In silico investigation of a KCNQ1 mutation associated with short QT syndrome.

Short QT syndrome (SQTS) is a rare condition characterized by abnormally 'short' QT intervals on the ECG and increased susceptibility to cardiac arrhythmias and sudden death. This simulation study investigated arrhythmia dynamics in multi-scale human ventricle models associated with the SQT2-related V307L KCNQ1 'gain-of-function' mutation, which increases slow-delayed rectifier potassium current (IKs). A Markov chain (MC) model recapitulating wild type (WT) and V307L mutant IKs kinetics was incorporated into a model of the human ventricular action potential (AP) for investigation of QT interval changes and arrhythmia substrates. In addition, the degree of simulated IKs inhibition necessary to normalize the QT interval and terminate re-entry in SQT2 conditions was quantified. The developed MC model accurately reproduced AP shortening and reduced effective refractory period associated with altered IKs kinetics in homozygous (V307L) and heterozygous (WT-V307L) mutation conditions, which increased the lifespan and dominant frequency of re-entry in 3D human ventricle models. IKs reductions of 58% and 65% were sufficient to terminate re-entry in WT-V307L and V307L conditions, respectively. This study further substantiates a causal link between the V307L KCNQ1 mutation and pro-arrhythmia in human ventricles, and establishes partial inhibition of IKs as a potential anti-arrhythmic strategy in SQT2.

Regulation of membrane KCNQ1/KCNE1 channel density by sphingomyelin synthase 1.

Sphingomyelin synthase (SMS) catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol. We previously showed that SMS1 deficiency leads to a reduction in expression of the K(+) channel KCNQ1 in the inner ear (Lu MH, Takemoto M, Watanabe K, Luo H, Nishimura M, Yano M, Tomimoto H, Okazaki T, Oike Y, and Song WJ. J Physiol 590: 4029-4044, 2012), causing hearing loss. However, it remains unknown whether this change in expression is attributable to a cellular process or a systemic effect in the knockout animal. Here, we examined whether manipulation of SMS1 activity affects KCNQ1/KCNE1 currents in individual cells. To this end, we expressed the KCNQ1/KCNE1 channel in human embryonic kidney 293T cells and evaluated the effect of SMS1 manipulations on the channel using whole cell recording. Application of tricyclodecan-9-yl-xanthogenate, a nonspecific inhibitor of SMSs, significantly reduced current density and altered channel voltage dependence. Knockdown of SMS1 by a short hairpin RNA, however, reduced current density alone. Consistent with this, overexpression of SMS1 increased the current density without changing channel properties. Furthermore, application of protein kinase D inhibitors also suppressed current density without changing channel properties; this effect was nonadditive with that of SMS1 short hairpin RNA. These results suggest that SMS1 positively regulates KCNQ1/KCNE1 channel density in a protein kinase D-dependent manner.

Assessment of the predictive accuracy of five in silico prediction tools, alone or in combination, and two metaservers to classify long QT syndrome gene mutations.

BACKGROUND: Long QT syndrome (LQTS) is an autosomal dominant condition predisposing to sudden death from malignant arrhythmia. Genetic testing identifies many missense single nucleotide variants of uncertain pathogenicity. Establishing genetic pathogenicity is an essential prerequisite to family cascade screening. Many laboratories use in silico prediction tools, either alone or in combination, or metaservers, in order to predict pathogenicity; however, their accuracy in the context of LQTS is unknown. We evaluated the accuracy of five in silico programs and two metaservers in the analysis of LQTS 1-3 gene variants. METHODS: The in silico tools SIFT, PolyPhen-2, PROVEAN, SNPs&GO and SNAP, either alone or in all possible combinations, and the metaservers Meta-SNP and PredictSNP, were tested on 312 KCNQ1, KCNH2 and SCN5A gene variants that have previously been characterised by either in vitro or co-segregation studies as either "pathogenic" (283) or "benign" (29). The accuracy, sensitivity, specificity and Matthews Correlation Coefficient (MCC) were calculated to determine the best combination of in silico tools for each LQTS gene, and when all genes are combined. RESULTS: The best combination of in silico tools for KCNQ1 is PROVEAN, SNPs&GO and SIFT (accuracy 92.7%, sensitivity 93.1%, specificity 100% and MCC 0.70). The best combination of in silico tools for KCNH2 is SIFT and PROVEAN or PROVEAN, SNPs&GO and SIFT. Both combinations have the same scores for accuracy (91.1%), sensitivity (91.5%), specificity (87.5%) and MCC (0.62). In the case of SCN5A, SNAP and PROVEAN provided the best combination (accuracy 81.4%, sensitivity 86.9%, specificity 50.0%, and MCC 0.32). When all three LQT genes are combined, SIFT, PROVEAN and SNAP is the combination with the best performance (accuracy 82.7%, sensitivity 83.0%, specificity 80.0%, and MCC 0.44). Both metaservers performed better than the single in silico tools; however, they did not perform better than the best performing combination of in silico tools. CONCLUSIONS: The combination of in silico tools with the best performance is gene-dependent. The in silico tools reported here may have some value in assessing variants in the KCNQ1 and KCNH2 genes, but caution should be taken when the analysis is applied to SCN5A gene variants.

Characterization of a novel KCNQ1 mutation for type 1 long QT syndrome and assessment of the therapeutic potential of a novel IKs activator using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Type 1 long QT syndrome (LQT1) is a common type of cardiac channelopathy associated with loss-of-function mutations of KCNQ1. Currently there is a lack of drugs that target the defected slowly activating delayed rectifier potassium channel (IKs). With LQT1 patient-specific human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs), we tested the effects of a selective IKs activator ML277 on reversing the disease phenotypes. METHODS: A LQT1 family with a novel heterozygous exon 7 deletion in the KCNQ1 gene was identified. Dermal fibroblasts from the proband and her healthy father were reprogrammed to hiPSCs and subsequently differentiated into hiPSC-CMs. RESULTS: Compared with the control, LQT1 patient hiPSC-CMs showed reduced levels of wild type KCNQ1 mRNA accompanied by multiple exon skipping mRNAs and a ~50% reduction of the full length Kv7.1 protein. Patient hiPSC-CMs showed reduced IKs current (tail current density at 30 mV: 0.33±0.02 vs. 0.92±0.21, P<0.05) and prolonged action potential duration (APD) (APD 50 and APD90: 603.9±39.2 vs. 319.3±13.8 ms, P<0.005; and 671.0±41.1 vs. 372.9±14.2 ms, P<0.005). ML277, a small molecule recently identified to selectively activate KV7.1, reversed the decreased IKs and partially restored APDs in patient hiPSC-CMs. CONCLUSIONS: From a LQT1 patient carrying a novel heterozygous exon7 deletion mutation of KCNQ1, we generated hiPSC-CMs that faithfully recapitulated the LQT1 phenotypes that are likely associated with haploinsufficiency and trafficking defect of KCNQ1/Kv7.1. The small molecule ML277 restored IKs function in hiPSC-CMs and could have therapeutic value for LQT1 patients.

In silico cardiac risk assessment in patients with long QT syndrome: type 1: clinical predictability of cardiac models.

OBJECTIVES: The study was designed to assess the ability of computer-simulated electrocardiography parameters to predict clinical outcomes and to risk-stratify patients with long QT syndrome type 1 (LQT1). BACKGROUND: Although attempts have been made to correlate mutation-specific ion channel dysfunction with patient phenotype in long QT syndrome, these have been largely unsuccessful. Systems-level computational models can be used to predict consequences of complex changes in channel function to the overall heart rhythm. METHODS: A total of 633 LQT1-genotyped subjects with 34 mutations from multinational long QT syndrome registries were studied. Cellular electrophysiology function was determined for the mutations and introduced in a 1-dimensional transmural electrocardiography computer model. The mutation effect on transmural repolarization was determined for each mutation and related to the risk for cardiac events (syncope, aborted cardiac arrest, and sudden cardiac death) among patients. RESULTS: Multivariate analysis showed that mutation-specific transmural repolarization prolongation (TRP) was associated with an increased risk for cardiac events (35% per 10-ms increment [p < 0.0001]; ≥upper quartile hazard ratio: 2.80 [p < 0.0001]) and life-threatening events (aborted cardiac arrest/sudden cardiac death: 27% per 10-ms increment [p = 0.03]; ≥upper quartile hazard ratio: 2.24 [p = 0.002]) independently of patients' individual QT interval corrected for heart rate (QTc). Subgroup analysis showed that among patients with mild to moderate QTc duration (<500 ms), the risk associated with TRP was maintained (36% per 10 ms [p < 0.0001]), whereas the patient's individual QTc was not associated with a significant risk increase after adjustment for TRP. CONCLUSIONS: These findings suggest that simulated repolarization can be used to predict clinical outcomes and to improve risk stratification in patients with LQT1, with a more pronounced effect among patients with a lower-range QTc, in whom a patient's individual QTc may provide less incremental prognostic information.

Combined assessment of sex- and mutation-specific information for risk stratification in type 1 long QT syndrome.

BACKGROUND: Men and women with type 1 long QT syndrome (LQT1) exhibit time-dependent differences in the risk for cardiac events. OBJECTIVE: We hypothesized that sex-specific risk for LQT1 is related to the location and function of the disease-causing mutation in the KCNQ1 gene. METHODS: The risk for life-threatening cardiac events (comprising aborted cardiac arrest [ACA] or sudden cardiac death [SCD]) from birth through age 40 years was assessed among 1051 individuals with LQT1 (450 men and 601 women) by the location and function of the LQT1-causing mutation (prespecified as mutations in the intracellular domains linking the membrane-spanning segments [ie, S2-S3 and S4-S5 cytoplasmic loops] involved in adrenergic channel regulation vs other mutations). RESULTS: Multivariate analysis showed that during childhood (age group: 0-13 years) men had >2-fold (P < .003) increased risk for ACA/SCD than did women, whereas after the onset of adolescence the risk for ACA/SCD was similar between men and women (hazard ratio = 0.89 [P = .64]). The presence of cytoplasmic-loop mutations was associated with a 2.7-fold (P < .001) increased risk for ACA/SCD among women, but it did not affect the risk among men (hazard ratio 1.37; P = .26). Time-dependent syncope was associated with a more pronounced risk-increase among men than among women (hazard ratio 4.73 [P < .001] and 2.43 [P = .02], respectively), whereas a prolonged corrected QT interval (≥ 500 ms) was associated with a higher risk among women than among men. CONCLUSION: Our findings suggest that the combined assessment of clinical and mutation location/functional data can be used to identify sex-specific risk factors for life-threatening events for patients with LQT1.

Arrhythmia formation in subclinical ("silent") long QT syndrome requires multiple insults: quantitative mechanistic study using the KCNQ1 mutation Q357R as example.

BACKGROUND: In subclinical or silent long QT syndrome, the QT interval is normal under basal conditions. The hypothesis that insults to the repolarization reserve may cause arrhythmias in silent mutation carriers but not in noncarriers has been proposed as a general principle, yet crucial aspects remain descriptive, lacking quantification. OBJECTIVE: To utilize accurate mathematical models of the human action potential and ß-adrenergic stimulation to quantitatively investigate arrhythmia-formation mechanisms peculiar to silent long QT syndrome, using mutation Q357R in KCNQ1 (α subunit of slow-delayed rectifier I(Ks)) as a paradigm. METHODS: Markov models were formulated to account for altered I(Ks) kinetics in Q357R compared with wild type and introduced into a detailed model of the human ventricular myocyte action potential. RESULTS: Dominant negative loss of I(Ks) available reserve accurately represents Q357R. Action potential prolongation with mutant I(Ks) was minimal, reproducing the silent phenotype. Partial block of rapid delayed rectifier current (I(Kr)) was needed in addition to fast pacing and isoproterenol application to cause early afterdepolarizations (EADs) in epicardial cells with mutant I(Ks), but this did not produce EADs in wild type. Reduced channel expression at the membrane, not I(Ks) kinetic differences, caused EADs in the silent mutant. With mutant I(Ks), isoproterenol plus partial I(Kr) block resulted in dramatic QT prolongation in the pseudo-electrocardiogram and EADs formed without I(Kr) block in mid-myocardial cells during simulated exercise onset. CONCLUSION: Multiple severe insults are needed to evince an arrhythmic phenotype in silent mutation Q357R. Reduced membrane I(Ks) expression, not kinetic changes, underlies the arrhythmic phenotype.

Development of a high resolution melting method for the detection of genetic variations in Long QT Syndrome.

BACKGROUND: Inherited Long QT Syndrome (LQTS) is a cardiac channelopathy associated with a high risk of sudden death. The prevalence has been estimated at close to 1:2000. Due to large cohorts to investigate, the size of the 3 prevalent mutated genes, and the presence of a large spectrum of private mutations, mutational screening requires an extremely sensitive and specific scanning method. METHODS: Efficiency of high resolution melting (HRM) analysis was evaluated for the most prevalent LQTS-causing genes (KCNQ1, KCNH2) using control DNAs and DNAs carrying previously identified gene variants. A cohort of 34 patients with a suspicion of LQTS was further blindly screened. To evaluate HRM sensitivity, this cohort was also screened using an optimized DHPLC strategy. RESULTS: HRM analysis was successfully optimized for KCNQ1 but optimisation of KCNH2 was more laborious as only 3 KCNH2 exons could be finally optimized. Remaining KCNH2 exons were analysed by direct sequencing. This molecular approach, which combined HRM and direct sequencing, was applied on the cohort of 34 cases and 9 putative mutations were identified. Using this approach, molecular investigation was completed faster and cheaper than using DHPLC strategy. CONCLUSIONS: This HRM/sequencing procedure represents an inexpensive, highly sensitive and high-throughput method to allow identification of mutations in the coding sequences of prevalent LQTS genes.

Genetic variation in the rhythmonome: ethnic variation and haplotype structure in candidate genes for arrhythmias.

AIMS: The 'rhythmonome' is the term we have adopted to describe the set of genes that determine the normal coordinated electrical activity in the heart. Elements of this set include pore-forming ion channels, function-modifying proteins and intracellular calcium control elements. Rare mutations in many of these genes are known to cause unusual congenital monogenic arrhythmia syndromes, and single common variants have been reported to modify arrhythmia phenotypes. Here, we report an evaluation of the variation and haplotype structure in six key components of the rhythmonome. MATERIALS & METHODS: SNPs were typed using DNA extracted from Coriell cell lines to survey allele frequencies and haplotype structure in six genes (ANK2, SCN5A, KCNE1 and 2 gene cluster, KCNQ1, KCNH2 and RYR2) across four human populations (African-American, European American, Han Chinese and Mexican American). RESULTS: A total of 307 SNPs were analyzed across the six genes, revealing significant allele-frequency differences between populations and clear differences in haplotype structure. CONCLUSIONS: The pattern of variation we report is an important step towards incorporating common variation across the rhythmonome in studies of arrhythmia susceptibility.

Functional assessment of compound mutations in the KCNQ1 and KCNH2 genes associated with long QT syndrome.

BACKGROUND: Long QT syndrome (LQTS) is a cardiovascular disorder characterized by prolonged QTc time, syncope, or sudden death caused by torsades de pointes and ventricular fibrillation. We investigated the clinical and electrophysiologic phenotype of individual mutations and the compound mutations in a family in which different genotypes could be found. OBJECTIVES: The purpose of this study was to determine the impact of genotype-based diagnostic assessment in LQTS. METHODS: We used cascade screening and functional analyses to investigate the phenotype in a family with LQTS. The contributions of the compound mutations in the KCNQ1 and KCNH2 genes (KCNQ1 R591H, KCNH2 R328C) were analyzed by heterologous expression in Xenopus laevis oocytes using two-electrode voltage clamp and by confocal imaging. RESULTS: KCNH2 R328C did not show any functional phenotype whereas KCNQ1 R591H resulted in severe reduction of current. Neither wild-type nor mutant channels affected each other functionally in coexpression experiments. Therefore, a direct interaction between KCNQ1 and KCNH2 was ruled out under these conditions. CONCLUSION: Assessment of novel mutational findings in LQTS should include accurate genetic and functional analysis. Notably, appropriate studies are needed if two or more mutations in different genes are present in one proband. Our findings prompt reconsideration of the impact of compound mutations in LQTS families and reinforce the need for thorough functional evaluation of novel ion channel mutations before assignment of pathogenic status.