Multifaceted analysis of noncoding and coding de novo variants implicates NOTCH signaling pathway in tetralogy of...

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect in neonates. While there is compelling evidence of genetic contribution to the etiology of TOF, the contribution of noncoding variants to the development of the defect remains unexplored. Potentially damaging noncoding de novo variants (NC DNVs) were detected from 141 Chinese nonsyndromic TOF trios (CHN-TOF) and compared with those detected in the Pediatric Cardiac Genomics Consortium (PCGC). Bioinformatic analyses on noncoding and previously detected coding DNVs were performed to identify developmental pathways affected in TOF. Chinese but not PCGC-TOF patients showed a notably increased burden of putative damaging NC DNVs (n = 249). In Chinese, NC and coding DNVs were predominantly associated with cardiomyocyte differentiation and with chamber/valve/aorta development, respectively, producing a combined enrichment in NOTCH signaling (p = 1.1 × 10^-6) and outflow tract morphogenesis (p = 2.2 × 10^-5). Genes with NC DNVs (e.g., EFNB2, HEY2, and PITX2) interacted with NOTCH1 and FLT4 in a tight STRING protein-protein interaction (PPI) network. During the in vitro cardiac differentiation process, these noncoding candidate genes, which harbored potentially damaging regulatory NC DNVs, exhibited co-expression with NOTCH signaling genes and demonstrated dysregulated gene expression at various differentiation stages following NOTCH1 downregulation. In summary, our findings highlight a significant contribution of NC DNVs to TOF and suggest the presence of population genetic heterogeneity. Integrative analyses implicate dysregulation of NOTCH signaling, with converging influences from both coding and noncoding variants, in TOF within the Chinese population.