【作者】 聂少芳；

【导师】 廖玉华； 程翔；

【作者基本信息】 华中科技大学 ， 内科学， 2013， 博士

【摘要】 目的冠心病和2型糖尿病都是由遗传因素和环境因素共同作用的复杂性疾病，两者有着类似的发生基础，严重危害人类健康并成为国民经济的重大负担。现阶段，以单核苷酸多态性（single nucleotide polymorphisms，SNPs）为基础的全基因组关联分析（genome-wide association analysis studies，GWAS）是当今复杂性疾病遗传学研究的重要方法。2007年GWAS发现位于人类9号染色体短臂2区1带3亚带（9p21.3）的SNPs可增加冠心病/心梗和2型糖尿病的发病风险，其中rs10116277与rs1333049位于两个相邻的单体型区域，分别是rs10116277与rs1333049之间44-kb的冠心病危险区域和rs10965243与rs10757283之间4kb的2型糖尿病危险区域。最近，两项以欧洲人群为基础的关联研究发现这两个单体型区域可能通过不同的机制来增加冠心病和2型糖尿病的发病风险。由于2型糖尿病与冠心病可能有着共同的遗传基础，因此我们假设上述2型糖尿危险区域在中国汉族人群中与冠心病也存在关联，并通过中国汉族人群大样本病例对照关联分析，以进一步复制9p21.3上2型糖尿病的GWAS易感位点，同时分析这一位点上的SNPs与冠心病的关联性。方法本研究以中国汉族人群为研究对象，共入选379例2型糖尿病患者，1093例冠心病患者和1695例对照个体，采用高分辨率溶解曲线（high resolution meltanalysis technology，HRM）进行两个阶段（two-stage）的关联分析研究：第一阶段，对包含379例2型糖尿病，496例冠心病和849例对照个体的中国中部人群进行分析，研究rs2383208，rs10811661和rs10757283与2型糖尿病、冠心病的关联性；第二阶段，对包含597例冠心病和846例对照个体的中国北部人群进行分析，以验证rs2383208，rs10811661和rs10757283与冠心病的关联性。最后，对1093例冠心病患者冠状动脉粥样硬化狭窄的严重程度进行Gensini评分，并通过数值变量关联分析和四分位法的病例对照关联分析，研究rs2383208，rs10811661和rs10757283与Gensini分值之间的关联性。采用多因素logistic回归分析和多元线性回归分析校正传统危险因素（如年龄、性别、吸烟、体重指数、血脂等）对关联分析结果的影响。结果在中国汉族人群中，两个SNPs（rs10811661和rs10757283）与2型糖尿病持续关联（rs10811661-T，P=0.020，OR=1.23，95%CI：1.03–1.47，P-adj=0.021；rs10757283-C，P=0.003，OR=1.30，95%CI:1.09–1.54，P-adj=0.004）；同时，它们也与冠心病相关（stage1：rs10811661-T，P=0.030，OR=1.19，95%CI：1.02–1.40，P-adj=0.048；rs10757283-C，P=0.026，OR=1.20，95%CI：1.02–1.40，P-adj=0.013；Stage2：rs10811661-T，P=0.028，OR=1.18，95%CI：1.02–1.38，P-adj=0.031；rs10757283-C，P=0.039，OR=1.17，95%CI:1.01–1.36，P-adj=0.035）。其中，rs10811661和rs10757283与冠心病的关联最显著（rs10811661-T，P=0.002，OR=1.19，95%CI：1.06–1.33, P-adj=0.003； rs10757283-C， P=0.003， OR=1.18，95%CI：1.06–1.32,P-adj=0.001）。而rs2383208与糖尿病不关联（P>0.1），与冠心病仅在合并群体中显示接近于阳性的关联性P值（rs2383208-G，P=0.058，OR=1.11，95%CI:1.00–1.24）。最后，三个SNPs中仅rs10757283与冠心病患者的冠状动脉粥样硬化严重程度的Gensini分值相关联（rs10757283-C：线性回归分析P=2.48×10-9，P-adj=0.002；四分位法病例对照关联分析，4thvs.1st，P=2.48×10-10，OR=2.09，95%CI：1.65–2.64，P-adj=0.006）。结论首先，我们发现9p21.3基因座上rs10811661和rs10757283在中国汉族人群中与2型糖尿病持续关联，且与之前研究报道的危险效应大小一致（OR=1.2-1.3）。其次，我们证实该糖尿病危险区域也是冠心病的一个新的易感位点，且可能通过不同机制参与冠心病的发生和发展。最后，我们发现rs10757283可能同时影响冠状动脉粥样硬化的进展和冠心病的发病风险。而进一步分析UCSC和Genevar数据库发现，以rs10811661和rs10757283为代表的9p21.3区域内没有已知基因，但却有大量的基因表达调控元件如增强子等存在，且rs10757283可调节其下游基因DMRTA1的表达。因此，我们可以推测以rs10811661和rs10757283为代表的9p21.3基因座可能通过调节DMRTA1基因的表达而参与冠心病和2型糖尿病的发生和发展，具体的分子遗传学机制有待进一步的研究探讨。综上，本研究在以往全基因组关联分析结果的基础上，采用大样本的病例对照关联分析验证方法，首次发现了中国汉族人群冠心病和2型糖尿病的共同易感位点——以rs10811661和rs10757283为代表的9p21.3区域，其中rs10757283与冠心病患者冠状动脉粥样硬化的严重程度密切相关。这是我国乃至世界上冠心病和2型糖尿病防治研究领域的一项重要突破，为进一步开展两重大复杂性疾病的防治研究提供了崭新的科学依据。更多还原

【Abstract】 Objective Leading by interactions among multiple genetic and environmental factors,coronary artery disease and type2diabetes are two main major disorders, which seriouslyreduce the people’s quality of life and become the great burden of the national economy. Atpresent, the genome-wide association analysis studies (GWAS) based on single nucleotidepolymorphisms (SNPs) become the important method of the genetic research for complexdisorders. In2007, GWAS have reported that the9p21.3locus associated with coronaryartery disease and myocardial infarction, as well as type2diabetes, and rs10116277andrs1333049located separately on the two adjacent haplotype blocks (44-kb coronary arterydisease block from rs10116277to rs1333049and4-kb type2diabetic block fromrs10965243to rs10757283). Most recently, two studies based on European populations alsoreported that the two blocks might contribute to the risk of the two disorders in differentways. As type2diabetes and coronary artery disease has possible common genetic basis,we hyperthesised that there might be common genetic antecedents between type2diabetesand coronary artery disease in the type2diabetic risk locus. Therefore, we investigated alarge scale case-control association analysis study in Chinese Han population, in order tofurther replicate type2diabetic GWAS SNPs on9p21.3, as well as to analysis theassociation between these SNPs and coronary artery disease.Methods Based on the Chinese Han population, we toltally selected379type2diabetes,1093coronary artery diseases and1695control subjects, used the high resolution meltanalysis technology (HRM) and finished2-stage association analysis: In stage-1, westudied379type2diabetes,496coronary artery diseases and849controls in theGeneID-Central-China cohort, to analysis the association of rs2383208, rs10811661andrs10757283with coronary artery disease, as well as type2diabets. In stage-2, we studied597coronary artery diseases and846controls in the GeneID-Northern-China cohort and tovalidate the association of the SNPs with coronary artery disease. We also estimated theseverity of atherosclerosis in all subjects with coronary artery disease by the Gensiniscoring system, and investigated the association between the selected SNPs and the Gensiniscores by liner regression analysis and quartile case-controal analysis. The traditional risk factors, such as age, sex, smoking, BMI, cholestroal and so on, were adjusted by multiplelogistic regression analysis and multiple linear regression analysis.Results Two SNPs (rs10811661and rs10757283) were continuously associated with type2diabetes (rs10811661-T, P=0.020, OR=1.23,95%CI:1.03–1.47, P-adj=0.021;rs10757283-C, P=0.003, OR=1.30,95%CI:1.09–1.54, P-adj=0.004). At the same time,theyalso assocated with coronary artery disease (stage-1, rs10811661-T, P=0.030, OR=1.19,95%CI:1.02–1.40, P-adj=0.048; rs10757283-C, P=0.026, OR=1.20,95%CI:1.02–1.40,P-adj=0.013; Stage-2, rs10811661-T, P=0.028, OR=1.18,95%CI:1.02–1.38, P-adj=0.031;rs10757283-C, P=0.039, OR=1.17,95%CI:1.01–1.36, P-adj=0.035). Here, the associationof rs10811661and rs10757283with coronary artery disease were the most significant(rs10811661-T, P=0.002, OR=1.19,95%CI:1.06–1.33, P-adj=0.004; rs10757283-C,P=0.003, OR=1.18,95%CI:1.06–1.32, P-adj=0.001). Though negatively asscoated withthe two diseases in the separate cohort with P values more than0.1, rs2383208gained anear signifcant association result in the combined population for coronary artery disease(rs2383208-G, P=0.058, OR=1.11,95%CI:1.00–1.24, P-adj=0.061). In the end, onlyrs10757283was significantly associated with the Gensini scors in the patients withcoronary artery disease (rs10757283-C: liner regression analysis, P=2.48×10-9, β=0.267,P-adj=0.002; case-control analysis based on Quartile analysis,4thvs.1st, P=9.50×10-10, OR=2.09,95%CI:1.65–2.64, P-adj=0.006).Conclusions Firstly, we found that rs10811661and rs10757283on9p21.3werecontinuously associated with type2diabetes in Chinese Han population, which was inaccordance with previous studies in risk effect (OR=1.2-1.3). Secondly, we demonstratedthat the same diabetic region was a novel risk locus for coronary artery disease, whichmight function in a different way. Finally, we found that rs10757283might both influencethe atherosclerosis development and increase coronary artery disease risk in Chinese Hanpopulation. Further studies based on the UCSC and Genevar database indicated the9p21.3locus represented by rs10757283and rs10811661has no known genes, but lots of geneexpression regulation elements, such as enhancer. And rs10757283could regulate theexpression of its downstream gene DMTRA1. Therefore, we may conclude that the9p21.3risk locus, represented by rs10811661and rs10757283, might influence the development of coronary artery diseases and type2diabetes by regulating the expression of DTMRA1.Further studies are needed to clarify the specific molecular biological mechanisms,Above all, based on the previous GWAS works, we performed the large scalecase-control association analysis, and for the first time found the common genetic risk locus(9p21.3represented by rs10811661and rs10757283) for coronary artery disease and type2diabetes. And rs10757283was also correlated with the severity of coronary atherosclerosis.This is an important breakthrough in the field of studies on the two complex disorders inChina even all over the world, which provides a new scientific basis for the furtherprevention and treatment research on coronary artery disease and type2diabetes.更多还原