Sr-bi Gene Polymorphism And Coronary Atherosclerotic Heart Disease Association Studies And The Mechanism Of Reverse Cholesterol Transport Function Of The Abca1 Discussed

BackgroundCAD has complex genetic traits,with multiple genetic and environmental components contributing to susceptibility.Association study is widely used in elucidating genetic basis of complex diseases.It has been proposed single nucleotide polymorphisms(SNPs) influence susceptibility to common disease.A candidate gene could be selected because of its'biological association with some disease or being congenerous to a known gene related disease.Appropriate application of intermediate phenotype of diseases and haplotype analyses may increase the opportunity to obtain meaningful findings in association studies.Accumulation of lipid-loaded macrophages(foam cells) within the vessel wall is an early hallmark of atherosclerosis.The atheroprotective effect of high-density lipoprotein cholesterol(HDL-C) is largely attributed to the ability of HDL to mediate the efflux of excess cholesterol from peripheral macrophages back to the liver,a process known as reverse cholesterol transport.The first step in this process is mediated by ABCA1 and SR-BI.ABCA1 are two membrane proteins that translocate phospholipids and cholesterol to the cell surface where it can be taken up by lipid-poor HDLand mature HDL.To date,the gene encoding ABCA1 is on chromosome 12q24,encompasses 50 exons and more than 100 mutations and single nucleotide polymorphisms(SNPs). Many SNPs in the promoter and coding region were genotyped in cases and controls. The -565C/T(previously designated as -477,rs2422493) polymorphism is a cytosine(C) to thymine(T) change in the promoter region of the ABCA1 gene.The R219K(rs2230806) polymorphism is a guanine(G) to adenine(A) substitution at position 1051 in 7exon.This results in changing an arginine to a lysine at residue 219. The -565C/T variant and the R219K variant were found associated with risk of CAD. However not all studies reveal the relationship between the genotype and the risk of CAD.The effect of genotype on HDL-C also remains controversial.The SR-BI gene is on chromosome 12q24,has 13 exons,common SR-B1 polymorphisms in exon 1, products for exon 1(G→A at cDNA position 4,Gly→Ser change at amino acid position 2).Association is found between SR-BI polymorphisms and high-density lipoprotein cholesterol(HDL) levels or severity of CAD,also remains controversial.At present,there are few studies focus on the associations between ABCA1, SR-BI and CAD in Chinese population.The purpose of this study is to find association of ABCA1,SR-BI with CAD in Han people.In addition,we will explore the molecular biological mechanism of the change on RCT associated with these 3 SNPs.Objectives:1.To study the traditional risk factors,the characteristics of lipid metabolism and pathological changes of coronary artery in CAD.2.To study the mutations and genetic characteristics of ABCA1 promoter and 7Exon and SR-BI lexon in CAD patients.3.To analyze ABCA1 mRNA and protein expression in the cultured monocyte-macrophages and apoA-I-mediated cholesterol efflux from the different genotype cultured macrophages.To explore the molecular biological mechanism of RCT dysfunction in CAD.Methods:1.Genetic and clinical analysis1.1 Patients were cases with angina pectoris and had＞50%stenosis in at least 1 segment of the coronary arteries verified by coronary angiography recruited from Peking Unin Medical Hospital and FuWai Cardiovascular Disease Hospital from 01/2007 to 04/2008.Normal controls were collected in regular health examination of normal people,who have no CAD,DM,and dyslipidemia.Demographic(age and sex),CAD risk factors(hypertension,hyperlipidemia,diabetes,smoking,and family history of CAD),the results of coronary angiography,the fasting lipid/lipoprotein profile during hospitalization formed the basis for assessment of intermediate (lipid/lipoprotein) phenotype.1.2 DNA extraction and genotyping at the time of enrollment.5 mL of blood was collected into EDTA tubes and stored at -40℃.DNA was extracted from the whole blood cells using the phenol-chloroform method.1.3 Genotyping for ABCA1 -565C/T,used high sensitive ligase detection reaction (LDR) sequencing polymerase chain reaction amplification and followed by restriction enzyme digestion of the polymorphic sites(ABCA1 with AciI),direct sequencing(DS).We measured haplotype construction at the same time.2.ABCA1-mediated cholesterol efflux2.1 Culturing primary monocyte-derived macrophages,foam cell was induced by acetyl-LDL and labeled with[~3H]cholesterol by incubating them for hours. Cholesterol efflux detected by radioactive scintilloscope.2.2 Quantitative PCR—real-time reverse-transcriptase polymerase chain reaction method was done to measure the relative abundance of ABCA1 transcripts.2.3 Western blot and ELISA were used to measure the abundance of ABCA1protein.3.Statistical analysis:The allele frequencies of polymorphisms were calculated.The HWE program was used to examine the observed genotype distribution deviated from Hardy-Weinberg equilibrium and haplotype were conducted.All analyses were performed using SPSS for Windows.Results1.Genetic characteristics of ABCA1 -565C/T,R219K and SR-BI Gly4Ser:1.1 Compare the characteristics between CAD case and control,the average age of CAD were 62±11 years,P＞0.05;74%were male,more than Controls'.Average total cholesterol in CAD was 4.46±1.03mmol/L,LDL 2.58±0.85 mmol/L and TG 1.86±1.54 mmol/L,which were higher than those of Control;HDL 1.58±0.53 mmol/L;and ApoA-I 1.25±0.22g/L,less than those of control;1.2 A total of 516 CAD patients and 544 Control were successfully genotyped for the-565C/T polymorphism.The frequencies of the CC,CT,and TT genotypes in CAD were 0.35(n=163),0.48(n=223),and 0.17(n=75),respectively.Case and control genotype distribution was consistent with Hardy-Weinberg equilibrium;1.3 The frequency of the TT genotype and T allele at the -565C/T locus had no significant alterations between CAD patients and Controls(0.1627 vs 0.1625;0.404 vs 0.414,P＞0.05).There had no significant alterations in the HDL profile in CC, CT and TT genotype;The proportion of TT genotype in myocardial infarction patients and with anterior wall or multi-regional walls was higher than CT,CC genotype,P＜0.05;1.4 The frequency of the AA and GA genotype at the R219K locus was lower in CAD patients compared with diabetes(0.65 vs 0.73,P=0.079).The AA genotype of the G1051A polymorphism with higher HDL-C,P＞0.05;1.6 Haplotype association with phenotype performed in the 2 SNPs of ABCA1 was not significant.1.7 Logistic regression analysis adjusted by age and sex did not reveal an association between -565C/T,R219K genotype and CAD;1.8 The frequencies of the less common A alleles for the polymorphisms at the SR-BI lexon locus in this Chinese population were 0.0023;2.In vitro cell culture:cholesterol efflux and ABCA1 expression2.1 Cholesterol efflux of foam cell induced by ac-LDL were the lowest in TT homozygotes compared with CC and CT.2.2 The western blot and ELISA assays showed the amounts of ABCA1 protein were the lowest in TT homozygotes and the highest in CC homozygotes,though the real time RT-PCR assays showed that the amounts of ABCA1 transcript were not the lowest in T/T homozygotes and the highest in C/C homozygotes,Conclusion1.ABCA1 the T allele of-565 C/T SNP has no significant association with CAD.TT genotype has relation with severity of myocardial infarction;2.R219K SNP predicted differences in angiographic CAD with diabetes.The AA genotype may protect against subclinical cardiovascular disease.The two SNPs were not associated with HDL level.3.The lowest cholesterol efflux to apoA-I in the TT genotype macrophages was relative to lower ABCA1 protein expression level.These results suggested that defective ABCA1 function in cholesterol-loaded macrophages should be one potential contributor to the dysfunction of RCT process and the increasing coronary heart disease.Strengths,Iimitations and developmentThe study has the advantage of especially for 3 SNPs,on the basis of previous literature.The use of high sensitive LDR and molecular biological technique such as western-blot,real-time RT-PCR had the strength of genetic and functional characteristics.Angiographic coronary are ensured of definition and quantifying coronary phenotype.This study has the limitations of all nonrandomized observational studies, including the possibility of selection bias and uncorrected confounding.Results of our genetic association studies should be regarded as population and SNP specific and might not be applied to other populations or to other SNPs.Finally,coronary heart disease risk also is influenced by many other factors.We will construct the eukaryotic expression carrier of ABCA1 and transfect the carrier into cells without 565C/T allele to observe theABCA1 expression and cholesterol efflux.We will enlarge sample capacity.