Genetic And Functional Variants Analysis Of The GATA6 Gene Promoter In Acute Myocardial Infarction

Background:Cardiovascular disease(CVD)has become a serious threat to human health.In China,the aging of the social population and the rise in the levels of various risk factors have led to a continuous increase in the prevalence of CVD,with an earlier age of onset.Acute myocardial infraction(AMI)is the most serious stage in the development of coronary atherosclerotic disease(CAD),with acute morbidity and high mortality.The cause of the disease is mainly caused by genetic factors and acquired environmental factors.Although some common genetic variations have been reported to contribute to the development of CAD and AMI,there are still many molecular genetic mechanisms to be explored.Based on previous epidemiological studies,we found that cardiovascular events and mortality were significantly higher in adults with congenital heart disease(CHD)than in the general population.Myocardial infraction(MI)is the main cause of death.Disorders in cardiac developmental genes may contribute to the onset of CAD or AMI.However,the GATA6 gene is expressed not only during cardiac development during human embryonic development,but also in vascular smooth muscle cells(VSMC)and different primary endothelial cells(EC)and vascular EC in mice.Therefore,we speculate that the variation of the promoter region of GATA6 gene may be involved as a risk factor in the occurrence and development of human AMIObjective:DNA sequence variants(DSVs)and single nucleotide polymorphisms(SNPs)of the promoter region of GATA6 gene in AMI and control groups were identified and statistical analysis was performed.Thereby,the study on the genetic variation of the GATA6 gene promoter was realized.Further explore whether the AMI-related DSVs and SNPs affect the binding to other transcription factors and confirm whether they alter the transcriptional activity of the GATA6 gene.To achieve study on the functional variation of the GATA6 gene promoter and provide a genetic basis for the treatment and prevention of AMI.Methods:In this study,we used a case-control study design to identify and analyze sequence variants of the GATA6 gene promoter region in AMI patients(352 patients)and controls(353 patients).A Hardy—Weinberg equilibrium(HWE)test was performed on each SNP frequency in AMI patients and controls by Fisher’s test.A Pearson chi-square test was performed to assess differences in allele and genotype frequencies between AMI patients and controls.The association of five genetic models with the onset of AMI was analyzed by the web-based software SNP-Stats(https://www.snpstats).Analysis of linkage disequilibrium(LD)and haplotypes was performed using the HaploView software and the SHE-sis software platform(http://analysis.bio-x.cn/SHEsisMain.htm).The interaction of SNP-SNPs was studied by using generalized multifactor dimensionality reduction(GMDR)software.A plasmid containing the gene fragment of interest was constructed and transfected into HEK293 and H9c2 cardiomyocytes cultured in vitro,and the expression level of luciferase was measured to perform functional analysis on the identified sequence variants.Prediction of the effect of transcription factor binding sites on variants identified only in the GATA6 gene promoter of AMI patients was performed using the JASPAR online program(http://jaspar.genereg.net/).In vitro,electrophoretic mobility shift assay(EMSA)was selected to detect DNA-protein interactions.Results:A total of 10 variants,including 7 SNPs,were identified in 705 subjects.A new heterozygous DSV(g.22168409 A>G)and two SNPs[g.22168362 C>A(rs1416421760)and g.22168521 G>T(rs 1445501474)]were only found in three AMI patients.Statistical analysis of relevant data,including allele and genotype frequencies,five genetic models,LD and haplotype analysis,and SNP-SNP interactions between AMI patients and controls,were not statistically significant(P>0.05).DSV(g.22168409 A>G)and SNP[g.22168362 C>A(rs1416421760)]significantly increased the transcriptional activity of the GATA6 gene promoter.EMSA results showed that DSV(g.22168409 A>G)and SNP[g.22168362 C>A(rs1416421760)]can affect 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.Meanwhile,our study provides a wealth of population genetic data from China on the GATA6 gene promoter region polymorphisms and laid the genetic basis for subsequent related studies.