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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a “pseudo” QRS complex suggesting reduced inward current(s). Voltage clamp analysis of ICa showed no difference in the magnitude of current. Measurements of INa reveal a 60% reduction in INa density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of INa was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of INa due to a mutation in PKP2 coupled with and a gain of function in IK,ATP due to a mutation in ABCC9.

Background: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. Methods: We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. Results: Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance ( P −8 ) near NOS1AP , KCNQ1 , and KLF12 , and 1 missense variant in KCNE1 (p.Asp85Asn) at the suggestive threshold ( P −6 ). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation ( h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (r g =0.40; P =3.2×10 −3 ). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls ( P P Conclusions: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.

Mutations in CACNA1C that increase current through the CaV1.2 L-type Ca2+ channel underlie rare forms of long QT syndrome (LQTS), and Timothy syndrome (TS). We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on CaV1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680) predicting a glycine>arginine substitution at residue 1911 (p.G1911R) of CaV1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of CaV1.2. We also observed a negative shift of V1/2 of activation and positive shift of V1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on CaV1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID), for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of CaV1.2 and increase window current expand our appreciation of mechanisms by which a gain of function of CaV1.2 can contribute to QT prolongation. Published version

Background Brugada syndrome (BrS) is a common heritable channelopathy. Mutations in the SCN5A -encoded sodium channel (BrS1) culminate in the most common genotype. Objective This study sought to perform a retrospective analysis of BrS databases from 9 centers that have each genotyped >100 unrelated cases of suspected BrS. Methods Mutational analysis of all 27 translated exons in SCN5A was performed. Mutation frequency, type, and localization were compared among cases and 1,300 ostensibly healthy volunteers including 649 white subjects and 651 nonwhite subjects (blacks, Asians, Hispanics, and others) that were genotyped previously. Results A total of 2,111 unrelated patients (78% male, mean age 39 ± 15 years) were referred for BrS genetic testing. Rare mutations/variants were more common among BrS cases than control subjects (438/2,111, 21% vs. 11/649, 1.7% white subjects and 31/651, 4.8% nonwhite subjects, respectively, P −53 ). The yield of BrS1 genetic testing ranged from 11% to 28% ( P = .0017). Overall, 293 distinct mutations were identified in SCN5A: 193 missense, 32 nonsense, 38 frameshift, 21 splice-site, and 9 in-frame deletions/insertions. The 4 most frequent BrS1-associated mutations were E1784K (14×), F861WfsX90 (11×), D356N (8×), and G1408R (7×). Most mutations localized to the transmembrane-spanning regions. Conclusion This international consortium of BrS genetic testing centers has added 200 new BrS1-associated mutations to the public domain. Overall, 21% of BrS probands have mutations in SCN5A compared to the 2% to 5% background rate of rare variants reported in healthy control subjects. Additional studies drawing on the data presented here may help further distinguish pathogenic mutations from similarly rare but otherwise innocuous ones found in cases.

Several studies have demonstrated an association between Brugada syndrome (BrS) and mutations in genes encoding ion channel subunits including SCN5A, CACNA1C, CACNB2b, SCN1B, and KCNE3. Mutations in SCN5A, encoding the voltage-gated sodium channel Nav1.5, represent the majority with greater than 293 mutations in SCN5A linked to the syndrome (Kapplinger et al, Heart Rhythm, In press 2009). We identified a missense mutation (G1408R) in SCN5A in a large BrS family. Intriguingly, this mutation had been reported earlier in two independent studies and has also shown to be associated with Sick Sinus Syndrome (SSS) and Cardiac Conduction Defect (CCD) (Kyndt et al, 2001; Benson et al, 2003). Nav1.5-G1408R channels heterologously expressed in CHO cells and studied using patch-clamp techniques failed to generate any sodium channel current (INa). Co-expression of the mutated channels with wild-type (WT) channels resulted in a 50% reduction of current amplitudes with no changes in kinetic properties when compared with WT channels. The residue resides in the DIII pore region and is conserved among species. We addressed the importance of this amino acid at position 1408 by replacing it with another small neutral amino acid (G1408A) and by substituting a negatively charged aspartic acid (G1408D). Our results show that substituting glycine with alanine retains WT behavior while exchange to the positively charged arginine (G1408R) or negatively charged aspartic acid leads to a complete loss-of-function. In conclusion, we describe a SCN5A mutation associated with BrS which results in a loss of function of INa important for action potential generation. Further, we show that the presence of a neutral hydrophobic amino acid at this position is crucial for normal channel function.

Introduction: Inherited loss of function mutations in SCN5A, the gene that encodes the α-subunit of the human cardiac sodium channel (hNav1.5), have been linked to overlapping syndromes including cardiac conduction disease (CCD) and Brugada syndrome (BrS). The mechanisms responsible for the development of one without the other are poorly understood.Methods: Direct sequencing analysis was performed in a family with CCD. Wild type (WT) and variant channels were co-expressed with CD8 cDNA in TSA201 cells for electrophysiological study. Green fluorescent protein (GFP)-fused WT or mutant SCN5A genes were used for confocal microscopy to assess channel trafficking.Results: A novel SCN5A missense mutation, P1008S, was identified in all family members displaying 1st degree AV block, but not in unaffected family members nor in 430 reference alleles. Peak P1008S current was 11.77% of WT (p