Effects Of The Polymorphisms In ABCG1 Promoter On The Transcription Activity And It's Correlation With Coronary Artery Disease

?Background? Coronary artery disease(CAD)is the leading cause of human health problem and its morbidity and mortality are the highest among all human diseases.CAD is mainly caused by atherosclerosis,and is closely associated with many genetic factors.High plasma cholesterol is one of the independent risk factors of CAD,and accumulation of cholesterol in cells is associated with regulation of cellular metabolism of cholesterol.ATP binding cassette transporter G1(ABCG1)is a member of the ATP-binding cassette(ABC)transporter superfamily,which is involved in the transport of cellular cholesterol and phospholipid to the high density lipoprotein cholesterol(HDL-C)and plays an important role in the reverse cholesterol transport(RCT).These roles of ABCG1 make serious cholesterol deposition be avoided in the peripheral cells.Single nucleotide polymorphisms(SNPs)within the ABCG1 gene or promoter region were reported to be associated with susceptibility to CAD.The SNPs in promoter region of ABCG1 may alter the transcription activity and impact on gene expression,which may lead to variation in susceptibility to CAD among individuals.SNPs induced a decrease in promoter activity was reported to reduce the risk of CAD,and the molecular mechanism might be apoptosis of macrophages induced by the decreased expression of ABCG1.ABCG1 has multiple transcriptional regulatory region(promoter region A,B,C,etc.).SNPs in promoter region A were also reported to be related to CAD susceptibility,and associated with early myocardial infarction and the number of coronary artery lesion.It was unclear whether rs1378577 affects the activity of ABCG1 promoter,and whether other SNPs existing in the promoter region-A are associated with susceptibility to CAD.This study purposed to elucidate the association between the SNPs in ABCG1 promoter-A and CAD by sequencing the proximal promoter-A of ABCG1 among all CAD and the control individuals,and to investigate the influence of the SNPs in ABCG1 promoter-A on the promoter activity using double luciferase reporter gene system.?Objectives?1.To investigate the influence of the polymorphisms in ABCG1 promoter A on the transcriptional activity.2.To investigate the correlation of the polymorphisms in ABCG1 promoter A with CAD in southern Chinese Han population.?Methods? 1.A case-control study was conducted,217 CAD patients and 142 controls were enrolled in this study.Genomic DNA was extracted from the peripheral blood,and clinical information for all subjects was collected.2.The fragment from the transcription start site to about 1000 bp upstream of ABCG1 promoter A was amplified by polymerase chain reaction(PCR)for each sample.The SNPs in the promoter of ABCG1 were identified by sequencing the PCR product among all individuals.3.Promoter vectors with different ABCG1 promoter haplotypes were constructed and transfected into Hela or HCAEC for analysis of the promoter activity by using dual luciferase reporter system.4.The allele frequency of SNPs and haplotypes were compared between CAD group and the controls,and between sub-groups,such as premature CAD and non-premature CAD,single vessel lesion and multivessel lesion.?Results? 1.Only 3 SNPs were found in ABCG1 promoter sequence of about 1000 bp upstream of the transcription start site,which are-384(A/G),-204(A/C)and-134(T/G),respectively.The 3 SNPs are in strong linkage disequilibrium,Tajima's D = 2.655(P<0.001),which constituted 3 haplotypes.2.Three luciferase reporter vectors containing different constitutive ABCG1 promoter haplotypes were contructed successfully,namely,PGL-ACG,PGL-GAT and PGL-GCG,and 2 promoter vectors with mutated ABCG1 haplotypes were constructed successfully,namely,PGL-GAG and PGL-GCT.No significant difference in the transcriptional activity was observed among three constitutive promoter haplotypes,but the transcriptional activity was notably elevated as the GAT haplotype was mutated into GAG(P<0.05).3.No significant difference in SNPs and haplotype frequency was observed between the CAD group and the normal control group.The severity of vascular disease and the early onset of coronary artery disease were not implied to be associated with the polymorphisms in ABCG1 promoter.?Conclusions? 1.The SNPs-384(A/G),-204(A/C)and-134(T/G)identified in ABCG1 proximal promoter A did not alter the promoter actitivity.2.The frequency distribution of the SNPs-384(A/G),-204(A/C)and-134(T/G)and the constitutive promoter haplotypes were not significantly associated with susceptibility to CAD.