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

Abstract

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.

Keywords: Atrial septal defects; GATA4; Genetics; Promoter; Regulatory variants.

Fig. 1

Regulatory variants of GATA4 gene. a Locations of regulatory variants upstream to the transcription start site of human GATA4 gene (NG_008177.2). b Sequencing chromatograms of GATA4 gene regulatory variants identified in ASD patients. Top panels are wild type and bottom heterozygous variants (marked with arrows). Sequencing orientations are forward

Fig. 2

Relative activities of GATA4 gene promoters with or without regulatory variants in H9c2 cells. All transfection experiments were performed three times independently, in triplicate. The results were represented as means ± SEM. Bar graph represented the mean and error bar indicated SEM. Empty pGL3-basic was used as a negative control. Activity of wild type GATA4 gene promoter was set as 100%. WT, wild type. *P < 0.01

Fig. 3

Effects of regulatory variants on transcription factor binding. EMSA was performed with biotin-labeled oligonucleotide and H9c2 cell nuclear extracts. Free probe was indicated at the bottom. Solid arrows indicated the affected binding for unknown transcription factors. WT, wild type. RV, regulatory variant.