Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): the Dutch Research Council: NWO Talent Scheme Background A large multigenerational family harboring a pathogenic KCNH2 variant (L69P) was identified. This gene encodes the hERG channel, responsible for the cardiac rapid delayed rectifier K+ current (IKr). Pathogenic variants in KCNH2 can cause Long QT syndrome type 2 (LQTS2). Interestingly, in addition to LQTS2 we observed a high incidence of bradycardia in this family. Bradycardia is a known feature of some types of LQTS. However, evidence of bradycardia in LQTS2 is limited to a few sporadic cases. Purpose This study aims to identify the genetic variants and biophysiological changes in ion channel function, explanatory of the phenotypes LQTS2 and bradycardia. Methods An overview of the phenotype and genetic information of the index and family members was generated , including symptoms and electrocardiogram (ECG) parameters. QTc was calculated using Bazetts’ correction. Segregation of the identified genetic variants with LQTS and bradycardia were determined by LOD score. Biophysiological properties of the encoded ion channels was measured by manual whole-cell patch-clamp experiments. Results On the basis of linkage analysis, the pathogenic variant KCNH2-p.L69P was found to be unrelated to the bradycardia. Therefore, Sanger sequencing of HCN4, encoding the channel responsible for the hyperpolarization-activated current (If), an important current for sinoatrial node automaticity, was performed. We identified the HCN4-p.R666W variant in multiple family members, which co-segregated with bradycardia (LOD-score 3.2). Patients carrying both variants had more severe phenotypes than carriers of a single variant. Patch-clamp experiments in HEK293A-cells expressing wild type, or KCNH2-p.L69P show a reduced current density, and an altered time component of the fast deactivation, explaining the observed LQTS2. Functional assays of HCN4-p.R666W will elucidate the biophysiological changes possibly underlying the bradycardia. Conclusion we present a large multigenerational family that harbors a likely pathogenic variant in HCN4 in conjunction with a pathogenic variant of KCNH2. Double carriers were more affected than single carriers, arguing for continued alertness and deep phenotyping even in families with known pathogenic variants. Furthermore, we identified functional changes in the kinetics of the hERG channel encoded by KCNH2-p.L69P, elucidating the molecular mechanism underlying LQTS2 in this family.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Dutch Research Council (NWO Talent Scheme) Background Variants in KCNH2, the gene encoding the hERG channel and responsible for the cardiac rapid delayed rectifier K+ current (IKr), have been linked to Long QT Syndrome type 2 (LQTS2). The KCNH2-p.S906L variant has been found in eight, small unrelated families with variable clinical pictures and incomplete penetrance of LQTS2. Purpose Due to the small size of the families, co-segregation analysis is limited. Functional studies were performed to evaluate the effect of the variant on channel functionality. Thereby assessing if KCNH2-p.S906L leads to functional changes explanatory for the phenotype. Methods Clinical data of the patients and family members were collected thereby, creating an overview of the phenotype. The biophysiological properties of the hERG channel were assessed by manual whole-cell patch-clamp, using HEK293a cells expressing (i) the wild type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, hm) or KCNH2-p.S906L in combination with WT (heterozygous, Hz). Results Assessment of the clinical data of the patients and family members shows an incomplete and low penetrance of the LQTS2. All carriers were heterozygous for the variant. In carriers of the KCNH2-p.S906L variant, LQTS2 severity was very variable, ranging from asymptomatic, to life-threatening arrhythmic events. The KCNH2-p.S906L variant reduced current density in a dosage-dependent manner compared to KCNH2-WT. The current density was reduced by 63, and 54% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. Changes in gating properties were absent, except for activation kinetics in KCNH2-p.S906L-Hm. In these cells, an increase in the time constant, and a positive shift in voltage dependency of activation were observed. Conclusions Both the reduced current density and changes in activation kinetics suggest the variant to result in a moderate loss-of-function of the hERG channel, fitting with the observed LQTS2. These observed functional changes in hERG combined with the reduced penetrance in the affected individuals suggest that this variant in KCNH2 is a risk factor for LQTS2.

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.

Abstract Background In the past decade, we and others have reported three families with rare genetic variants in TNNI3K, encoding the cardiac-specific troponin-I interacting kinase (TNNI3K), co-segregating with a mixed, but highly penetrant, cardiac phenotype that features predominant atrial/junctional tachycardia occurring in combination with cardiac conduction disease and dilated cardiomyopathy. We demonstrated that while the p.Thr539Ala and p.Gly526Asp TNNI3K variants had decreased auto-phosphorylation activity the p.Glu768Lys variant, present in 3 independent families, leads to increased auto-phosphorylation levels, in line with the finding that increased levels of Tnni3k expression are associated with slower atrial-ventricular conduction in mice. Objective Identifying new genetic variants in the TNNI3K gene associated with cardiac disease and assessing their impact on TNNI3K auto-phosphorylation levels. Methods Through next generation sequencing of a panel of genes associated with cardiac disease we assessed TNNI3K in patients with cardiac arrhythmias and cardiomyopathies. All variants identified were assessed in vitro for effects on auto-phosphorylation. Briefly, wild-type and mutant TNNI3K constructs were transfected into HEK293 cells, protein was extracted after 48 hours and analyzed with anti-flag and anti-phospho-tyrosine antibodies on Western blot. Results We identified 7 novel and rare variants in TNNI3K in 11 additional probands, with predominantly cardiac conduction disease, with or without dilated cardiomyopathy, and atrial-ventricular-re-entry-tachycardia (AVNRT). Of these, multiple variants were found to have aberrant auto-phosphorylation including almost absent auto-phosphorylation capacity for one (TNNI3K-p.Val510Leu). All three-independent wild type TNNI3K transfected HEK293 cell lysates showed similar phosphorylated TNNI3K levels and the kinase-dead negative control demonstrated no phosphorylation activity. Conclusion We here present 7 novel genetic variants in TNNI3K in patients with a remarkable overlap in cardiac phenotype consisting mainly of AVNRT and cardiac conduction disease. We further show that some of these variants alter the auto-phosphorylation of TNNI3K. These results indicate a more prevalent role of variants in TNNI3K in human cardiac disease and a possible in vitro functional assay to assess the pathogenicity of such variants. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The Dutch Research Council (NWO Talent Scheme VIDI-91718361)

Congenital heart disease (CHD) is the most common congenital defect, with an estimated prevalence of 1%. Although the heritability of CHD is well demonstrated, the underlying genetic basis and molecular mechanisms remain largely unknown. Transposition of the great arteries of the dextro type (D-TGA) is a rare critical form of CHD that results in neonatal death if untreated. We here conducted a case-control genome-wide association study to identify genetic loci associated with D-TGA.

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With aging and in cardiac disease increasing fibrosis due to collagen deposition disturbs cell-cell coupling of cardiomyocytes, providing a substrate for arrhythmia. In this study we set out to identify genetic modifiers of collagen deposition in heart. We exploited the genetic variability among F2 progeny of 129P2 and FVBN/J mice carrying the Scn5a-1798insD/+ mutation to identify genes that influence the amount of collagen deposition in left ventricular (LV) myocardium. Relative amounts of collagen were determined in 65 F2 mice and combined with genome-wide genotypic and expression data to identify collagen-QTLs (cQTLs) and the underlying expression QTLs (eQTLs). In both collagen-QTL mapping as well as eQTL mapping we identified significant co-factors throuhgh multiple QTL mapping (MQM). A significant cQTLs was identified on mouse chr8 and after correction for cofactors using MQM an additional cQTL was found on mouse chr2. For both these loci a significant co-factor was identified on chr18. Of the 24 eQTLs mapping to the chr8-cQTL, 8 transcripts correlated to relative collagen amount. Similarly, of the 6 chr2-cQTL eQTLs only Gpr158 (significantly) and of the 12 eQTLs of chr18-co-factor-region only Fgf1 (suggestively) correlated with the relative collagen amount. Furthermore, two cQTL-eQTLs, Pdlim3 (chr8) and Itga6 (chr2), had a significant co-factor on chr18 that coincided with the chr18 collagen cofactor. We validated the interaction of Fgf1 with these transcripts and collagen production in vitro in isolated cardiac fibroblasts. In conclusion, we mapped, for the first time, a genetic network that modulates collagen deposition in mouse LV myocardium.

We set out to identify the genetic defect underlying multiple cardiac manifestations in a Dutch family. Multiple family members presented with bradycardia in combination with hypertrabeculation of the myocardium. Atrial fibrillation, mitral valve complications and sudden cardiac death also occurred in the pedigree. We undertook a strategy consisting of first reducing the genome space by identifying chromosomal regions shared among affected individuals, followed by exome sequencing for identification of sequence variants within these regions. In our gene discovery effort we focused on the bradycardia phenotype as this could be assessed in most pedigree members and multiple affected individuals were available. Genome-wide SNP genotyping was carried out using the Illumina HumanOmni2.5 array. This genotypic data was used for identification of chromosomal regions shared identical-by-descent (IBD) among individuals affected with bradycardia, uncovering a total of 21 loci shared IBD. Exome sequencing was carried out on the 2 most distantly related individuals with bradycardia. The coding region of the genome was captured using the Agilent SureSelect Target enrichment system followed by sequencing on the Illumina Hiseq2000 platform. The SOAPsnp (for single nucleotide variants, SNVs) and the GATK (for copy number variants, CNVs) genome analysis algorithms were used for genotype calling. Variants were filtered against multiple exome and genome sequencing databases (e.g. dbSNP132, Exome Variant Server, 1000Genomes, Genome of the Netherlands). Seven novel and potentially malignant variants located within the 21 IBD regions were identified. Of these only one variant, p.G482R in HCN4, segregated with the combined bradycardia and hypertrabeculation phenotype in the family. HCN4 encodes the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4, underlying the cardiac pacemaker funny current (If). In line with the role of If in pacemaker activity of the sinus node, mutations in HCN4 are an established cause of bradycardia and it is therefore highly likely that the identified variant underlies the bradycardia in the family. However, the association of an HCN4 variant with hypertrabeculation of the myocardium is novel. Investigation of HCN4 in additional families with this combined phenotype is currently ongoing in order to explore further the possible link between mutation in HCN4 and myocardial hypertrabeculation