A Functional Assay for Sick Sinus Syndrome Genetic Variants.

BACKGROUND/AIMS: Congenital Sick Sinus Syndrome (SSS) is a disorder associated with sudden cardiac death due to severe bradycardia and prolonged pauses. Mutations in HCN4, the gene encoding inward Na+/K+ current (If), have been described as a cause of congenital SSS. The objective of this study is to develop an SSS model in embryonic zebrafish, and use zebrafish as a moderate-throughput assay to functionally characterize HCN4 variants. METHODS: To determine the function of hcn4 in zebrafish, embryos were either bathed in the If -specific blocker (ZD-7288), or endogenous hcn4 expression was knocked down using splice-blocking morpholinos. To assess whether the zebrafish model discriminates benign from pathogenic variants, we tested four HCN4 mutations known to cause human SSS and four variants of unknown significance (VUS). RESULTS: Pharmacological blockade and knockdown of hcn4 in zebrafish phenocopied human SSS, displaying bradycardia and cardiac pauses in intact embryos and explanted hearts. The zebrafish assay correctly identified all disease-causing variants. Of the VUS, the assay predicted 2 as benign and 2 as hypomorphic variants. CONCLUSIONS: We conclude that our embryonic zebrafish assay is a novel and effective tool to functionally characterize human HCN4 variants, which can be translated into important clinical prognostic information.

Novel mutation in the α-myosin heavy chain gene is associated with sick sinus syndrome.

BACKGROUND: Recent genome-wide association studies have demonstrated an association between MYH6, the gene encoding α-myosin heavy chain (α-MHC), and sinus node function in the general population. Moreover, a rare MYH6 variant, R721W, predisposing susceptibility to sick sinus syndrome has been identified. However, the existence of disease-causing MYH6 mutations for familial sick sinus syndrome and their underlying mechanisms remain unknown. METHODS AND RESULTS: We screened 9 genotype-negative probands with sick sinus syndrome families for mutations in MYH6 and identified an in-frame 3-bp deletion predicted to delete one residue (delE933) at the highly conserved coiled-coil structure within the binding motif to myosin-binding protein C in one patient. Co-immunoprecipitation analysis revealed enhanced binding of delE933 α-MHC to myosin-binding protein C. Irregular fluorescent speckles retained in the cytoplasm with substantially disrupted sarcomere striation were observed in neonatal rat cardiomyocytes transfected with α-MHC mutants carrying delE933 or R721W. In addition to the sarcomere impairments, delE933 α-MHC exhibited electrophysiological abnormalities both in vitro and in vivo. The atrial cardiomyocyte cell line HL-1 stably expressing delE933 α-MHC showed a significantly slower conduction velocity on multielectrode array than those of wild-type α-MHC or control plasmid transfected cells. Furthermore, targeted morpholino knockdown of MYH6 in zebrafish significantly reduced the heart rate, which was rescued by coexpressed wild-type human α-MHC but not by delE933 α-MHC. CONCLUSIONS: The novel MYH6 mutation delE933 causes both structural damage of the sarcomere and functional impairments on atrial action propagation. This report reinforces the relevance of MYH6 for sinus node function and identifies a novel pathophysiology underlying familial sick sinus syndrome.

An in vivo cardiac assay to determine the functional consequences of putative long QT syndrome mutations.

RATIONALE: Genetic testing for Long QT Syndrome is now a standard and integral component of clinical cardiology. A major obstacle to the interpretation of genetic findings is the lack of robust functional assays to determine the pathogenicity of identified gene variants in a high-throughput manner. OBJECTIVE: The goal of this study was to design and test a high-throughput in vivo cardiac assay to distinguish between disease-causing and benign KCNH2 (hERG1) variants, using the zebrafish as a model organism. METHODS AND RESULTS: We tested the ability of previously characterized Long QT Syndrome hERG1 mutations and polymorphisms to restore normal repolarization in the kcnh2-knockdown embryonic zebrafish. The cardiac assay correctly identified a benign variant in 9 of 10 cases (negative predictive value 90%), whereas correctly identifying a disease-causing variant in 39/39 cases (positive predictive value 100%). CONCLUSIONS: The in vivo zebrafish cardiac assay approaches the accuracy of the current benchmark in vitro assay for the detection of disease-causing mutations, and is far superior in terms of throughput rate. Together with emerging algorithms for interpreting a positive long QT syndrome genetic test, the zebrafish cardiac assay provides an additional tool for the final determination of pathogenicity of gene variants identified in long QT syndrome genetic screening.