【作者】 王红；

【导师】 张向阳；

【作者基本信息】 新疆医科大学 ， 内科学， 2008， 博士

【摘要】 目的:研究同型半胱氨酸(homocysteine,Hcy)代谢酶亚甲基四氢叶酸还原酶(MTHFR)A1298C多态性、甲硫氨酸合成酶(MS)A2756G多态性,以及MTHFR A1298C和MS A2756G组成的联合基因型在新疆哈萨克族人群中的分布特点,探讨Hcy水平及MTHFR A1298C、MS A2756G多态性与新疆哈萨克族原发性高血压的关系;探讨MTHFR A1298C、MS A2756G单个基因突变及联合基因突变对血浆Hcy的影响。方法:采用随机整群抽样的方法,以生活在新疆塔城、伊犁、阿勒泰地区定居的哈萨克族人群为研究对象,进行标准化的问卷调查、体格检查。经流行病学问卷调查和体格检查后,按照2005年中国高血压防治指南制定的高血压诊断标准,收缩压(SBP)≥140mmHg和或舒张压(DBP)≥90mmHg,以及曾确诊高血压目前正在服降压药物者为高血压病例组(EH组),同一地区的的正常血压者为对照组(NT组),采用酶标免疫吸附检测法检测所有研究对象的血浆Hcy浓度,采用基因组DNA提取试剂盒及酚-氯仿法提取外周血白细胞基因组DNA,应用聚合酶链反应-限制性片断长度多态性(PCR-RFLP)方法,检测研究对象MTHFR基因A1298C、MS基因A2756G多态位点的基因型。分析EH组和NT组中基因型、等位基因、联合基因型的频率分布特点,比较上述两个多态位点各基因型携带者之间血浆Hcy水平的差别及其与高血压的相关性。结果:1)新疆哈萨克族人群存在MTHFR基因A1298C位点多态性,存在AA、AC、CC三种基因型和A、C两种等位基因。基因型AA、AC、CC分布频率分别为64.72%、30.84%、4.44%,等位基因A、C分布频率分别为80.14%、19.86%,符合Hardy-Weinberg平衡( x 2=0.414,P=0.813)。MTHFR基因A1298C多态位点的AA、AC、CC三种基因型在高血压组分布频率分别为67.98%、28.08%和3.94%,在正常对照组分布频率分别为61.78%、33.33%和4.89%;A、C两种等位基因在高血压组频率分别为82.02%和17.98%,在正常对照组等位基因分布频率分别为78.44%和21.56%,两组基因型和等位基因频率分布差异无统计学意义( x 2=1.806,P=0.405和x 2=1.714,P=0.190)。按性别分层,男性EH组和NT组间、女性EH组和NT组间MTHFR基因A1298C多态位点的AA、AC、CC三种基因型和A、C两种等位基因均无统计学差异。新疆哈萨克族高血压组血浆Hcy水平明显高于同族正常对照组(14.87±8.76 vs 11.15±5.21),有统计学差异(P<0.01)。按性别分类比较,男性EH组和NT组血浆Hcy水平均高于同组女性,分别为17.26±9.07(μmol/L)vs 12.65±5.04(μmol/L)和12.75±6.13 vs 9.82±5.09,差异有统计学意义(P<0.05)。男性、女性EH组Hcy水平均高于NT组,分别为17.26±9.07(μmol/L)vs12.75±6.13(μmol/L)和12.65±5.04(μmol/L)vs9.82±5.09(μmol/L),两组比较差异有统计学意义(P<0.05)。MTHFR A1298C多态位点AA、AC、CC三种基因型的血浆Hcy水平无统计学差异,分别为13.19±8.90(μmol/L)、12.24±4.77(μmol/L)、11.99±5.40(μmol/L)。AA、AC、CC三种基因型之间在年龄、BMI、SBP、DBP、FBG、BUN、Cr、UA、TC、TG、HDL、LDL、脂蛋白a各临床资料间均无统计学差异。年龄、BMI是新疆哈萨克族高血压发病的独立危险因素,而MTHFR基因A1298C位点多态性、血浆Hcy水平不是新疆哈萨克族高血压发病的独立危险因素。2)新疆哈萨克族人群存在MS基因A2756G位点多态性,存在AA、AG、GG三种基因型和A、G两种等位基因。AA、AG、GG分布频率分别为78.15%、20.19%、1.66%,A、G等位基因频率分别为88.24%、11.76%,符合Hardy-Weinberg平衡( x 2=0.3072,P=0.8576)。MS基因A2756G多态位点的AA、AG、GG三种基因型在高血压组分布频率分别为76.12%、21.39%和2.49%,在正常对照组分布频率分别为80.00%、19.09%和0.91%;A、G两种等位基因在高血压组频率分别为86.82%和13.18%,在正常对照组等位基因分布频率分别为89.55%和10.45%,两组基因型和等位基因频率分布差异无统计学意义( x 2=0.926,P=0.336和x 2=1.508,P=0.219)。按性别分层,男性EH组和NT组间、女性EH组和NT组间MS基因A2756G多态位点的AA、AG、GG三种基因型和A、G两种等位基因均无统计学差异。MS A2756G多态位点AA、AG、GG三种基因型的血浆Hcy水平分别为12.74±7.88(μmol/L)、12.95±5.04(μmol/L)、13.38±5.84(μmol/L),三种基因型血浆Hcy水平的差异无统计学意义。MS基因A2756G多态位点的AA、AG、GG三种基因型之间在年龄、BMI、SBP、DBP、FBG、BUN、Cr、UA、TC、TG、HDL、LDL、脂蛋白a各临床资料间均无统计学差异。年龄、BMI是新疆哈萨克族高血压发病的独立危险因素,而MS基因A2756G位点多态性、血浆Hcy水平不是新疆哈萨克族高血压发病的独立危险因素。3)新疆哈萨克族MTHFR A1298C和MS A2756G联合基因型存在8种联合基因型:AA/AA、AA/AG、AA/GG、AC/AA、AC/AG、CC/AA、CC/AG、CC/GG,但未发现AC/GG联合基因型。各联合基因型分别在EH组和NT组之间无统计学差异。以MTHFR 1298CC/MS 2756 AA基因型为对照,用非条件Logistic回归模型进行MTHFR A1298C基因型与MS A2756G基因型的联合作用与高血压的关系分析,提示MTHFR 1298AA/MS 2756 GG发生高血压的风险最高,OR值为1.80,MTHFR 1298CC/MS 2756 AG发生高血压的风险最低,OR值为0.240,但与MTHFR 1298CC/MS 2756 AA基因型比较无统计学差异。其余各联合基因型与MTHFR 1298CC/MS 2756 AA基因型比较均无统计学差异。MTHFR A1298C、MS A2756G各联合基因型的血浆Hcy水平之间进行比较,MTHFR 1298AA/MS 2756GG基因型的血浆Hcy水平最高,为13.30±3.56(μmol/L);MTHFR 1298CC/MS 2756AA基因型的血浆Hcy水平最低,为12.08±4.88(μmol/L),但两者比较差异无统计学意义(P>0.05)。联合基因型各型之间比较血浆Hcy水平均无统计学差异。结论:1)新疆哈萨克族人群MTHFR基因A1298C、MS基因A2756G位点存在多态性表现。MTHFR 1298位点存在AA、AC、CC三种基因型和A、C两种等位基因;MS 2756位点存在AA、AG、GG三种基因型和A、G两种等位基因。基因型的分布均符合Hardy-Weinberg平衡;2)新疆哈萨克族MTHFR基因A1298C位点多态性与新疆哈萨克族高血压的发生无明显的相关关系。MTHFR基因A1298C点突变可能不是影响血浆Hcy水平的重要遗传因素。3)新疆哈萨克族MS基因A2756G位点多态性与新疆哈萨克族高血压的发生无明显的相关关系。MS基因A2756G点突变可能不是影响血浆Hcy水平的重要遗传因素。4)新疆哈萨克族高血压组血浆Hcy明显高于正常对照组,且血浆Hcy水平有性别差异,男性血浆Hcy水平明显高于女性;血浆Hcy可能是新疆哈萨克族高血压发生的危险因素,但不是独立的危险因素。5)新疆哈萨克族MTHFR 1298、MS 2756联合基因型与新疆哈萨克族高血压的发生无明显的相关关系。MTHFR A1298C、MS A2756G联合突变可能不是影响血浆Hcy水平的重要遗传因素。更多还原

【Abstract】 Objective:To investigate distributed characteristic of the methylenetetrahydrofolate reductase (MTHFR) A1298C polymorphism and the methionine synthase (MS) A2756G polymorphism and their united genotypes of MTHFR A1298C and MS A2756G in Xinjiang Kazakhs people. And to research the effects of MTHFR A1298C and MS A2756G genetic mutation and united two genotypes on plasma homocysteine level in Xinjiang Kazakhs with essential hypertension (EH). Methods: More than 400 Xinjiang Kazakhs aged more than 30 years old were recruited in this population-based case-control study by cluster sampling who lived in pasture area of Tacheng, Yili, Aretai region in Xinjiang. After questionnaire and physical examination, they were divided into hypertensives group (EH) whose systolic blood pressure (SBP)≥140mmHg and/or diastolic blood pressure (DBP)≥90mmHg and normotensives group (NT) whose blood pressure<140/90mmHg according to the guideline of the hypertensive prevention and treatment of China in 2005. We extracted the genome DNA of peripheral leucocyte by kits or phenol-chloroform, and measured their gene polymorphisms of MTHFR A1298C and MS A2756G by Polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) method. The level of plasma homocysteine were measured using enzymoimmunoassay. We studied the relationship of the polymorphisms and the united genotypes with EH and plasma homocysteine level and researched the association of the genetypes with EH by SPSS15.0 statistic software. Results: 1) There were three kinds of genotypes and two kinds of alleles of MTHFR gene A1298C polymorphism be detected in Xinjiang Kazakhs. In this population, the frequencies of the genotypes AA, AC, CC were 64.72%, 30.84%, 4.44% and the frequencies of alleles A, C were 80.14%, 19.86%, respectively. The x 2 test for the Hardy-Weinberg equilibrium with polymorphism was not statistically significant ( x 2=0.414, P=0.813). The distributed genotypic frequencies of AA, AC, CC were 67.98%, 28.08%, 3.94% in EH group and 61.78%, 33.33%, 4.89% in NT group, respectively. The distributed allelic frequencies of A, C were 82.02%, 17.98% in EH group and 78.44%, 21.56% in NT group, respectively. The distribution of genotypic and allelic frequencies had no significant difference between EH group and NH group ( x 2=1.806, P=0.405 and x 2=1.714, P=0.190). Through to analyze the data stratified with gender (male and female), the distribution of genotypic and allelic frequencies had no significant difference between two groups (P>0.05). There was significant difference in levels of plasma homocysteine between EH group and NT geoup, (14.87±8.76 vs 11.15±5.21, P<0.01). Either EH group or NT group, the level of plasma homocysteine in male was higher than that of female, There was significant difference between male and female (17.26±9.07μmol/L vs 12.65±5.04μmol/L, P<0.01, and 12.75±6.13 vs 9.82±5.09, respectively, P<0.05). Either in male or in female, the level of plasma homocysteine in EH group was higher than that of NT group (17.26±9.07μmol/L vs 12.75±6.13μmol/L, P<0.01, and 12.65±5.04μmol/L vs 9.82±5.09μmol/L, respectively, P<0.05). There was no significant difference in the level of plasma homocysteine among subjects with the AA, AC and CC genotypes (13.19±8.90μmol/L, 12.24±4.77μmol/L and 11.99±5.40μmol/L, P>0.05). There was no significant difference in age, BMI, SBP, DBP, FBG, BUN, Cr, UA, TC, TG, HDL, LDL, Apo-a among subjects with the AA, AC and CC genotypes (P>0.05). Age and BMI were independent risk factors for essential hypertension in Xinjiang Kazakhs. The genotypes of the MTHFR A1298C polymorphism and the level of plasma homocysteine were excluded as independent variables which related to the blood pressure of subjects after controlling the confounding factors like age, gender, BMI by multiple logistic analysis. 2) There were three kinds of genotypes and two kinds of alleles of MS gene A2756G polymorphism be detected in Xinjiang Kazakhs. In this population, the frequencies of the genotypes AA, AG, GG were 78.15%, 20.19%, 1.66% and the frequencies of alleles A, G were 88.24%, 11.76%, respectively. The x 2 test for the Hardy-Weinberg equilibrium with polymor- phism was not statistically significant ( x 2=0.3072, P=0.8576). The distributed genotypic frequencies of AA, AG, GG were 76.12%, 21.39%, 2.49% in EH group and 80.00%, 19.09%, 0.91% in NT group, respectively. The distributed allelic frequencies of A, G were 86.82%, 13.18% in EH group and 89.55%, 10.45% in NT group, respectively. The distribution of genotypic and allelic frequencies had no significant difference between EH group and NH group ( x 2=0.926, P=0.336 and x 2=1.508, P=0.219). Through to analyze the data stratified with gender (male and female) , the distribution of genotypic and allelic frequencies had no significant difference between two groups (P>0.05). There was no significant difference in the level of plasma homocysteine among subjects with the AA, AG and GG genotypes (12.74±7.88μmol/L, 12.95±5.04μmol/L and 13.38±5.84μmol/L, respectively, P>0.05). There was no significant difference in age, BMI, SBP, DBP, FBG, BUN, Cr, UA, TC, TG, HDL, LDL, Apo-a among subjects with the AA, AG and GG genotypes (P > 0.05). Age and BMI were independent risk factors for essential hypertension in Xinjiang Kazakhs. The genotypes of the MS A2756G polymorphism and the level of plasma homocysteine were excluded as independent variables which related to the blood pressure of subjects after controlling the confounding factors like age, gender, BMI by multiple logistic analysis. 3) There were eight kinds united genotype between MTHFR A1298C and MS A2756G: AA/AA, AA/AG, AA/GG, AC/AA, AC/AG, CC/AA, CC/AG, CC/GG, but there was no AC/GG united genotype be detected. There was no significant difference in united genotypes distribution between EH group and NT group (P>0.05). We used an unconditional logistic regression model to analyze the relation between the united genotypes and essential hypertension. When the MTHFR 1298CC/MS 2756AA was defined as the reference, the MTHFR 1298AA/MS 2756GG showed a higher adjusted OR (1.80) , and MTHFR 1298CC/MS 2756AG showed a lower adjusted OR (0.240), but statistical significance were not be found for each combination of alleles (P>0.05). Comparing the level of plasma homocysteine among each combination of alleles, the level of plasma homocysteine in MTHFR 1298AA/MS 2756GG was highest (13.30±3.56μmol/L); the level of plasma homocysteine in MTHFR 1298CC/MS 2756AA was lowest (12.08±4.88μmol/L); but there was no significant difference between the each combination group. Conclusions : 1) There existed A1298C polymorphisms of MTHFR gene and A2756G of MS gene in Xinjiang Kazakhs people. There were three kinds of genotypes AA, AC, CC and two kinds of alleles A, C in A1298C, polymorphisms of MTHFR gene, respectively. There were three kinds of genotypes AA, AG, GG and two kinds of alleles A, G in A2756G polymorphism of MS gene. The genotypic frequencies were in Hardy-Weinberg equilibrium. 2) There was probably no relation between MTHFR A1298C genetic polymorphism and essential hypertension in Xinjiang Kazakhs. The MTHFR A1298C genetic polymorphism probably was not important hereditary factor at affecting the level of plasma homocysteine in Xinjiang Kazakhs. 3) There was probably no relation between MS A2756G genetic polymorphism and essential hypertension in Xinjiang Kazakhs. The MS A2756G genetic polymorphism probably was not important hereditary factor at affecting the level of plasma homocysteine in Xinjiang Kazakhs. 4) The level of plasma homocysteine were significantly higher in EH group than NT group in Xinjiang Kazakhs, and there was significant difference in gender, the level of plasma homocysteine in male was higher than that in female. Higher level of homocysteine was probably an risk factor for hypertension in Xinjiang Kazakhs, but it was not an independent risk factor for hypertension in Xinjiang Kazakhs. 5) There was probably no relation between the combination of MTHFR A1298C and MS A2756G genetic polymorphism and essential hypertension in Xinjiang Kazakhs. The combinations of MTHFR A1298C and MS A2756G genetic polymorphism probably were not important hereditary factors at affecting the level of plasma homocysteine in Xinjiang Kazakhs.更多还原