汉族人群高血压易感基因研究及血压反应性的遗传度分析

【作者】 顾东风；

【导师】 强伯勤；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 高血压是人类最重要的健康问题之一,也是心脑血管疾病和肾脏病的主要危险因素。随着生活方式和饮食结构的改变以及期望寿命的增加,高血压的患病率也大幅度增加。我国以往调查资料显示,在过去的几十年中,高血压的患病率和绝对数正在快速增长。2002年全国营养和健康调查结果显示成年人高血压的患病率为18.8%,患者达到1.6亿多人。原发性高血压是一种复杂性疾病,受遗传和环境两方面因素的影响。研究表明血压变异的30%-50%受到遗传因素的影响,而这些遗传因素包括多个基因的作用。高血压的生物学进程涉及到多种调节机制,而每种机制又涉及多种基因产物,因此高血压潜在的遗传机制不仅依赖于多个微效基因的单独作用,同时也依赖于基因-基因之间的交互作用。为了验证这一假设,我们从涉及高血压发生和发展的相关基因中选择具有代表性11个高血压候选基因（ACE、AGTR1、CYP11B2、ADRA1A、ADRB2、TH、LPL、GNB3、NOS3、GRK4、WNK4）,综合运用PCR-PFLP和基因组测序等常用的基因型检测技术,采用病例对照研究方法分析基因单个位点以及单体型与高血压发生的关联关系（本论文将重点阐述ADRA1A、CYP11B2和TH基因与高血压的关系）,同时评价11个基因在高血压发生中可能存在的致病交互作用。此外,为了从分子水平揭示TH基因rs2070762多态在高血压发病过程中的作用,我们对该多态位点进行初步的生物学功能研究。目前遗传流行病学研究已经发现了一些与高血压发生相关的候选基因,然而,每个基因只能解释非常小的血压变异。所以认为遗传因素对血压变异的作用可能不是独立的,而会受到环境暴露的影响。盐作为重要的环境因素,与高血压的发生发展密切相关。流行病学研究结果显示膳食钠摄入与血压呈正相关。然而血压对钠摄入的反应性在个体中的变异非常大。遗传因素可能影响着个体血压对膳食钠摄入的反应性。为了评价遗传因素的效应,我们设计了“盐敏感性遗传流行病学网络研究（GenSalt Study）”,测量我国人群血压对膳食钠、钾摄入反应性的遗传度。本研究结果对于进一步阐明高血压发生的分子遗传学机制以及可能存在的基因-基因/基因-环境交互作用等具有重要的理论意义。本研究的结果支持高血压病因学的多基因本质,同时也为今后的研究提供了检测复杂性疾病基因-基因交互分析的模型。本研究评价了遗传因素在个体血压对膳食钠摄入的反应性中所起的重要作用,这将有助于发现那些新的血压盐敏感性基因,对高血压的个体化防治具有积极的意义。第一部分肾上腺素受体α_1A基因变异与高血压的关联研究肾上腺素受体α₁(α_1A^-,α_1B^-,α_1D^-)是一个G蛋白偶联跨膜受体的家族,通过结合儿茶酚胺、肾上腺素和去甲肾上腺素来调节交感神经系统的活动。α₁肾上腺素受体调节心血管功能,在血压的形成过程中可能起到重要作用。我们以前报道了人染色体8p22与原发性高血压和血压水平存在显著的连锁,而这一区段与肾上腺素受体α_1A基因（ADRA1A）定位相邻。为此,我们采用病例对照研究来检验ADRA1A多态与中国北方汉族人群原发性高血压的关联关系。本研究所有DNA样本和临床资料均来自亚洲国际心血管病协作研究（TheInternational Collaborative Study of Cardiovascular Disease in Asia,InterASIA）中国部分。我们选取了480对2期高血压患者和年龄（相差2岁以内）、性别和地区匹配的健康对照,2期高血压定义为收缩压（SBP）≥160mmHg和/或舒张压（DBP）≥100mmHg。对照组血压要求SBP低于140mmHg且DBP低于90mmHg。排除了有继发性高血压、冠心病和糖尿病的患者。通过对随机选择的48个高血压病例和48个对照的基因测序确定了7个多态位点,并对所有研究对象进行基因型鉴定。采用McNemar检验来估计每个多态位点引起高血压的OR值。条件logistic回归分析时控制传统高血压危险因素,如BMI、胆固醇、血糖和甘油三酯,结果用OR的95%可信区间表示。估计每个单体型的频率并用单体型趋势回归分析各个单体型与高血压的关联。病例组中347Arg和2457G等位基因频率显著高于对照组（P=0.04和0.007）。McNemar检验显示2457G等位基因携带者发生高血压的危险性较高,OR为3.00（95%CI 1.23-8.35）。条件Logistic回归分析结果显示调整了高血压的传统危险因素后2457G等位基因携带者发生高血压相对危险度为2.84（95%CI1.15-6.99）。用PHASE估计单体型频率并且进行单体型趋势回归分析,发现调整了高血压的传统危险因素后单体型7与原发性高血压关联（P=0.02）我们的结果显示ADRA1A基因变异与高血压存在显著关联,可能在中国人群高血压的发生中起到重要作用。第二部分醛固酮合酶基因与高血压的关联研究人类醛固酮合酶（CYP11B2）是一种类固醇11β羟化酶或18-羟化酶和18-氧化酶,催化肾上腺球状带细胞中醛固酮的终末合成。本研究旨在了解醛固酮合酶基因CYP11B2（T-344C,Lys173Arg和一个内含子转换[IC]多态）与中国北方汉族人群2期高血压的关系。我们共收集了503对高血压病人和经年龄、性别和地区匹配的正常对照。结果显示女性高血压病人中-344T,173Lys和IC-转换等位基因的频率显著高于正常对照组（P值分别为0.002,0.002和0.014）。女性高血压病例组中由-344T,173Lys和IC-转换等位基因组成的单体型4的频率显著高于对照组（P=0.007）。在使用计分检验（Score Test）调整相关协变量之后,单体型4与女性高血压的关系依然显著（P=0.003）。而由T-Arg-WT组成的单体型3则在男性和女性中均具有保护作用（P值分别为0.03和0.006）。结果显示CYP11B2基因的173Lys和IC-转换多态能增加中国北方女性人群2期高血压的发病风险。第三部分酪氨酸羟化酶基因与高血压关联研究及初步功能分析酪氨酸羟化酶（TH）是儿茶酚胺合成的限速酶,在交感神经系统中发挥重要的生理作用。虽然TH是原发性高血压的候选基因之一,但仍没有关于TH与高血压发生的系统研究。我们选择了503名高血压患者和在年龄、性别和地区上均匹配的490名对照,对TH基因与高血压进行关联分析。根据HapMap和dbSNP数据,选择了TH基因的4个多态位点:rs6356、rs6357、rs2070762和rs1800033,进行基因分型。rs1800033位点在我们的研究人群中不存在多态性。rs6356和rs6357多态在高血压和非高血压人群中分布没有显著差别。但是rs2070762位点的基因型（P＜0.001）和等位基因频率（P=0.005）在病例组和对照组中均存在显著差异。rs6356和rs6357位点与rs2070762存在弱连锁不平衡。单体型分析显示,研究人群中共有8种单体型,单体型频率总的分布在高血压人群和非高血压人群中存在显著差别。单体型A-A-C（rs6356-rs6357-rs2070762）和A-G-C在高血压人群中频率显著高于非高血压人群（P值分别为0.003和0.013）,且A-A-C仅在高血压人群中存在。校正协变量后,单体型G-G-C携带者发生高血压风险比最常见单体型A-G-T携带者高1.83倍（P=0.0049）。功能分析结果表明,与T等位基因相比,C等位基因使启动子转录活性增强2倍;未知核蛋白因子特异性结合到T等位基因,而不结合C等位基因。这提示,可能存在一种转录抑制因子,协同T等位基因抑制启动子转录活性。功能研究结果为TH基因rs2070762多态与高血压的关联关系提供了重要证据,并提示rs2070762多态C等位基因由于不结合转录抑制因子而使TH基因表达上调,可能参与高血压的发病过程。第四部分11个基因的33SNPs与中国人群高血压的关联研究原发性高血压是一种典型的复杂性疾病,是环境因素和多个基因共同作用的结果。单个位点的结果很难重复,其中一个原因就是高血压潜在的遗传机制不仅依赖于多个微效基因的单独作用,同时也依赖于基因-基因之间的交互作用。为了检验这种假设,我们在中国人群中进行了一项病例对照研究来检测单位点及多位点的效应。从涉及高血压发生和发展的相关基因中,挑选了11个候选基因和33个多态位点,并在503名高血压患者和490名年龄、性别和地区匹配的对照中对这些位点进行基因型鉴定。本研究既采用了传统的logistic回归进行单位点的关联分析,又使用了分类回归树（classification and regression trees,CART）和多元适应性回归样条（multivariate adaptive regression splines,MARS）进行多位点关联分析。使用Bonferroni校正或交叉验证来选择最终的模型。我们发现TH^*rs2070762、ADRB2^*Q27E和GRK4^*A486V三个多态与中国人群的原发性高血压的发生独立相关。另外,发现CYP11B2和AGTR1基因在高血压的发病中存在交互作用。这些发现支持原发性高血压病因学的多基因本质,同时也为今后提供了一个研究基因-基因交互作用的模型。第五部分血压对膳食钠和钾摄入反应性的遗传度研究目前缺乏血压对膳食干预反应性的遗传度的研究报道。本研究通过膳食干预,在中国北方农村的1906个研究对象中检测了血压对膳食钠、钾摄入反应的遗传度。膳食干预包括7天低钠饮食（钠摄入量为每天51.3mmol）,7天高钠饮食（钠摄入量为每天307.8mmol）,和7天高钠补钾（钾摄入量为每天60mmol）饮食。在干预前三天的基线期测量9次血压,在每个干预期的最后3天也测量9次血压。血压测量采用“随机零点血压计”。遗传度采用方差组分模型下的最大似然比法来计算,所有的计算使用SOLAR程序来完成。基线期收缩压的遗传度为0.31,舒张压的遗传度为0.32,平均动脉压的遗传度为0.34。膳食干预下遗传度显著增加,低钠期上述三种血压的遗传度分别为0.49、0.49和0.51;高钠期为0.47、0.49和0.51;高钠补钾期为0.51、0.52和0.53。对于低钠膳食干预,收缩压、舒张压和平均动脉压改变百分比的遗传度为0.20、0.21和0.23;对于高钠膳食干预,其遗传度分别为0.22、0.33和0.33;对于高钠补钾膳食干预,遗传度分别为0.24、0.21和0.25。我们的研究表明控制膳食钠、钾摄入时血压的遗传度显著高于通常饮食时（未特意控制钠、钾摄入）血压的遗传度。此外,在本研究人群中血压对膳食钠、钾摄入反应的遗传度是适度的。更多还原

【Abstract】 Hypertension is an important worldwide public-health challenge because of it is one of the most important modifiable risk factors for CVD and renal disease.With changes in lifestyle and diet and an increase in life expectancy,the morbidity and mortality of hypertension have greatly increased.According to previous national surveys conducted in China,the prevalence and absolute numbers of hypertension have increased dramatically during the past several decades.It was estimated that 18.8%of the adult population in 2002 had hypertension,and the total number of adults with hypertension was 160 million.Essential hypertension is considered to be a typical complex disease and is influenced by both genetic and environmental factors.The genetic contribution to blood pressure（BP） variation ranges from 30 to 50%.It is likely that a number of genes with smaller effects account for the heritability of this complex disorder.Since the biological process of hypertension involves multiple physiological pathways,each of which may be affected by multiple gene products,the underlying genetics of hypertension is not only based on multiple genes with minor effects,but also on gene-gene interactions.To test this hypothesis,we focused on 11 representive candidate genes involved in multiple biochemical pathways that have been implicated in the development and progression of hypertension（ACE,AGTR1,CYP11B2, ADRA1A,ADRB2,TH,LPL,GNB3,NOS3,GRK4,WNK4）,performed PCR-PFLP or direct sequencing for the genotyping,and tested their associations with hypertension in a case-control study（in this paper,the associations of three genes, ADRA1A,CYP11B2 and TH,with hypertension were shown）.We also evaluated the influence of gene-gene interactions on the risk of hypertension.Furthermore,the biological relevance of the significantly associated rs2070762 in TH gene was studied by various functional assays in vitro.Genetic epidemiology studies have identified many biological candidate genes that were related to BP.However,it is generally found that association of any of these genes accounts for a very little variance.Thus,genetic factors may not independently affect BP,and the effect on BP is influenced by environmental factors.The increased dietary intake of salt as an importantly environmental factor plays a pathogenic role in essential hypertension.Epidemiological studies have shown that dietary sodium intake was positively associated with blood pressure.However,BP response to dietary sodium intake varies considerably among individual subjects.Genetic factors might play an important role in determining the BP responses of an individual subject to dietary sodium intake.To evaluate the effect of genetic factors,the Genetic Epidemiology Network of Salt Sensitivity（GenSalt） Study was designed to examine the genetic influence on BP responses to dietary sodium and potassium intake in Chinese families.Our study improve our understanding of mechanisms of hypertension and multiple gene-gene/gene-environment interactions.The findings support the multigenic nature of the etiology of essential hypertension and propose a potential gene-gene interactive model for future studies.Our study also measures the heritability of BP response to dietary sodium intake.Understanding the genetic effects has important implications for identifying important novel genes for salt sensitivity of BP,and contributes to individualized prevention and therapy on hypertension.SECTION ONE ADRA1A gene variants and hypertensionα₁-adrenergic receptors(α_1A-,α_1B-,α_1D-) are a family of G protein-coupled transmembrane receptors that mediate actions in the sympathetic nervous system through binding the catecholamines,epinephrine and norepinephrine,α₁-adrenergic receptors regulate cardiovascular function and may play a role on the development of hypertension.Our previous report linked human chromosome 8p22,which is near the location of humanα_1Aadrenergic receptor（ADRA1A） gene,to essential hypertension and blood pressure.Upon this,here we present a case-control study on the relationship between ADRA1A gene variants and hypertension in northern Han Chinese.All DNA samples and clinical data were collected from the International Collaborative Study of Cardiovascular Disease in Asia（InterASIA）.The measurements and interviews were taken under standard conditions.We enrolled 480 unrelated stage-2 hypertensive patients and their individually age-（within 2 years）, gender-and area-matched healthy control subjects.Stage-2 hypertension was defined as an average SBP≥160 mm Hg and/or DBP≥100 mm Hg.Control subjects had SBP and DBP＜140 mm Hg and＜90 mm Hg.Subjects with a clinical history of secondary hypertension,coronary heart disease and diabetes were excluded from the study.The interview included questions related to the diagnosis and treatment of hypertension.Anthropometrical measurements,including height,weight,waist and hip circumferences were taken.Concentrations of serum lipids were also determined by standard protocols.Seven polymorphisms were identified by direct sequencing of genomic DNA derived from 48 randomly recruited hypertensives and 48 healthy subjects.We used McNemar’s test to estimate the odds ratio（OR） for hypertension for each polymorphism.Conditional logistic regression analysis was used to assess whether the genetic variation was associated independently with hypertension after adjustment for covariates.The extent of pairwise linkage disequilibrium was expressed in terms of D’,and the pairwise correlation was presented as r².Haplotype Trend Regression（HTR） was used to test the association between each haplotype and hypertension..We observed significantly higher frequencies of the 347Arg allele and 2547G alleles in the cases compared to their controls（P=0.04 and 0.007,respectively）. McNemar’s test revealed carriers of 2547G alleles were at a higher risk for EH with an OR of 3.00（95%CI:1.23-8.35）.We then performed a conditional logistic regression to adjust the effects of conventional risk factors,revealing an OR of 2.84 for carriers of 2547G allele（95%CI:1.15-6.99）.With the haplotypic probabilities estimated using the PHASE software,we performed the Haplotype Trend Regression analysis,showing a significant association between haplotype 7 and EH（P=0.02）, after adjustment for conventional risk factors.Our findings suggest that the genetic variations in the ADRA1A gene are significantly associated with EH and may play an important role in the development of essential hypertension in this Chinese population. SECTION TWO CYP11B2 gene haplotypes and hypertensionHuman aldosterone synthase（CYP11B2）,a steroid 11β-hydroxylase as well as an 18-hydroxylase and an 18-oxidase,catalyzes the terminal steps of aldosterone biosynthesis in the zona glomerulosa cells of the adrenal.The objective of this study was to investigate the association of polymorphisms in the aldosterone synthase gene CYP11B2（T-344C,Lys173Arg,and an intronic conversion[IC]） with stage-2 hypertension in northern Han Chinese.A total of 503 hypertensives and their age-, gender- and area-matched controls were included in this study.The female hypertensives had significantly higher frequencies of the -344T,173Lys and IC-conversion alleles（p=0.002,0.002 and 0.014,respectively）.The estimated frequency of haplotype composed of the -344T,173Lys and IC-conversion alleles （haplotype 4） was significantly higher in the female hypertensives compared with their controls（p=0.007）.Using a multivariate score test,we found that haplotype 4 remained associated with female hypertension after the adjustment for covariates （p=0.003）,while the haplotype 3 of T-Arg-WT showed a protective effect both in the males and in the females（p=0.03 and 0.006,respectively）.These results indicate that the 173Lys and the IC-conversion allele of the CYP11B2 gene confer an increased risk for stage-2 hypertension in northern Han Chinese women.SECTION THREE Association study of TH Gene with Hypertension and Primary Functional AnalysisThe tyrosine hydroxylase（TH） gene encodes the rate-limiting enzyme of catecholamine biosynthesis,thus it plays a pivotal role in the physiology of sympathetic nervous system.However,as one of the candidate genes of essential hypertension,the relation between the variants of TH gene and hypertension had not been extensively studied.We designed a case-control study consisting of 490 normotensive（NT） controls and 503 hypertensive（HT） cases matched in area,age and gender to systematically investigate the relationship between TH gene and hypertension.Based on the HapMap and dbSNP data,four SNPs,rs6356,rs6357, rs2070762 and rs1800033 in the TH gene were selected for genotyping.The SNP rs1800033 did not present polymorphic in our studied population.No significant distribution differences were observed for rs6356 and rs6357 between HT and NT groups.However,both the genotype and allele frequencies of rs2070762 showed significant differences between cases and controls（P＜0.001 and P=0.005, respectively）.The SNP rs6356 and rs6357 were both in weak linkage disequilibrium with rs2070762.In haplotype analysis,total eight haplotypes are observed in the entire population and the overall frequency distributions differ significantly between HT group and NT group.Specifically,haplotype A-A-C（in the order of rs6356-rs6357-rs2070762） occurs only in HT group and A-G-C occurs more often in HT subjects than in NT subjects（P=0.003 and P=0.013,respectively）.Moreover, haplotype G-G-C carriers get about 1.83-fold higher risk for hypertension than non-carriers（p=0.0049） after adjustment for covariates.Functional analyses showed that C allele confered a two-fold higher promoter activity when fused to a heterologous promoter than the T allele,and unidentified nuclear factor（s） binded specifically to T allele,but not C allele.This suggested the existence of transcriptional repressor（s） responsible for the lower promoter activity regulated by T allele.These results provide evidence for an association of the functional SNP rs2070762 of TH gene with essential hypertension and suggest that the up-regulated expression of TH gene by rs2070762 C allele without binding transcriptional repressor（s） might be involved in the pathogenesis of hypertension.SECTION FOUR Association Study with 33 SNPs in 11 Candidate Genes for Hypertension Essential hypertension is considered to be a typical complex disease with multifactorial etiology,which leads to inconsistent findings in genetic studies.One possibility of failure to replicate some single-locus results is that the underlying genetics of hypertension is not only based on multiple genes with minor effects,but also on gene-gene interactions.To test this hypothesis,a case-control study was constructed in Chinese,detecting both single locus and multilocus effects.Eleven candidate genes were selected from related genes that have been implicated in the development and progression of hypertension and thirty-three polymorphisms were evaluated in 503 hypertension patients and 490 age-,gender-matched controls. Single-locus associations,using traditional logistic regression analyses,and multi-locus associations,using classification and regression trees（CART） and multivariate adaptive regression splines（MARS） were both explored in this study. Final models were selected using either Bonferroni correction or cross-validation. Three polymorphisms,TH^*rs2070762,ADRB2^*Q27E and GRK4^*A486V,were found to be independently associated with essential hypertension in Chinese.In addition to these individual predictors,a potential interaction of CYP11B2-AGTR1 is also involved in the etiology of hypertension.These findings support the multigenic nature of the etiology of essential hypertension and propose a potential gene-gene interactive model for future studies.SECTION FIVE Heritability of Blood Pressure Responses to Dietary Sodium and Potassium IntakeThe heritability of blood pressure responses to dietary intervention has not been well studied.We examined the heritability of blood pressure responses to dietary sodium and potassium intake in a family feeding study among 1906 study participants living in rural North China.The dietary intervention included a 7-day low-sodium feeding（51.3mmol per day）,a 7-day high-sodium feeding（307.8 mmol per day）,and a 7-day high-sodium plus potassium supplementation（60 mmol per day）.Blood pressure was measured 9 times during the 3-day baseline period preceding the intervention and also during the last 3 days of each intervention phase using a random-0 sphygmomanometer.Heritability was computed using maximum likelihood methods under a variance components model as implemented in the computer program SOLAR.The heritabilities of baseline blood pressure were 0.31 for systolic, 0.32 for diastolic,and 0.34 for mean arterial pressure.The heritabilities increased significantly under dietary intervention and were 0.49,0.49,and 0.51 during low sodium;0.47,0.49,and 0.51 during high sodium;and 0.51,0.52,and 0.53 during potassium supplementation for systolic,diastolic,and mean arterial pressure, respectively.The heritabilities for percentage of blood pressure responses to low sodium were 0.20,0.21,and 0.23;to high-sodium were 0.22,0.33,and 0.33;and to potassium supplementation were 0.24,0.21,and 0.25 for systolic,diastolic,and mean arterial pressure,respectively.Our study indicated that the heritabilities of blood pressure under controlled dietary sodium and potassium intake were significantly higher than those under a usual diet.In addition,the heritabilities of blood pressure responses to dietary sodium and potassium intake were moderate in this study population.更多还原