Genetic and Functional Variants Analysis of the GATA6 Gene Promoter in Acute Myocardial Infarction

Abstract

Background: Acute myocardial infarction (AMI) which is a specific type of coronary artery disease (CAD), is caused by the combination of genetic factors and acquired environment. Although some common genetic variations have been recorded to contribute to the development of CAD and AMI, more genetic factors and potential molecular mechanisms remain largely unknown. The GATA6 gene is expressed in the heart during embryogenesis and is also detected in vascular smooth muscle cells (VSMCs), different human primary endothelial cells (ECs), and vascular ECs in mice. To date, no studies have directly linked GATA6 gene with regulation of the CAD. Methods: In this study, we used a case-control study to investigate and analyze the genetic variations and functional variations of the GATA6 gene promoter region in AMI patients and controls. A variety of statistical analysis methods were utilized to analyze the association of single nucleotide polymorphisms (SNPs) with AMI. Functional analysis of DNA sequence variants (DSVs) was performed using a dual luciferase reporter assay. In vitro, electrophoretic mobility shift assay (EMSA) was selected to examine DNA-protein interactions. Results: A total of 705 subjects were enrolled in the study. Ten DSVs were found in AMI patients (n = 352) and controls (n = 353), including seven SNPs. One novel heterozygous DSV, (g.22168409 A > G), and two SNPs, [g.22168362 C > A(rs1416421760) and g.22168521 G > T(rs1445501474)], were reported in three AMI patients, which were not found in controls. The relevant statistical analysis, including allele and genotype frequencies between AMI patients and controls, five genetic models, linkage disequilibrium (LD) and haplotype analysis, and SNP-SNP interactions, suggested no statistical significance (P > 0.05). The transcriptional activity of GATA6 gene promoter was significantly increased by the DSV (g.22168409 A > G) and SNP [g.22168362 C > A(rs1416421760)]. The EMSA revealed that the DSV (g.22168409 A > G) and SNP [g.22168362 C > A(rs1416421760)] evidently influenced the binding of transcription factors. Conclusion: In conclusion, the DSV (g.22168409 A > G) and SNP [g.22168362 C > A(rs1416421760)] may increase GATA6 levels in both HEK-293 and H9c2 cell lines by affecting the binding of transcription factors. Whether the two variants identified in the GATA6 gene promoter can promote the development and progression of human AMI by altering GATA6 levels still requires further studies to verify.

Keywords: GATA binding protein 6; acute myocardial infarction; gene expression regulation; genetic variants; promoter; single nucleotide polymorphisms.

Figure 1

The DSVs and SNPs within the GATA6 gene promoter identified in AMI patients and controls. Schematic representation of the identified GATA6 gene DSVs. The DSVs were named according to their locations in the GATA6 genomic sequences (NCBI: NC_000018.10). The transcription starts at 22169437 in the first exon that is untranslated.

Figure 2

Sequencing chromatograms of the DSVs and SNPs within the GATA6 gene promoter. (A). Sequencing chromatograms of the DSV and SNPs in AMI patients. (B). Sequencing chromatograms of the DSV and SNP in healthy controls. All sequence orientations of the DSVs and SNPs are forward. Top panels show wild type and bottom panels heterozygous DSVs or SNPs, which are marked with arrows.

Figure 3

LD analysis of the six SNPs in AMI. Standard color schemes indicate different levels of LD. Bright red: LOD > 2, D' = 1; Purple: LOD < 2, D' < 1; White: LOD < 2, D' < 1; LOD: Log of the odds of there being LD between two loci.

Figure 4

Relative transcriptional activity of wild-type and variant GATA6 gene promoters. Wild-type and variant GATA6 gene promoters were cloned into reporter gene vector pGL3 and transfected into cultured cells. The transfected cells were collected, and dual-luciferase activities were assayed. Empty vector pGL3-basic was used as a negative control. Transcriptional activity of the wild-type GATA6 gene promoter was designed as 100%. Relative activities of GATA6 gene promoters were calculated. (A) Relative activities of wild-type and variant GATA6 gene promoters in HEK-293 cells. Lanes 1, pGL3-basic; 2, pGL3-WT; 3, pGL3-22168362A; 4, pGL3-22168409G; 5, pGL3-22168438A; 6, pGL3-22168521T+22168944A+22169265A; 7, pGL3-22168521T; 8, pGL3-22168780G. (B) Relative activities of wild-type and variant GATA6 gene promoters in H9c2 cells. Lanes 1, pGL3-basic; 2, pGL3-WT; 3, pGL3-22168362A; 4, pGL3-22168409G; 5, pGL3-22168438A; 6, pGL3-22168521T+22168944A+22169265A; 7, pGL3-22168521T; 8, pGL3-22168780G. WT, wild-type. *, P 0.05; **, P 0.01.

Figure 5

EMSA of biotin-labeled oligonucleotides containing DSV and SNP. Wild-type and variant oligonucleotides (31 bp) were designed and labeled with biotin for the DSV and SNP identified in AMI patients, including g.22168362C>A (rs1416421760) and g.22168409A>G. EMSA was conducted with biotinylated oligonucleotides and the nuclear extracts from HEK-293 and H9c2 cells. Free probe was marked with an arrow at the bottom. The affected binding for transcription factors was marked with an open arrow.