ObjectiveCongenital 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.MethodsWe 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 investigatedin silico, and the functional effects of the variants were confirmedin vitrousing luciferase assays. The combinatorial effect of gene alterations of the putative responsible genes was testedin vivousing genetically engineered mutant mice.ResultsNGS-based gene panel analyses revealed two heterozygous rare variants inNODALand inTBX20common to the siblings and to just one of parents. Both variants were suspected pathogenicin silico,and decreased transcriptional activities of downstream signaling pathways were observedin vitro. The analyses ofNodalandTbx20double mutant mice demonstrated thatNodal+/−Tbx20−/−embryos showed more severe defects thanNodal+/+Tbx20−/−embryos during early heart development. The expression ofPitx2, a known downstream target ofNodal, was downregulated inTbx20−/−mutants.ConclusionsTwo rare variants onNODALandTBX20genes detected in this family were considered to be loss-of-function mutations. Our results suggest thatNODALandTBX20may be complementary for the cardiac development, and a combinatorial loss-of-function ofNODALandTBX20could be implicated in digenic inherence as the etiology of complex CHD associated with single ventricle defects in this family.