Minoru Horie

SCN5A-E1784K (c.5350G>A) is the most common variant associated with the long QT (LQTS) and Brugada syndromes (BrS). It can manifest variably as LQTS, BrS and/or conduction disorders. This presents a challenge for risk stratification. We aimed to describe clinical and ECG characteristics and identify risk markers that associate with arrhythmic events. We undertook a retrospective observational multicentre study of a large cohort of 231 subjects with SCN5A-E1784K from Europe, USA and Japan. Comprehensive demographic and clinical data, including initial presentation ECG and follow-up, were collected. ‘Lethal events’ were defined as sudden death, non-fatal cardiac arrest, and documented sustained VT or VF. ‘Cardiac events’ were defined as arrhythmic syncope plus any lethal events. Clinical characteristics and ECG parameters corrected for age were investigated for association with lethal and/or cardiac events. Fourteen (6%) subjects experienced a lethal event and 45 (19%) a cardiac event. PR interval and QRS duration were associated with lethal and cardiac events. In multivariable models, both PR interval and QRS duration were associated with lethal events but only QRS duration was associated with cardiac events. Only age-corrected QRS (rQRS) was associated with lethal and cardiac event-free survival from birth after correction for multiple testing. Ventricular myocardial conduction appears likely to play a role in the risk of arrhythmic events in patients with SCN5A-E1784K. This provides an important opportunity for the personalisation of management and has the potential to guide preventative therapies.

BACKGROUND: Pathogenic SCN5A variants are associated with inherited arrhythmias such as long-QT syndrome, Brugada syndrome, and sick sinus syndrome. While Na _v 1.5, an α-subunit of the cardiac sodium channel encoded by SCN5A , has been considered to function as a monomer, recent studies reveal that a reduction of sodium current in wild-type Na _v 1.5 can be caused by dimerization with loss-of-function mutated Na _v 1.5 through dominant-negative effects. However, the clinical significance of the dominant-negative effect remains unclear. METHOD: We genetically screened a family who presented with sick sinus syndrome and sudden cardiac death. Whole-cell patch-clamp study using HEK293 (human embryonic kidney) cells coexpressing wild-type and variant SCN5A was performed. Channel dimerization was assessed by coimmunoprecipitation and proximity ligation assays. Also, the effects of difopein, a high-affinity inhibitor of Na _v 1.5 interaction via 14-3-3 proteins, were evaluated. RESULTS: The proband carried compound heterozygous variants p.T1396P and p.G833R. The whole-cell mode patch-clamp techniques demonstrated that the p.T1396P showed a dominant-negative effect on the peak sodium currents (37% decrease in I _Na ) and altered gating properties (5.6-mV shift in steady-state inactivation) when expressed with wild-type SCN5A . These effects were abolished by difopein. p.G833R showed no dominant-negative or coupled gating effect but still formed dimers. The proband developed earlier and more severe bradycardia than the mother, who only carries p.T1396P, suggesting that loss of coupled gating effect contributed to the severe phenotype. CONCLUSIONS: Our findings suggest that coupled gating may be physiologically important for normal Na _v 1.5 function, and its loss can exacerbate disease severity.

Background and Aims: Patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) are at risk for potentially life-threatening arrhythmic events (AEs) even while treated with β-blockers. The aim was to develop a model for individualized prediction of AEs in patients with RYR2-mediated CPVT on β-blocker monotherapy. Methods: The derivation and independent validation cohorts included 743 and 129 patients, respectively. AEs were defined as arrhythmic syncope, appropriate implantable cardioverter–defibrillator shock, sudden cardiac arrest (SCA), and sudden cardiac death. Near-fatal or fatal AEs (nf/fAEs) included all AEs except for arrhythmic syncope. Prediction models using Cox regression were developed and internally and externally validated. Results: A total of 102 (13.7%) patients in the derivation cohort and 24 (18.6%) patients in the validation cohort experienced ≥1 AE over a median follow-up of 5.1 [interquartile range (IQR), 7.7] and 2.4 (IQR, 4.4) years, respectively. Predictors of AE were arrhythmic syncope or SCA prior to diagnosis and age at β-blocker initiation. In the derivation and validation cohorts, the optimism-corrected C-indices of the models for AE were 0.67 [95% confidence interval (CI) 0.62–0.72] and 0.59 (95% CI 0.48–0.71), respectively. For nf/fAEs, ventricular arrhythmia severity before β-blocker initiation was a fourth independent predictor, and C-indices of the models in the derivation and validation cohorts were 0.74 (95% CI 0.68–0.80) and 0.60 (95% CI 0.47–0.72), respectively. In the derivation cohort, calibration slopes were 1.00 (95% CI 0.59–1.41) for AE and 1.00 (95% CI 0.69–1.32) for nf/fAE. Conclusions: These externally validated risk prediction models using clinical parameters accurately distinguished CPVT patients on β-blocker monotherapy at low and high risk for future AEs while treated with β-blockers. These models provide guidance for implementation of clinical management therapies to prevent AEs in patients with CPVT.

Sudden unexpected death in epilepsy (SUDEP) is one of the most frequent causes of death in patients with epilepsy, though the pathogenesis of SUDEP has not been well elucidated. Here, we report novel heterozygous KCND3 variants, p.V401L and p.V401M, identified in young patients with refractory epilepsy (RE) and neurodevelopmental disorders, and the functional properties of these variants. We aimed to investigate the electrophysiological changes in de novo KCND3 variants and analyse the pharmacological effects of quinidine on these variants. Chinese hamster ovary (CHO) cells were transiently co-transfected with wild-type (WT) and/or variant KCND3 and Kcnip2. Transient outward potassium currents (I_to) were recorded using the whole-cell patch-clamp method. The inhibitory effect of quinidine on I_to was evaluated. In electrophysiological analysis, CHO cells expressing the variant channels showed a significant increase in current density compared with those expressing WT channels. The I_to activation curves were shifted significantly to the left, and significantly slower inactivation time constants were observed in both variant channels. Recovery from inactivation of the variant channels was significantly slower than that of WT. Quinidine suppressed I_to in a concentration-dependent manner and accelerated the slow inactivation of variant channels. In conclusion, de novo KCND3 variants identified in patients with RE and neurodevelopmental disorders showed gain and loss of function effects on I_to. These patients may be at risk of developing early repolarization syndrome, leading to SUDEP. Increased I_to was suppressed by quinidine, suggesting that it may be an effective therapy for RE and possibly for preventing SUDEP.

Background: Pathogenic SCN5A variants are associated with inherited arrhythmias such as long QT syndrome, Brugada syndrome, and sick sinus syndrome (SSS). While Na _v 1.5, an α-subunit of the cardiac sodium channel encoded by SCN5A , has been considered to function as a monomer, recent studies reveal that a reduction of sodium current in wild-type (WT) Na _v 1.5 can be caused by dimerization with loss-of-function (LOF) mutated Na _v 1.5 through dominant-negative (DN) effects. However, the clinical significance of the DN effect remains unclear. Method: We genetically screened a family who presented with SSS and sudden cardiac death (SCD). Whole-cell patch-clamp study using HEK293 cells co-expressing WT- and variant SCN5A was performed. Channel dimerization was assessed by co-immunoprecipitation and proximity ligation assays. Also, the effects of difopein, a high affinity inhibitor of Na _v 1.5 interaction via 14-3-3 proteins, were evaluated. Results: The proband carried compound heterozygous variants p.T1396P and p.G833R. The whole-cell mode patch-clamp techniques demonstrated that the p.T1396P showed a DN effect on the peak sodium currents (37% decrease in I _Na ) and altered gating properties (+5.6 mV shift in steady-state inactivation) when expressed with WT SCN5A . These effects were abolished by difopein. p.G833R showed no DN or coupled gating effect but still formed dimers. The proband developed earlier and more severe bradycardia than her parent, who only carries p.T1396P, suggesting that loss of coupled gating effect contributed to the severe phenotype. Conclusion: Our findings suggest that coupled gating may be physiologically important for normal Na _v 1.5 function, and that its loss can exacerbate disease severity.

Aims: The variant in SCN5A with the loss of function (LOF) effect in the cardiac Na+ channel (Nav1.5) is the definitive cause for Brugada syndrome (BrS), and the functional analysis data revealed that LOF variants are associated with poor prognosis. However, which variant types (e.g. missense or non-missense) affect the prognoses of those variant carriers remain unelucidated. Methods and results: We defined SCN5A LOF variants as all non-missense and missense variants that produce peak INa < 65% of wild-type previously confirmed by patch-clamp studies. The study population consisted of 76 Japanese BrS patients (74% patients were male and the median age [IQR] at diagnosis was 28 [14–45] years) with LOF type of SCN5A variants: 40 with missense and 36 with non-missense variants. Non-missense variant carriers presented significantly more severe cardiac conduction disorder compared to the missense variant carriers. During follow-up periods of 9.0 [5.0–14.0] years, compared to missense variants, non-missense variants were significant risk factors of lifetime lethal arrhythmia events (LAEs) (P = 0.023). When focusing only on the missense variants that produce no peak INa, these missense variant carriers exhibited the same clinical outcomes as those with non-missense (log-rank P = 0.325). After diagnosis, however, both variant types were comparable in risk of LAEs (P = 0.155). Conclusion: We identified, for the first time, that SCN5A non-missense variants were associated with higher probability of LAE than missense variants in BrS patients though it did not change significantly after diagnosis.

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.