Polymorphism Of ENOS And MTHFR Genes,Environmental Factors And Their Interaction Involved In The Pathogenesis Of Essential Hypertension In Kazakh Ethnic Of Xinjiang

Background and Objective Essential hypertension(EH) is a disease with polygenic complex trait, as well as with many environmental risk factors and genetic-environmental interaction involved. The huge social burden caused by EH has brought about serious concerns in the public. In the early studies on EH, environmental risk factors were the major concern. Recently however, with the progress in molecular biological technology and Human Genome project, the genes susceptible to EH has greatly been interested by researchers at home and abroad. The association between EH and 27 bp repeat polymorphism in intron 4 of endothelial nitric oxide synthase gene(eNOS27bpVNTR) and Methylenetetrahydrofolate reductase C667T(MTHFR C667T) polymorphism have been broad investigated, but few studies were done on their genetic-environmental interaction in EH subjects with genetic homogeneous population.Therefore,this study was designed to investigate the polymorphisms of eNOS27bpVNTR and MTHFR C667T in a genetic homogeneous population, the Kazakh ethnic of Xinjiang, the environmental risk factors for their EH and the interaction between genetic polymorphisms and environmental factors, in order to provide experimental datas for a deep understanding of pathogenesis of EH, facilitate the prevention and control of EH. Materials and Methods1.Cross-sectional study with epidemiologic investigation and relevant clinical indices determination.Epidemiologic investigation was conducted in 289 Kazakhs including 151 essential hypertensive and 138 healthy Kazakhs aged from 20 to 60 in Xinjiang Uygur Autonomous Region of China in 2006.They lived in Boertonggu countryside of Shawan region at least for three generations by random cluster sampling.The comprehensive investigation questionnaire,essential auxiliary examination and clinical survey were completed for each individual and their blood specimens were collected for biochemical and genetic determinations with informed consent, including anthropometry, blood pressure, body indices and other blood indices(folic acid, endothelial nitric oxide synthase(eNOS) activity, endothelial nitric oxide(NO) et al.)2. eNOS27bp VNTR and MTHFR C677T genotypes were detected by polymerase chain reaction(PCR)eNOS 27bpVNTR repeat in intron 4 and MTHFR C677T genotypes were detected by a PCR assay in 151 essential hypertensive patients and 138 healt hy Kazakhs population.PCR products were separated on 2～2.5% agarose gel stained with ethidium bromide, and verified by DNA sequencing. MTHFR C6 77T genotypes were detected by a PCR-based RFLP assay.3. Statistical analysisContinuous variables were presented as mean±SD and differences between two groups were tested using t-test and differences of discrete variables were tested using theχ~2 test. The Hardy-Weinberg equilibrium of polymorphism was assessed by theχ~2 test.The linear regression and correlation analysis was used in numerical variables.Risk factors for EH by Logistic-regression analysis.Differences were considered statistically significant at P<0.05. Result1. Environmental factors predisposing to EHOur results showed that major risk factors predisposing to EH in Kazakh population included obesity and few vegetables in their diet.Logistic regression analysis stratified by age and gender showed that EH associated with obesity(OR=8.670) and folic acid(0R=0.125) in men and associated with concentric obesity(OR=9.050) in women aged under 45.While EH associated with concentric obesity(OR=2.26) and folic acid(0R=0.466) in men and with overweight(OR=3.842) and obesity(OR=4.488) in women aged over 45. No significant impact of cigarette smoking,drinking liquor on EH was observed in this study.The clinical characteristics of the 151 hypertensive and 138 normotensive subjects were compared; There were significant differences for body mass index, systolic blood pressure, diastolic blood pressure, waist to hip ratio, total plasma cholesterol level, insulin level, insulin resistance index(IRI), creatinine level, eNOS activity, NO level and plasma folic acid level between the hypertensive and control groups(P<0.05). The plasma folic acid level is lower in male Kazakh than in the female(P<0.05).Logistic-regression analysis exposed that the obesity and short of vegetables are major environment predisposing factors for Kazakh EH in Boertonggu countryside of Shawan region of Xinjiang Uygur Autonomous Region;and we did not find that smoking, drink alcohol liquor are environment predisposing factors in the Kazakh EH of this region.2.Interaction of eNOS27bp VNTR polymorphism and environmental factors(1) The allele and genotype frequency distribution of eNOS 27bpVNTR meet Hardy-Weinberg balance test, the study population embodies good representation. b/b,b/a and a/a genotypic frequency distribution of eNOS 27bpVNTR are 84.06%, 15.22%,0.72% respectively in NT group. The genotypic frequency distribution are 81.46%,15.89%,2.65% respectively in EH group,b and a allele frequency distribution are 89.40%, 10.60% respectively in EH group. Comparing EH and NT no difference between either genotypic or allele frequency distribution was found. Major genetypes of Kazakh in this region is bb, secondly is ab, the least is aa. b allele frequency is more than a allele frequency, all these embody race specificity. There is no association between eNOS27bpVNTR polymorphism and Kazakh EH in Xinjiang.(2) There is no difference among BMI,WHR,SBP,DBP,fasting blood glucose(FBG),cholesterol and blood folic acid levels in different eNOS27bpVNTR genetype(P>0.05); Then we divide EH group into two subgroups according gender, and there was also no difference found in above indexes between different genetypes in male group or in female group(all P>0.05).(3) Logistic-regression analysis did not show any interaction between eNOS27bp VNTR polymorphism and environment predisposing factor such as age,gender,smoking,drink liquor andobesity(all P>0.05)et al.3.Interaction beween MTHFR C677T gene polymorphism and environment factors(1) The allele and genotype frequency distribution of MTHFR C677T meet Hardy-Weinberg balance test, the study population embodies good representation. CC,,TC and TT genotypic frequency distribution of MTHFR C677T are 65.9 %,29.0%,5.11% and 71.5%,23.2%,5.3% in NT group and EH group respectively. C and T allele frequency distribution are 80.4%,19.6% and 83.0%,16.9% in NT group and EH group respectively.There is no difference between allele and genotype frequency distribution in two groups(P>0.05). Major genetype of Kazakh in this region is CC, secondly is TC, the least is TT. T allele frequency is more than C allele frequency, all these embody race specificity.There is no association between MTHFR C677T polymorphism and Kazakh EH in Xinjiang.(2) There is no difference among age,BMI,WHR,SBP,DBP,FBS,cholesterol level,blood folic acid level et al in different MTHFR C677T genetype(all p>0.05). Then we divide EH group into two subgroups according gender, and there was also no difference found in above indexes between different genetypes in male group or in female group(all P>0.05).(3) Logistic-regression analysis did not show any interaction beween MTHFRC677T polymorphism and environment predisposing factor such as age,gender,smoking,drink liquor and obesity(all P>0.05).4. The possible synergistic effects of gene eNOS27bpVNTR and MTHFRC677T polymorphisms on predisposing factorsLogistic-regression analysis with combining eNOS27bpVNTR and MTHFRC677T gene polymorphism did yet not exposed any significant differences in above indexes between any comparing groups(all p>0.05).Conclusion1. Obesity and short of vegetables are major environmental predisposing factor for Kazakh EH in Xinjiang Shawan region; and we did not find that smoking, drink liquor are environment predisposing factor in the region.2. The low plasma folic acid level may be caused by diet shortage of vegetables, and the plasma folic acid level is lower in male Kazakh than in the female.3. Majority of eNOS 27bpVNTR genetype of Kazakh in this Xinjiang region is bb, secondly is ab, the least is aa, and b allele frequency is more than a allele frequency, all these embodies race specificity. There is no association between eNOS27bpVNTR polymorphism and Kazakh EH in Xinjiang.4. Majority of MTHFR C677T genetype of Kazakh in this Xinjiang region is CC, secondly is TC, the least is TT, and T allele frequency is more than C allele frequency, all these embodies race specificity. There is no association between MTHFR C677T polymorphism and Kazakh EH in Xinjiang. 5. There is no synergistic effect among eNOS27bpVNTR polymorphism, MTHFRC677T polymorphism and environment predisposing factor on Kazakh EH in Xinjiang.6. It is possible that high incidence of Kazakh EH in Xinjiang is associated mainly with environmental factors. Increasing the vegetables intake in Kazakh of Xinjiang Autonomous Region may prove to be a convenient and very helpful way for reducing the high incidence of essential hypertension in Kazakh ethnic.