Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations

Abstract

Background. Variants of several genes encoding transcription modulators, signal transduction, and structural proteins are known to cause Mendelian congenital heart disease (CHD). NKX2-5 and GATA4 were the first CHD-causing genes identified by linkage analysis in large affected families. Mutations of TBX5 cause Holt-Oram syndrome, which includes CHD as a clinical feature. All three genes have a well-established role in cardiac development. Design. In order to investigate the possible role of multiple mutations in CHD, a combined mutation screening was performed in NKX2-5, GATA4, and TBX5 in the same patient cohort. Samples from a cohort of 331 CHD patients were analyzed by polymerase chain reaction, double high-performance liquid chromatography and sequencing in order to identify changes in the NKX2-5, GATA4, and TBX5 genes. Results. Two cases of multiple heterozygosity of putative disease-causing mutations were identified. One patient was found with a novel L122P NKX2-5 mutation in combination with the private A1443D mutation of MYH6. A patient heterozygote for a D425N GATA4 mutation carries also a private mutation of the MYH6 gene (V700M). Conclusions. In addition to reporting two novel mutations of NKX2-5 in CHD, we describe families where multiple individual mutations seem to have an additive effect over the pathogenesis of CHD. Our findings highlight the usefulness of multiple gene mutational analysis of large CHD cohorts.

Figure 1

Sequence traces showing the nonsynonymous nucleotide changes causing amino acid replacements in the NKX2.5, GATA4, and TBX5 proteins. The changes are indicated with arrows.

Figure 2

nnpredict output showing the prediction of the structural consequence of the L122P mutation in NKX2.5. The panels show the sequence of the wild-type (A) and mutant (B) NKX2.5 proteins. The relevant residues at position 122 are underlined. The secondary structure prediction is shown for each sequence. The fourth helix predicted in the wild-type sequence is 18 residues long (in bold). An eight-residue stretch within that segment is predicted to adopt a different configuration in the mutant peptide (H, helix, E, strand, -. no prediction).

Figure 3

Pedigrees of the families CHD where multiple heterozygosity for the NKX2.5, GATA4, and MYH6 genes was detected. ASD, atrial septal defect; PFO, patent foramen ovale.

Figure 4

Multiple alignment of amino acid sequences of segments of the NKX2-5, GATA4, and TBX5 proteins from every vertebrate species available. (A) The NKX2-5 G232R mutation introduces a positively charged arginine residue in a position where only noncharged amino acids are present in NKX2.5 vertebrate orthologs. (B) The TBX5 D111Y mutation replaces a negatively charged aspartic acid residue with a noncharged tyrosine residue. Both amino acids forming a salt bridge predicted by the human TBX5 tridimensional structure (PDB: 2X6U) are universally conserved among TBX5 vertebrate orthologs. (C) The D425N GATA4 mutation introduces a noncharged residue in a position where only negatively charged residues are present in every vertebrate GATA4 ortholog sequence available.

Figure 5

(A) Molecular model of human TBX5 protein free from nucleic acid based on PDB file 2X6U showing the salt bridge between the D111 and the K126 residues. (B) The D111Y change disrupts the salt bridge, as the negatively charged aspartic acid (D) residue is replaced by an uncharged tyrosine residue (Y). (C) Model of the T-box motif bound to nucleic acid based on PBD file 1H6F showing that during interaction with its target promoter, the side-chains of the D111 and K126 residues point to opposite directions, canceling the salt bridge.