Utility of in vivo zebrafish cardiac assay to predict the functional impact of KCNQ1 variants

Abstract Background Genetic testing for inherited arrhythmias and discriminating pathogenic from benign variants are integral for the gene-based medicine. However, the high throughput in vivo functional analysis for the rare variants of the KCNQ1 potassium channel is scarce. Purpose We tested the utility of the in vivo zebrafish cardiac assay for determining the pathogenicity of the KCNQ1 variants identified in patients with long QT syndrome (LQTS) and atrial fibrillation (AF). Methods We generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the KCNQ1 homolog in zebrafish (kcnq1+/+). To test the utility of the cardiac assay, we used five KCNQ1 variants identified in patients with LQTS or familial AF. Human wild-type or mutant KCNQ1 cRNA (Q1) was co-injected with human KCNE1 cRNA (E1) into the F3 generation embryos with homozygous deletions. We dissected the hearts from the thorax at 72 hour-post-fertilization and measured transmembrane potential in zebrafish heart using the disrupted patch technique. Action potential duration was calculated as the time interval between the peak maximum upstroke velocity and 90% of repolarization (APD90). We compared the APD90s with patients' clinical phenotype and IKs density measured by patch-clamp technique in heterologous system. Results The mean APD90 of embryos with kcnq1del/del was 279±48 ms, which was restored by injecting Q1 WT and E1 (159±29 ms) to that with kcnq1+/+ (167±28 ms). We tested if the mean APD90 of embryos with kcnq1del/del was restored (shortened) by injecting the KCNQ1 variants. First we tested the dominant negative variant p.S277L and the trafficking deficient variant p.T587M. Patients with these variants showed significant prolonged QT intervals, and patch clamp study showed both variants caused the non-functional channels. Zebrafish cardiac assay showed the mean APD90 of embryos with kcnq1del/del+ Q1 S277L+E1 or Q1 T587M+E1 was significantly longer than that with kcnq1del/del+Q1 WT+E1 (Table). Next we tested in-frame variant c.1472_1473 ins GGACCT, which was identified from a patient with AF and normal QT interval. Patch clamp study showed the current density of the mutant KCNQ1 channel with KCNE1 was comparable to that of wild-type KCNQ1 channel with KCNE1. Zebrafish assay showed the mean APD90 of embryos with kcnq1del/del shortened by injecting Q1 insACCTGG +E1 (Table). Finally we tested a missense variant p.R451Q, which was identified from a patient with LQTS. Patch clamp study showed the currents in the cells transfected with R451Q+KCNE1 were similar to those with WT+KCNE1. Zebrafish assay showed the mean APD90 of embryos with kcnq1del/del+Q1 R451Q+E1 was longer than that with kcnq1del/del+Q1 WT+E1 (Table). Conclusions Functional analysis of in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with LQTS. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): The Grant-in-Aid for Scientific Research (C)