| Objective: As a risk factor of coronary heart disease and stroke,essential hypertension(EH)takes more and more interests.Many epidemic studies about EH exhibit it`s high prevalence.Total prevalence rate in our prevalent study about EH in FUXIN rural country in China is 37.8%.EH is the result of interactions between gene and environment.Many micro-effective genes account for 30-60% genetic heterogeneity of EH.The causes of EH are not yet entirely clear at present.With the completion of the Human Genome Project,the gene-disease interaction becomes a research hotspot.Some new biotechnologies also accelerate the pace of people proceeding to the reality,gene chip is one of the outstanding representatives.However,its high price constraints its use for epidemiology in large population-study.SNPMa P is the genome-wide association study(GWAS)of pooled DNA with 50-100 individial DNA.It saves hundreds and thousands of moneys.We screened susceptibile genes for Chinese Han EH patients in northeast rural,and then validated the results from GWAS in individual samples with the method of HRM(High resolution melting).Methods: A large cross-sectional survey including BP measurements was conducted from 2004 to 2006 in the rural areas of Fuxin county in Liaoning province,China,where there are 35 towns and approximately 640,000 people.A second survey with collection of blood samples was performed from 2008 to 2009.Surveys and administration of ques-tionnaires were carried out by well-trained local doctors.Demographic data(age,sex,and ethnicity),smoking status,use of alcohol,marriage status,family medical history,use of antihypertensive medications,disease history at baseline,and living and dietary habits were recorded.Anthropometric measurements,including body mass index(BMI)and waist circumference(WC),were made.Hypertension was defined according to the criteria of AHA definition as the presence of at least 1 of the following: 1)self-reported history of hypertension and currently taking any antihypertensive medication(s),and 2)systolic blood pressure (SBP)≥ 140 mm Hg and/or diastolic blood pressure(DBP)≥ 90 mm Hg.The hypertension group in the second and third studies had the following inclusion criteria:1)age ≥ 45 years and ≤ 75 years,and 2)SBP ≥ 160 mm Hg and/or DBP ≥ 90 mm Hg or a history of stage 2 or 3 hypertension.Controls were recruited with normal BP(SBP < 140 mm Hg and DBP < 90 mm Hg)and no personal or family history of hypertension.Exclusion criteria were secondary hypertension,pregnancy,severe renal or liver dysfunction,and cancer.All individuals were of Han Chinese ethnicity and were determined not to be biological relatives within 4 generations.All individuals provided written informed consent.All procedures of this study involving patients were approved by the Ethics Committee of China Medical University.Fasting blood samples were collected between 7:00 am and 9:00 am and were anticoagulated with heparin or EDTA.Heparin anticoagulated samples were used for analyzing lipid profile,liver function,renal function,levels of blood sodium(Na+)and potassium(K+),fasting blood sugar(FBS),and routine blood tests in the Clinical Laboratory of General Hospital of the Fuxin Mining Bureau.Genomic DNA was extracted from EDTA anticoagulated peripheral blood leukocytes using a TIANamp Blood DNA kit,according to the manufacturer’s instructions.The quality and quantity of extracted DNA were validated spectrophotometrically.All DNAs were pooled in several pools and scanned for GWAS on Affymetrix SNP 6.0 microarray platforms.The results were analyzed with R language.Then the associations between CORIN/KIF6 and hypertension were evaluated by genotyping with PCR-HRM.All results were produced in SPSS.Results: After rigorous screening,361 controls(male 158,female 203)and 740 hypertensives(male 476,female 264)were enrolled for the first part of this study.According to blood pressure and age,their DNA were mixed into 14 pools.Epidemiological findings: hypertension weight,waist circumference,body mass index(BMI),low density lipoprotein cholesterol(LDL-C),serum sodium,potassium levels were significantly higher in hypertension group(p < 0.05);high-density lipoprotein cholesterol(HDL-C)was significantly lower in hypertension group than in control group(p = 0.025).But there were no differences between two groups in FBS and total cholesterol.Age and gender were different in two groups for the prevalent selection in younger cases and older control to form obvious genetic base.And gender as male is one of the risk factors for EH.In this study,the relative risk ratio between female and male to EH was 0.432.GWAS results: rs4540261 and rs9625367 were two most highly different SNPs between two groups,with p values of 2.69328*10-6 and 7.22879*10-5 respectively.Meanwhile,genes around the two SNPs were investigsted.Successful validations were done in genotyping KIF6(rs6930913 and rs20456)and CORIN(rs2271037 and rs3749585).As a result,mutant T allele in rs2271037(odds ratio [OR] 1.693,95% confidence [CI] [1.528-1.877],p < 0.001)and C allele in rs3749585(OR 1.114,95% CI [1.011-1.227],p = 0.029)increased the risk of hypertension,comparing with wild G allele and T allele,respectively.Patients with genotype TT(OR 10.209,95% CI [6.414-16.250],p < 0.001)and GT(OR 1.730,95% CI [1.226-2.443],p = 0.002)have higher risk of hypertension than those with genotype GG.SNP rs2271037 was significantly associated with susceptibility to hypertension in all genetic models(dominant model: OR 2.879,95% CI [ 2.080–3.986],p < 0.001;recessive model: OR 7.159,95% CI [4.779-10.724],p < 0.001;additive model: OR 1.535,95% CI [1.163-2.027],p = 0.002).SNP rs3749585 was significantly correlated with hypertension susceptibility only in dominant model(OR 1.533,95% CI [1.073-2.189],p = 0.019),but not in recessive model(OR 1.220,95% CI [0.906-1.644],p = 0.191)or additive model(OR 0.915,95% CI [0.694-1.205],p = 0.527).After adjusting for age,gender,body mass index(BMI),smoking,low-density lipoprotein cholesterol(LDL-C),and serum sodium level in logistic models,the same statistical results were obtained.Interaction study showed the association between CORIN polymorphisms and hypertension could be changed by overweight(BMI ≥ 25 kg/m2).In silico analyses implicated hsa-mi R-495 as a target mi RNA that potentially interacts with the 3′ UTR of CORIN.Mutant T allele(OR 0.733,95% CI [0.664-0.809],p < 0.001)and genotype TT(OR 0.222,95% CI [0.137-0.357],p < 0.001)in rs6930913 of KIF6 were shown to be related to a decreased risk of hypertension.Genotype CC decreased the risk to hypertension(OR 0.630,95% CI [0.447-0.887],p = 0.008),comparing with genotype TT.Under dominant and additive genetic models of rs20456,and dominant and recessive models of rs6930913,the susceptibility of hypertension was significantly changed.Interaction analyses showed 1)modifications of gender on the relationships of hypertension and KIF6 genotypes(TT and CT within rs20456,TT and CC within rs6930913);2)modifications of age on the relationships of hypertension and rs20456 genotypes(TT and CT).Conalusions: 1.SNPMaP is a good method for screening of susceptible genes of EH.We found that KIF6,CHEK2 and PIPNβ genes may be susceptible genes of EH in rural northeast China;2.Polymorphisms of rs2271037 and rs3749585 in CORIN were significantly associated with hypertension in a Han population of northeastern China.The mutant-type T allele of rs2271037 and C allele of rs3749585 might increase the susceptibility to hypertension in this population;3.KIF6 polymorphisms might relate to the susceptibility of hypertension,and genotype TT of rs6930913 and genotype CC of rs20456 were associated with decreased risk of hypertension.The associations could be modified by gender and age;4.The method of GWAS combined with DNA pooling has good positive predictability,but excessively negative results. |
PDF Full Text Request