NKX2-5 is a pivotal transcription factor in heart development. Previous studies on lymphocytic DNA provided evidence of familial NKX2-5 gene mutations in cardiac malformations. Common mutations are rare in unrelated families. We analysed, by direct sequencing, the gene encoding NKX2-5 in the diseased heart tissues of 68 patients with complex congenital heart disease, focussing particularly on atrial, ventricular, and atrioventricular septal defects. We identified 35 non-synonymous NKX2-5 mutations in the diseased heart tissues of patients. These mutations were mainly absent in normal, for example, unaffected, heart tissue of the same patient, indicating the somatic nature and mosaicism of mutations. We also observed multiple mutations and multiple haplotypes, as well as mutations in Down's syndrome patients with cardiac malformations. Taken collectively, the above results suggest the somatic nature of NKX2-5 mutations associated with complex cardiac malformations. Somatic mutations in transcription factor genes of cardiac progenitor cells provide a novel mechanism of disease

GATA4 is localised on human chromosome 8p23.1-p22 and codes for a zinc finger transcription factor. Mice lacking Gata4 suffer from defective ventral morphogenesis and heart tube formation.1,2 This factor therefore targets various genes important for heart development and basic cardiac function. Recently, three GATA4 mutations have been detected in families with congenital heart disease.3,4 A mutation affecting a residue next to the C-terminal zinc finger has been identified in family members with atrial septal defects, and resulted in the disruption of a physical interaction between Gata4 and TBX5.3 There is growing evidence that the two zinc fingers of Gata4 display distinct functions so as to facilitate protein–DNA and protein-protein interactions. To further our understanding of the specific role of GATA4 in human congenital heart disease, we searched for mutations in the sequences coding for the zinc fingers in diseased heart tissues of 68 patients with complex heart malformations, encompassing atrial (ASD), ventricular (VSD), and atrioventricular septal defects (AVSD). These patients mostly died in early infancy as a result of the severity of the cardiac abnormalities. We isolated genomic DNA from the formalin fixed malformed hearts, amplified exons 3 and 4 from DNA fragments of about 2 kb, and carried out double strand direct sequencing. Mutations were confirmed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) or cloning and subsequent sequencing to separate alleles (fig 1AGo-F)

Congenital heart disease (CHD) is among the most prevalent and fatal of all birth defects. Deciphering its causes, however, is complicated, as many patients affected by CHD have no family history of the disease. There is also widespread heterogeneity of cardiac malformations within affected individuals. Nonetheless, there have been tremendous efforts toward a better understanding of the molecular and cellular events leading to CHD. Notably, certain cardiac-specific transcription factors have been implicated in mammalian heart development and disruption of their activity has been demonstrated in CHD. The homeodomain transcription factor NKX2-5 is an important member of this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. Thus, NKX2-5 appears to be hypermutable, yet the overall detection frequency in sporadic CHD is about 2% and NKX2-5 mutations are one-time detections with single-positives or private to families. Furthermore, there is lack of genotype-phenotype correlation, in which the same cardiac malformations have been exhibited in different NKX2-5 mutations or the same NKX2-5 mutation associated with diverse malformations. Here, we summarize published NKX2-5 germline mutations and explore different avenues in disease pathogenesis to support the notion of a multifactorial cause of CHD where possibly several genes and associated pathways are involved.

Hypoplasia of the human heart is the most severe form of congenital heart disease (CHD) and usually lethal during early infancy. It is a leading cause of neonatal loss, especially in infants diagnosed with hypoplastic left heart syndrome (HLHS), a condition where the left side of the heart including the aorta, aortic valve, left ventricle (LV) and mitral valve are underdeveloped. The molecular causes of HLHS are unclear, but the basic helix-loop-helix (bHLH) transcription factor heart and neural crest derivatives expressed 1 (Hand1), may be a candidate culprit for this condition. The absence of Hand1 in mice resulted in the failure of rightward looping of the heart tube, a severely hypoplastic LV and outflow tract abnormalities. Nonetheless, no HAND1 mutations associated with human CHD have been reported so far. We sequenced the human HAND1 gene in heart tissues derived from 31 unrelated patients diagnosed with hypoplastic hearts. We detected in 24 of 31 hypoplastic ventricles, a common frameshift mutation (A126fs) in the bHLH domain, which is necessary for DNA binding and combinatorial interactions. The resulting mutant protein, unlike wild-type (wt) HAND1, was unable to modulate transcription of reporter constructs containing specific DNA-binding sites. Thus, in hypoplastic human hearts HAND1 function is impaired.

NKX2-5 is a homeodomain-containing transcription factor important in cardiac development. Familial mutations in the NKX2-5 gene are associated with cardiac abnormalities, but mutations are rare in sporadic cases. We studied the pathology and molecular genetics of NKX2-5 in diseased heart tissues of 68 patients with complex congenital heart disease (CHD), particularly atrial (ASD), ventricular (VSD), and atrioventricular septal defects (AVSD). We also studied DNA extracted from 16 normal hearts, as well as lymphocytic DNA from 50 healthy volunteers, 7 families, and 4 unrelated individuals with CHD. Direct sequencing revealed 53 NKX2-5 mutations in the diseased heart tissues, including nonsynonymous substitutions in the homeodomain of NKX2-5. We found common mutations among unrelated patients, but certain mutations were specific to VSDs and AVSDs. Many patients had multiple NKX2-5 mutations, up to 14 nonsynonymous mutations per patient in VSDs. Importantly, these nonsynonymous mutations were mainly absent in normal heart tissues of the same CHD patients, thus indicating somatic origin and mosaicism of mutations. Further, observed mutations were completely absent in normal hearts and lymphocytic DNA of healthy individuals. Our findings provide new insights for somatic NKX2-5 mutations to be of importance in congenital heart disease.