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

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

JOURNAL=Frontiers in Cardiovascular Medicine

VOLUME=10

YEAR=2023

URL=https://www.frontiersin.org/journals/cardiovascular-medicine/articles/10.3389/fcvm.2023.1135141

DOI=10.3389/fcvm.2023.1135141

ISSN=2297-055X

ABSTRACT=<sec><title>Objective</title><p>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.</p></sec><sec><title>Methods</title><p>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 <italic>in silico</italic>, and the functional effects of the variants were confirmed <italic>in vitro</italic> using luciferase assays. The combinatorial effect of gene alterations of the putative responsible genes was tested <italic>in vivo</italic> using genetically engineered mutant mice.</p></sec><sec><title>Results</title><p>NGS-based gene panel analyses revealed two heterozygous rare variants in <italic>NODAL</italic> and in <italic>TBX20</italic> common to the siblings and to just one of parents. Both variants were suspected pathogenic <italic>in silico,</italic> and decreased transcriptional activities of downstream signaling pathways were observed <italic>in vitro</italic>. The analyses of <italic>Nodal</italic> and <italic>Tbx20</italic> double mutant mice demonstrated that <italic>Nodal<sup>+/−</sup>Tbx20<sup>−/−</sup></italic> embryos showed more severe defects than <italic>Nodal<sup>+/+</sup>Tbx20<sup>−/−</sup></italic> embryos during early heart development. The expression of <italic>Pitx2</italic>, a known downstream target of <italic>Nodal</italic>, was downregulated in <italic>Tbx20<sup>−/−</sup></italic> mutants.</p></sec><sec><title>Conclusions</title><p>Two rare variants on <italic>NODAL</italic> and <italic>TBX20</italic> genes detected in this family were considered to be loss-of-function mutations. Our results suggest that <italic>NODAL</italic> and <italic>TBX20</italic> may be complementary for the cardiac development, and a combinatorial loss-of-function of <italic>NODAL</italic> and <italic>TBX20</italic> could be implicated in digenic inherence as the etiology of complex CHD associated with single ventricle defects in this family.</p></sec>