Yuko Wada

Background: KCNQ1 loss of function variants are thought to cause type 1 long QT syndrome by reducing I _Ks . However, we have recently reported that pharmacologic block of I _Ks in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) produced minimal increases in action potential duration at 90% repolarization (APD ₉₀ ), while genetic loss of KCNQ1 markedly prolonged APD ₉₀ . We sought here to define mechanisms underlying APD prolongation by genetic loss of KCNQ1 . Methods: We studied iPSC-CMs from population controls, an isogenic KCNQ1 knock out (KO) line created by a homozygous edit for the R518X loss of function variant, and 2 unrelated patients with the Jervell and Lange-Nielsen syndrome (JLN) due to compound heterozygosity for loss of function KCNQ1 variants. Results: In both JLN and the KCNQ1-KO lines, I _Ks was absent, APD ₉₀ was markedly prolonged, and L-type Ca channel (LTCC) current ( I _Ca-L ) was significantly increased, 2-3-fold, compared to the control cells with no change in kinetics or gating. RNA-sequencing identified 298 and 584 genes that were up– and down-regulated, respectively, by KCNQ1-KO compared to the isogenic control cells. Gene ontology analysis identified down-regulation of 6 Ca ²⁺ channel negative regulatory genes (p=0.0002, FDR=0.02), and in knockdown experiments in wild-type iPSC-CMs, three of these, CBARP , FKBP1B , and RRAD , increased I _Ca-L , and RRAD increased APD ₉₀ . A therapeutic low concentration (1 μM) of the Ca channel antagonist diltiazem significantly shortened APD ₉₀ in the two JLN cell lines and in KCNQ1-KO cells. A single low dose of intravenous diltiazem in one of the JLN patients shortened QTc. Conclusions: These data further support the concept that delayed repolarization in JLN cannot be explained solely by loss of I _Ks . Our findings demonstrate that KCNQ1 mutations lead to down-regulation of Ca ²⁺ channel inhibitory genes, with resultant increased I _Ca-L that underlies delayed repolarization in JLN. We further propose that diltiazem can be repurposed for treatment of patients with JLN.

BACKGROUND: Long QT syndrome is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2 . Variant classification is difficult, often because of lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes. METHODS: We quantified cell-surface trafficking of 18 796 variants in KCNH2 using a multiplexed assay of variant effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping. We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian long QT syndrome penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events. RESULTS: Variant MAVE trafficking scores and automated patch clamping peak tail currents were highly correlated (Spearman rank-order ρ=0.69; n=433). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian long QT syndrome penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and corrected QT interval (QTc); however, MAVE data became nonsignificant when peak tail current and penetrance estimates were also available. The area under the receiver operator characteristic curve for 20-year event outcomes based on patient-specific sex and QTc (area under the curve, 0.80 [0.76–0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (area under the curve, 0.86 [0.83–0.89]) or attainable automated patch clamping peak tail current data (area under the curve, 0.84 [0.81–0.88]). CONCLUSIONS: High-throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale, whereas long QT syndrome penetrance estimates and automated patch clamping peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.

While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small I_Ks in cardiomyocytes from human induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss of function KCNQ1 variants. We compared the effects of pharmacologic I_Ks block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 loss of function allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of I_Kr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small I_Ks was readily recorded but pharmacologic I_Ks block produced no change in action potential duration at 90% repolarization (APD₉₀). By contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD₉₀ was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16 ms). JLN cells displayed increased sensitivity to acute I_Kr block: the concentration (μM) of moxifloxacin required to prolong APD₉₀ 100 msec was 237.4 (median, IQR 100.6-391.6, n = 7) in population cells versus 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300μM) mildly prolonged APD₉₀ (10%) and increased I_Ks, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in I_Ks. However, in the siRNA-treated cells, moxifloxacin did not increase I_Ks, and markedly prolonged APD₉₀. Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to I_Kr block, through mechanisms beyond simply generating I_Ks. Mutations in KCNQ1 – whose expression generates I_Ks – are the major cause of long QT syndrome. We report here that while pharmacologic I_Ks block in human cardiomyocytes generates minimal change in repolarization, suppressing KCNQ1 expression markedly increases both baseline repolarization duration and sensitivity to some (but not all) specific I_Kr blockers. Thus, beyond simply generating I_Ks, KCNQ1 subserves critical additional role(s) in repolarization control at baseline and in response to I_Kr block. Our findings imply that assessment of arrhythmic risk in individual patients and by drugs requires a framework that extends beyond a simple one gene_-one ion current paradigm.

Background: We identified a novel SCN5A variant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide. This clinical picture is consistent with multifocal ectopic Purkinje-related premature contractions (MEPPC). Most previous reports of MEPPC have implicated SCN5A variants such as R222Q that neutralize positive charges in the S4 voltage sensor helix of the channel protein Na _V 1.5 and generate a gating pore current. Methods and Results: E171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of Na _V 1.5 domain I. E171 is a key component of the Gating Charge Transfer Center, a region thought to be critical for normal movement of the S4 voltage sensor helix. We used heterologous expression, CRISPR-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and molecular dynamics simulations to demonstrate that E171Q generates a gating pore current, which was suppressed by a low concentration of flecainide (IC50 = 0.71±0.07 µM). R222Q shifts voltage dependence of activation and inactivation in a negative direction but we observed positive shifts with E171Q. E171Q iPSC-CMs demonstrated abnormal spontaneous activity and prolonged action potentials. Molecular dynamics simulations revealed that both R222Q and E171Q proteins generate a water-filled permeation pathway that underlies generation of the gating pore current. Conclusion: Previously identified MEPPC-associated variants that create gating pore currents are located in positively-charged residues in the S4 voltage sensor and generate negative shifts in the voltage dependence of activation and inactivation. We demonstrate that neutralizing a negatively charged S2 helix residue in the Gating Charge Transfer Center generates positive shifts but also create a gating pore pathway. These findings implicate the gating pore pathway as the primary functional and structural determinant of MEPPC and widen the spectrum of variants that are associated with gating pore-related disease in voltage-gated ion channels.

More than one-third of type 2 long QT syndrome (LQT2) patients carry KCNH2 non-missense variants that can result in haploinsufficiency (HI), leading to mechanistic loss-of-function. However, their clinical phenotypes have not been fully investigated. The remaining two-thirds of patients harbour missense variants, and past studies uncovered that most of these variants cause trafficking deficiency, resulting in different functional changes: either HI or dominant-negative (DN) effects. In this study, we examined the impact of altered molecular mechanisms on clinical outcomes in LQT2 patients. We included 429 LQT2 patients (234 probands) carrying a rare KCNH2 variant from our patient cohort undergoing genetic testing. Non-missense variants showed shorter corrected QT (QTc) and less arrhythmic events (AEs) than missense variants. We found that 40% of missense variants in this study were previously reported as HI or DN. Non-missense and HI-groups had similar phenotypes, while both exhibited shorter QTc and less AEs than the DN-group. Based on previous work, we predicted the functional change of the unreported variants—whether they cause HI or DN via altered functional domains—and stratified them as predicted HI (pHI)- or pDN-group. The pHI-group including non-missense variants exhibited milder phenotypes compared to the pDN-group. Multivariable Cox model showed that the functional change was an independent risk of AEs (P = 0.005). Stratification based on molecular biological studies enables us to better predict clinical outcomes in the patients with LQT2. AE, arrhythmia event; CI, confidence interval; CNBHD, cyclic nucleotide-binding homology domain; DN, dominant-negative; EAGD, ether-à-go-go domain; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; HR, hazard ratio; HI, haploinsufficiency; LOF, loss-of-function; LQT2, long QT syndrome type 2; mRNA, messenger RNA; NMD, nonsense mediated mRNA decay; PD, pore domain; pDN, predicted DN; pHI, predicted HI; PTC, premature termination codon; QTc, corrected QT interval; VSD, voltage sensor domain; WT, wild-type.

Background: Rare protein-altering variants in SCN5A , KCNQ1 , and KCNH2 are major causes of Brugada syndrome and the congenital long QT syndrome. While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described. We implemented 2 functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance and 1 likely pathogenic variant without functional data observed in Brugada syndrome and long QT syndrome probands. Methods: We deployed minigene assays to assess the splicing consequences of 10 variants. Three variants incompatible with the minigene approach were introduced into control induced pluripotent stem cells by CRISPR genome editing. We differentiated cells into induced pluripotent stem cell–derived cardiomyocytes and studied splicing outcomes by reverse transcription-polymerase chain reaction. We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants. Results: We identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in induced pluripotent stem cell-derived cardiomyocytes. We reclassified 8 variants of uncertain significance to likely pathogenic, 1 variant of uncertain significance to likely benign, and 1 likely pathogenic variant to pathogenic. Conclusions: Functional assays reclassified splice-altering variants outside canonical splice sites in Brugada Syndrome- and long QT syndrome-associated genes.