Functional Characterization Of Genomic Variant Rs6903956 In ADTRP,a Susceptibility Gene For Coronary Artery Disease In The Chinese Han Population

Coronary artery disease(CAD)is the most common form of cardiovascular diseases.CAD is also known as atherosclerotic coronary heart disease.CAD is caused by the complex interactions among genetic susceptibility factors and environmental risk factors,and is currently one of the leading causes of morbidity and mortality worldwide.Genome-wide association studies(GWAS)developing in recent years,have identified >60 genetic loci for CAD in populations of European ancestry,which have provided important insights into the genetic and molecular mechanisms of CAD.In 2011,our laboratory reported the first GWAS for CAD in the Chinese Han population and identified a SNP(single nucleotide polymorphism),rs6903956,on chromosome 6p24.1 which was significantly associated with susceptibility to CAD.The minor risk allele A of SNP rs6903956 is associated with a decreased mRNA expression level of ADTRP,which regulates the expression level of the tissue factor pathway inhibitor(TFPI)gene.However,it is still unclear about the detailed molecular mechanisms by which SNP rs6903956 regulates the expression of the ADTRP gene and increase the risk of CAD.Therefore,this thesis research project focuses on the molecular and functional characterization of SNP rs6903956 for its role in ADTRP gene expression and CAD pathogenesis using an integrated strategy of bioinformatic analysis,cell biology and molecular biology.In this study,by searching the Ensembl and UCSC databases,we found that SNP rs6903956 is located in the intron 1 of ADTRP and the substitution of the G allele to the A allele occurs at a potential GATA site for binding the GATA transcriptional factors.Moreover,the GATA2 gene was previously shown to be involved in the pathogenesis of CAD.Based on these data,we proposed a hypothesis for this project: SNP rs6903956 may affect allele specific expression of ADTRP by affecting the potential GATA transcription factor binding sites,resulting in increased susceptibility to CAD.To test the hypothesis,we first cloned the 525 bp DNA sequence containing rs6903956 into a dual luciferase reporter and performed luciferase assays.The data showed that GATA1,GATA2 and GATA4 can all significantly increase the luciferase activities;The wild allele G of rs6903956 increases significantly more luciferase activities than the minor A allele;The effects of GATA2 on luciferase activities are the most prominent compared with the other two GATA transcriptional factors.Moreover,we performed similar gene regulation studies by determining ADTRP mRNA expression by qRT-PCR and ADTRP protein expression by Western blot analysis,respectively.We found that ADTRP mRNA and protein expression levels were regulated by GATA1/2/4,and the effect of GATA2 was the greatest.These data suggest that genomic variant rs6903956 in ADTRP decreases the expression of ADTRP by affecting regulation of ADTRP by GATA transcriptional factors,in particular,the GATA2 factor.In conclusion,we have found that SNP rs6903956,the first identified susceptibility genomic variant to CAD in the Chinese Han population by GWAS,can regulate ADTRP gene expression by affecting the regulation of ADTRP by transcription factors GATA1/2/4,thereby affecting the risk of CAD.Our data provide a molecular mechanism by which ADTRP SNP rs6903956 affects the expression level of ADTRP and the risk of CAD.Further studies may provide important molecular basis and important insights into the pathogenesis of CAD,which may be of importance to the prevention and treatment of CAD.