Relative Study Of The G894T Polymorphism Of Endothelial Nitric Oxide Synthase Gene And Coronary Heart Disease

BackgroundCoronary heart disease(CHD)is one of principal disease threaten our healthy today.It is multigenic disease,caused by the interaction diverse genetic factors and environmental factors.Age,Hypertension,smoking,obesitas,etc,as tradition Risk factors were widely accepted.To fix and discriminate CHD related genes become hot spot now.Endothelial Nitric Oxide Synthase(eNOS)gene is one, of them.Nitric Oxide(NO)is a important bioactivitive medium in cell,participating the development of CHD.It is eNOS that regulate the NO production in physiological condition.ENOS gene encodes 1203 amino acids in the human being,fixed on chromosome 7q35-q36,with 21²2kb total length,consist of 25 introns and 26 exon.In varied Polymorphisms of eNOS,G894T Polymorphism is considered important to CHD.G894T Polymorphism means amino acids Glu298Asp Polymorphism。This alteration result in depression of eNOS activity,descend NO level,promote atherosclerosis. The aims of this study were to investigate the possible relationship beteen the eNOS G894T polymorphism and the presence,the severity of CHD concerning with tradition Risk factors. SubjectCHD(+)group:196 CHD patients(121 smokers and 75 no-smokers)were consecutively collected from the hospitalized in the department of cardiology,the second affiliated hospital of medical college,Zhejiang University.All patients were confirmed by coronary angiography.CHD(-)group:189 CHD(-)object(82 smokers and 107 no-smokers)served as a control group.All subjects were consecutively collected from the hospitalized in the department of cardiology,the second affiliated hospital of medical college,Zhejiang University.All patients were confirmed have no CHD by coronary angiography.methodDocumentation of CHD severityThe severity of coronary stenosis was determined by the number of significantly stenosed coronary arteries as follows.Each angiogram was classified as revealing either coronary lesion with more than 50%luminal stenosis or one,two,or three major epicardial coronary arteries with more than 50%luminal obstructions.Laboratory methods for eNOS gene polymorphism1)DNA was extracted from the peripheral blood leukocytes by standard phenol and chloroform method. 2)The primers used were 5' -CATGAGGCTCAGCCCCAGAAC-3'(forward) and 5' -AGTCAATCCCTTTGGTGCTCAC-3'(reverse).PCR was carried out using a 25ul reaction mix containing 10×Reaction buffer(without MgCl₂) 2.5ul,25mM MgCl₂ 1.5ul,10mM dNTP 0.5ul,genomic DNA 2.5ul,Taq DNA polymerase 0.25ul and 10pmol each primer.The PCR cycles were modified as follows:initial denaturation at 95℃for 5 minutes,then DNA is amplified for 30 cycles,each cycle comprising denaturation at 95℃for 45 second, annealing at 60℃for 45 second,extention at 70℃for 1 min,with final extention time of 5 min at 70℃.PCR products were digested with therestriction enzyme Mbol at 37℃for 16 h.In the presence of T at nucleotide 894 which corresponds to Asp 298,the 206 base pair(bp)PCR product is cleaved into two fragments of 119 and 87 bp. Digested product were separated on 2%agarose gels and visualized with ethidium bromide.Statistical analysisStatistical analysis was carried out with SPSS for Windows version 10.0 statistical software.Count Data were performed by Student' s t-test.To test for independent relationships between variables,the x² test were performed.We calculated the genotypic odds ratio(OR)for premature CHD and their 95%confidence interveal(Cl)with two-tailed p values.P-value of 0.05 or less was considered as significant.Results1.There is a polymorphism of G894T in Zhejiang Han population.The G894T genotype distribution of the subject was in Hardy-Weinberg equilibrium(x²=0.12,p>0.05).2.The distribution of genotype GT and TT in CHD group was significant more than in control group(x¹=7.97,p<0.01).The T allele frequency in CHD group is significant higher than in control group x²=10.58,p=<0.01)3.There was a significant difference in the genotype distribution and allele frequency of G894T comparing patients with and without smoking in CHD group.I4.The distribution of genotype GT and TT in CHD group was no significant difference between single lesion group and others;but it is different in CHD group with smoking.5.The T allele frequency in CHD group with smoking was significant difference between with hypertension or not.6.The distribution of genotype GT and TT,T allele frequency in CHD group with smoking was significant difference between single lesion group and others,also was those with hypertension in them.Conclusion1.There is a polymorphism of G894T in Zhejiang Han population.2.T allele may be a susceptibility gene of CHD.G894T gene polymorphism may be not affect the severity of CHD.3.Smoking may be lead to G→T,G894T gene polymorphism may be affect the severity of CHD.4.The more risk factor,magbe the more G→T happened,the more sever atherosclerosis of coronary artery.