(A) 26 de novo mutated genes from CHD study participants selected as being of particular interest (Zaidi et al., 2013) based upon sequence quality, mutation type, the expression level of the mouse homolog during heart development on embryonic day 14.5, and previously reported involvement in CHD or heart development. The 26 corresponding Drosophila homologs are shown with protein function (Flybase), and Mortality Rate (Mortality Index). (B–D) The proportions of Drosophila gene homologs that, when silenced by cardiac cell specific RNAi expression, induce developmental lethality at normal/background levels (blue); low levels (green); medium levels (orange); high levels (red) based on Mortality Index values. The Mortality Index is determined by crossing homozygous UAS-RNAi transgenic flies with flies bearing a 4XHand-Gal4 ‘driver’ (four repeats of the cardioblast cell-specific Hand enhancer element 5’ of Gal4) balanced over CyO. Progeny flies that emerge as adults with curly wings (CyO, no transgene expression) vs. straight wings (expressing 4XHand-Gal4 driven UAS-RNAi transgene in cardioblasts) are recorded and the developmental mortality attributable to RNAi heart expression (Mortality Index) is calculated as (Curly – Straight) / Curly X 100. Divergence from 1:1 ratio (Normal, blue) ≥ 7% was considered a lethal phenotype. A Normal range of divergence from a 1:1 ratio of <6% based on analysis of 400 progeny from control crosses. Varying degrees of phenotype severity were observed (Low = 7–30%, green; Medium = 31–60%, orange; High = 61–100%, red). (B) Left: chart (PCGC top 26) shows proportions of RNAi silencing effects on lethality for Drosophila homologs of 26 genes identified as being of particular interest based exclusively on bioinformatics-based criteria. Right: chart (PCGC total list) shows the results of 134 fly homologs of all de novo mutated genes (with available RNAi silencing lines) identified in pediatric CHD study participants (Zaidi et al., 2013) (Supplementary file 1). (C) Comparison of silencing-induced lethality for Drosophila homologs of 134 candidate CHD-associated genes as a function of high (HHE) versus low (LHE) levels of expression of murine homologs in embryonic mouse heart. (D) Comparison of silencing-induced lethality for Drosophila homologs of 134 candidate CHD-associated genes as a function of fly-to-human gene conservation (High Conservation, score 6 to 10; Low Conservation, score 2 to 5).