A genetic and developmental biological approach for a family with complex congenital heart diseases-evidence of digenic inheritance

Yu Yoshida; Keiko Uchida; Kazuki Kodo; Reina Ishizaki-Asami; Jun Maeda; Yoshinori Katsumata; Shinsuke Yuasa; Keiichi Fukuda; Kenjiro Kosaki; Yusuke Watanabe; Osamu Nakagawa; Hiroyuki Yamagishi

doi:10.3389/fcvm.2023.1135141

A genetic and developmental biological approach for a family with complex congenital heart diseases-evidence of digenic inheritance

Front Cardiovasc Med. 2023 Apr 25:10:1135141. doi: 10.3389/fcvm.2023.1135141. eCollection 2023.

Authors

Affiliations

¹ Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan.
² Health Center, Keio University, Kanagawa, Japan.
³ Department of Cardiology, Keio University School of Medicine, Tokyo, Japan.
⁴ Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan.
⁵ Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan.

Abstract

Objective: Congenital heart disease (CHD) is caused by cardiovascular developmental defects and has a global prevalence of ∼1%. The etiology of CHD is multifactorial and remains generally unknown, despite advances in analytical techniques based on next-generation sequencing (NGS). The aim of our study was to elucidate the multi-genetic origin and pathogenesis of an intriguing familial case with complex CHD.

Methods: We performed an original trio-based gene panel analysis using NGS of the family, including two siblings with CHD of single ventricular phenotype, and their unaffected parents. The pathogenicity of the detected rare variants was investigated in silico, and the functional effects of the variants were confirmed in vitro using luciferase assays. The combinatorial effect of gene alterations of the putative responsible genes was tested in vivo using genetically engineered mutant mice.

Results: NGS-based gene panel analyses revealed two heterozygous rare variants in NODAL and in TBX20 common to the siblings and to just one of parents. Both variants were suspected pathogenic in silico, and decreased transcriptional activities of downstream signaling pathways were observed in vitro. The analyses of Nodal and Tbx20 double mutant mice demonstrated that Nodal^+/-Tbx20^-/- embryos showed more severe defects than Nodal^+/+Tbx20^-/- embryos during early heart development. The expression of Pitx2, a known downstream target of Nodal, was downregulated in Tbx20^-/- mutants.

Conclusions: Two rare variants on NODAL and TBX20 genes detected in this family were considered to be loss-of-function mutations. Our results suggest that NODAL and TBX20 may be complementary for the cardiac development, and a combinatorial loss-of-function of NODAL and TBX20 could be implicated in digenic inherence as the etiology of complex CHD associated with single ventricle defects in this family.

Keywords: complex congenital heart disease; nodal; rare variants; tbx20; trio gene analysis.

Grants and funding

This work was supported by the Japan Agency for Medical Research and Development (AMED) under grant number JP20ek0109487. KU, JM, YW, ON, and HY were supported by JSPS KAKENHI [grant numbers JP17K10153 to KU, JP19K08352 to JM, JP19H03398 to YW, JP21H02890 to ON, and JP16H05359 to HY]. YW was supported by a grant from The Japan Foundation for Pediatric Research.