Genetic analysis of the SIRT1 gene promoter in ventricular septal defects.

Mutations in cardiac transcription factor genes, such as GATA-4, NKX2-5 and TBX5 genes, have been associated to the patients with familial and isolated congenital heart disease (CHD). Little work has been done on the epigenetic causes for CHD. Sirtuis are highly conserved NAD-dependent class III deacetylases. In mammals, there are seven members of surtuin family, SIRT1-SIRT7. SIRT1, the closest to yeast Sir2, has deacetylase activity and ADP-ribosyltransferase activity. SIRT1 has been involved in many cellular processes and implicated in human diseases, such as obesity, type 2 diabetes, cancer and neurodegenerative diseases. We hypothesized that altered levels of SIRT1 gene expression, rather than mutations in SIRT1 gene, may contribute to the human diseases. In this study, we genetically analyze the SIRT1 gene promoter in patients with ventricular septal defects (VSD) (n=333) and ethic-matched healthy controls (n=348). In all, six single-nucleotide polymorphisms (SNPs) and twelve heterozygous sequence variants were identified. Four novel heterozygous variants, g.69643693A>G, g.69643963A>T, g.69643971G>A and g.69644366Ins, were found in six VSD patients, but in none of controls. Six SNPs and variants, g.69643707A>C (rs35706870), g.69643874C>A, g.69644209C>G, g.69644213G>A, g.69644268T>A and g.69644441G>A, were only identified in controls. The other SNPs and variants were found in both groups with similar frequencies. Therefore, the variants within the SIRT1 gene promoter identified in VSD patients may alter the transcriptional activities of SIRT1 gene promoter. Changed SIRT1 protein levels may contribute to the VSD etiology by affecting the activities of its substrates.

Genetic and functional analysis of the NKX2-5 gene promoter in patients with ventricular septal defects.

The ventricular septal defect (VSD) is the most common type of congenital heart disease (CHD). The morbidity and mortality of CHD patients are significantly higher due to late cardiac complications, likely caused by genetic defects. Mutations in cardiac transcription factor genes such as GATA-4, TBX5, and NKX2-5 have been implicated in CHD cases. The NKX2-5 gene, a homeobox gene, is expressed in the developing heart and the adult heart. Because NKX2-5 is a dosage-sensitive regulator during embryonic development, the authors hypothesized that the expression levels of the NKX2-5 gene rather than the mutant protein may play important roles in CHD. In this study, the promoter regions and exon regions of the NKX2-5 gene were bidirectionally sequenced in large cohorts of VSD patients and healthy control subjects. The results showed that a novel sequence variant (g.4574c>deletion), found only in one VSD patient, and a single nucleotide polymorphism (rs118026695), the frequency of which was significantly higher in VSD patients, were identified within the promoter region. Functional analysis confirmed that these sequence variants significantly enhanced the transcriptional activities of the NKX2-5 gene promoter, altering the expression of the NKX2-5 gene and the cardiac gene regulatory network. In addition, a synonymous mutation in the second exon of the NKX2-5 gene was identified in one VSD patient, which may affect the translation process. Therefore, the authors' data provide supportive evidence that mutations in the coding region of the NKX2-5 gene and sequence variants within its promoter region may be among the contributors to the CHD etiology.

Genetic analysis of the TBX20 gene promoter region in patients with ventricular septal defects.

Congenital heart disease (CHD) is the most common human birth defect. The morbidity and mortality of CHD patients are significantly higher than normal population even after surgical correction of cardiac defects, which is likely caused by genetic defects. To date, genetic causes for CHD remain largely unknown. TBX20 gene encodes a T-box transcription factor that plays pivotal roles in cardiac morphogenesis and is required for maintaining adult heart function and maturation. Mutations in TBX20 gene have been reported in familiar and sporadic CHD patients. However, the promoter region of TBX20 gene has not been genetically analyzed in CHD patients. As TBX20 functions as a dosage-dependent regulator during the heart development, we hypothesized that the sequence variants within the promoter region of the TBX20 gene may contribute to CHD. In this study, we bi-directionally sequenced the promoter region of the TBX20 gene in 265 patients with ventricular septal defects (VSD) and 242 controls. Within the promoter region of the TBX20 gene, one single-nucleotide polymorphism (SNP), rs336284 (g.4740T>C), and one novel heterozygous variant g.4741 G>A, which was linked with rs336284 (g.4740 T>C), were found in both VSD patients and controls with similar frequencies. A novel heterozygous variant, g.4932 G>A, was found in one VSD patient, but in none of controls, which significantly inhibited the transcriptional activities of TBX20 gene promoter, suggesting that the variant may contribute to the VSD etiology. Therefore, our data provides new information with respect to TBX20 gene mutations in CHD patients.