【作者】 王红梅；

【导师】 李南方；

【作者基本信息】 新疆医科大学 ， 临床医学， 2010， 博士

【摘要】 目的：高血压是世界范围内的公共卫生问题。在我国,18岁及以上居民高血压患病率为18.8%,据此估计全国患病人数为1.6亿；人群患病特点是北方高于南方,农村已接近城市,但按我国人口分布特征来看,估计农村高血压患者已达1.16亿。高血压是多种遗传因素和环境因素共同作用的结果,其中遗传因素在高血压的发病机制中起重要作用。随着国际人类基因组计划测序完成和基于单核苷酸多态性(single-nucleotide polymorphisms; SNPs)的单体型图谱构建完成,人类遗传学研究正在进入一个新时代—全基因组关联研究。但是在特殊的人群中,尤其是在稀有突变及低频率SNPs方面,传统的候选基因研究策略在高血压等复杂疾病的遗传机制研究中仍是不可替代的。众所周知,水盐代谢失衡是高血压的原因之一,而上皮细胞钠通道(ENaC)在调节水盐代谢平衡过程中发挥着重要作用；神经前体细胞表达发育调控样蛋白4 (neural precursor cell expressed developmentally downregulated 4-like, NEDD4L)因介导ENaC的表达及降解过程而参与血压调节,是高血压的候选基因。新疆地处祖国西北边陲,新疆哈萨克族人是高血压的高发民族,而且是一个遗传背景单一、影响血压的环境因素较为一致的相对隔离群体,是进行高血压遗传研究的理想人群。故了解新疆农牧区哈萨克族人群高血压患病情况并研究该人群高血压高发的遗传机制是十分必要的。所以本课题对新疆阜康牧区哈萨克族牧民进行了高血压流行病学调查,以了解高血压病流行现状,以期为制定该民族特异性的高血压防治策略提供依据；然后采用候选基因研究策略,研究NEDD4L基因变异与该人群高血压及其中间表型的关系,以期探讨高血压可能的遗传学机制,从而为早期预防高血压及其相关疾病提供科学依据。方法：(1)采用多级随机抽样法对新疆阜康牧区哈萨克族人群进行了高血压流行病学调查,研究对象30-70岁,三代内无异族通婚史,收集人口学、病史及高血压相关表型资料,采集空腹(至少10小时)及服糖后2小时静脉血,测血脂、血糖等生化指标。(2)在总体样本中随机选择94例30-60岁高血压患者,利用美国ABI公司3100XL基因分析仪对NEDD4L基因编码区和启动子区直接测序筛查该基因的变异位点,测序结果通过Sequencher 4.7基因序列分析软件与NEDD4L基因组标准序列进行比对,找出变异位点并明确其在基因序列中的位置及变异性质,利用专业遗传学分析软件SNPAlyze Ver.7.0.1 pro (日本)确定该人群代表性、低频率SNPs和稀有突变。(3)在883例30-60岁新疆阜康牧区哈萨克族人中采用TaqMan PCR技术对筛查出的NEDD4L基因代表性、低频率SNPs和稀有突变进行基因分型：按病例-对照设计来分析NEDD4L基因变异与该人群高血压的关系。(4)遗传学分析如Hardy-Weinberg平衡检验、关联分析、单体型分析采用专业遗传学分析软件SNPAlyze Ver.7.0.1 pro(日本)进行分析。其他统计学分析采用SPSS(version 16; SPSS Inc., Chicago, Illinois, USA)统计软件包进行数据处理。计量资料用均数±标准差描述数据的集中趋势。所有资料进行分析之前先进行正态性检验,如有非正态性分布的数据经转换为对数并满足正态分布条件后再进行分析。正常对照组和高血压组临床表型定量资料在进行两组比较时应先进行方差齐性检验,以决定是采用t检验或采用Wilcoxon秩和检验等；分类资料用构成比、率描述其基本特征。高血压组、血压正常组间临床表型计量资料比较采用t检验,分类资料采用卡方检验。NEDD4L变异对高血压的发病风险分析采用logistic回归模型,并校正了吸烟、饮酒、年龄、肥胖等影响因素,关联程度用优势比(odd ratio, OR)及其95%可信区间(95% confidence interval,95% CI)表示。不同基因型间临床表型资料比较采用协方差分析(校正年龄、吸烟、饮酒等)。检验标准α=0.05。结果：(1)30-70岁新疆阜康牧区哈萨克族居民中高血压年龄标化患病率为46.8%；高血压年龄标化患病率男性高于女性；高血压患病率随年龄增长而增加。(2)94例高血压患者中,在NEDD4L基因31个外显子、部分内含子以及转录起始点上游约1000bp测序范围内共检出11个变异位点,除了位于第6内含子区的271420T>C (rs2288774)、271454A>G(rs2288775)变异与NCBI公共数据库中报道一致外,其余9个均为新发现的变异位点；在筛查出的11个变异位点中,271420T>C(rs2288774)、271454A>G(rs2288775)和296921-296923delTTG为高频率SNPs,其余为低频率SNPs或稀有突变；采用SNPalyzer软件(日本)分析11个变异位点的LD,结果提示此11个变异位点无LD关系。因为6个低频率SNPs在内含子区,另外2个是稀有突变,虽然在外显子区,但是为同义突变,因此最终选择271420T>C(rs2288774)、271454A>G(rs2288775)和296921-296923delTTG三个SNPs进入下一步在大样本新疆阜康牧区哈萨克族人群中的关联研究。(3)应用TaqMan技术对入选样本进行NEDD4L基因271420T>C (rs2288774)、271454A>G(rs2288775)和296921-296923delTTG三个SNPs位点的扩增和基因分型成功,三个SNPs在新疆阜康牧区哈萨克族牧民中的基因型、等位基因的频率分布符合均Hardy-Weinberg平衡。①单个位点关联分析结果：在女性中,rs2288774多态性的显性模型(AA vs. AG+GG,P=0.040),等位基因(P=0.038)频率在高血压组和血压正常组中的分布差异具有统计学意义,高血压组AG+GG基因型、G等位基因频率高于血压正常组；296921-3delTTG多态性的三种基因型模型(additive model, P=0.024)、隐形模型(II+ID vs DD, P=0.007)及等位基因频率(P=0.028)分布均有统计学意义。校正了年龄、肥胖、吸烟、饮酒等影响因素后,Logistics回归分析显示：rs2288775的AG+GG基因型仍与女性哈族人高血压相关(OR=1.479,95% CI1.011-2.064,P=0.044)；296921-3delTTG多态性的DD基因型仍与女性哈族人高血压相关(OR=1.908,95% CI 1.020-3.568,P=0.043)。②单体型分析：NEDD4L基因三个SNPs在该人群中构建了4种主要单体型,分别为ITA,DCG, ICA和DCA(H 1-4),其中H2(DCG)单体型频率在病例组和对照组中分布有差异。③校正了年龄、吸烟、饮酒、肥胖等混杂因素后,协方差分析结果提示在总体及女性中,29692 1-296923delTTG多态性的DD基因型的尿钠排泄率低于II+ID基因型,差异具有统计学意义。④271420T>C (rs2288774)、271454A>G(rs2288775)和296921-296923delTTG三个SNPs的基因型和等位基因频率在超重肥胖、高血糖、血脂紊乱、中心性肥胖组及其相应的对照组的分布无统计学意义。结论：(1)新疆阜康牧区30-70岁哈萨克族人高血压年龄标化患病率高；高血压年龄标化患病率男性高于女性；高血压患病率随年龄增长而增加。提示应该针对特殊人群制定个体化治疗方案和民族特异性的高血压防治策略,这对于该人群的高血压成功防治具有重要意义。(2)在NEDD4L基因启动子区、31个外显子、部分内含子内共检出11个变异位点,其中9个为新发现变异位点；3个为高频SNPs,6个低频率SNPs,2个稀有突变,但为同义突变；本研究没有在NEDD4L基因编码区发现错义突变,这种高度保守性进一步说明了NEDD4L在维持机体正常生理功能的过程中可能起着至关重要的作用。(3)NEDD4L基因的296921-296923delTTG及rs2288775变异与新疆阜康牧区哈族女性自然人群的高血压病相关,296921-296923delTTG的DD基因型以及rs2288775变异的AG+GG基因型、G等位基因可能是新疆阜康牧区哈族女性高血压的易患因素。更多还原

【Abstract】 Objective:Hypertension is the most prevalent risk factor for cardiovascular morbidity and mortality worldwide. Indeed, hypertension affects> 18.8% of the adult population in China. According to the data of China National Nutrition and Health Survey in 2002, there are 160 million hypertensive patients in China and most of them (116 million) live in rural. The prevention of hypertension has been an important public health issue of rural area in the underdeveloped regions of China. Hypertension is a complex disease resulted from the interaction of the cumulative effect of multiple genetic and environmental factors and genetic factors play an important role in it. Along with the achievement of human genome project and human haplotype map project, the genetic study of complex disease get into a new era-genome-wide association study (GWAT). Nevertheless, there may still be a need for candidate gene approaches, especially when looking at single-nucleotide polymorphisms (SNPs) with low allele frequencies and rare mutations, especially in special ethnic population. By systemically screening SNPS and rare mutation in coding and regulating region of candidate gene and study the relationship between these variations and hypertension, it is possibal to find variations are related to diseases. This will play an essential role in searching the genetic mechanisms of some complex polygenic diseases. A region on chromosome 18q21-22 has been identified in a number of genome-wide scans on hypertension, therefore making the locus a suitable candidate for harboring gene/genes involved in blood pressure (BP) regulation. The 18q21-22 locus harbors an important BP-regulating gene (the neural precursor cell expressed developmentally down-regulated 4-like [NEDD4L] gene). NEDD4L regulates BP through its ubiquitinating effects on the epithelial sodium channel (ENaC) which is a critical component of the pathway maintaining salt and water balance at the luminal cell membrane in the renal collecting ducts, resulting in reduction of the number of ENaC present on the plasma membrane. Thus, NEDD4L is a candidate gene for hypertension, both functionally and genetically. Epidemiological data indicates that the prevalence of hypertension varies greatly between different ethnic populations. Kazakh, a nomad population which dwells north of Xinjiang in northwest China and in which 99% are herdsman, is characterized by a higher prevalence of hypertension and higher BP levels compared to other ethnic populations residing in the same area. Moreover, very few Kazakh marry people of other ethnicities because the unique customs, cultural background, and food habits are different from other ethnicities. Therefore, Kazakh is a relatively isolated population with a pure genetic background and is an ideal population to study genetic mechanisms of complex diseases, such as hypertension. However, there are no reports regarding the relationship between genetic variations in the human NEDD4L gene and essential hypertension in Kazakh. The purpose of the current study was to investigate the prevalence of hypertension in Kazakh, and the relationship between the variation of NEDD4L and essential hypertension in Kazakh from the pasture area of Xinjiang Fukang.Methods:(1) A population-based, cross-sectional study in a Kazakh general population in the pasture area of Xinjiang Fukang was performed which included 1083 participants (response rate 95.5%) aged 30-70 years and with no mixed marriages within the past three generations by cluster sampling. In this study, a multi-stage sampling method was used to select a representative sample of the population. In addition to performing routine blood testing that included lipid profiles, blood electrolytes, glucose levels, and /urine electrolytes, anthropometric measurements were obtained and a questionnaire was completed.(2) All the sequence variants located promoter and exon regions of NEDD4L were identified by directly sequencing of PCR product. Blood samples were obtained from 94 (47 males and 47 females)hypertensive patients, which were randomly chosen from the hypertensive group of the study population, and genomic DNA was isolated from peripheral blood leukocytes using a PAXgene Blood DNA kit (PreAnalytiX). All exons with their flanking sequences and approximately 500 bp of the upstream region of the promoter were directly sequenced by an ABI 3130X1 genetic analyzer (Applied Biosystems, Foster City, CA, USA) using 35 sets of primers. The obtained sequences were examined for the presence of variations using Sequencher 4.7 software (Gene Codes Corporation, Ann Arbor, MI, USA), followed by visual inspection. The A of the ATG of the initiator Met codon is denoted nucleotide +1. The nucleotide sequence [NCBI:NM-015277] was used as a reference sequence.(3) The TaqMan SNP Genotyping Assays were performed in 883 subjects aged 30-60 years for genotyping using the method of Taq amplification in the 7900HT Fast Real-Time PCR system (Applied Biosystems). The primers and probes (Applied Biosystems) used in the TaqMan SNP Genotyping Assays were chosen based on information available on the Applied Biosystems Inc. website (http://myscience. appliedbiosystems.com). Finally, all of the three selected representative SNPs were successfully genotyped in 883 subjects participating in the study.(4) Data analyses were performed by SPSS for Windows (version 16; SPSS Inc., Chicago, IL, USA). Values are expressed as the means±SD. The distribution of patient characteristics between the normotensive and hypertensive groups in the Kazakh general population was analyzed using a Student’s t-test or a chi-square test. The differences in distributions of genotypes and alleles between the essential hypertension patients and control individuals were analyzed using a chi-square test. In addition, logistic regression analysis was performed to assess the contribution of the major risk factors (including smoking, drinking, age, and obesity). Covariate variance analysis was performed to compare the UNa rate and plasma Na level between the different genotypes after adjusting for age, smoking, and drinking. A case-control-based haplotype study, linkage disequilibrium, and the Hardy-Weinberg equilibrium were analyzed using SNPAlyze, version 7.0 Pro (DYNACOM Co. Ltd., Mobara, Japan). Statistical significance was established at P values< 0.05.Results:(1) Epidemiology research indicates:The age-adjusted prevalence of hypertension in Kazakh was 46.8%. In male, the age-adjusted prevalence of hypertension was higher than female. The prevalence of hypertension was increased with age.(2) Eleven genetic variations in NEDD4L were identified by sequencing 94 hypertensive individuals, among which there were 3 common SNPs with a minor allele frequency> 10%(271420T>C,271454A>G, and 296921-296923delTTG).296921-296923delTTG is a new genetic common variation in NEDD4L which is not found in the NCBI SNP-database. No missense mutations in NEDD4L were identified, but we identified the following two synonymous mutations:R423 (297071G>A in exon 14) with a minor allele frequency of 0.91%; and A944 (351555C>T in exon 30) with a minor allele frequency of 0.56%. None of the variations were in tight linkage disequilibrium with an r2< 0.5. After considering the function and linkage disequilibrium relationships among the identified genetic variations,3 common SNPs with a minor allele frequency of> 10% were selected as representatives for genotyping in Kazakh general population.(3) The distribution of the dominant model (AA vs. AG+GG) of rs2288775, the additive model, and the recessive model (II+ID vs. DD) of 296921-296923delTTG differed significantly between the cases and controls in females (P=0.040, P=0.024, and P=0.007, respectively). After adjusting for confounding factors, logistic regression analysis showed that rs2288775 (in the dominant model) and 296921-296923delTTG (in the recessive model) were significantly associated with hypertension (rs2288775:OR=1.479,95% CI= 1.011-2.064, P=0.044; and 296921-296923delTTG:OR=1.908,95% CI= 1.020-3.568, P =0.043) in females. The frequency of the D-C-G haplotype was significantly higher for cases than for controls in females (P=0.020). For females and total participants, the urinary Na excretion rate was significantly lower in the DD than the I/I+I/D individuals (P=0.032 and P=0.027 respectively). The associations between the SNPs and overweight, dyslipidemia, hyperglycemia, and central obesity were also examined, and no significant associations were found.Conclusions:(1) The age-adjusted prevalence of hypertension was high among Kazakh (aged 30 to 70 years old) from the pasture area of Xinjiang Fukang. Attention should be paid to establishment the ethnic-specific and cost-effective strategy to prevent and treat hypertension efficiently.(2) Eleven genetic variations in NEDD4L were identified, among which there were 3 common SNPs with a minor allele frequency> 10%(271420T>C,271454A>G, and 296921-296923delTTG).296921-296923delTTG is a new genetic common variation in NEDD4L which is not found in the NCBI SNP-database. No missense mutations in NEDD4L were identified. This may be indicative of the relatively high conservation of NEDD4L and the importance of this molecule in regulating salt and water balance and BP. (3) The genetic variations of NEDD4L may be associated with essential hypertension infemales in the Kazakh general population. This reconfirms that genetic variations of NEDD4L might be involved in the pathogenesis of essential hypertension.更多还原