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. 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. 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. Among currently recognized genetic and non-genetic risk factors for Brugada syndrome, functionally validated loss-of-function SCN5A mutations are associated with worse prognosis with earlier manifestation of lethal arrhythmic events (7.9%/year) than in silico algorithm-predicted SCN5A rare variations (5.1%/year) or Brugada-type ECG (2.5%/year).

Background—: Few rare variants in atrial fibrillation (AF)–associated genes have been functionally characterized to identify a causal relationship between these variants and development of AF. We here sought to determine the clinical effect of rare variants in AF-associated genes in patients with lone AF and characterized these variants electrophysiologically and bioinformatically. Methods and Results—: We screened all coding regions in 12 AF-associated genes in 90 patients with lone AF, with an onset of 47±11 years (66 men; mean age, 56±13 years) by high-resolution melting curve analysis and DNA sequencing. The potassium and sodium currents were analyzed using whole-cell patch clamping. In addition to using 4 individual in silico prediction tools, we extended those predictions to an integrated tool (Combined Annotation Dependent Depletion). We identified 7 rare variants in KCNA5 , KCNQ1 , KCNH2 , SCN5A , and SCN1B genes in 8 patients: 2 of 8 probands had a family history of AF. Electrophysiological studies revealed that 2 variants showed a loss-of-function, and 4 variants showed a gain-of-function. Five of 6 variants with electrophysiological abnormalities were predicted as pathogenic by Combined Annotation Dependent Depletion scores. Conclusions—: In our cohort of patients with lone AF, 7 rare variants in cardiac ion channels were identified in 8 probands. A combination of electrophysiological studies and in silico predictions showed that these variants could contribute to the development of lone AF, although further in vivo study is necessary to confirm these results. More than half of AF-associated rare variants showed gain-of-function behavior, which may be targeted using genotype-specific pharmacological therapy.

Japan's largest platform for academic e-journals: J-STAGE is a full text database for reviewed academic papers published by Japanese societies

Background: The human oxytocin receptor (hOXTR) is implicated in the etiology of autism spectrum disorders (ASDs) and is a potential target for therapeutic intervention. Several studies have reported single-nucleotide polymorphisms (SNPs) of the OXTR gene associated with ASDs. These SNPs, however, reside outside the protein-coding region. Not much is known about genetic variations that cause amino acid substitutions that alter receptor functions. Methods: Variations in the OXTR gene were analyzed in 132 ASD patients at Kanazawa University Hospital in Japan and 248 unrelated healthy Japanese volunteers by re-sequencing and real-time polymerase chain reaction-based genotyping. Functional changes in variant OXTRs were assessed by radioligand binding assay and measurements of intracellular free calcium concentrations ([Ca²⁺]_i) and inositol 1,4,5-trisphosphate (IP₃) levels. Results: Six subjects (4.5%) in the ASD group and two in the control group (0.8%) were identified as heterozygotes carrying the R376G variation (rs35062132; c.1126C>G); one individual from the ASD group (0.8%) and three members of the control group (1.2%) were found to be carrying R376C (c.1126C>T). The C/G genotype significantly correlated with an increased risk of ASDs (odds ratio (OR) = 5.83; 95% confidence interval (CI) = 1.16 to 29.33; P = 0.024, Fisher’s exact test). Consistently, the G allele showed a correlation with an increased likelihood of ASDs (OR = 5.73; 95% CI = 1.15 to 28.61; P = 0.024, Fisher’s exact test). The frequencies of the C/T genotype and the T allele in the ASD and control groups did not differ significantly. We also examined changes in agonist-induced cellular responses mediated by the variant receptors hOXTR-376G and hOXTR-376C. OXT-induced receptor internalization and recycling were faster in hOXTR-376G-expressing HEK-293 cells than in cells expressing hOXTR-376R or hOXTR-376C. In addition, the elevation in [Ca²⁺]_i and IP₃ formation decreased in the cells expressing hOXTR-376G and hOXTR-376C tagged with enhanced green fluorescent protein (EGFP), in comparison with the cells expressing the common-type hOXTR-376R tagged with EGFP. Conclusions: These results suggest that the rare genetic variation rs35062132 might contribute to the pathogenesis of ASDs, and could provide a molecular basis of individual differences in OXTR-mediated modulation of social behavior.

LQTS (long QT syndrome) is caused by mutations in cardiac ion channel genes; however, the prevalence of LQTS in the general population is not well known. In the present study, we prospectively estimated the prevalence of LQTS and analysed the associated mutation carriers in Japanese children. ECGs were recorded from 7961 Japanese school children (4044 males; mean age, 9.9±3.0 years). ECGs were examined again for children who had prolonged QTc (corrected QT) intervals in the initial ECGs, and their QT intervals were measured manually. An LQTS score was determined according to Schwartz's criteria, and ion channel genes were analysed. In vitro characterization of the identified mutants was performed by heterologous expression experiments. Three subjects were assigned to a high probability of LQTS (3.5≤ LQTS score), and eight subjects to an intermediate probability (1.0< LQTS score ≤3.0). Genetic analysis of these II subjects identified three KCNH2 mutations (M124T, 547–553 del GGCGGCG and 2311–2332 del/ins TC). In contrast, no mutations were identified in the 15 subjects with a low probability of LQTS. Electrophysiological studies showed that both the M124T and the 547–553 del GGCGGCG KCNH2 did not suppress the wild-type KCNH2 channel in a dominant-negative manner. These results demonstrate that, in a random sample of healthy Japanese children, the prevalence of a high probability of LQTS is 0.038% (three in 7961), and that LQTS mutation carriers can be identified in at least 0.038% (one in 2653). Furthermore, large-scale genetic studies will be needed to clarify the real prevalence of LQTS by gene-carrier status, as it may have been underestimated in the present study.