Identification and functional study of GATA4 gene regulatory variants in atrial septal defects.

BACKGROUND: Congenital heart disease (CHD) is the leading cause of mortality from birth defects. In adult CHD patients with successful surgical repair, cardiac complications including heart failure develop at late stage, likely due to genetic causes. To date, many mutations in cardiac developmental genes have been associated with CHD. Recently, regulatory variants in genes have been linked to many human diseases. Although mutations and splicing variants in GATA4 gene have been reported in CHD patients, few regulatory variants of GATA4 gene are identified in CHD patients. METHODS: GATA4 gene regulatory region was investigated in the patients with atrial septal defects (ASD) (n = 332) and ethnic-matched controls (n = 336). RESULTS: Five heterozygous regulatory variants including four SNPs [g.31360 T>C (rs372004083), g.31436G>A, g.31437C>A (rs769262495), g.31487C>G (rs1053351749) and g.31856C>T (rs1385460518)] were only identified in ASD patients. Functional analysis indicated that the regulatory variants significantly affected the transcriptional activity of GATA4 gene promoter. Furthermore, two of the five regulatory variants have evidently effected on transcription factor binding sites. CONCLUSIONS: Our data suggested that GATA4 gene regulatory variants may confer ASD susceptibility by decreasing GATA4 levels.

Novel and functional DNA sequence variants within the GATA5 gene promoter in ventricular septal defects.

BACKGROUND: Congenital heart disease (CHD) is the most common human birth defect. Genetic causes for CHD remain largely unknown. GATA transcription factor 5 (GATA 5) is an essential regulator for the heart development. Mutations in the GATA5 gene have been reported in patients with a variety of CHD. Since misregulation of gene expression have been associated with human diseases, we speculated that changed levels of cardiac transcription factors, GATA5, may mediate the development of CHD. METHODS: In this study, GATA5 gene promoter was genetically and functionally analyzed in large cohorts of patients with ventricular septal defect (VSD) (n=343) and ethnic-matched healthy controls (n=348). RESULTS: Two novel and heterozygous DNA sequence variants (DSVs), g.61051165A>G and g.61051463delC, were identified in three VSD patients, but not in the controls. In cultured cardiomyocytes, GATA5 gene promoter activities were significantly decreased by DSV g.61051165A>G and increased by DSV g.61051463delC. Moreover, fathers of the VSD patients carrying the same DSVs had reduced diastolic function of left ventricles. Three SNPs, g.61051279C>T (rs77067995), g.61051327A>C (rs145936691) and g.61051373G>A (rs80197101), and one novel heterozygous DSV, g.61051227C>T, were found in both VSD patients and controls with similar frequencies. CONCLUSION: Our data suggested that the DSVs in the GATA5 gene promoter may increase the susceptibility to the development of VSD as a risk factor.

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 promoter region of the GATA4 gene in patients with ventricular septal defects.

Ventricular septal defects (VSDs) are the most common type of congenital heart diseases (CHDs). To date, the genetic causes for sporadic VSDs remain largely unknown. GATA transcription factor 4 (GATA4) is a zinc-finger transcription factor that is expressed in developing heart and adult cardiomyocytes. Mutations in the coding region of the GATA4 gene have been identified in CHD patients, including VSD. As the GATA4 factor is a dosage-sensitive regulator, we hypothesized that the promoter region variants of the GATA4 gene may be genetic causes of VSD. In this study, we analyzed the promoter region of the GATA4 gene by bidirectional sequencing in 172 VSD patients and 171 healthy controls. The results showed that 5 heterozygous sequence variants (NG_008177:g.4071T>C, NG_008177:g.4148C>A, NG_008177:g.4566C>T, NG_008177:g.4653G>T, and NG_008177:g.4690G>deletion) within the promoter region of the GATA gene were identified in 5 VSD patients, but in none of controls. One heterozygous sequence variant (g.4762C>A) was found only in one control, which may have no functional significance. A functional analysis revealed that the transcriptional activity of variant NG_008177:g.4566C>T was reduced significantly, whereas the transcriptional activities of the variants (NG_008177:g.4071T>C, NG_008177:g.4148C>A, NG_008177:g.4653G>T, and NG_008177:g.4690G>deletion) were increased significantly compared with the wild-type GATA4 gene promoter. As GATA4 is a dosage-sensitive regulator during development, our data suggest that these sequence variants within the promoter region of the GATA4 gene may contribute to the VSD etiology by altering its gene expression. Additional studies in experimental animals will deepen our understanding of the genetic basis of VSD and shed light on designing novel molecular therapies for adult VSD patients carrying these variants.

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.