Abstract 14320: The Utility of Zebrafish Cardiac Arrhythmia Model to Predict the Pathogenicity of KCNQ1 Variants

Abstract

Introduction: Genetic testing for inherited arrhythmias and discriminating pathogenic or benign variants from variants of unknown significance (VUS) is essential for gene-based medicine. KCNQ1 is a causative gene of type 1 long QT syndrome (LQTS) and familial atrial fibrillation (AF), and approximately 30% of the variants found in type 1 LQTS are classified as VUS. Aims: We studied the role of zebrafish cardiac arrhythmia model in determining the clinical significance of KCNQ1 variants. Methods and Results: We generated homozygous kcnq1 deletion zebrafish ( kcnq1 del/del ) using the CRISPR/Cas9 technique and expressed human Kv7.1/MinK channels in kcnq1 del/del embryos. We dissected the hearts from the thorax at 72 h postfertilization and measured the transmembrane potential of the ventricle or the atrium in the zebrafish heart. Action potential duration was calculated as the time interval between peak maximum upstroke velocity and 90% repolarization (APD90). The APD90 of the kcnq1 del/del embryonic ventricle was 280 ± 47 ms (n = 63), which was significantly shortened by injecting KCNQ1 wild-type (WT) cRNA and KCNE1 cRNA (162 ± 28 ms, n = 34, P < 0.01 vs. kcnq1 del/del ). A study of two pathogenic variants (S277L and T587M) and one VUS (R451Q) associated with clinically definite LQTS showed that the APD90 of kcnq1 del/del embryonic ventricles with mutant Kv7.1/MinK channels (292 ± 40 ms for S277L, n = 27; 297 ± 63 ms for T587M, n = 25; 218 ± 49 ms for R451Q, n = 29, respectively) was significantly longer than that of Kv7.1 WT/MinK channels (P < 0.01). Regarding the pathogenic variant (S140G) associated with familial AF, the APD90 of the kcnq1 del/del embryonic atrium with mutant Kv7.1/MinK channel was 124 ± 21 ms (n=13), which was significantly shorter than that with Kv7.1 WT/MinK (148 ± 17 ms, n = 8, P < 0.05 vs. kcnq1 del/del with S140G Kv7.1/MinK channel). Conclusions: Functional analysis using in vivo zebrafish cardiac arrhythmia model may be useful for determining the pathogenicity of both loss-of-function variants in patients with LQTS and gain-of-function variants in patients with familial AF.