【作者】 汤纳平；

【导师】 王斌；

【作者基本信息】 南京医科大学 ， 药理学， 2008， 硕士

【摘要】 冠心病是心血管疾病中常见的致死性疾病之一。冠心病的发生是多因素、多步骤的,是遗传因素和环境因素共同作用的结果。许多传统的危险因素如高血压、糖尿病、高血脂、肥胖、吸烟等早已被证实与冠心病的发生和发展密切相关。一些新的生物危险因子如C反应蛋白,血清淀粉样蛋白A以及8异前列烷等也被证实参与了冠心病的发生和发展。另外,遗传学研究发现一些基因或基因的一些位点可能也参与了冠心病的发病过程。已有研究证实,抵抗素(Resistin)、Preproghrelin和内皮脂肪酶(Endothelial lipase)等因子均参与了动脉粥样硬化的形成过程,而编码这些因子基因的单核苷酸多态性如RETN-420C＞G、PreproghrelinLeu72Met以及EL 584C/T也与冠心病传统的危险因素如肥胖,糖尿病等密切相关。然而,据我们所知,目前还没有研究关注这些单核苷酸多态性与冠心病遗传易感性的联系。因此,本研究通过采用病例对照研究,应用单因素和多因素的分析方法来分析RETN-420C＞G、Preproghrelin Leu72Met以及EL 584C/T单核苷酸多态性与冠心病的相关性。第一部分抵抗素(RETN)基因启动子区多态性与冠心病遗传易感性研究Resistin是一个新发现的脂肪细胞衍生的多肽。最近的研究显示Resistin可能参与动脉粥样硬化的形成过程。-420C＞G突变位于Resistin基因RETN的启动子区。之前的研究显示RETN-420G等位基因与肥胖、胰岛素抵抗、2型糖尿病以及脑血管疾病密切相关。在本研究中,我们用PCR-RFLP的方法检测了RETN-420C＞G基因多态性在冠脉造影确诊的冠心病病人中的发生率。1.RETN-420C＞G基因型在冠心病组和对照组中的分布及风险估计野生型纯合子CC基因型在冠心病组和对照组中的分布频率分别为43.1%和54.7%,杂合子CG基因型的分布频率分别为47.6%和40.4%,而变异基因型纯合子GG的分布频率在两组间分别为9.3%和4.9%。对照组基因型分布符合Hardy-Weinberg平衡(χ~2=1.277,P=0.258),表明没有遗传漂移或对RETN特定等位基因没有选择优势。两组之间基因型的分布存在显著性差异(χ~2=7.491,P=0.024)。与对照组相比,病例组G等位基因频率较高(病例组:33.1%,对照组:25.1%),且差异具有统计学意义(χ~2=6.978,P=0.008)。在所有研究对象中,相对于CC基因型携带者而言,变异基因型(CG+GG)携带者患冠心病的分险增高(未校正OR=1.59,95%CI=1.10-2.31)。而携带GG基因型者,其患冠心病的风险增加更多(未校正OR=2.42,95%CI=1.11-5.26)。经校正年龄、性别、体重指数(BMI)、吸烟状态、高血压、糖尿病和高血脂后,GG基因型和变异基因型(CG+GG)的OR值分别为3.01(95%CI=1.29-7.03)和1.62(95%CI=1.09-2.41)。2.在冠心病患者中,RETN-420C＞G基因型与体重、BMI、血脂水平以及冠脉病变的严重程度之间的相关性由于GG基因型较少,故将变异基因型(CG+GG)整合在一起进行计算。我们发现无论是体重、BMI还是脂质水平(TC、TG、HDL-C以及LDL-C),在变异基因型和野生型基因型患者间均无统计学差异。另外,我们也没发现RETN-420C＞G基因型与冠脉病变的严重程度(由病变冠脉的支数表示)相关。第二部分:Proproghrelin外显子区基因突变与冠心病相关性研究Ghrelin是新近发现的生长激素促分泌激素受体的内源性配体。研究显示ghrelin能抑制炎症反应,阻断氧化还原反应介导的细胞信号通路,并能产生舒血管效应和改善内皮功能,提示其具有抗动脉粥样硬化功能。Preproghrelin是编码Ghrelin前体蛋白的基因。以往研究显示,Preproghrelin Leu72Met变异与肥胖、糖尿病、代谢综合症以及厌食症密切相关,但未见有关于Preproghrelin Leu72Met突变与冠心病相关性的报道。因此我们通过病例对照研究来确定在中国人群中Preproghrelin Leu72Met基因多态性与冠心病的相关性。1.Preproghrelin Leu72Met基因型在冠心病组和对照组的分布及风险估计野生型纯合子Leu72Leu基因型在病例与对照组中的频率分别为61.8%和60.4%,杂合子Leu72Met基因型的频率分别为34.4%和34.7%,而变异基因型纯合子Met72Met的频率为3.8%和5.0%。病例组和对照组基因型分布均符合Hardy-Weinberg平衡(P=0.509,P=0.987),表明没有遗传漂移或对Preproghrelin Leu72Met特定等位基因没有选择优势。两组之间基因型的分布未见统计学差异(χ~2=0.559,P=0.756)。变异等位基因Met72频率的分布在两组间亦未见统计学差异(病例组:21.0%,对照组:22.3%,P=0.568)。多因素logistic回归分析结果显示,在模型中加入年龄、性别、BMI、吸烟状况、高血压、糖尿病以及高血脂等危险因素后,变异基因型(Leu72Met+Met72Met)并不是冠心病的独立危险因素(校正后OR=1.09,95%CI=0.76-1.55)。2.在冠心病患者中,Preproghrelin Leu72Met基因型与冠心病表型的相关性在冠心病患者中,携带变异基因型者(Leu72Met+Met72Met)其BMI水平低于携带Leu72Leu基因型者(24.4±0.3 kg/m~2 vs.25.4±0.2kg/m~2,P=0.033)。而TC、TG、HDL-C、Glucose水平在两者间的差异无统计学意义.高血压、糖尿病以及高血脂在这两种基因型间的分布也无统计学差异。另外,对冠心病患者按病变程度(冠脉病变支数)分层分析结果表明Preproghrelin Leu72Met基因型与冠心病的严重程度无关。第三部分:內皮脂肪酶(EL)基因多态性与高密度脂蛋白胆固醇水平以及冠心病遺传易感性关系的研究内皮脂肪酶(Endothelial lipase,EL)是甘油三酯脂肪酶基因家族的一员,它与脂蛋白脂肪酶、肝脂肪酶高度同源。与脂蛋白脂肪酶和肝脂肪酶一样,EL在脂质代谢,特别是HDL-C的代谢过程中也起到至关重要的作用。以往研究显示,EL 584C/T基因多态性与HDL-C代谢相关,且EL 584T等位基因与急性冠脉综合征的高发生风险密切相关。在本研究中,我们以PCR-RFLP方法检测EL 584C/T在冠心病患者中的发生率,进而探讨在中国人群中,EL 584C/T基因多态性与血浆HDL-C水平以及冠心病发生风险之间的相关性。1.EL 584C/T基因型在冠心病组和对照组的分布EL 584CC基因型在病例与对照组中的频率分别为65.7%和47.2%,杂合子CT基因型的频率分别为32.1%和46.0%,而变异基因型纯合子TT的频率为2.3%和6.8%。病例组和对照组基因型分布均符合Hardy-Weinberg平衡(χ~2=1.397,P=0.237;χ~=2.656,P=0.103),表明没有遗传漂移或对EL 584C/T特定等位基因没有选择优势。两组之间基因型的分布存在显著性差异(χ~2=20.644,P＜0.001)。在冠心病组中,EL 584T等位基因分布频率明显低于对照组(病例:18.3%,对照:29.8%,P＜0.001)。2.EL 584C/T基因型与血脂水平的相关性在校正了年龄、性别以及降血脂药的影响后,我们发现,在病例组和对照组中,变异基因型(CT+TT)携带者血浆HDL-C水平显著高于CC基因型携带者(病例组:1.06±0.03 vs.0.98±0.02mmol/1,P=0.013;对照组:1.16±0.03 vs.1.05±0.03 mmol/l,P=0.010)。但在其他血脂如TC、TG以及LDL-C中未发现显著性差异。3.EL 584C/T基因型与冠心病发生风险的相关性单因素分析显示,EL 584T等位基因(CT+TT)携带者与CC基因型携带者相比,其冠心病发生风险较低(OR=0.467,95%CI=0.329-0.663)。把年龄、性别、BMI、吸烟状况、高血压、糖尿病、高血脂以及LDL-C纳入回归模型后,OR值虽有所增高(OR=0.496,95%CI=0.341-0.723),但仍有统计学意义。当仅把基因型和HDL-C纳入回归模型,结果显示,T等位基因对冠心病保护作用独立于HDL-C(OR=0.501,95%CI=0.351-0.715)。把年龄、性别、BMI、吸烟状况、高血压、糖尿病、高血脂以及LDL-C再加入模型后,此结果仍有统计学意义(OR=0.528,95%CI=0.361-0.773)。更多还原

【Abstract】 Coronary artery disease(CAD)is a major cause of morbidity and mortality among cardiovascular disease(CVD).It has a multifactorial aetiology,and is thought to result from an interaction between genetic backgrounds and environmental factors.Many conventional risk factors for CAD,including hypertension,diabetes,dyslipidemia,obesity and smoking have been identified.Recent studies,including ours,have also revealed some new biological risk factors for human CAD,including C-reactive protein,serum amyloid A protein and 8-isoprostane.In addition,genetic studies have demonstrated a series of susceptibility loci and candidate genes that may also contribute to the pathogenesis of CAD.Recently,Resistin、Preproghrelin and Endothelial lipase has been linked to the pathogenesis of atherosclerosis.Single nucleotide polymorphisms(SNPs)of these genes such as RETN-420C＞G、Preproghrelin Leu72Met and EL 584C/T has been reported to be associated with obesity and diabetes,the traditional risk factors of coronary artery disease(CAD).However,to our knowledge,there is no study concerning association between these SNPs and susceptibility to CAD.Thus,we conducted a case-control study to assess the association of these SNPs with the risk of CAD by univariate and multivariate analysis.Part 1:Association between Genetic Polymorphism in Resistin Gene Promoter and Susceptibility to Coronary Artery DiseaseResistin,a novel adipocyte-derived peptide,has been linked to the pathogenesis of atherosclerosis.Recently,-420C＞G,a variant located in the promoter region of the resistin gene(RETN)was identified.Previous studies have shown that this polymorphism was related to obesity, insulin resistance,Type 2 diabetes and cerebrovascular disease.In this case-control study,we used PCR-RFLP protocols to examine the prevalence of RETN-420C＞G polymorphism in CAD.1.The distribution of genotypic characteristics in the cases and controls and the risk estimates for the variant RETN-420C＞G genotypes.The genotype frequencies in cases were 43.1%for wild type CC, 47.6%for CG and 9.3%for GG,while in controls were 54.7%,40.4% and 4.9%respectively.The genotype distribution were in Hardy-Weinberg equilibrium in the control population(χ~2=1.277, P=0.258),indicating that there was no genetic drift or any selective advantage for particular RETN alleles.A significantly different distribution of the RETN-420C＞G genotype was demonstrated among the cases and controls(χ~2=7.491,P=0.024).A higher frequency of G alleles was found in cases compared with the controls(33.1%vs.25.1%,χ~2=6.978,P=0.008).Among total subjects,the crude OR for subjects with the variant genotypes(CG + GG)was 1.59(95%CI= 1.10-2.31)relative to CC carriers.And we noted a more increased risk for CAD in subjects with the GG genotype(crude OR=2.42,95%CI=1.11-5.26).After adjustment for age,sex,BMI,smoking status,hypertension,diabetes and dyslipidemia,the ORs for subjects with GG genotype and those with the variant genotypes(CG + GG)were 3.01(95%CI=1.29-7.03)and 1.62 (95%CI=1.09-2.41),respectively.2.Association of RETN-420C＞G polymorphism with weight,BMI, lipid profiles and the severity of CAD among casesBecause of the lower occurrence of GG carriers,the G allele carriers (CG + GG)were ascribed to a single group.However,there were no significant differences between the genotypes with respect to weight, BMI and lipid profiles.Further stratified analyses by the severity of CAD showed no significant association between the RETN-420C＞G genotype and the severity of CAD. Ghrelin is a novel endogenous ligand for the growth hormone secretagogue receptor.Recent studies have reported a protective effect of ghrelin against the development of atherosclerosis via inhibiting pro-inflammatory response,blocking the redox-mediated cellular signaling,producing vasodilation and improving endothelial function. Preproghrelin is the gene coding for the ghrelin precursor.Previous studies have reported significant associations of preproghrelin Leu72Met polymorphism with obesity,diabetes,metabolic syndrome and eating disorder.However,it is unclear whether this polymorphism is associated with the susceptibility to CAD.Therefore,we conducted a case-control study to examine the association between the risk of CAD and preproghrelin Leu72Met polymorphism.1.The distribution of genotypic characteristics in the cases and controls and the risk estimates for the variant Leu72Met genotypes.The genotype frequencies in cases were 61.8%for wild type Leu72Leu,34.4%for Leu72Met and3.8%for Met72Met,while in controls were 60.4%,34.7%and 5.0%respectively.The genotype distribution were in Hardy-Weinberg equilibrium in the both case and control population(P=0.509,P=0.987),indicating that there was no genetic drift or any selective advantage for particular RETN alleles.No significant difference in the Leu72Met genotype frequency was observed between CAD patients and controls(χ~2=0.559,P=0.756).The distribution of Met72 alleles was not significantly different between the two groups(case:21.0%,controls:22.3%,P=0.568).Multiple logistic regression analysis was further used to assess the association between the Leu72Met polymorphism and CAD with the risk factors including age,sex,BMI,smoking status,hypertension,diabetes and dyslipidemia under control.However,no statistical significance was noted between the Leu72Met polymorphism and the presence of CAD(adjusted OR -1.09,95%CI = 0.76-1.55)2.Association between Preproghrelin Leu72Met genotype and CAD related phenotypes.Among CAD patients,those with variant genotypes(Leu72Met + Met72Met)had lower BMI than Leu72Leu carriers(24.4±0.3 kg/m~2 vs. 25.4±0.2 kg/m~2,adjusted P=0.033).However,the Leu72Met polymorphism was not associated with hypertension,diabetes, dyslipidemia,plasma total cholesterol,triglyceride,high density lipoprotein cholesterol,low density lipoprotein cholesterol or fasting glucose levels in CAD patients.Furthermore,no association was noted between the variant genotypes and the severity of CAD classified according to the number of affected arteries. Endothelial lipase(EL)is a newly discovered member of the triglyceride lipase gene family,which is highly homologous to LPL and HL.Like LPL and HL,EL plays an important regulatory role in lipid metabolism,in particular,HDL-C metabolism.Previous studies have reported that EL 584C/T was related to HDL-C metabolism. Furthermore,studies have confirmed that EL 584T allele was associated with increased risk of acute coronary syndrome.In this case-control study,we used PCR-RFLP protocols to evaluate the prevalence of EL 584C/T polymorphism in CAD.1.The distribution of genotypic characteristics in the cases and controls and the risk estimates for the variant EL 584C/T genotypes.The genotype frequencies in cases were 65.7%for wild type CC, 32.1%for CT and 2.3%for TT,while in controls were 47.2%,46.0% and 6.8%respectively.The genotype distribution were in Hardy-Weinberg equilibrium in both cases and controls(χ~2=1.397, P=0.237;χ~2=2.656,P=0.103),indicating that there was no genetic drift or any selective advantage for particular EL 584C/T alleles.A significantly different distribution of the EL 584C/T genotype was demonstrated among the cases and controls(χ~2=20.644,P＜0.001).The frequency of the variant T allele was significantly lower among CAD patients compared with control subjects(18.3%vs.29.8%,P＜0.001).2.Association between EL 584C/T genotype and lipid profiles After adjustment for age,sex,and lipid-lowering medications,the T allele carriers(CT + TT)had significantly higher plasma HDL-C levels compared with the wild-type CC carriers(case,1.06±0.03 vs.0.98±0.02 mmol/1,P = 0.013;control,1.16±0.03 vs.1.05±0.03 mmol/1,P= 0.010).However,no significant differences were noted between the 584C/T variant and other lipid levels(TC,TG and LDL-C).3.Association between EL 584C/T polymorphism and risk of CADIn univariate analysis,subjects with the T allele(CT + TT)had a significantly decreased risk for CAD(OR=0.467,95%CI=0.329-0.663). In multiple logistic regression model 1,which included 584C/T genotype,age,sex,BMI,smoking,hypertension,diabetes,dyslipidemia, and LDL-C but not HDL-C levels,we noted that the OR for subjects with the T allele was(OR=0.496,95%CI=0.341-0.723).The protective effect of the T allele was reduced but remained significant(OR=0.501, 95%CI=0.351-0.715)in multiple logistic regression model 2,which included 584C/T genotype and HDL-C only.In multiple logistic regression model 3,the P value remained significant after the full adjustment for age,sex,BMI,smoking,hypertension,diabetes, dyslipidemia,LDL-C and HDL-C(OR=0.528,95%CI=0.361-0.773).更多还原