The utility of zebrafish cardiac arrhythmia model to predict the pathogenicity of KCNQ1 variants

Shihe Cui; Kenshi Hayashi; Isao Kobayashi; Kazuyoshi Hosomichi; Akihiro Nomura; Ryota Teramoto; Keisuke Usuda; Hirofumi Okada; Yaowen Deng; Jingjing Kobayashi-Sun; Tetsuo Nishikawa; Hiroshi Furusho; Takekatsu Saito; Hiroaki Hirase; Kunio Ohta; Manabu Fujimoto; Yuki Horita; Takashi Kusayama; Toyonobu Tsuda; Hayato Tada; Takeshi Kato; Soichiro Usui; Kenji Sakata; Noboru Fujino; Atsushi Tajima; Masakazu Yamagishi; Masayuki Takamura

doi:10.1016/j.yjmcc.2023.03.001

The utility of zebrafish cardiac arrhythmia model to predict the pathogenicity of KCNQ1 variants

J Mol Cell Cardiol. 2023 Apr:177:50-61. doi: 10.1016/j.yjmcc.2023.03.001. Epub 2023 Mar 8.

Authors

Shihe Cui¹, Kenshi Hayashi², Isao Kobayashi³, Kazuyoshi Hosomichi⁴, Akihiro Nomura¹, Ryota Teramoto⁵, Keisuke Usuda¹, Hirofumi Okada¹, Yaowen Deng¹, Jingjing Kobayashi-Sun⁶, Tetsuo Nishikawa¹, Hiroshi Furusho⁷, Takekatsu Saito⁸, Hiroaki Hirase⁹, Kunio Ohta¹⁰, Manabu Fujimoto¹¹, Yuki Horita¹², Takashi Kusayama¹, Toyonobu Tsuda¹, Hayato Tada¹, Takeshi Kato¹, Soichiro Usui¹, Kenji Sakata¹, Noboru Fujino¹³, Atsushi Tajima¹⁴, Masakazu Yamagishi¹⁵, Masayuki Takamura¹

Affiliations

¹ Department of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan.
² Department of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan; School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan. Electronic address: kenshi@med.kanazawa-u.ac.jp.
³ Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kanazawa, Japan.
⁴ Laboratory of Computational Genomics, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
⁵ Laboratory for Comprehensive Genomic Analysis, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
⁶ Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kanazawa, Japan; Department of Clinical Engineering, Faculty of Health Sciences, Komatsu University, Komatsu, Japan.
⁷ Department of Cardiology, Ishikawa Prefectural Central Hospital, Kanazawa, Japan.
⁸ Department of Pediatrics, Minamigaoka Hospital, Kanazawa, Japan.
⁹ Department of Cardiology, Takaoka Minami Heart Center, Takaoka, Japan.
¹⁰ Department of Pediatrics, School of Medicine, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kanazawa, Japan; Medical Education Research Center, Graduate School of Medical Sciences, Kanazawa University, Japan.
¹¹ Department of Cardiology, Kouseiren Takaoka Hospital, Takaoka, Japan.
¹² Department of Cardiology, Kanazawa Cardiovascular Hospital, Kanazawa, Japan.
¹³ Department of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan; School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan.
¹⁴ Department of Bioinformatics and Genomics, Graduate School of Advanced Preventive Medical Sciences, Kanazawa University, Kanazawa, Japan.
¹⁵ Osaka University of Human Sciences, Settsu, Osaka, Japan.

PMID: 36898499
DOI: 10.1016/j.yjmcc.2023.03.001

Abstract

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 approximately 30% of the variants found in type 1 LQTS are classified as VUS. We studied the role of zebrafish cardiac arrhythmia model in determining the clinical significance of KCNQ1 variants. We generated homozygous kcnq1 deletion zebrafish (kcnq1^del/del) using the CRISPR/Cas9 and expressed human Kv7.1/MinK channels in kcnq1^del/del embryos. We dissected the hearts from the thorax at 48 h post-fertilization and measured the transmembrane potential of the ventricle 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 kcnq1^del/del embryos was 280 ± 47 ms, which was significantly shortened by injecting KCNQ1 wild-type (WT) cRNA and KCNE1 cRNA (168 ± 26 ms, 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 embryos with these mutant Kv7.1/MinK channels was significantly longer than that of Kv7.1 WT/MinK channels. Given the functional results of the zebrafish model, R451Q could be reevaluated physiologically from VUS to likely pathogenic. In conclusion, functional analysis using in vivo zebrafish cardiac arrhythmia model can be useful for determining the pathogenicity of loss-of-function variants in patients with LQTS.

Keywords: Action potential duration; CRISPR/Cas9; KCNQ1; Long QT syndrome; Variants of unknown significance; Zebrafish.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Humans
KCNQ1 Potassium Channel / genetics
Long QT Syndrome* / genetics
Mutation
RNA, Complementary
Virulence
Zebrafish* / genetics

Substances

KCNQ1 Potassium Channel
KCNQ1 protein, human
RNA, Complementary