Objective To determine the spectrum and prevalence of mutations in the RYR2-encoded the cardiac ryanodine receptor in cases with exertional syncope and normal QTc. Background Mutations in the RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by < 40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. Methods Mutational analysis of all RYR2 exons was performed using PCR, DHPLC, and DNA sequencing on 155 unrelated patients (49% females, 96% white, age at diagnosis 20 ± 15 years, mean QTc 428 ± 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome (LQTS) but with QTc < 480 ms and a subsequent negative LQTS genetic test (n = 45). Results Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two thirds of the CPVT1-positive patients had mutations that localized to one of 16 exons. Conclusions Possible CPVT1 mutations in RYR2 were identified in nearly half of this cohort. 45 of the 105 translated exons are now known to host possible mutations. Considering that ~65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal Mendelian Long QT Syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals we identified 35 common variant QT interval loci, that collectively explain ∼8-10% of QT variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 novel QT loci in 298 unrelated LQTS probands identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode for proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies novel candidate genes for ventricular arrhythmias, LQTS,and SCD.

Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for Ϸ75-80% of cases (LQT1-11). Direct sequencing of SNTA1, the gene encoding ␣1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTSsusceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane CaATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused Snitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.cardiac arrhythmia ͉ ion channel ͉ nitrosylation ͉ plasma membrane Ca-ATPase ͉ sodium current

Background-Congenital long-QT syndrome (LQTS) is potentially lethal secondary to malignant ventricular arrhythmias and is caused predominantly by mutations in genes that encode cardiac ion channels. Nearly 25% of patients remain without a genetic diagnosis, and genes that encode cardiac channel regulatory proteins represent attractive candidates. Voltage-gated sodium channels have a pore-forming ␣-subunit associated with 1 or more auxiliary ␤-subunits. Four different ␤-subunits have been described. All are detectable in cardiac tissue, but none have yet been linked to any heritable arrhythmia syndrome. Methods and Results-We present a case of a 21-month-old Mexican-mestizo female with intermittent 2:1 atrioventricular block and a corrected QT interval of 712 ms. Comprehensive open reading frame/splice mutational analysis of the 9 established LQTS-susceptibility genes proved negative, and complete mutational analysis of the 4 Na v␤ -subunits revealed a L179F (C535T) missense mutation in SCN4B that cosegregated properly throughout a 3-generation pedigree and was absent in 800 reference alleles. After this discovery, SCN4B was analyzed in 262 genotype-negative LQTS patients (96% white), but no further mutations were found. L179F was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells that contained the stably expressed SCN5A-encoded sodium channel ␣-subunit (hNa V 1.5). Compared with the wild-type, L179F-␤4 caused an 8-fold (compared with SCN5A alone) and 3-fold (compared with SCN5A ϩ WT-␤4) increase in late sodium current consistent with the molecular/electrophysiological phenotype previously shown for LQTS-associated mutations. Conclusions-We provide the seminal report of SCN4B-encoded Na v␤ 4 as a novel LQT3-susceptibility gene. (Circulation.