血管紧张素原基因部分多态性指标与原发性高血压的发病风险:社区病例对照

背景:血管紧张素原基因是第一个被发现的原发性高血压候选基因,T174M5和M235T多态均位于AGT基因第二外显子,且存在连锁不平衡。启动子区域A-6G和G-217A位点多态对其基因表达起重要调节作用,且血管紧张素原基因的表达产物与血压水平的维持密切相关。目的:探讨血管紧张素原基因A-6G,T174M和G-217A位点多态性与中国汉族人群原发性高血压发病风险的关系。设计:整群抽样,病例-对照分析。单位:南京医科大学第一附属医院老年医学科与心血管科,国家人类基因组南方研究中心,江苏省东台市人民医院心血管科。对象:实验于2005-09/10在江苏省东台市农村完成。①263例实验对象均来自江苏省盐城市东台县农村,其中原发性高血压组177例为未经药物治疗的原发性高血压患者,高血压的诊断参照1999年WHO/ISH高血压诊断标准(收缩压≥140mmHg和/或舒张压≥90mmHg);正常对照组86例。②纳入标准:实验对象为汉族;长期居住本地非外来人口;能清楚回答问题;经病史、临床症状、体征及辅助检查确诊;统一问卷面访调查资料完整。③排除标准:原发性高血压组排除继发性高血压,正常对照组排除高血压家族史,同时两组人群均排除肝、肾慢性疾病和糖尿病。方法:采集外周静脉血3mL,用FlexiGeneDNAKit(250)提取人外周血中DNA。应用primer3软件进行引物设计,并排除引物序列中的多态位点。多重聚合酶链反应扩增后,取3μL反应产物用琼脂糖凝胶电泳检测扩增结果,扩增成功的聚合酶链反应产物用QIAquickPCRPurifica-tionKit纯化,纯化后的产物用DNaseⅠ片段化,片段化的酶切产物以脱氧核苷酸末端转移酶进行荧光素标记。每个单核苷酸多态各设计2条等位基因特异性探针和1条错配探针,芯片用OmniGridTM100点样仪制备,每个探针重复3次,形成3个阵列。杂交液95℃变性10min后,立即置于冰上冷切,取10μL杂交液加入到芯片矩阵上,50℃杂交2h,然后洗涤,甩干。GenePix4000B共聚焦激光扫描仪进行芯片扫描,利用GenePixPro提取得到每条探针的荧光信号强度值,通过计算每个单核苷酸多态的等位基因分数判断基因型。主要观察指标:①两组血管紧张素原基因各多态位点基因型分布频率的比较。②血管紧张素原基因A-6G和T174M位点多态性与原发性高血压发病风险的相关分析。③两组血管紧张素原基因A-6G,T174M和G-217A位点多态性对血压的影响。结果:按意向处理分析,263例实验对象均进入结果分析。①血管紧张素原基因A-6G位点AA,AG,GG基因型(P=0.014)以及A,G等位基因频率(P=0.004,OR=0.44)差异明显;T174M位点CC,CT,TT基因型(P=0.031)以及C,T等位基因频率(P=0.014,OR=0.55)差异有显著性意义;未发现G-217A位点GG,AG,AA基因型(P=0.722)以及G,A等位基因频率(P=0.403,OR=0.80)有明显差异。②携带A-6G多态AA基因型和T174M多态CC基因型的个体发生原发性高血压的风险分别减少57%(95%可信区间=0.23～0.82,P=0.010)和56%(95%可信区间=0.25～0.79,P=0.006)。③两组血管紧张素原基因A-6G,T174M和G-217A位点各基因型的收缩压、舒张压和平均动脉压的差异均无显著性意义(F=0.100～2.911,P均>0.05)。结论:血管紧张素原基因A-6G位点AA基因型和T174M位点CC基因型可能会减少中国汉族人群原发性高血压发病风险,未发现G-217A多态基因型与其有显著相关性。

Association between partial indexes of angiotensinogen gene polymorphisms and the risk of essential hypertension:A community case-control study

BACKGROUND: Angiotensinogen (AGT) gene is the firstly discovered candidate gene for essential hypertension, both the T174M and M235T polymorphisms locate at the second exons of AGT gene, and there is existence of linkage disequilibrium. The polymorphism at A-6G and G-217A sites in promotor region plays an important role in regulating the gene expression, and the products of keep close correlation with the level of blood pressure. OBJECTIVE: To investigate the association between the polymorphism of AGT gene at A-6G, T174M and G-217A sites and the risk for the attack of essential hypertension in Chinese Han population, DESIGN: A cluster sampling and case-control analysis. SETTINGS: Department of Geriatrics and Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University; Southern Research Center of National Human genome; Department of Cardiology, Dongtai People's Hospital of Jiangsu Province. PARTICIPANTS: The experiment was carried out in the countryside of Dongtai county, Yancheng city, Jiangsu province. All the subjects were selected from the countryside of Dongtai county, Yancheng city, Jiangsu province. Totally 177 patients with essential hypertension who had never accepted any drug treatment, were taken as the essential hypertension group, and hypertension was diagnosed according to the diagnostic standard of hypertension set by WHO/ISH in 1999 (systolic blood pressure ≥ 140 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg); Another 86 normal person were taken as the normal control group. ② Inclusive criteria: The enrolled subjects should be Han nationality; long-term local residents but not from other places; able to answer questions clearly; diagnosed by disease history, clinical symptoms, physical signs and assistant examinations; have complete data of investigation of uniform questionnaires by face-to-face interview (including demographic information, profession history, family history and life styles of smoking, drinking, drinking tea, etc.). ③ Exclusive criteria: The patients with secondary hypertension in the essential hypertension group, subjects having family hisory of hypertension in the normal control group, and those with chronic diseases of liver and kidney, and diabetes mellitus in both groups were excluded. METHODS: Peripheral venous blood samples (3 mL) were collected, and DNA was extracted from human peripheral blood with FlexiGene DNA Kit (250). The Primer3 software was applied to design primers, and the polymorphism sites in the primer sequence were excluded. After multiplex polymerase chain reaction (PCR), 3 μL products were selected to detected the amplified results by agarose gel electrophoresis. The successfully amplified PCR products were purified with the QIAquick PCR Purification Kit, and the purified products were fragmentized with Dnase Ⅰ . The fragmentized products of enzyme digestion were labeled with fluorescein by deoxynucleotide terminal transferase. Two allele specific probes and one mismatched probe were designed respectively for each single nucleotide polymorphism. The chips were prepared with the OmniGridTM 100 TLC samler, each probe was repeated for three times to form three matrix. The hyridization solution was degenerated at 95 ℃ for 10 minutes, and then immediately cut on ice. 10 μL hybridization solution was added onto the chip matrix, hybridized at 50 ℃ for 2 hours, then washed and dried. The chips were scanned with the GenePix 4000B laser confocal scanner (Figure 2),and the intensity of the fluorescent signal for each probe was extracted with GenePix Pro, and the allele score of each single nucleotide polymorphism was calculated to judge the genotype. MAIN OUTCOME MEASURES: ① Comparison of the frequencies of genotype distribution at each polymorphism site of AGT gene in both groups; ② Correlation analysis of the polymorphism of AGT gene at A-6G and T-174M sites with the risk for the attack of essential hypertension; ③ Effects of the polymorphism of AGT gene at A-6G, T-174M and G-217A sites on blood pressure.RESULTS: According to the intention-to-treat analysis,all the 263 subjects were involved in the analysis of results. ① At the A-6G site of AGT gene, the frequencies of AA, AG and GG genotypes (P=0.014) and A and G alleles (P=0.004, OR=0.44) had significant differences between the essential hypertension group and normal control group; At the T174M site, the frequencies of CC, CT and TT genotypes (P=0.031) and A and G alleles (P=0.014, OR=0.55) were significantly different; At the G-217A site, no obvious differences were found in the GG, AG and AA genotypes (P=0.722) and G and A alleles (P=0.403, OR=0.80). ② The risk of essential hypertension in the individuals carrying AA genotype of A-6G polymorphism and CC genotype of T174M polymorphism was reduced by 57% (95%CI= 0.23-0.82, P= 0.010) and 56% (95%CI= 0.25-0.79, P= 0.006) respectively. ③ There were no significant differences in the systolic blood pressure, diastolic blood pressure and mean arterial pressure among different genotypes at the A-6G, T174M sites and G-217A sites (F=0.100- 2.911, P ＞ 0.05). CONCLUSION: The AA genope at A-6G and the CC genotype at T174M site of AGT gene may reduce the risk for the attack of essential hypertension in Chinese Hun population, and no significant correlation was found between the genotype of G-217A polymorphism and the attack of essential hypertension.