The ABCG1 Gene Polymorphism And Coronary Heart Disease Association Studies And ABCG1 Promoter Transcriptional Activity Of Research

BackgroundCoronary Artery Disease (CAD) is not only the most common disease in our country, but also one of the most important culprits for morbidity and mortality. ABCA1 and ABCG1 both belong to the ABC superfamily, a group of cellular transmembrane proteins that mediate the ATP-dependent lipid transport. Previous studies identified both transporters as regulators of cholesterol efflux, although they may have different roles in the process. ABCA1 was showed to transport cholesterol to lipid-depleted apoA-l, forming pre-β-HDL, whereas ABCG1 transports cellular cholesterol to the major HDL fractions (HDL3 and HDL2). ABCG1 plays an important role in mediating cholesterol efflux to HDL Recent studies have shown that ABCG1 involved in the apoptosis of macrophages and endothelial cells dysfunction, indicating that ABCG1 plays an important role in the formation of atherosclerosis. But overall, its functional significance in the development of atherosclerosis remains unknown. Studies have found that ABCG1 promoter-257T> G polymorphism was associated with increased risk of CAD. In this study, we investigate whether the SNPs in the ABCG1 promoter region (rs2234714, rs2234715 and rs57137919) and 3'-UTR (rs1044317) are associated with the development of CAD in a Chinese Han population. We explore the molecular biological mechanism of RCT dysfunction in CAD.Objective:1. In this study, we investigate whether the SNPs in the human ATP binding cassette transporter G1 (ABCG1) gene promoter region (rs2234714, rs2234715 and rs57137919) and 3'-UTR (rs1044317) are associated with the development of atherosclerotic coronary artery disease (CAD) in a Chinese Han population.2. According to the results of genetic analysis, we select the-768G>A and-367G> A to construct the two loci of wild type and mutant luciferase reporter plasmids, and explore wild type and mutant reporter plasmids of the ABCG1 gene transcription activity.3. Compare different genotype-367AA/AG/GG patients with coronary heart disease in primary monocyte-derived macrophages ABCG1 expression. Methods:1.1021 patients with CAD and 1013 unaffected control subjects were enrolled. PCR-based ligation detection reaction (PCR-LDR) method was used to genotype four SNPs of ABCG1, three (rs2234714, rs2234715 and rs57137919) in the promoter region and one (rs1044317) in the 3' untranslated region (UTR). We investigate the association between genotype frequencies of these loci and serum lipids in control group, CAD and Ml group.2. Select-768G> A and-367G> A to construct the two loci of wild type and mutant luciferase reporter plasmids. Culture THP-1, RAW264.7,293T cells and human peripheral blood monocyte-macrophage cells, dual-luciferase reporter system detects the transcriptional activity of the report of wild-type and mutant luciferase plasmid (pGL3-basic/P-GG, pGL3-basic/P-GA, pGL3-basic/P-AG and pGL3-basic/P-AA).3. In vitro we examin peripheral blood mononuclear macrophage ABCG1 protein expression in different genotypes (-367AA/AG/GG) of CAD patients.4. Statistical Methods:Differences in study characteristics were tested through non-parametric Mann-Whitney U tests for continuous variables. Differences between categorical variables, genotype/allele frequencies and Hardy-Weinberg equilibrium (HWE) were tested by chi-squared analysis or Fisher's exact test. Independence of associations between SNPs and CAD or Ml were assessed after adjustment for other potentially confounding factors (age, gender, hypertension, type 2 diabetes, smoking, HDL-C and TG) using multiple logistic regression analysis and calculating the adjusted odds ratios and their 95%confidence intervals. All data were analyzed using SPSS version 13.0 software package. Results were considered to be statistically significant if bilateral p-values were less than 0.05. The pattern of pair-wise linkage disequilibrium (LD) between the selected SNPs was measured by D' and r2 using the SHEsis software. Haplotype frequency was determined by means of the algorithms implemented in the PHASE program (version 2.1). Retrospective statistical powers were calculated using Epi InfoTM software (version 3.5.1). The study had more than 80%power to detect the differences between case and control subjects with an OR of less than 1.45 at a significant level of 0.05.Results:1. Genotype distributions of all studied SNPs in both groups followed HWE (p>0.05).2. The minor AA genotype for the rs57137919 polymorphism was associated with a significantly reduced risk for CAD (OR=0.77,95%CI 0.60-0.97, p=0.026). The minor A allele frequency of rs57137919 was significantly lower in CAD or Ml patients than that in controls (p=0.006 and p=0.004, respectively). Further analysis using a dominant model revealed that A allele carriers (AG+AA) appeared to be at a significantly lower risk for CAD or Ml when compared with that in GG homozygote (adjusted OR=0.73, p=0.033 for CAD and adjusted OR=0.65, p=0.014 for Ml, respectively).3. The minor A allele frequency of the rs2234714 in the CAD patients was significantly lower than that in the control group (p=0.05 for CAD, and p=0.0046 for Ml), indicating that rs2234714 was associated with a reduced risk for CAD or Ml in a recessive model after adjustment (adjusted OR=0.64, p=0.015 for CAD and adjusted OR=0.49, p=0.001 for Ml, respectively). But the ABCG1-768G>A polymorphism (rs2234714) did not demonstrate a functional influence on reporter gene expression in the luciferse reporter assay.4. For the rs2234715 and rs1044317 polymorphisms, we found no significant difference on either allele frequency or genotype distribution between CAD patients and controls.5. We analyzed individuals who did not take cholesterol-lowering medications and found no significant difference between the rs2234714 and rs57137919 polymorphisms and plasma lipid levels.6. The haplotype A-A-A-A (rs2234714-rs2234715-rs57137919-rsl044317) was found to be associated with a decreased risk for MI (adjusted OR=0.59,p=0.023).Conclusions:The transcriptional activity and ABCG1 protein expression were significantly lower in macrophages from patient with AA genotype than that in patients with GG genotype. The SNP rs57137919 in the ABCG1 promoter region is functionally associated with a reduced risk of CAD in a Chinese Han population. This is the first human study found that ABCG negatively correlated with atherosclerotic disease, suggesting that ABCG1 may contribute to coronary heart disease.