Whole Exome Sequencing Reveals the Major Genetic Contributors to Non-Syndromic Tetralogy of Fallot.

RATIONALE Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD) phenotype. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. OBJECTIVE We used whole exome sequencing (WES) to assess the prevalence of unique, deleterious variants in the largest cohort of non-syndromic TOF patients reported to date. METHODS AND RESULTS 829 TOF patients underwent WES. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database (gnomAD) and a scaled combined annotation-dependent depletion (CADD) score of {greater than or equal to}20. The clustering of variants in two genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P<5x10) after correction for multiple comparisons. NOTCH1 was most frequently found to harbor unique, deleterious variants. 31 changes were observed in 37 probands (4.5%; 95% confidence interval [CI]:3.2-6.1%) and included seven loss-of-function variants 22 missense variants and two in-frame indels. Sanger-sequencing of the unaffected parents of seven cases identified five de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y and p.N1875S) were subjected to functional evaluation and two showed a reduction in Jagged1-induced NOTCH signalling. FLT4 variants were found in 2.4% (95% CI:1.6-3.8%) of TOF patients, with 21 patients harboring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy Disease. In addition to NOTCH1, FLT4 and the well-established TOF gene, TBX1, we identified potential association with variants in several other candidates including RYR1, ZFPM1, CAMTA2, DLX6 and PCM1. CONCLUSIONS The NOTCH1 locus is the most frequent site of genetic variants predisposing to non-syndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients.