Assays of Variant Effect and Automated Patch Clamping Improve KCNH2-LQTS Variant Classification and Cardiac Event Risk Stratification.

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 adjusted 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.

Brugada syndrome in Japan and Europe: a genome-wide association study reveals shared genetic architecture and new risk loci.

BACKGROUND AND AIMS: Brugada syndrome (BrS) is an inherited arrhythmia with a higher disease prevalence and more lethal arrhythmic events in Asians than in Europeans. Genome-wide association studies (GWAS) have revealed its polygenic architecture mainly in European populations. The aim of this study was to identify novel BrS-associated loci and to compare allelic effects across ancestries. METHODS: A GWAS was conducted in Japanese participants, involving 940 cases and 1634 controls, followed by a cross-ancestry meta-analysis of Japanese and European GWAS (total of 3760 cases and 11 635 controls). The novel loci were characterized by fine-mapping, gene expression, and splicing quantitative trait associations in the human heart. RESULTS: The Japanese-specific GWAS identified one novel locus near ZSCAN20 (P = 1.0 × 10-8), and the cross-ancestry meta-analysis identified 17 association signals, including six novel loci. The effect directions of the 17 lead variants were consistent (94.1%; P for sign test = 2.7 × 10-4), and their allelic effects were highly correlated across ancestries (Pearson's R = .91; P = 2.9 × 10-7). The genetic risk score derived from the BrS GWAS of European ancestry was significantly associated with the risk of BrS in the Japanese population [odds ratio 2.12 (95% confidence interval 1.94-2.31); P = 1.2 × 10-61], suggesting a shared genetic architecture across ancestries. Functional characterization revealed that a lead variant in CAMK2D promotes alternative splicing, resulting in an isoform switch of calmodulin kinase II-δ, favouring a pro-inflammatory/pro-death pathway. CONCLUSIONS: This study demonstrates novel susceptibility loci implicating potentially novel pathogenesis underlying BrS. Despite differences in clinical expressivity and epidemiology, the polygenic architecture of BrS was substantially shared across ancestries.

Flecainide Is Associated With a Lower Incidence of Arrhythmic Events in a Large Cohort of Patients With Catecholaminergic Polymorphic Ventricular Tachycardia.

BACKGROUND: In severely affected patients with catecholaminergic polymorphic ventricular tachycardia, beta-blockers are often insufficiently protective. The purpose of this study was to evaluate whether flecainide is associated with a lower incidence of arrhythmic events (AEs) when added to beta-blockers in a large cohort of patients with catecholaminergic polymorphic ventricular tachycardia. METHODS: From 2 international registries, this multicenter case cross-over study included patients with a clinical or genetic diagnosis of catecholaminergic polymorphic ventricular tachycardia in whom flecainide was added to beta-blocker therapy. The study period was defined as the period in which background therapy (ie, beta-blocker type [beta1-selective or nonselective]), left cardiac sympathetic denervation, and implantable cardioverter defibrillator treatment status, remained unchanged within individual patients and was divided into pre-flecainide and on-flecainide periods. The primary end point was AEs, defined as sudden cardiac death, sudden cardiac arrest, appropriate implantable cardioverter defibrillator shock, and arrhythmic syncope. The association of flecainide with AE rates was assessed using a generalized linear mixed model assuming negative binomial distribution and random effects for patients. RESULTS: A total of 247 patients (123 [50%] females; median age at start of flecainide, 18 years [interquartile range, 14-29]; median flecainide dose, 2.2 mg/kg per day [interquartile range, 1.7-3.1]) were included. At baseline, all patients used a beta-blocker, 70 (28%) had an implantable cardioverter defibrillator, and 21 (9%) had a left cardiac sympathetic denervation. During a median pre-flecainide follow-up of 2.1 years (interquartile range, 0.4-7.2), 41 patients (17%) experienced 58 AEs (annual event rate, 5.6%). During a median on-flecainide follow-up of 2.9 years (interquartile range, 1.0-6.0), 23 patients (9%) experienced 38 AEs (annual event rate, 4.0%). There were significantly fewer AEs after initiation of flecainide (incidence rate ratio, 0.55 [95% CI, 0.38-0.83]; P=0.007). Among patients who were symptomatic before diagnosis or during the pre-flecainide period (n=167), flecainide was associated with significantly fewer AEs (incidence rate ratio, 0.49 [95% CI, 0.31-0.77]; P=0.002). Among patients with ≥1 AE on beta-blocker therapy (n=41), adding flecainide was also associated with significantly fewer AEs (incidence rate ratio, 0.25 [95% CI, 0.14-0.45]; P<0.001). CONCLUSIONS: For patients with catecholaminergic polymorphic ventricular tachycardia, adding flecainide to beta-blocker therapy was associated with a lower incidence of AEs in the overall cohort, in symptomatic patients, and particularly in patients with breakthrough AEs while on beta-blocker therapy.

Clinical presentation of calmodulin mutations: the International Calmodulinopathy Registry.

AIMS: Calmodulinopathy due to mutations in any of the three CALM genes (CALM1-3) causes life-threatening arrhythmia syndromes, especially in young individuals. The International Calmodulinopathy Registry (ICalmR) aims to define and link the increasing complexity of the clinical presentation to the underlying molecular mechanisms. METHODS AND RESULTS: The ICalmR is an international, collaborative, observational study, assembling and analysing clinical and genetic data on CALM-positive patients. The ICalmR has enrolled 140 subjects (median age 10.8 years [interquartile range 5-19]), 97 index cases and 43 family members. CALM-LQTS and CALM-CPVT are the prevalent phenotypes. Primary neurological manifestations, unrelated to post-anoxic sequelae, manifested in 20 patients. Calmodulinopathy remains associated with a high arrhythmic event rate (symptomatic patients, n = 103, 74%). However, compared with the original 2019 cohort, there was a reduced frequency and severity of all cardiac events (61% vs. 85%; P = .001) and sudden death (9% vs. 27%; P = .008). Data on therapy do not allow definitive recommendations. Cardiac structural abnormalities, either cardiomyopathy or congenital heart defects, are present in 30% of patients, mainly CALM-LQTS, and lethal cases of heart failure have occurred. The number of familial cases and of families with strikingly different phenotypes is increasing. CONCLUSION: Calmodulinopathy has pleiotropic presentations, from channelopathy to syndromic forms. Clinical severity ranges from the early onset of life-threatening arrhythmias to the absence of symptoms, and the percentage of milder and familial forms is increasing. There are no hard data to guide therapy, and current management includes pharmacological and surgical antiadrenergic interventions with sodium channel blockers often accompanied by an implantable cardioverter-defibrillator.

An International Multicenter Cohort Study on ß-Blockers for the Treatment of Symptomatic Children With Catecholaminergic Polymorphic Ventricular Tachycardia.

BACKGROUND: Symptomatic children with catecholaminergic polymorphic ventricular tachycardia (CPVT) are at risk for recurrent arrhythmic events. ß-Blockers decrease this risk, but studies comparing individual ß-blockers in sizeable cohorts are lacking. We aimed to assess the association between risk for arrhythmic events and type of ß-blocker in a large cohort of symptomatic children with CPVT. METHODS: From 2 international registries of patients with CPVT, RYR2 variant-carrying symptomatic children (defined as syncope or sudden cardiac arrest before ß-blocker initiation and age at start of ß-blocker therapy <18 years), treated with a ß-blocker were included. Cox regression analyses with time-dependent covariates for ß-blockers and potential confounders were used to assess the hazard ratio (HR). The primary outcome was the first occurrence of sudden cardiac death, sudden cardiac arrest, appropriate implantable cardioverter-defibrillator shock, or syncope. The secondary outcome was the first occurrence of any of the primary outcomes except syncope. RESULTS: We included 329 patients (median age at diagnosis, 12 [interquartile range, 7-15] years, 35% females). Ninety-nine (30.1%) patients experienced the primary outcome and 74 (22.5%) experienced the secondary outcome during a median follow-up of 6.7 (interquartile range, 2.8-12.5) years. Two-hundred sixteen patients (66.0%) used a nonselective ß-blocker (predominantly nadolol [n=140] or propranolol [n=70]) and 111 (33.7%) used a ß1-selective ß-blocker (predominantly atenolol [n=51], metoprolol [n=33], or bisoprolol [n=19]) as initial ß-blocker. Baseline characteristics did not differ. The HRs for both the primary and secondary outcomes were higher for ß1-selective compared with nonselective ß-blockers (HR, 2.04 [95% CI, 1.31-3.17]; and HR, 1.99 [95% CI, 1.20-3.30], respectively). When assessed separately, the HR for the primary outcome was higher for atenolol (HR, 2.68 [95% CI, 1.44-4.99]), bisoprolol (HR, 3.24 [95% CI, 1.47-7.18]), and metoprolol (HR, 2.18 [95% CI, 1.08-4.40]) compared with nadolol, but did not differ from propranolol. The HR of the secondary outcome was only higher in atenolol compared with nadolol (HR, 2.68 [95% CI, 1.30-5.55]). CONCLUSIONS: ß1-selective ß-blockers were associated with a significantly higher risk for arrhythmic events in symptomatic children with CPVT compared with nonselective ß-blockers, specifically nadolol. Nadolol, or propranolol if nadolol is unavailable, should be the preferred ß-blocker for treating symptomatic children with CPVT.

Continuous Bayesian variant interpretation accounts for incomplete penetrance among Mendelian cardiac channelopathies.

PURPOSE: The congenital Long QT Syndrome (LQTS) and Brugada Syndrome (BrS) are Mendelian autosomal dominant diseases that frequently precipitate fatal cardiac arrhythmias. Incomplete penetrance is a barrier to clinical management of heterozygotes harboring variants in the major implicated disease genes KCNQ1, KCNH2, and SCN5A. We apply and evaluate a Bayesian penetrance estimation strategy that accounts for this phenomenon. METHODS: We generated Bayesian penetrance models for KCNQ1-LQT1 and SCN5A-LQT3 using variant-specific features and clinical data from the literature, international arrhythmia genetic centers, and population controls. We analyzed the distribution of posterior penetrance estimates across 4 genotype-phenotype relationships and compared continuous estimates with ClinVar annotations. Posterior estimates were mapped onto protein structure. RESULTS: Bayesian penetrance estimates of KCNQ1-LQT1 and SCN5A-LQT3 are empirically equivalent to 10 and 5 clinically phenotype heterozygotes, respectively. Posterior penetrance estimates were bimodal for KCNQ1-LQT1 and KCNH2-LQT2, with a higher fraction of missense variants with high penetrance among KCNQ1 variants. There was a wide distribution of variant penetrance estimates among identical ClinVar categories. Structural mapping revealed heterogeneity among "hot spot" regions and featured high penetrance estimates for KCNQ1 variants in contact with calmodulin and the S6 domain. CONCLUSIONS: Bayesian penetrance estimates provide a continuous framework for variant interpretation.

Non-missense variants of KCNH2 show better outcomes in type 2 long QT syndrome.

AIMS: 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. METHODS AND RESULTS: 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). CONCLUSION: Stratification based on molecular biological studies enables us to better predict clinical outcomes in the patients with LQT2.

Holter Electrocardiographic Approach to Predicting Outcomes of Pediatric Patients With Long QT Syndrome.

BACKGROUND: This study was performed to clarify the clinical findings of pediatric patients diagnosed with long QT syndrome (LQTS) through electrocardiographic screening programs and to predict their outcome using Holter electrocardiographic approaches.Methods and Results: This retrospective study included pediatric patients with a Schwartz score of ≥3.5 who visited the National Hospital Organization Kagoshima Medical Center between April 2005 and March 2019. Resting 12-lead and Holter electrocardiograms were recorded at every visit. The maximum resting QTc and maximum Holter QTc values among all recordings were used for statistical analyses. To test the prognostic value of QTc for the appearance of cardiac events after the first hospital visit, receiver operating characteristic curves were used to calculate the area under the curve (AUC). Among 207 patients, 181 (87%) were diagnosed through screening programs. The prevalence of cardiac events after the first hospital visit was 4% (8/207). Among QTc at diagnosis, maximum resting QTc, and maximum Holter QTc, only maximum Holter QTc value was a predictor (P=0.02) of cardiac events after the hospital visit in multivariate regression analysis. The AUC of the maximum Holter QTc was significantly superior to that of maximum resting QTc. CONCLUSIONS: The maximum Holter QTc value can be used to predict the appearance of symptoms in pediatric patients with LQTS.

Calmodulinopathy in Japanese Children　- Their Cardiac Phenotypes Are Severe and Show Early Onset in Fetal Life and Infancy.

BACKGROUND: Cardiac calmodulinopathy, characterized by a life-threatening arrhythmia and sudden death in the young, is extremely rare and caused by genes encoding calmodulin, namely calmodulin 1 (CALM1), CALM2, and CALM3.Methods and Results: We screened 195 symptomatic children (age 0-12 years) who were suspected of inherited arrhythmias for 48 candidate genes, using a next-generation sequencer. Ten probands were identified as carrying variants in any of CALM1-3 (5%; median age 5 years), who were initially diagnosed with long QT syndrome (LQTS; n=5), catecholaminergic polymorphic ventricular tachycardia (CPVT; n=3), and overlap syndrome (n=2). Two probands harbored a CALM1 variant and 8 probands harbored 6 CALM2 variants. There were 4 clinical phenotypes: (1) documented lethal arrhythmic events (LAEs): 4 carriers of N98S in CALM1 or CALM2; (2) suspected LAEs: CALM2 p.D96G and D132G carriers experienced syncope and transient cardiopulmonary arrest under emotional stimulation; (3) critical cardiac complication: CALM2 p.D96V and p.E141K carriers showed severe cardiac dysfunction with QTc prolongation; and (4) neurological and developmental disorders: 2 carriers of CALM2 p.E46K showed cardiac phenotypes of CPVT. Beta-blocker therapy was effective in all cases except cardiac dysfunction, especially in combination with flecainide (CPVT-like phenotype) and mexiletine (LQTS-like). CONCLUSIONS: Calmodulinopathy patients presented severe cardiac features, and their onset of LAEs was earlier in life, requiring diagnosis and treatment at the earliest age possible.

School-based routine screenings of electrocardiograms for the diagnosis of long QT syndrome.

AIMS: School-based routine screenings of electrocardiograms (ECGs) have been performed upon admission to primary school (PS), junior high school (JHS), and high school (HS) in Japan. Though ECGs with prolonged QT intervals are occasionally found, the role of regular ECG screening tests in identifying long QT syndrome (LQTS) remains to be determined. We investigated the usefulness of the ECG screenings by comparing the results of genetic tests between students who showed QT-prolongation in the screenings and patients with LQTS. METHODS AND RESULTS: We genetically screened 341 students (106 PS, 173 JHS, and 62 HS). Of these, 230 subjects showed QT-prolongation during regular screenings (S-S group), and the other 111 patients were clinically consulted with suspected LQTS by paediatricians (C-C group). Genotype-phenotype relationships were compared between the two groups. The positive rates in the genetic tests were comparable among the two groups; however, symptomatic subjects were significantly fewer in the S-S group than the C-C group (3% vs. 70%). Compared to the genotype-negative subjects, the positive subjects showed significantly longer QTc (P < 0.0001) and more frequently presented LQTS risk scores with ≥3.5 points (P < 0.0001). Lethal arrhythmic events (LAE) occurred only in the C-C group; 18 subjects experienced LAE and 83% of them were found to carry variant(s) in the LQTS-related genes. CONCLUSION: The school-based ECG screenings are effective in identifying young patients with LQTS who require genetic analysis. If individuals are screened at a younger age, we can identify patients at risk earlier and provide preventative treatments.

Loss-of-function mutations in cardiac ryanodine receptor channel cause various types of arrhythmias including long QT syndrome.

AIMS: Gain-of-function mutations in RYR2, encoding the cardiac ryanodine receptor channel (RyR2), cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Whereas, genotype-phenotype correlations of loss-of-function mutations remains unknown, due to a small number of analysed mutations. In this study, we aimed to investigate their genotype-phenotype correlations in patients with loss-of-function RYR2 mutations. METHODS AND RESULTS: We performed targeted gene sequencing for 710 probands younger than 16-year-old with inherited primary arrhythmia syndromes (IPAS). RYR2 mutations were identified in 63 probands, and 3 probands displayed clinical features different from CPVT. A proband with p.E4146D developed ventricular fibrillation (VF) and QT prolongation whereas that with p.S4168P showed QT prolongation and bradycardia. Another proband with p.S4938F showed short-coupled variant of torsade de pointes (scTdP). To evaluate the functional alterations in these three mutant RyR2s and p.K4594Q previously reported in a long QT syndrome (LQTS), we measured Ca2+ signals in HEK293 cells and HL-1 cardiomyocytes as well as Ca2+-dependent [3H]ryanodine binding. All mutant RyR2s demonstrated a reduced Ca2+ release, an increased endoplasmic reticulum Ca2+, and a reduced [3H]ryanodine binding, indicating loss-of-functions. In HL-1 cells, the exogenous expression of S4168P and K4594Q reduced amplitude of Ca2+ transients without inducing Ca2+ waves, whereas that of E4146D and S4938F evoked frequent localized Ca2+ waves. CONCLUSION: Loss-of-function RYR2 mutations may be implicated in various types of arrhythmias including LQTS, VF, and scTdP, depending on alteration of the channel activity. Search of RYR2 mutations in IPAS patients clinically different from CPVT will be a useful strategy to effectively discover loss-of-function RYR2 mutations.

Functionally validated SCN5A variants allow interpretation of pathogenicity and prediction of lethal events in Brugada syndrome.

AIMS: The prognostic value of genetic variants for predicting lethal arrhythmic events (LAEs) in Brugada syndrome (BrS) remains controversial. We investigated whether the functional curation of SCN5A variations improves prognostic predictability. METHODS AND RESULTS: Using a heterologous expression system and whole-cell patch clamping, we functionally characterized 22 variants of unknown significance (VUSs) among 55 SCN5A mutations previously curated using in silico prediction algorithms in the Japanese BrS registry (n = 415). According to the loss-of-function (LOF) properties, SCN5A mutation carriers (n = 60) were divided into two groups: LOF-SCN5A mutations and non-LOF SCN5A variations. Functionally proven LOF-SCN5A mutation carriers (n = 45) showed significantly severer electrocardiographic conduction abnormalities and worse prognosis associated with earlier manifestations of LAEs (7.9%/year) than in silico algorithm-predicted SCN5A carriers (5.1%/year) or all BrS probands (2.5%/year). Notably, non-LOF SCN5A variation carriers (n = 15) exhibited no LAEs during the follow-up period. Multivariate analysis demonstrated that only LOF-SCN5A mutations and a history of aborted cardiac arrest were significant predictors of LAEs. Gene-based association studies using whole-exome sequencing data on another independent SCN5A mutation-negative BrS cohort (n = 288) showed no significant enrichment of rare variants in 16 985 genes including 22 non-SCN5A BrS-associated genes as compared with controls (n = 372). Furthermore, rare variations of non-SCN5A BrS-associated genes did not affect LAE-free survival curves. CONCLUSION: In vitro functional validation is key to classifying the pathogenicity of SCN5A VUSs and for risk stratification of genetic predictors of LAEs. Functionally proven LOF-SCN5A mutations are genetic burdens of sudden death in BrS, but evidence for other BrS-associated genes is elusive.

Oral Adrenergic Agents Produced Ventricular Fibrillation and QT Prolongation in an Elderly Patient Carrying an RYR2 Variant.

Mutant cardiac ryanodine receptor channels (RyR2) are "leaky," and spontaneous Ca2+ release through these channels causes delayed afterdepolarizations that can deteriorate into ventricular fibrillation. Some patients carrying RYR2 mutations in type 1 catecholaminergic polymorphic ventricular tachycardia exhibit QT prolongation and are initially diagnosed with long QT syndrome. However, none have been reported to cause drug-induced ventricular fibrillation in patients with RYR2 variants. We describe the first case of an elderly woman with drug-induced QT prolongation and ventricular fibrillation who carried a novel RYR2 variant but no other mutations related to long QT syndrome. Oral adrenergic agents might induce QT prolongation and subsequent ventricular fibrillation in patients carrying an RYR2 variant. Screening for RYR2 could be valuable in patients with suspected drug-induced long QT syndrome.

Identification of transmembrane protein 168 mutation in familial Brugada syndrome.

Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na+ current and Nav 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na+ current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Nav 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Nav 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.

Novel electrocardiographic criteria for short QT syndrome in children and adolescents.

AIMS: Although shortening of the corrected QT interval (QTc) is a key finding in the diagnosis of short QT syndrome (SQTS), there may be overlap of the QTc between SQTS patients and normal subjects in childhood and adolescence. We aimed to investigate electrocardiographic findings for differentiation of SQTS patients. METHODS AND RESULTS: The SQTS group comprised 34 SQTS patients <20 years old, including 9 from our institutions and 25 from previous reports. The control group comprised 61 apparently healthy subjects with an QTc of <360 ms who were selected from 13 314 participants in a school-based screening programme. We compared electrocardiographic findings, including QT and Jpoint-Tpeak intervals (QT and J-Tpeak, respectively), those corrected by using the Bazett's and Fridericia's formulae (cB and cF, respectively) and early repolarization (ER) between the groups. QT, QTc by using Bazett's formula (QTcB), QTc by using Fridericia's formula (QTcF), J-Tpeak, J-Tpeak cB, and J-Tpeak cF were significantly shorter in the SQTS group than in the control group. On receiver operating characteristic curve analysis, the area under the curve (AUC) was largest for QTcB (0.888) among QT, QTcB, and QTcF, with a cut-off value of 316 ms (sensitivity: 79.4% and specificity: 96.7%). The AUC was largest for J-Tpeak cB (0.848) among J-Tpeak, J-Tpeak cB, and J-Tpeak cF, with a cut-off value of 181 ms (sensitivity: 80.8% and specificity: 91.8%). Early repolarization was found more frequently in the SQTS group than in the control group (67% vs. 23%, P = 0.001). CONCLUSION: A QTcB <316 ms, J-Tpeak cB < 181 ms, and the presence of ER may indicate SQTS patients in childhood and adolescence.

Electrocardiographic Diagnosis of Hypertrophic Cardiomyopathy in the Pre- and Post-Diagnostic Phases in Children and Adolescents.

BACKGROUND: The usefulness of electrocardiographic (ECG) voltage criteria for diagnosing hypertrophic cardiomyopathy (HCM) in pediatric patients is poorly defined.Methods and Results:ECGs at the 1st grade (mean [±SD] age 6.6±0.3 years) were available for 11 patients diagnosed with HCM at around the 7th grade (13.2±0.3 years). ECGs were available for another 64 patients diagnosed with HCM in the 1st (n=15), 7th (n=32), and 10th (n=17) grades. Fifty-one voltage criteria were developed by grade and sex using 62,841 ECGs from the general population. Voltage criteria were set at the 99.95th percentile (1/2,000) point based on the estimated prevalence of childhood HCM (2.9 per 100,000 [1/34,483]) to decrease false negatives. Conventional criteria were from guidelines for school-aged children in Japan. Of 11 patients before diagnosis, 2 satisfied conventional criteria in 1st grade; 5 (56%) of the remaining 9 patients fulfilled 2 voltage criteria (R wave in limb-lead I [RI]+S wave in lead V3 [SV3] and R wave in lead V3 [RV3]+SV3). Robustness analysis for sensitivity showed RV3+SV3 was superior to RI+SV3. For all patients after diagnosis, RI+SV4 was the main candidate. However, conventional criteria were more useful than voltage criteria. CONCLUSIONS: Early HCM prediction was possible using RV3+SV3 in >50% of patients in 1st grade. Voltage criteria may help diagnose prediagnostic or early HCM, and prevent tragic accidents, although further prospective studies are required.

Diagnostic value of P-waves in children with idiopathic restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare myocardial disease with an impaired diastolic function and poor prognosis. Almost all RCM patients are reported to have abnormal P-waves due to atrial overloading. This study aimed to reveal the characteristics of the P-waves in RCM patients and to suggest the diagnostic index of RCM in children with a 12-lead electrocardiogram (ECG). We retrospectively investigated 17 ECGs of children with idiopathic RCM during the initial visit at 15 institutes in Japan between 1979 and 2013. The RCM group was divided into four groups based on the age (elementary school [ES] and junior high school [JHS] students) and inception of the diagnosis (abnormal ECG on school-heart-screening [e-RCM] and some cardiovascular symptoms [s-RCM]), the ES/e-RCM (n = 5), ES/s-RCM (n = 4), JHS/e-RCM (n = 4), and JHS/s-RCM (n = 4) groups. As an aged-match control group, school-heart-screening ECGs of 1st-grade ES students (16,770 students) and 1st-grade JHS students (18,126 students) from Kagoshima in 2016 were adopted. For a comparison between the groups, we used the effect size "Hedge's g" by calculating the mean and standard deviation of the two groups. An effect size of 0.8 (or above) had an overlap of 53% (or less). The effect sizes of the sum of the absolute values of the forward and backward amplitudes in lead V1 (P1 + P2 V1) was the largest, and the ES/e-RCM, ES/s-RCM, JHS/e-RCM, and JHS/s-RCM were 15.8, 22.1, 9.4, and 10.3, respectively. A P1 + P2 V1 > 200 µV was able to rule in all RCM patients, thus, we proposed 200 µV as the cutoff value for screening purposes. In conclusion, the P1 + P2 V1 in the school-heart-screening may be useful for detecting asymptomatic or early-stage RCM in school-age children.

Sudden death after inappropriate shocks of implantable cardioverter defibrillator in a catecholaminergic polymorphic ventricular tachycardia case with a novel RyR2 mutation.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic syndrome and a cause of exercise-related sudden death. CPVT has been reported to be caused by gain of function underlying a mutation of cardiac ryanodine receptor (RyR2). METHODS: In a family with a CPVT patient, genomic DNA was extracted from peripheral blood lymphocytes, and the RyR2 gene underwent target gene sequence using MiSeq. The activity of wild-type (WT) and mutant RyR2 channel were evaluated by monitoring Ca2+ signals in HEK293 cells expressing WT and mutant RyR2. We investigated a role of a RyR2 mutation in the recent tertiary structure of RyR2. RESULTS: Though a 17-year-old man diagnosed as CPVT had implantable cardioverter defibrillator (ICD) and was going to undergo catheter ablation for the control of paroxysmal atrial fibrillation, he suddenly died at the age of twenty-one because of ventricular fibrillation which was spontaneously developed after maximum inappropriate ICD shocks against rapid atrial fibrillation. The genetic test revealed a de novo RyR2 mutation, Gln4936Lys in mosaicism which was located at the α-helix interface between U-motif and C-terminal domain. In the functional analysis, Ca2+ release from endoplasmic reticulum via the mutant RyR2 significantly increased than that from WT. CONCLUSION: A RyR2 mutation, Gln4936Lys, to be documented in a CPVT patient with exercise-induced ventricular tachycardias causes an excessive Ca2+ release from the sarcoplasmic reticulum which corresponded to clinical phenotypes of CPVT. The reduction of inappropriate shocks of ICD is essential to prevent unexpected sudden death in patients with CPVT.

Mutant KCNJ3 and KCNJ5 Potassium Channels as Novel Molecular Targets in Bradyarrhythmias and Atrial Fibrillation.

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.

An NGS-based genotyping in LQTS; minor genes are no longer minor.

Mutations in KCNQ1, KCNH2, and SCN5A are the major cause of long QT syndrome (LQTS). More than 90% of the genotyped patients have been reported to carry mutations in any of these three genes. Thanks to increasing popularity of next generation sequencer (NGS), novel CACNA1C mutations have been identified among LQTS patients without extra-cardiac phenotypes. We aimed to clarify the frequency of genotypes in LQTS patients in the era of NGS. The study comprised 160 congenital LQTS patients (71 males) registered from November 2015 to September 2018. Inclusion criteria was QTc > 460 ms and Schwartz score ≥ 3. We performed genetic analysis using target gene method by NGS and confirmed the mutations by Sanger method. The median age for genetic screening was 13 (0-68) years. Sixteen patients suffered cardiac arrest, 47 syncope, and 97 were asymptomatic. We identified genetic mutations in 111 (69.4%) patients including 6 CACNA1C (5.4% of the genotyped patients) with 4 asymptomatic patients. Five (3.1%) patients carried double mutations; three out of them with RYR2 and KCNQ1 or KCNH2. In conclusion, CACNA1C screening would be recommended even if the patient is asymptomatic to elucidate the genetic background of the LQTS patients.