Background Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, affecting 3–6 infants for every 10 000 births. TOF is phenotypically well defined; it consists of four heart abnormalities: a VSD, an over-riding aorta, a narrowed pulmonary valve and right ventricular hypertrophy. During heart development two heart fields can be distinguished. The first one gives origin to the left ventricle and contributes to the right and left atria. The secondary heart field gives origin to the right ventricle and the outflow tract. Each of these fields can be identified by the expression of specific markers. As TOF is a malformation of the outflow tract, we hypothesised genes involved in the regulatory network of the secondary heart field were particularly good candidates for TOF susceptibility.

Methods We examined by standard Sanger method the full exonic and intron boundary regions of 14 secondary heart field genes, namely NKX2-5, GATA4, TBX20, MEF2C, BOP, HAND2, FOXC1, FOXC2, TBX1, FOXA2, FGF10, FGF8, ISL1 and FOXH1, in a panel of 93 TOF patients. All newly discovered rare variants were checked in a panel of 1000 control chromosomes by multiplex Sequenom assays. When available, parents of cases were screened to assess inheritance of the rare variant.

Results We re-sequenced a total of 80 exons and ∼30 Kb. Among the 14 genes studied we found a total of 50 new variants, of which 23 were exclusive to the patient population, ie, were absent from 1000 normal chromosomes. Nine of these variants cause change in the aminoacid sequence. We found a functional 19aa deletion of a highly conserved region of TBX1. In FOXC1 we found a contraction of both alanine and glycine tracts. An alanine expansion, usually known to be deleterious, was found in HAND2. Four non-synonymous changes were found in FOXA2. Most patients presented just one variant, however 3 patients presented two, and one patient presented up to 3 variants. All patients were heterozygotes for the variants, and had inherited them from one of their phenotypically normal parents (when parental information was available). In addition, 75% of the variants were inherited from the mother.

Conclusions Although genes of the secondary heart field seemed good candidates for TOF susceptibility, thus far we have not found any strong indication of unique causal effect, as all variation found in probands was also present in their unaffected parents. However, the presence of multiple variants in the same proband may result in the disruption of gene-gene interactions in the secondary heart field pathway, which in turn may lead to outflow tract defects. Based on our results, it would seem more likely that susceptibility to TOF be determined by a larger number of small genetic contributions which are also modified by environmental factors. It is evident that larger scale analysis of significant numbers of whole genomes/exomes will be necessary to better understand the molecular aetiology of TOF.