were affected or probably affected by an autosomal dominant SND + AVB. The mean age of cardiac pacemaker implantation was 42 years old, the youngest of them had at 25 years old. Among the family WGS was performed in two first cousins. We exclude the presence of rare variants among the gene previously associated with SND or AVB. Our results suggest the presence of the mutation in a new gene. Thanks to the linkage analysis we may isolate a region associated with the phenotype and then identify candidate rare variant shared by the 2 affected members whole genome sequenced. Such candidate variants will be tested in other affected family members, prioritized according tissue expression and pathogenicity prediction. [ABSTRACT FROM AUTHOR]

Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease, affecting preferentially people under 40 years old with ventricular arrhythmia to sudden death. To date, the rate of successful genotyping is about 50%. Desmosomal genes (PKP2, DSC2, DSG2, DSP, JUP) are the most frequently mutated, the implication of the other genes associated with other cardiomyopathies is still debated. The pathogenicity of variants among these genes should be assessed not only based on the rarity because of the presence of rare variants in individuals in the general population. Assess the pathogenicity of variants and the relevance of genes involved in ACM. DNA from 179 patients with ACM were sequenced on a panel of 71 heart disease genes including 11 genes most often described in ACM. The pathogenicity of variants is assessed according to international guidelines (ACMG score) with 2 bioinformatic tools. We performed burden (CAST) and dispersion tests (SKAT, SKAT-O) on the 71 genes between patients and a control population. Among the 179 patients, 34.6% have a pathogenic variant with PKP2 in 42 patients. Furthermore, 17.3% of patients have uncertain significance variants in genes involved in ACM. Burden tests showed a significant enrichment in pathogenic variants in PKP2, DSP, DSC2 and DSG2 (P < 0.001) and in uncertain significance variants in the PKP2, DSG2 and DSC2 genes (P < 0.01). No enrichment was observed among other genes. Patients with a variant in genes involved in ACM are diagnosed earlier than patients without variant (P < 0.05). Only desmosomal genes count a significate proportion of pathogenic variants in patients with ACM compared to control population. Uncertain significance variants in PKP2, DSC2, DSG2 genes seem to contribute to ACM and should be investigated. The age of symptom onset is correlated with the presence of pathogenic variant, thus justifying early family screening for better clinical management. [ABSTRACT FROM AUTHOR]

Background: The Brugada syndrome (BrS) is considered a rare mendelian disorder with autosomal dominant transmission. BrS is associated with an increased risk of sudden cardiac death and specific ECG features consisting of ST-segment elevation in the right precordial leads. Loss-of-function mutations in SCN5A, encoding the pore-forming subunit of the cardiac sodium channel (Nav1.5), are identified in ~20% of patients. However, studies in families harboring mutations in SCN5A have demonstrated low disease penetrance and, in some instances, absence of the familial SCN5A mutation in some affected members. These observations suggest a more complex inheritance model. Methods: To identify common genetic factors modulating disease risk, we conducted a genome-wide association study on 312 individuals with BrS and 1115 ancestry-matched controls. Results: Two genomic loci displayed significant association. Replication testing on 2 independent case/control sets from Europe (598/855) and Japan (208/1016) confirmed both associations and revealed a third locus. The cumulative effect of the 3 loci on disease susceptibility was large, with an odds ratio of 21.5 in the presence of more than 4 risk alleles vs less than 2. Two of 3 loci had previously been shown to influence ECG conduction parameters in the general population. The third locus encompasses a transcription factor that has not previously been implicated in cardiac electrical function and arrhythmia. Functional studies in mice heterozygous knockout for this transcription factor identified differences in expression of Nav1.5 in addition to differences in conduction of the cardiac electrical impulse in the right ventricular outflow tract. Conclusions: Our findings (1) indicate that common genetic variation may have a strong impact on predisposition to BrS and (2) identify a new gene involved in the pathogenesis of the disease. [Copyright &y& Elsevier]

Introduction: Brugada Syndrome (BrS) is a disorder associated with an increased risk of sudden cardiac death and specific ECG features consisting of ST-segment elevation in the right precordial leads. Loss-of-function mutations in SCN5A underlie the disorder in ∼25% of patients. While other genes have been implicated, these account for <1% of subjects. Thus the genetic basis of BrS remains unknown in the majority of cases. We here set out to identify the genetic defect underlying BrS in a 3 generation family from Ireland with a case of spontaneous type 1 ECG who died suddenly at 24 years old.Methods: Exome sequencing was carried out in 3 individuals from the pedigree using Agilent SureSelect Target Enrichment 50Mb capture followed by sequencing on an Illumina HiSeq 2000. These 3 individuals were selected based on their genetic distance and the presentation of a BrS ECG at baseline.Results: Comparison of 3 exomes from affected individuals identified 22,171 shared variants. Filtering out of variants occurring at a minor allele frequency (MAF) of >1% in public and in-house exome/genome databases, and considering all types of exonic variants and splice site variants, resulted in 10 rare variants shared among the 3 affected. Two of the variants are found in all afffected family members and not in the unaffected and reside in 2 different genes, namely HOOK3 and PXDNL. These 2 variants have been found at a MAF of 0.02% and 0.008%, respectively, in the NHLBI Exome Variant Project population (n=6500 individuals). The function of PXDNL, which appears to be specifically expressed in heart, is yet unknown. The PXDNL variant is predicted to be deleterious and probably damaging by SIFT and Polyphen2 algorithms, respectively.Conclusions: We identified by exome sequencing a chromosomal interval and a possible novel gene for BrS. Ongoing functional studies are aimed at investigating further the involvement of this gene in the BrS. [ABSTRACT FROM PUBLISHER]

Objective: The aim of this study was to identify the genetic defect underlying the combined clinical presentation of bradycardia and left ventricular noncompaction cardiomyopathy (LVNC), hypothesizing that these two clinical abnormalities have a common genetic cause.Background: Familial forms of primary sinus bradycardia have in some cases been attributed to mutations in HCN4, SCN5A and ANK2. In these studies no structural cardiac alterations were reported in mutation carriers. However a cluster of reports in the literature describe patients presenting with sinus bradycardia in association with LVNC pointing to a shared genetic cause.Methods: Exome sequencing was carried out in two cousins from the index family that were affected by the combined bradycardia-LVNC phenotype; shared variants thus identified were subsequently overlaid with the chromosomal regions shared among five affected family members that were identified using SNP-array analysis.Results: The combined linkage analysis and exome sequencing in the index family identified 11 novel variants shared among the two affected cousins. One of these, p.G482R in HCN4, segregated with the combined bradycardia and LVNC phenotype in the entire family. Subsequent screening of HCN4 in three additional families, with the same clinical combination of bradycardia and LVNC, identified HCN4 mutations in all three. In electrophysiological studies, all found HCN4 mutations showed a more negative voltage-dependency of activation, consistent with the observed bradycardia.Conclusions: While mutations in HCN4 have been previously linked to bradycardia, our study provides the first evidence that mutations in this ion channel gene may also associate with structural abnormalities of the myocardium. [ABSTRACT FROM PUBLISHER]

Background: Brugada syndrome (BrS) is an inherited arrhythmia syndrome with an increased risk of sudden death resulting from polymorphic ventricular tachycardia (VT) and/or ventricular fibrillation (VF) in the absence of gross structural abnormalities. Mutations in SCN5A, encoding the pore-forming subunit α of the cardiac voltage-gated sodium channel, are identified in about 20-30 % of probands affected by Brugada syndrome (BrS). SCN5A mutations may also lead to progressive cardiac conduction defects (PCCD). The causality of SCN5A mutations in PCCD was proven by linkage analysis. In contrast, SCN5A mutations in BrS were discovered by a candidate gene approach19 and linkage data are still lacking. The aim of this study was to investigate the association of SCN5A mutations and BrS in a group of large genotyped families. Methods and Results: Families were included if at least 4 family members were carriers of the SCN5A mutation identified in the proband. Thirteen large families composed by 115 mutation-carriers were studied. The signature type I ECG was present in 54 mutation-carriers (BrS-ECG+) (47 %). In 5 families, we found 7 individuals affected by BrS, but with a negative genotype (mutation-negative BrS-ECG+). Among these 7 mutation-negative BrS+ individuals, 3, belonging to 3 different families, had a spontaneous type I ECG, while 4 had a type I ECG after administration of Na+ channel blockers. EPS was performed in 5 BrS-ECG non-mutation patients. Ventricular tachyarrhythmias were inducible in 3. An ICD was implanted in these 3 patients. Mutation carriers (n=115) had, on average, longer PR and QRS intervals than non-carriers (n=148) demonstrating that these mutations exerted functional effects. Conclusions: Our results suggest that SCN5A mutations might not be sufficient to cause BrS and that genetic background may play a powerful role in the pathophysiology of BrS. However, this study confirms the role of SCN5A mutations in PCCD. These findings add further complexity to concepts regarding the causes of BrS, and are consistent with the emerging notion that the pathophysiology of BrS includes various elements beyond mutant sodium channels. [Copyright &y& Elsevier]

Short QT syndrome (SQTS) emerged as a new inherited channelopathy characterized by constantly short QT interval (QTc<360ms) associated with atrial fibrillation, syncopal episodes, and/or sudden cardiac death in patients with no underlying structural heart disease. It has been associated with a gain of function in 3 distinct potassium channels (KCNH2, KCNQ1, and KCNJ2) or a loss-of-function of a calcium channel Cav1.2. We identified a 6-year-old male proband who experienced a syncope episode and presented a QTc of 346ms. In addition, a triangular T wave was observed in V2, V3 leads. His 34-year-old mother was also symptomatic (QTc: 359 ms), with Ventricular fibrillation. The grandmother was asymptomatic (QT: 405ms). After a screen for ion channel mutations, we found a variant of CACNA1C, gene encoding the α1c of the cardiac L-type calcium channel, leading to c.667G>C transition caused a p.A223P substitution of a mammalian highly conserved residue. This variant was absent in 312 healthy controls. Cardiac Ca2+ channels are complexes including α1,β, and α2δ subunits allowing the Ca2+ influx (ICaL) essential for excitationcontraction coupling. To evaluate the incidence of this substitution on Cav1.2 function,α1c,β2a, and α2δ1b rat subunits were transfected in HEK-tsA201 cells. The human A223P mutation corresponds to A253P in the rat sequence. Whole-cell patch-clamp experiments were performed to study the mutation effects on ICaL biophysical parameters. A253P Cav1.2 generated a reduced ICaL (-5.48±0.85Pa/ pF, n=54) in comparison with WT Cav1.2 (−12.24±1.54Pa/pF, n=51). The voltage dependence of the Ca2+ current activation did not show any change (V1/2act: −11.54±1.15mV, n=9, versus −12.94±1.03mV, n=19 ; slope: 6.37±0.28mV versus 6.46±0.27mV, for mutant and WT channels, respectively). Second, we studied WT and mutant Cav1.2 expression levels. We quantified channels expression by Western blot. The mutated protein was expressed at the same level as WT-Cav1.2. We are currently investigating Cav1.2 addressing to the membrane. In conclusion, we have shown that A253P mutation reduces the ICaL amplitude but the mechanism behind this loss of function remains to be determined. [Copyright &y& Elsevier]