The Relationship Between Genetic Variants Of Chromosome9p21and Expression Of Adjacent Genes And Coronary Heart Disease

Coronary heart diseas（eCHD）is the most common and the most frequent chroniccardiovascular diseases (CVD). It is the main cause of morbidity and mortality indeveloped countries and most developing countries. Until now, the etiology andpathogenesis of CHD has not been clarified. Following by the deep research inetiology and pathogenesis of CHD, it was found that40%~50%of CHD due to thegenetic factors. However, decisive genetic factors of CHD are not clear. Therefore, toexplore the genetic susceptibility pathogenesis of CHD from gene level is becoming ahot spot of current researches.In recent years, scholars found many susceptibility regions and sites of CHDwith genome-wide association studies (GWAS), among which chromosome9p21areas have attracted the most attention. Patients with CHD and healthy controls ofnorthern Chinese Han were recuited. We collected their blood samples. To discussrelationship between the genetic variations in chromosome9p21region and theexpression of adjacent genes and CHD, we detected the gene polymorphisns in theregion and the expression of adjacent genes. To study the possible mechanism of theeffects of genetic variation in the region on CHD of the northern Chines Hanpopulation, we detected the levels of CDKN2A, CDKN2B, ANRIL and MTAPtranscription and translation in patients with different genotypes. This study is of greatsignificance to reveal the etiology and pathogenesis of CHD. It is divided into thefollowing three parts:PartⅠ the relationship between genetic variations in chromosome9p21regionand CHDPurpose:The present study was undertaken to genotype single nucleotide polymorphisms (SNPs) rs2383206、rs2383207、rs10757274、rs10757278and rs1333049present inChromosome9p21region for their association with CHD in a northern Chinese Hanpopulation.Methods:A total of583unrelated patients with CAD and540controls were recruited fromthe First Hospital of Jilin University between2009and2012. All of them are northernChinese Han people, and without bood relationship. Both gender and age are matchedbetween cases and controls. All the subjects were given an informed consent, andwere well told of the study protocol. Peripheral venous blood was collected in tubescontaining anticoagulant to extract genomic DNA. We selected rs2383206, rs2383207,rs10757274, rs10757278and rs1333049sites in chromosome9p21region ascandidate loci. SNPs were genotyped using SEQUENOM Mass-ARRAY system. Wealso collected the epidemiological data. SPSS16.0software was used to analyze theassociation of general informations, alleles, genotypes and CHD, and the associationof SNPs and the risk factors of CHD. The Haploview program was applied to estimatethe linkage disequilibrium (LD). Haplotypes and combined genotypes analysis wasperformed with the UNPHASED program.Results:1. Smoking, waist-to-hip ratio (WHR), history of hypertension and diabetes arestatistically significant different between cases and controls (P＜0.05). The proportionof smoking, history of hypertension and diabetes in patients with CHD is significantlyhigher than that in controls, and the patients’ WHR is higher than controls’.2. The distribution of rs1333049alleles is significant difference between casesand controls（P＜0.05）; the risk of CHD of G allele carriers is0.923times that of theC allele carriers. The distribution of rs2383206alleles is significant differencebetween cases and controls（P＜0.05）; the risk of CHD of G allele carriers is1.097times that of the A allele carriers. The distributions of rs2383207alleles andgenotypes are significant difference between cases and controls（P＜0.05）; the risk ofCHD of AG genotype carriers and GG genotype carriers is1.104and1.122times that of the AA genotype carriers; the risk of CHD of G allele carriers is1.081times that ofthe A allele carriers. The distributions of rs10757274alleles and genotypes aresignificant difference between cases and controls（P＜0.05）; the risk of CHD of GGgenotype carriers is1.287times that of the AA genotype carriers; the risk of CHD ofG allele carriers is1.126times that of the A allele carriers. The distributions ofrs10757278alleles and genotypes are significant difference between cases andcontrols（P＜0.05）; the risk of CHD of AG genotype carriers and GG genotypecarriers is1.123and1.229times that of the AA genotype carriers; the risk of CHD ofG allele carriers is1.111times that of the A allele carriers.3. Rs2383206, rs2383207, rs10757274, rs10757278and rs1333049inchromosome9p21region are linkage disequilibrium (LD). When2risk allelesexistence, the risk of CHD will increase; rs10757274and rs10757278loci orrs2383206and rs2383207loci exist two kinds of risk genotypes, the risk of CHD willalso improve.4. In the controls, The TC level of rs10757274and rs10757278GG genotypecarriers is significantly lower than that of AA genotype carriers（P＜0.05）, and TClevel of rs1333049sites GG genotype carriers is significantly higher than the CCgenotype carriers（P＜0.05）, while there is no difference in patients with CHD（P＞0.05）; In cases, TC level of rs2383206GG genotype carries is significantly lowerthan that of AG genotype carriers （P＜0.05）, while there is no difference in thecontrols（P＞0.05）.5. In cases and controls, there is no significant difference of history of diabetesand hypertension in different genotype carriers（P＞0.05）.6. There is no significant difference in rs2383207, rs10757274, rs10757278,rs1333049and rs2383206loci and genetic variation between early-onset CHD andlate onset CHD（P＞0.05）.Conclusions:1. Smoking, WHR, history of diabetes and hypertension may be risk factors ofCHD. 2. Rs2383207, rs10757274, rs10757278, rs1333049and rs2383206loci in Chromosome9p21area are susceptibility loci for coronary heart disease of north China Han population; Callele of rs1333049and G allele of rs2383207, rs10757274, rs10757278, rs2383206are risk alleles of CHD; AG genotype and GG genotype of rs2383207, GG genotypeof rs10757278, AG genotype and GG genotype of rs10757274are risk genotypes ofCHD.3. When2risk alleles existence, the risk of CHD will increase; rs10757274andrs10757278loci or rs2383206and rs2383207loci exist two kinds of risk genotypes,the risk of CHD will also improve.4. GG genotype of rs10757274and rs10757278, CC genotype of rs1333049, AGgenotype of rs2383206may influence total cholesterol (TC) levels of the carriers toaffect the occurrence of CHD.5. Genetic variants of rs2383207, rs10757274, rs10757278, rs1333049and rs2383206affect the occurrence of CHD is not through the way to affect TG and GLUmetabolism and the history of hypertension and diabetes.6. There is no significant difference in rs2383207, rs10757274, rs10757278,rs1333049and rs2383206loci between early-onset CHD and late onset CHD.Part Ⅱthe relationship between mRNA expression of chromosome9p21CHDrisk sites adjacent genes and the CHDPurpose:To indentify the relationship between mRNA expressions of chromosome9p21CHD risk sites adjacent genes and CHD; to clarify the effect of genetic variations inchromosome9p21on its adjacent gene mRNA expression levels.Methods:A total of91unrelated patients with CAD and30controls were recruited fromthe First Hospital of Jilin University in2012. Both gender and age are matchedbetween cases and controls. All the subjects were given an informed consent, andwere well told of the study protocol. Peripheral venous blood was collected in tubes containing anticoagulant to extract total mRNA. CDKN2A, CDKN2B, MTAP andANRIL are selected as candidate genes, and real time fluorescence quantitative PCRwas used to test the mRNA levels. SPSS16.0software was used to analyze the data.Results:1. Compared with controls, the CDKN2A, CDKN2B mRNA levels are significantlow in cases（P＜0.05）, while MTAP mRNA level is significant high（P＜0.05）.There is no difference of ANRIL mRNA between cases and controls（P＞0.05）.2. Patients with different genotypes of rs2383207have different CDKN2AmRNA leve（lP＜0.05）, while CDKN2B, MTAP, ANRIL are not different（P＞0.05）.Patients with different genotypes of rs2383206, rs1333049, rs10757274andrs10757278do not have different CDKN2A, CDKN2B, MTAP, ANRIL mRNA levels（P＞0.05）.Conclusions:1. The lower mRNA levels of CDKN2A and CDKN2B, the higher mRNA level ofMTAP may be related to CHD of northern Chinese Han population.2. The possible mechanisms of genetic variant of rs2383207affected theoccurrence of CHD is by influencing the patients’ CDKN2A mRNA expression levelin the peripheral blood.3. Genetic variantss of rs10757274, rs10757278, rs1333049and rs2383207affected the occurrence of CHD is not by influencing the patients’ CDKN2A,CDKN2B, MTAP and ANRIL mRNA expression levels in the peripheral blood.Part Ⅲthe relationship between protein expression of chromosome9p21CHDrisk sites adjacent genes and the CHDPurpose:To indentify the relationship between protein expressions of chromosome9p21CHD risk sites adjacent genes and CHD; to clarify the effect of genetic variations inchromosome9p21on its adjacent gene protein expression levels.Methods: A total of55unrelated patients with CAD and15controls were recruited fromthe First Hospital of Jilin University in2012. Both gender and age are matchedbetween cases and controls. All the subjects were given an informed consent, andwere well told of the study protocol. Peripheral venous blood was collected in tubescontaining anticoagulant to extract total protein. CDKN2A, CDKN2B and MTAP areselected as candidate genes, and western blot was used to test the protein levels.Semi-quantitative analysis was carried with Image J2x software. SPSS16.0softwarewas used to analyze the data.Results:1. There are no differences of p14ARF, p15INK4b, MTAP between cases andcontrols（P＞0.05）.2. Patients with different genotypes of rs2383207have different MTAP levels（P＜0.05）, while p14ARF、p15INK4bare not different（P＞0.05）.3. Patients with different genotypes of rs2383206, rs1333049, rs10757274andrs10757278do not have differentp14ARF, p15INK4b, MTAP levels（P＞0.05）.Conclusions:1. Patients with CHD p14ARF, p15INK4b, MTAP levels did not significantlychange in the peripheral blood.2. The possible mechanisms of genetic variant of rs2383207affected theoccurrence of CHD is by up-regulated the patients’ MTAP level in the peripheralblood.3. Genetic variantss of rs10757274, rs10757278, rs1333049and rs2383207affected the occurrence of CHD is not by influencing the patients’ p14ARF, p15INK4b,MTAP levels in the peripheral blood.