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