Background:Tetralogy of Fallot (TOF)—the most common cyanotic heart defect in newborns—has evidence of multiple genetic contributing factors. Identifying variants that are clinically relevant is essential to understand patient-specific disease susceptibility and outcomes and could contribute to delineating pathomechanisms.Methods:Using a clinically driven strategy, we reanalyzed exome sequencing data from 811 probands with TOF, to identify rare loss-of-function and other likely pathogenic variants in genes associated with congenital heart disease.Results:We confirmed a major contribution of likely pathogenic variants inFLT4(VEGFR3 [vascular endothelial growth factor receptor 3]; n=14) andNOTCH1(n=10) and identified 1 to 3 variants in each of 21 other genes, includingATRX,DLL4,EP300,GATA6,JAG1,NF1,PIK3CA,RAF1,RASA1,SMAD2, andTBX1. In addition, multiple loss-of-function variants provided support for 3 emerging congenital heart disease/TOF candidate genes:KDR(n=4),IQGAP1(n=3), andGDF1(n=8). In total, these variants were identified in 63 probands (7.8%). Using the 26 composite genes in a STRING protein interaction enrichment analysis revealed a biologically relevant network (P=3.3×10⁻¹⁶), with VEGFR2 (vascular endothelial growth factor receptor 2;KDR) and NOTCH1 (neurogenic locus notch homolog protein 1) representing central nodes. Variants associated with arrhythmias/sudden death and heart failure indicated factors that could influence long-term outcomes.Conclusions:The results are relevant to precision medicine for TOF. They suggest considerable clinical yield from genome-wide sequencing, with further evidence forKDR(VEGFR2) as a congenital heart disease/TOF gene and for VEGF (vascular endothelial growth factor) and Notch signaling as mechanisms in human disease. Harnessing the genetic heterogeneity of single gene defects could inform etiopathogenesis and help prioritize novel candidate genes for TOF.