Whole Exome Sequencing Reveals the Major Genetic Contributors to Nonsyndromic Tetralogy of Fallot

Abstract

Rationale: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, nonsyndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease phenotype. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied to date.

Objective: We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date.

Methods and results: Eight hundred twenty-nine TOF patients underwent whole exome sequencing. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database and a scaled combined annotation-dependent depletion score of ≥20. The clustering of variants in 2 genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P<5×10^-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbor unique, deleterious variants. Thirty-one changes were observed in 37 probands (4.5%; 95% CI, 3.2%-6.1%) and included 7 loss-of-function variants 22 missense variants and 2 in-frame indels. Sanger sequencing of the unaffected parents of 7 cases identified 5 de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y, and p.N1875S) were subjected to functional evaluation, and 2 showed a reduction in Jagged1-induced NOTCH signaling. 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 nonsyndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients.

Keywords: Tetralogy of Fallot; genes; genetic variation; heart diseases; whole exome sequencing.

Figure 1

The top genes, in order of significance, in which non-syndromic TOF patients carry unique, deleterious variants. Bars indicate the respective significance levels of variant clustering for each gene, represented as –log P values. Circles represent the number of variants. The -log10(p) column for NOTCH1 (P<2.22 x 10^-16) goes towards infinity and is shown as arbitrarily high.

Figure 2

Unique, deleterious NOTCH1 variants in TOF patients. Diagrammatic representation of the NOTCH1 protein with known protein domains indicated. The location of NOTCH1 variants identified in our TOF cohort is shown. p.P143L, p.G193A and p.R2004L discussed in the main text are indicated (bold). ANK, ankyrin repeats; EGF, epidermal growth factor; HD, heterodimerisation domain; LBR, ligand binding region; LNR, Lin/Notch repeats; PEST, PEST domain; RAM, RBPJ-associated molecule domain; TAD, transactivation domain; TM, transmembrane domain.

Figure 3

(a) Immunoblot for FLAG to determine the expression and S1 cleavage of NOTCH1 variants p.G200R, p.C607Y and p.N1875S in comparison to WT NOTCH1 following overexpression in HeLa cells. The two bands at 300 kDa (P300) and 120 kDa (P120) represent the full length and the S1-cleaved NOTCH1 protein. β-actin was used as a loading control. (b) Quantification of the percentage of S1 cleaved versus uncleaved NOTCH1 protein for WT NOTCH1 and NOTCH variants p.G200R, p.C607Y and p.N1875S. Error bars: mean ±SEM from three biological replicates and statistical significance was determined using two-tailed paired t-tests. (c) The effect of rare, deleterious NOTCH1 variants on Jagged-induced NOTCH signalling levels. NOTCH signalling activity was measured using a luciferase-based reporter system (RBPJ). HeLa cells were cultured with or without immobilised JAG1 ligand and co-transfected with RBPJ reporter constructs and WT NOTCH1, p.G200R, p.C607Y or p.N1875S. Firefly luciferase readings were normalised to Renilla luciferase readings to control for transfection efficiency and cell number. RBPJ activity was expressed relative to WT NOTCH1 for comparison. Error bars: mean ±SEM from four biological replicates, each with three technical replicates. Statistical significance was assessed using two-tailed paired t-tests and the Hochberg step-up procedure to control for family-wise error rate.

Figure 4

Unique, deleterious FLT4 variants in TOF patients. Schematic representation of FLT4 structure with immunoglobulin (Ig) domains and protein kinase domain, indicated. Top: FLT4 variants identified in our TOF cohort (black) and those previously reported (grey). Bottom: FLT4 missense or in-frame mutations reported in Milroy disease, all located in the protein kinase domain.