【作者】 王海鱼；

【导师】 陈爱华；

【作者基本信息】 南方医科大学 ， 内科学， 2013， 博士

【摘要】 前言和背景目前认为,冠心病是一种多基因遗传复杂疾病,肥胖是公认的冠心病危险因素之一。脂肪量与肥胖相关(fat mass and obesity associated, FTO)基因是至今为止研究证实最强最确定的肥胖易感基因。自2007年Frayling首次报道,FTO基因与肥胖相关以来,大量全基因组关联研究证实：在全世界大多数不同人种人群中,FTO基因多态性与体质量指数(BMI)、肥胖、胰岛素抵抗及糖尿病(DM)、代谢综合征、高血压、动脉粥样硬化及C-反应蛋白显著关联。冠心病的病理基础是冠状动脉粥样硬化,其发生发展与遗传和环境因素密切相关,肥胖、糖尿病、炎症等均参与其发病及病情进展,而从某种意义上讲,糖尿病等于冠心病。因此,我们假设冠心病与肥胖、糖尿病可能具有共同的遗传学基础,FTO基因多态性可能与冠心病患病风险相关。据文献检索,目前国内外仅有少数几个学者在FTO基因的单核苷酸多态性(single nucleotide polymorphism, SNP)与2型糖尿病的关联研究中发现：FTO基因SNP与DM合并冠心病有相关性。2010年Hubacek等首次研究表明：在男性白色人种中FTO基因rs17817449多态性与急性冠脉综合征显著相关。而对于非洲印第安人的研究表明：急性心肌梗死并代谢综合征与肥胖相关基因多态性并不相关。对于FTO基因SNP与冠心病的关联研究尚处于不确定阶段,国内尚未见关于FTO基因多态性与冠心病相关性研究的报道,且不同人种具有遗传异质性,全基因组关联研究(GWAS)发现的冠心病易感基因,需要在不同人种大样本研究中得到重复验证。近十年来的研究表明瘦素(leptin)与肥胖、血脂异常、胰岛素抵抗、高血压、动脉粥样硬化等关联,是冠心病的易患因素之一。LEP基因也是肥胖相关基因,编码瘦素,Lep基因与FTO基因同是通过作用于下丘脑,而抑制饮食,调节能量平衡,其基因多态性变异导致肥胖。研究亦证实瘦素与FTO基因存在关联,具体机制尚不清楚。FTO基因与肥胖相关的SNP均大致定位于第1内含子,第1内含子在遗传上被证实具有高度保守性。研究最为深入的位点rs9939609、 rs17817449与其他位点呈强连锁不平衡,且基因型分型研究成功率很高。GWAS目前研究热点,主要是通过疾病易感基因单核苷酸多态性与疾病的关联,研究复杂疾病的遗传机制。因此本实验选择典型位点FTO rs9939609, rs17817449以及LEP rs10954174对其SNPs与冠心病的关系进行研究。本研究采用病例对照的全基因组关联研究方法,首次对中国汉族冠心病患者肥胖相关基因3个SNPs多态性进行分析,采用基因多态性研究可靠性精确度最高的直接测序方法确定FTO、LEP基因SNP位点的基因型,探讨FTO、 LEP基因单核苷酸多态性与冠心病及其相关临床表型(高血压、高血脂、高血糖、高同型半胱氨酸等)的相关性,进一步验证冠心病FTO基因与LEP基因SNP位点风险等位基因,为从基因水平筛查冠心病高危人群提供依据。目的1.重复和验证中国汉族冠心病患者肥胖相关基因FTO基因位点rs9939609(A/T)、rs17817449(G/T)和LEP基因位点rs10954174(G/A)多态性；2.探讨上述3个肥胖相关基因单核苷酸多态性SNPs和冠心病的相关性；3.探讨上述3个SNPs多态性与冠心病相关临床性状(肥胖、高血压、高血脂、糖尿病、高尿酸、高同型半胱氨酸等)的相关性,比较不同基因型的临床代谢、生化指标的差异是否具有统计学意义。对象和方法1.随机选择2012年1月至2012年12月深圳市龙岗中心医院心内科住院冠心病患者147例,男127例,女20例,年龄40～72岁,平均年龄(57.22±10.56)岁。同期住院的98例非CAD患者为对照组,男88例,女10例,年龄38～72岁,平均年龄(54.24±8.49)岁。入选研究对象均为汉族且均签署知情同意书。对所有符合入选标准的研究对象均常规收集下列临床资料：年龄、性别、早发心血管病家族史、高血压、糖尿病、脑卒中等相关病史、饮酒、吸烟史、体力活动和主要饮食习惯等。空腹测量身高、体重、腹围(WC)及血压(blood pressure, BP),计算体质量指数(BMI)=体重(公斤)/身高(米)2(kg/m2)。实验室检查以下生化指标：空腹血糖(fasting plasma glucose, FPG)、总胆固醇(total cholesterol, TC)、甘油三酯(triglyceride, TG)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol, HDL-C)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-C)、尿酸(uric acid, UA)、同型半胱氨酸(Homocysteine,HCy)等,采用美国罗氏全自动生化分析仪测定。2.肥胖相关基因基因多态性检测：①基因组DNA的制备：清晨空腹用EDTA抗凝管抽取外周静脉血3ml,采用亚能生物技术(深圳)有限公司全血DNA快速提取试剂盒(25次量)抽提全血基因组DNA。电泳鉴定基因组DNA纯度。②聚合酶链反应(PCR)扩增：设计FTO基因位点rs9939609(A/T)、rs17817449(G/T)和LEP基因位点rs10954174(G/A)各自双向引物。通过PCR扩增相应的目的基因片段,PCR反应总体系30ul,包括:10×rTaq buffer(10×缓冲液)3ul,dNTP(2.5mM)2ul,正向、反向引物(10mM)各1ul, DNA模板2ul,Taq DNA聚合酶(r Taq)0.2ul,50%DMSO1ul,去离子水19.8ul。PCR反应条件：95℃预变性5min后进行PCR循环,PCR循环参数为：95℃变性20sec,55℃退火20sec,72℃延伸30sec,进行30个循环,最后72℃总延伸5min,12℃保存。③测序前DNA模板处理：PCR反应产物电泳鉴定,PCR扩增的目的基因片段,rs9939609扩增的片段长度为226bp; rs17817449为253bp; rs10954174为257bp。PCR产物纯化,按照Millipore公司96纯化板操作流程操作处理。纯化后方可作为测序前DNA模板。④基因型检测采用美国ABI3730型(Applied Biosystem)基因分析仪全自动化测序进行基因多态分型。建立单引物PCR测序反应体系：rs9939609、 rs17817449、rs10954174相应的PCR纯化后产物作为测序模板DNA3μl (30-50ng),单向引物(rs9939609R, rs17817449R, rs10954174F)(3pM)1μl, Bigdye Mix0.5μl, ddH2O0.5μl,总反应体积5μl。反应条件：95℃预变性2min,95℃变性10s,51℃退火10s,60℃延伸190s,25次循环。按照分析仪操作说明,按编程设定程序后自动次序进样,毛细管电泳,激光检测器检测荧光,分析DNA碱基序列,仪器自动数据分析得出样本FTOrs9939609、rs17817449和LEP rs10954174基因型。本实验使用的测序试剂盒是Bigdye荧光标记终止底物循环测序试剂盒,由北京六合华大基因科技股份有限公司协助完成。3.统计学处理：统计分析比较冠心病组和对照组之间基本临床资料的差异,分析FTO基因rs9939609、rs17817449, LEP基因rs10954174各位点等位基因及基因型频率分布,以及FTO、LEP不同基因型频率分布之间各生化、代谢指标的分布差异。采用SPSS17.0统计软件进行统计学处理,计量资料用均数±标准差(x±s)表示,两组间比较用独立样本t检验,三组间比较采用one-way ANOVA分析。计数资料以率比较,组间比较用x2检验。以x2检验基因型分布是否符合Hardy-Weinberg平衡。采用logistic回归分析不同基因型频率分布与冠心病的关系。P<0.05为差异有统计学意义。结果1.一般临床资料比较：147例冠心病(病例组)以及98例非冠心病患者(对照组)的一般临床资料比较,年龄、吸烟、高血压病史、高脂血症病史、腰围(WC)、空腹血糖(FPG)、总胆固醇(TC)及同型半胱氨酸(HCY),冠心病组均高于对照组,差异具有统计学意义(P<0.05),而高密度脂蛋白胆固醇(HDL-C)低于对照组,差异具有统计学意义(P<0.05)。性别、既往糖尿病、痛风史、体质量指数(BMI)、收缩压(SBP)、舒张压(DBP)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)以及尿酸(UA),在两组间差异无统计学意义(P>0.05)。2.CAD组和对照组中,FTO基因FTOrs9939609、rs17817449基因型和LEP rs10954174基因型分布频率符合Hardy-Weinberg(H-W)遗传平衡(P>0.05)。3.CAD组和对照组中FTO rs9939609等位基因及基因型频率比较：CAD组147例中,FTO rs9939609TT基因型108例,AT型39例,表达频率分别为73.47%、26.53%,未检测到AA基因型。A等位基因频率为13.27%,T等位基因频率为86.73%。98例对照组中,TT基因型72例,AT型26例,频率分别为73.50%、26.50%,未检测到AA基因型；A等位基因频率为13.30%,T为86.70%。两组基因型及A、T等位基因分布频率比较,差异无统计学意义(P>0.05)。4.CAD组和对照组中FTO rs17817449等位基因及基因型频率比较：CAD组147例中,rs17817449GG基因型7例,GT型35例,TT型105例,表达频率分别为4.8%、23.8%和71.4%,G等位基因频率为16.67%,T为83.33%。对照组98例中,TT基因型83例,占84.7%,GT基因型14例,占14.3%,GG基因型1例,占1%。等位基因频率：T为91.8%,G为8.2%。携带G等位基因的基因型与不携带的纯合子TT基因型比较,冠心病风险增高,是TT基因型的4.85倍(P=0.038, OR=4.85,95%CI:0.587～40.053)携带至少一个G等位基因患冠心病的风险是不携带者的2.250倍(P=0.007,OR=2.250,95%CI:1.239～4.084)。5.CAD组和对照组中LEP rs10954174等位基因及基因型频率比较：147例CAD组LEP基因rs10954174GG基因型137例,占93.2%, GA基因型10例,占6.8%,未检测到AA基因型。G等位基因频率为96.6%,A为3.4%；对照组98例,GG型90例,占91.8%,GA型8例,占8.2%,未检测到AA基因型。等位基因频率：G为95.92%,A为4.08%。CAD组LEP rs10954174A等位基因及GA+AA基因型分布频率低于对照组,两组间差异无统计学意义(P>0.05)。6.FTO基因SNP位点rs9939609和rs17817449呈强连锁不平衡：连锁不平衡检验(D=3.4%,r2=0.97>0.33)。7.3个SNPs不同基因型的临床代谢生化指标比较：FTO基因rs9939609AT+AA基因型的高血压病史、高脂血症病史、BMI、WC、SBP、DBP、 FPG、TC、LDL-C水平均高于TT基因型的,二者比较差异显著(P<0.05)；FTO rs17817449GG基因型的BMI、WC、SBP、LDL-C水平均高于TT基因型的,性别、吸烟史、高血压病史,二者比较均差异显著(P<0.05); LEP基因rs10954174GG基因型与GA+AA基因型比较,所有研究指标包括：性别、年龄、吸烟史、高血压病史、糖尿病史、痛风及高脂血症史、BMI、 WC、SBP、DBP、UA、FPG、TC、TG、LDL-C、HDL-C及同型半胱氨酸,差异均无统计学意义(P>0.05)。8. Logistic回归分析结果提示,影响冠心病发病的因素有年龄、腰围(WC)、 BMI、空腹血糖(FPG)、甘油三酯(TG)、HDL-C、吸烟、高脂血症史以及FTO基因rs17817449G等位基因。其中危险因素有体质量指数(BMI)、空腹血糖(FPG)、甘油三酯(TG)、HDL-C、高脂血症史和rs17817449G等位基因。rs17817449G等位基因与冠心病呈相关性(P<0.05,OR值25.81,95%CI：2.348-285),且独立于BMI、WC及高血压病、糖尿病及血清总胆固醇、低密度脂蛋白胆固醇、血尿酸、血同型半胱氨酸等风险因素。结论①FTO基因位点rs9939609(A/T)多态性与冠心病无明显关联,rs9939609A等位基因携带者的高血压病史、高脂血症史、BMI、WC、SBP、DBP、FPG、 TC、LDL-C水平均高于TT基因型的,携带A等位基因可能是冠心病的易感因素。②FTO基因位点rs17817449(G/T)多态性与冠心病显著关联,G等位基因是冠心病的风险基因,携带GG基因型的中国汉族人冠心病患病风险高于TT基因型者。③在中国汉族人群中重复证实FTO rs9939609和rs17817449呈强连锁不平衡。④初步研究得出LEP rs10954174(A/G)多态性与冠心病无明显关联。⑤本研究重复和验证了在中国汉族人中肥胖相关基因FTOrs9939609, rs17817449SNPs多态性与BMI、WC显著相关。LEPrs10954174则与冠心病及冠心病相关性状无明显相关。首次在中国汉族冠心病患者中,对3个SNPs与冠心病患病风险的相关性进行了初步探讨。更多还原

【Abstract】 BackgroundAt present, coronary artery disease is a polygenic complex diseases and obesity is one of the risk factors of coronary artery disease. The pathological basis of coronary artery disease is coronary atherosclerosis, the occurrence and development of CAD are closely related with genetic and environmental factors. FTO (fat mass and obesity associated, FTO) gene is by far the strongest obesity susceptibility gene. In2007, Frayling first reported the FTO gene. Genome-wide association studies have identified FTO gene common variants that are robustly associated with obesity, body mass index (BMI) and the risk of diabetes, hypertension, the metabolic syndrome, atherosclerosis and C-reactive protein. Since these cardiovascular risk factors play an important role in the etiology of coronary artery disease(CAD), obesity, diabetes, inflammation are involved in the onset and severity of CAD. Obesity is related to chronic subclinical inflammation which plays an essential role in the development of atherosclerosis. In a certain sense, diabetes is equal to the coronary artery disease. Therefore, We assume that the coronary artery disease and obesity, diabetes may have a common genetic basis, FTO gene polymorphisms may be associated with the risk of coronary artery disease. According to the literature retrieval, studies on obesity related gene polymorphisms and CAD are very little, only a few scholars in the study of type2diabetes mellitus. Otherwise subgroup analysis of the relationship between diabetes with coronary heart disease and FTO gene polymorphisms were taken. In2010Hubacek first reported that there were a significant association between the FTO gene rs17817449polymorphism and risk of acute coronary syndrome (ACS) in Caucasian males. But research on Africa Indian showed that FTO variant was not related with metabolic syndrome with acute myocardial infarction. The association of FTO gene single nucleotide polymorphism (SNP) and CAD is still uncertainty. Up to now, there is no direct study on the association between FTO gene polymorphism and CAD in China. Different races have genetic heterogeneity, susceptibility gene of CAD found by genome-wide association study (GWAS) need to be duplicated in a large sample of different races. Researches in the last ten years had shown that leptin was associated with obesity, dyslipidemia, insulin resistance, hypertension and atherosclerosis, it was one of the risk factors of CAD.The LEP gene encoding leptin was one of obesity-associated gene. Lep gene and FTO gene play an important part mainly by hypothalamus, inhibiting the food intake to keep energy balance in the same way. Researches also confirmed the existence of correlation of leptin and FTO gene, the specific mechanism is still not clear. Obesity related SNP in FTO gene are mainly located in the first intron and it was confirmed in the highly conserved gene sequence.The most well-studied locus rs9939609were in strong linkage disequilibrium with rs17817449and other loci, and its genotyping was high success rate. A large number of studies have reported a significant association of the intronic SNP rs9939609of FTO gene with obesity and with a number of obesity-related features. Therefore our experiment chooses the typical sites of FTO rs9939609, rs17817449 and LEP rs10954174as tagging SNP. We evaluated the relationships between the3SNPs and CAD.This was a case-control study, It was the first time that we studied the relationship between the3SNPs polymorphisms and risk of CAD in Chinese Han populations. It is well-known that DNA sequencing is the highest accuracy method for detecting gene polymorphisms. So we took direct sequencing method to determine the genotypes of FTO and LEP gene and analyze the correlation between gene variant and CAD-related subclinical phenotype (hypertension, high blood lipid, high blood sugar and high homocysteine, etc.). We aim to investigate the association between FTO, LEP gene polymorphisms and CAD in Chinese Han populations.Objective①To assay the allele and genotypes of fat mass and obesity-associated(FTO) gene rs9939609(A/T)、rs17817449(G/T) and LEP gene rs10954174(A/G) in Chinese Han CAD and Control patients.②To explore the correlation between FTO rs9939609(A/T)、rs17817449(G/T) and LEP rs10954174(A/G) polymorphisms and coronary artery disease(CAD).③To evaluate the relationship between the polymorphisms of FTO rs9939609(A/T)、rs17817449(G/T) and LEP rs10954174(A/G) and a number of CAD-related clinical parameters.Methods1. Subjects A total of245subjects,147CAD and98matched non-CAD(Control) patients, were randomly enrolled in this case-control study. All participants were from the department of cardiology, Shenzhen Longgang Central Hospital. The cases group included127male and20female, The control group included88male and10female. They were all Chinese Han populations by self-report. Informed written consent was obtained from all subjects before participation. The studies were approved by the regional ethical committees and were in accordance with the principles of the Declaration of Helsinki.2. Biochemical and anthropometrical measurements Anthropometrics including weight, height, waist circumference were measured at the baseline examination by trained persons with standardized methods. and Cardiometabolic risk factors, like blood pressure, fasting plasma glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, homocysteine and uric acid were measured by routine laboratory methods. Risk factors of CAD questionnaires were administered in both groups. Medical history were obtained from medical records of the subjects. Information about smoking, personal and family history of cardiovascular disease, and history of hypertension, diabetes mellitus, gout, hyperlipidemia,myocardial infarction, stroke and physical activity was recorded3. Genotyping Genomic DNA was isolated from peripheral whole blood cells by standard methods according to the manufacturer’s instructions and stored at-20℃. Genotyping of FTO gene (rs9939609, rsl7817449) and LEP rs10954174polymorphisms for all subjects were carried out using sequences retrieval method through the ABI3730sequencing equipment (Applied Biosystem) and genotypes were read using automated software. Reactions were run in5μL volumes using an amplification protocol of95℃for2minutes, followed by25cycles of95℃for10seconds,51℃for10seconds, then60℃for190seconds.4. Statistical analysis Statistical analysis was performed by SPSS version17.0statistical software. Data were presented as means±standard deviation (SD) or as percentages for discrete variables. Differences in quantitative traits between the two groups were analyzed by the independent sample T Test, the three groups by one-way ANOVA analysis. Differences in discrete variables were analyzed using χ2analysis and odds ratios (OR) were calculated using the "case-control" method. Frequency distribution of genotypes and alleles in the3SNPs were consistent with the Hardy-Weinberg equilibrium evaluating with χ2tests. Association between3SNPs and CAD and CAD-related phenotypes(BMI, WC, FPG and TC, TG, LDL-C, HDL-C, et al.) were analyzed by using the logistic regression analysis. The3SNPs were included as a dichotomous variable in dominant models. Statistical significance was taken at a P value<0.05for all comparisons.Results1. Basic characteristics of the participants The basic characteristics of the two groups under study were summarized. Gender, BMI, Systemic blood pressure and Diastolic blood pressure, TG, LDL-C, and UA were similar to the both groups (P>0.05). History of diabetes mellitus (DM) and gout were no significance in the two groups (P>0.05). The CAD patients have higher prevalence of smoking, hypertension and hyperlipidemia (P<0.05). Age, BMI, WC, FPG,TC and HCY were higher in CAD group than in Control. And HDL-C lower than that of the control group, the difference was statistically significant (P<0.05).2. In both analyzed groups, The genotype frequencies of FTO rs17817449were in accordance with the Hardy-Weinberg equilibrium. Distributions of FTO rs9939609and LEP rs10954174genotypes did not deviate from the Hardy-Weinberg equilibrium (P>0.05).3. The genotype frequencies of AT+AA and TT were26.53%and73.47%, and the A allele frequency of rs9939609was13.27%in CAD group and13.30%in control groups (P>0.05). 4. G allele frequency at rs17817449was16.67%in CAD and8.2%in Control group. The frequency of the GG genotype and G allele was significantly higher in CAD group than in control(4.8%vs.1.0%,16.67%vs8.2%, P<0.05). Carriers of rs17817449GG+GT genotype had the increased risk of CAD,4.85times higher than that of homozygous TT genotype (P=0.038, OR=4.85,95%CI:0.587～40.053). rs17817449at least one G allele carriers had a higher risk of CAD than non-G allele carriers (P=0.007, OR=2.250,95%CI:1.239～4.084).5. The LEP rs10954174A allele frequency was3.4%and4.08%, respectively in CAD and Control group (P>0.05).The frequency of the GA+AA genotype was lower in CAD group than in control(6.8%vs.8.2%, P>0.05).6. The2SNPs rs9939609and rs17817449in FTO gene were strongly in linkage disequilibrium (D=3.4%, r2=0.97>0.33).7. There was a significant association between rs9939609(A/T) variation and BMI, WC, SBP, DBP, FPG, TC, LDL-C in both CAD and control group (P<0.05). There was a higher prevalence of hypertension and hyperlipidemia history in AA+AT genotypes than that of TT genotypes (P<0.05). There was no significant association between FTO rs9939609(A/T)) polymorphisms and TG, HDL-C, UA, HCY and medical history of smoking, diabetes and gout in both groups (P>0.05).8. There was no significant association between FTO rs17817449(G/T) polymorphisms and DBP, UA,FPG, TG, TC, HDL-C and HCY in CAD and Control group (P>0.05). The history of diabetes, gout and hyperlipidemia were similar in the both groups(P>0.05). BMI,WC, SBP, LDL-C were higher in FTO rs17817449GG homozygotes than that of TT genotype. Difference were significant in the two kinds of genotypes(P<0.05).9. All of the CAD-related parameters were no any significant differences in LEP rs10954174GG genotype and GA+AA genotypes in the both groups (P>0.05).10. The results of Logistic regression analysis showed, The risk factors of CAD were body mass index (BMI), fasting blood glucose (FPG), triglyceride (TG), HDL-C, hyperlipidemia history and rs17817449G allele. The rs17817449G allele was significantly associated with coronary artery disease risk (P<0.05, OR=25.81,95%CI:2.348～285). The association was independent of BMI, WC, smoking, history of hypertension, history of diabetes and serum total cholesterol, low density lipoprotein cholesterol, uric acid, homocysteine.Conclusions1. Association of FTO gene rs9939609(A/T) polymorphisms with coronary artery disease(CAD) in Chinese Han nationality was not observed. the allele A may be a risk factor of CAD in Chinese Han populations.2. The single nucleotide polymorphism (SNP) of rs17817449(G/T) in FTO gene was associated with CAD, GG genotypes carriers have higher risk of CAD than TT carriers in Chinese Han populations.3. No any association was found between LEP rs10954174polymorphisms and CAD-related quantitative traits and obesity-associated parameters.4. These results replicate the association of FTO rs9939609(A/T) and rs17817449(G/T) variants with BMI in Chinese Han populations. And FTO rs9939609and rs17817449were strongly in linkage disequilibrium.5. Our data provide preliminary evidence that FTO and LEP polymorphisms may lead to increased risk of CAD in Chinese Han populations. The influence of these genotypes on CAD risk warrants further investigation.更多还原