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离子通道基因SLC12A3多态性和内蒙古地区蒙古族原发性高血压的关联研究
The Association Study of Ion-Channel Gene SLC12A3Variants with Essential Hypertension of Mongolian Population in the Inner Mongolia Region
【作者】 梁俊青;
【导师】 苏秀兰;
【作者基本信息】 首都医科大学 , 细胞生物学, 2014, 博士
【摘要】 目的:原发性高血压是最常见的危害人类健康的心血管疾病,是由遗传和环境因素共同作用而引起的复杂性疾病,寻找原发性高血压的致病相关基因和危险因素对高血压的防治具有重要意义。本研究以流行病学调查结合遗传学分析,探索影响内蒙古地区蒙古族血压特征的遗传变异,揭示原发性高血压发病机制,特别是内蒙古地区蒙古族高血压的发病机制,进一步缩小高血压这种复杂疾病可能的致病区域,并为当地蒙古族人群高血压的防治提供科学依据。方法:1.以横断面调查的方法开展了四次高血压流行病学调查,调查地点为内蒙古自治区锡林郭勒盟,包括东乌珠穆沁旗3个苏木(乌拉盖苏木、宝拉格苏木和巴彦霍布尔苏木)、镶黄旗,共采集蒙古族牧民样本908例。经过筛查,排除伴发冠心病、脑卒中、肾病及其他相关疾病的样本,并排除具有亲缘关系的个体后,符合标准并有足够的血液学标本受试者合计754名,其中蒙古族高血压病患者508名和血压正常受试者246名。2.使用Haploview软件对少见等位基因频率(minor allele frequency, MAF)≥0.05,r2≥0.5的常见SNP位点进行筛选,根据block选择15个tagSNP(rs4784733、rs2304478、rs13306673、rs2289119、rs8043560、rs2304483、rs5803、rs7187932、rs6499858、rs11644728、rs8049280、rs7204044、rs2010501、rs2399594和rs711746)。采用连接酶检测反应(ligase detectionreaction,LDR)技术分别对入选样本的15个tagSNP进行基因分型。3.单因素方差分析(SPSS13.0软件)用于高血压患者和正常血压者一般资料的比较;UNPHASED3.0.13软件和Haploview4.0软件应用于单个SNP位点与蒙古族血压特征(高血压、收缩压和舒张压)之间的关联分析及15个被选取SNPs之间的连锁不平衡关系和单倍型关联分析。分别探讨单个SNP位点及其构成的单倍型与原发性高血压的相关性,并探讨了高血压特征:收缩压(Systolicblood pressure,SBP)和舒张压(Diastolic blood pressure,DBP)与SLC12A3的15个常见遗传变异之间的相关性。;G-power软件应用于所有研究样本的统计效力分析。结果:1.对高血压患者和正常血压者的一般临床资料进行比较后显示,在蒙古族人群中,高血压患者与正常血压者在性别、平均年龄、BMI、腰围、臀围、SBP、DBP和TG等方面均具有显著性差异,且高血压组的平均年龄、BMI、腰围、臀围、SBP、DBP和TG均高于正常血压组(P<0.01);空腹血糖、心率、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇和总胆固醇在蒙古族高血压组和蒙古族正常血压组均无显著性差异(P>0.05);在生活习惯方面,饮酒在蒙古族高血压组和正常血压组的差异具有统计学意义(P<0.01),提示饮酒对蒙古族高血压患者的发病起到关键作用,而吸烟在两组中不具有显著性差异(P>0.05)。2.单位点关联分析结果显示,SLC12A3基因上的3个SNPs位点与血压特征(HTN,SBP和DBP)显著相关,这三个位点分别是rs2399594(HTN,p=0.030;SBP,P=0.029;DBP,P=0.050),rs7187932(HTN,p=0.048)和rs2304478(SBP,P=0.028)。3.配对连锁不平衡分析显示,SLC12A3基因上的11个SNPs位点处于3个具有较高LD值的连锁不平衡区(Block): Block1(rs4784733、 rs2304478和rs13306673)、Block2(rs8043560、rs2304483、rs5803、rs7187932、rs6499858和rs11644728);Block3(rs2399594和rs711746),因此,对它们展开单倍型关联分析。4.单倍型关联分析结果显示,SLC12A3基因上具有较高LD值的连锁不平衡区Block3的单倍型GG与高血压特征显著相关(P=0.029),是蒙古族高血压患病的遗传风险因子。结论:1.在蒙古族人群中,高血压组与正常血压组在性别、平均年龄、BMI、腰围、臀围、SBP、DBP和TG等方面均具有显著性差异,且高血压组的平均年龄、BMI、腰围、臀围、SBP、DBP和TG均高于正常血压组(P<0.01)。在生活习惯方面,饮酒在蒙古族高血压组和正常血压组之间的差异具有统计学意义(P<0.01),提示饮酒对蒙古族高血压患者的发病起到关键作用;2. SLC12A3基因上的3个SNPs位点与血压特征(HTN,SBP和DBP)显著相关,其中SNP位点rs2399594与HTN(p=0.030)、SBP(P=0.029)和DBP(P=0.050)均显著相关;SNP位点rs7187932仅与HTN显著相关(p=0.048);SNP位点rs2304478仅与SBP显著相关(P=0.028);3. SLC12A3基因上具有较高LD值的连锁不平衡区Block3(由SNPs位点rs2399594和rs711746组成)的单倍型GG与高血压特征显著相关(P=0.029),是蒙古族高血压患病的遗传风险因子。
【Abstract】 Objective: Essential hypertension (EH) is one of the most common cardiovasculardiseases threatening human health. The pathogenesis of EH stems from a complexinteraction between environmental and genetic factors. Identifying the related genesand risk factors of EH is of great significance for the prevention and treatment of EH.Using epidemiological investigation combined with genetic study, we aimed to findthe variants affecting blood pressure traits of Mongolian population in InnerMongolia regions, clarify the underlying molecular mechanisms of essentialhypertension, especially the molecular mechanisms of Mongolian hypertension in theInner Mongolia region, further narrow these regions to identify the causal gene(s),and provide scientific basises for the prevention and treatment of local Mongolianhypertention.Methods:1. Epidemiological researches were carried out by cross-sectional survey for fourtimes. Totally,908blood samples were collected from three villages and involvedin Dongwuzhumuxin county (including three investigation townships of Wulagai,Baolage and Bayanhuobuer), the Xianghuang county of Xilin Gol League in InnerMongolia Autonomous Region. After excluding the subjects with coronary heartdisease, stroke, the kidney disease, the other related disease as well as the subjects having the relative relationship,754subjects were ultimately selected in our study,including508unrelated Mongolian essential hypertension patients and246unrelated Mongolian normotensive subjects.2. TagSNPs were identified from the Chinese HapMap database based on pairwiser2≥0.5and minor allele frequency (MAF)≥0.05, and15tagSNPs were selectedaccording to the block of LD in our study, including rs4784733、rs2304478、rs13306673、rs2289119、rs8043560、rs2304483、rs5803、rs7187932、rs6499858、rs11644728、rs8049280、rs7204044、rs2010501、rs2399594and rs711746.Genotyping was performed using the PCR/ligase detection reaction assay.3. The basic clinical data of hypertensive and normotensive subjects were assessedby ANOVA (SPSS13.0software). The association study of single SNPs, theanalysis of LD and haplotype in selected SNPs were performed byUNPHASED3.0.13software and Haploview4.0software. We researched theassociation of15single selected SNPs of SLC12A3and their haplotypes withessential hypertension. We also analyzed the association of15single selectedSNPs of SLC12A3with the other blood traits SBP and DBP. G power softwarewas used to assess the statistical power of our study.Results:1. The analysis of basic clinical data showed that there are significant differences insex, average age, BMI, waistline, hipline, SBP, DBP and TG betweenhypertensive and normotensive subjects of Mongolian population. Compared tonormotensive group, the cases are higher in average age, BMI, waistline, hipline,SBP, DBP and TG (P<0.01). However, there are no significant differences infasting blood glucose, heart beat, HDL-C, LDL-C and TC between the twogroups(P>0.05). In the habits of life, drinking showed statistical difference(P<0.01), however smoking didn’t showed statistically significant differences between the two groups (P>0.05), suggesting that drinking may play animportant role in the development of Mongolian hypertension.2. The association study of single SNPs showed that three SNPs of SLC12A3geneare significantly associated with the traits of blood pressure respectively:rs2399594(HTN,p=0.030;SBP,P=0.029;DBP,P=0.050),rs7187932(HTN,p=0.048)and rs2304478(SBP,P=0.028).3. LD analysis showed that11SNPs of SLC12A3gene composed three high LDblocks: block1(rs4784733, rs2304478and rs13306673), block2(rs8043560,rs2304483, rs5803, rs7187932, rs6499858and rs11644728) and block3(rs2399594and rs711746); Therefore we further performed a haplotypeanalysis.4. The results of Haplotype analysis showed that the GG haplotype of block3issignificantly associated with hypertension (P=0.029), suggesting it is likely to bea genetic risk factor of Mongolian hypertension.Conclusion:1. In Mongolian population, there are significant differences in sex, average age,BMI, waistline, hipline, SBP, DBP and TG between hypertensive andnormotensive groups. Compared to normotensive subjects, the cases are higher inaverage age, BMI, waistline, hipline, SBP, DBP and TG. In the habits of life,drinking showed statistical difference, suggesting that drinking may play animportant role in the development of Mongolian hypertension.2. Three SNPs of SLC12A3gene are significantly associated with blood pressuretraits respectively: rs2399594(HTN, SBP and DBP),rs7187932(HTN)andrs2304478(SBP).3. A haplotype GG of the block3(rs2399594and rs711746) is significantly associatedwith hypertension, suggesting it is likely to be a genetic risk factor of Mongolian hypertension.
【Key words】 Essential hypertension; TagSNP; SLC12A3; Mongolian population; Haplotype;