延边地区朝鲜族和汉族高血压遗传与环境危险因素的研究(遗传学报2).doc

Investigation of genetic and environmental risk factors on Essential hypertension in Yanbian Korean and Han Chinese populationsZibo Zhang , Kangjuan Yang , Tianxin Sheng , Liangwei Xu , Ping Hao , Yuping Wang , Fanping Meng , Yuanzhe JinDepartment of Cell Biology and Medical Genetics, Yanbian University College of Basic Medicine, Yanji 133000, Jilin, ChinaAbstract This study was designed to analyze the genetic and environment risk factors on essential hypertension (EH) in Yanbian Korean- and Han-Chinese populations. We enrolled a total of 488 subjects from Yanbian, including 240 Korean-Chinese (112 EH patients and 128 healthy individuals) and 248 Han Chinese (108 EH patients and 140 healthy individuals). More than 20 nature and environment factors were investigated and 10 clinical biochemical indicators were measured. we also researched their genealogy. PCR and sequencing were used for screening of 5 single nucleotide polymorphisms (SNPs) (-11426AG、-11391GA、-11377CG、-11156insCA、-11043CT）of the promoter of adiponectin gene. Multiple factor logistic regression , ANOVA , 2 test were used for analysis of data. The results indicated that hypertension family history ,body mass index (BMI),drinking , intake of Salt, the level of plasma adiponectin had significantly associated with EH in Yanbian Korean -Chinese and Han- Chinese populations（P0.001）. The frequency of G allele -11426AG in EH group （19.91%）was obviously higher than in normal group（12.50%）in Yanbian Han- Chinese（P=0.025）. The frequency of haplotype -11426G -11377C in EH was higher than in normal group in Yanbian Han- Chinese（P=0.031）. The SNPs of -11426A and -11156N (no insert of CA) or -11426G and -11156I (an insert of CA) were always exist together with the same chromosome. There is a perfect linkage disequilibrium (LD) between -11426AG and -11156insCA in Yanbian Korean- and Han-Chinese populations. The heritability of hypertension in Yanbian Korean- and Han- Chinese populations were 62.60% and 60.60%（P0.05）. The prevalence rate of the EH first- degree relative was significantly higher than in normal group in both Yanbian Korean- and Han- Chinese populations. So the genetic factor is important in the mechanism of EH. These results indicated that hypertension family medical history ,overweigh or obesity, drinking , high salt diet, lower plasma adiponectin were the major risk factors of EH in Yanbian Korean- and Han-Chinese populations. The SNP of -11426AG might be associated with EH in Yanbian Han- Chinese. The allele -11426G and haplotype -11426G -11377C of the promoter of adiponectin gene were important risk factors with EH in Yanbian Han-Chinese populations. Prevent measure should be constituted to the people who have risk factors of EH.Keywords: essential hypertension; risk factors; polymorphisms of adiponectin gene; Yanbian Korean and Han Chinese populationsIntroductionEssential hypertension (EH) is a kind of syndrome being characterized with increasing systemic arterial pressure. Long term hypertension could be an important risk factor in various cardiovascular diseases and be harmful to life and health. The prevalence rate of EH is increasing year by year all of the world. Its mechanism is complicated and unknown up to now. Presently, a majority of scholars have thought EH was caused by coactions and/or interaction of genetic and environmental factors. But the level and pattern of the genetic factor in EH is unknown. And the reports about EH are various with different nationalities, ethnicities and regions. Recently, adiponectin gene is recognized as candidate gene, which is for scholars detecting mechanism of obesity(Panagopoulou P,2009), diabetes(Foucan L,2009; Mohammadzadeh G,2009; Hwang JY,2010), cardiovascular diseases(Chang YC,2009), hypertension(Iwashima Y,2004) and the like. According to the samples survey about EH in 1991, the prevalence rate of EH in Korean-Chinese populations was 22.95%（Cheng-bin ZHU,2005）, taking the first place in China. A survey in 2006 (Lan CUI, 2006)had showed that in Yanbian the prevalence rates of EH of males in Korean and Han-Chinese populations were 53.8% and 39% respectively, and of females were 44.9% and 31.4% respectively, obviously higher than before 15 years. So we will carry out an epidemiological and genetic investigation in Yanbian Korean and Han-Chinese populations, Investigate the genetic and environment risk factors , the heritability, The prevalence rate of the first degree relative of EH, providing reliable materials for prevention and interfering of EH in Yanbian Korean and Han Chinese populations.Materials and methodsStudy subjects According to the principle of knowing fact and freewill, a total of 220 patients were involved in this study, who had been diagnosed as EH in Yanbian University Hospital and other hospitals in Yanbian from 2007 to 2009, including 112 Korean-Chinese and 108 Han-Chinese( ages from 40 to 70 years old, 120 men and 100 women). The control group involved 268 healthy individuals matching with case group in age, sex, nationality and so on, including 118 Korean-Chinese and 140 Han-Chinese. MethodsAfter system training, investigators including teachers, doctors, graduates, questioned 20 surplus environment factors by the unified table, and the disease condition of probands and their first degree relatives including parents, brethren and children . We measured blood pressure of all the subjects , three constant times and 10 minutes interval at each two times, then calculated their means. We also measured their body height, weight, waist-to-hip circumference ratio(WHR) ,body fat rate et.al, and calculated body mass index (BMI). All individuals were taken blood with empty stomach in the morning for biochemical test and extracting genomic DNA. Diagnosis criteriaAll EH patients were diagnosed by , systolic blood pressure (SBP) 140 mmHg and diastolic blood pressure (DBP) 90 mmHg. According to the criteria of WHO, body mass index (BMI) 25 could diagnose as overweigh and BMI 28 could diagnose as obesity. Criteria of abdominal obesity: waist-to-hip circumference ratio (WHR) in men 0.9 and WHR in women 0.85. Drinking definition was that drinking 50 grams or more of distillate spirit (or the same alcoholic amount of beer) average per day , and continuing 1 year or more. 1 or more cigarette average per day and continuing 1 year or more was definited smoking. The amount of salt-eating exceeded 8 grams was definited high salt diet. The correlation factors and evaluate of EH (see table 1).Extraction of genomic DNA Genomic DNA was extracted with Axyprep DNA Blood Mini Kit ( Biao Biotech-nology company limited provided ,Changchun). Its purity and density was tested by DNA/RNA protein determinator .PCR amplificationPCR amplification was performed on GeneAmp PCR System 9700(Applied Biosystems Corp., Foster City, USA). The primers are F: 5-GCACCTGACCTGAAGTTTAT -3and R: 5- TGACCTGGACCCTGGATTTA -3(SHENG Tian-xin,2009),which are synthesized by Shanghai Shensu Biological Engineering Technology & Services Co., Ltd (Shanghai, China).Reaction system of 25L involves 2.5L of genomic DNA, 2.5L of dNTPs (2.5mM),2.5L of 10Buffer I(with Mg2+),0.5L of forward primer(12.5M),0.5L of reverse primer(12.5M),0.5L of TansTaq HiFi DNA polymerase (Beijing TransGen Biotech Co., Ltd., Beijing, China),and 16L of ddH2O.The reaction program is: initial denaturation at 94 for 5 min; denaturation at 94 for 45 sec, annealing at 57 for 45 sec, polymerization at 72 for 45 sec ,35 cycles; final polymerization at 72 for 5 min. Sequencing and Screening for SNPsAll amplification products were sequenced on Applied Biosystems 3730xl DNA Analyzer(Life Technologies Corporation, Foster City, USA)by Beijing Genomics Institute (Beijing, China). SNPs were screened with Sequencher 4.8 Demo(Gene Codes Corp., Ann Arbor, USA) and Chromas Lite 2.01 (Technelysium Pty., Ltd., Tewantin, Australia).Statistical analysisMultiple factor logistic regression, MeanSD，ANOVA, 2 test were used for risk factors of EH and treatment of clinical data of subjects with SPSS 17.0 for Windows(SPSS Inc., Chicago, USA).Hardy-Weinberg equilibrium was determined with Hardy-Weinberg equilibrium calculator (.uk/software/hardy-weiberg.shtml). Linkage disequilibrium and haplotype frequencies were analysis with SHEsis (/my Analysis.php).ResultsThe Analysis result of multiple factor logistic regression for EH (See table 2)Multivariate Logistic regression analysis showed that family medical history, drinking, high salt diet were statistically significant factors on EH.Clinical data of EH and control group were calculated (See table 3)The level of SBP, DBP, BMI, Body fat rate in EH group were obviously higher than that in normal group in both Yanbian Korean and Han Chinese populations（P0.001）. The level of plasma adiponectin in EH group was lower than that in normal group in Yanbian（P0.001）. These results indicated that BMI, Body fat rate, plasma adiponectin were relative to EH .Other clinical data were analyzed .But no any statistical difference between EH and normal group or between Yanbian Korean and Han Chinese had been found.The results and analysis of PCR and SNPsThe amplification product is a DNA fragment of 993 base pair by 1.5% agarose gel- electrophoresis. There are -11426AG(rs16861194),-11391GA(rs17300539),-11377CG(rs266729),-11156insCA,-11043CT of the promoter of adiponectin gene .But only SNPs of-11426AG , -11377CG,-11156insCA were found in Yanbian Korean and Han Chinese populations. Of them, the SNPs of -11426A and -11156N(no insert of CA) or -11426G and -11156I(an insert of CA) were always exist together within the same chromosome. -11391GA and -11043CT were rare SNP in Yanbian .So it was enough to just examine the SNPs of -11426AG and -11377CG.(See table 4).The genotypes and alleles with the promoter of adiponectin gene in Yanbian Korean and Han Chinese populations were analyzed. The frequency of allele -11426G in EH was greater than in normal group in Yanbian Han Chinese（P=0.025）. The SNPs of -11426AG might be associated with EH in Yanbian Han Chinese. The allele -11426G of the promoter of adiponectin gene was a risk gene with EH in Yanbian Han Chinese populations. The genotypes and alleles of -11377CG was not any statistical difference between EH and normal group or between Yanbian Korean and Han Chinese populations.Analysis of haplotypes The SNPs of -11426AG and-11377CG were determined to be in complete linkage disequilibrium (D0.85)and in Hardy-Weinberg equilibrium(P0.05).The haplotypes of the SNPs of -11426AG and-11377CG could be examined(See table 5).A haplotype of -11426G-11377C was discovered has statistical difference between EH and normal in Yanbian Han Chinese. The frequency of haplotype of -11426G-11377C in EH group was higher than in normal group in Yanbian Han Chinese(P=0.031).Relationship between genotypes of -11426AG and clinical data（See table 6）No any statistical difference between different genotype and clinical data in Yanbian Han Chinese had been found.Estimation of heritability of Yanbian Korean and Han Chinese (See table 7)From Table7, the total heritability of EH in Yanbian was 61.40%, including 62.60% in Korean-Chinese and 60.60% in Han-Chinese. So the genetic factor is important in the mechanism of EH. There was not statistical difference of EH heritability between Korean and Han-Chinese populations.Comparing of prevalence rate of EH fist degree relatives(See table 8) The prevalence rate of first degree relatives in case group was statistically higher than in control group(P 0.001) in both Yanbian Korean and Han Chinese populations. There was not statistically difference between Yanbian Korean and Han Chinese. Table 1Correlation factor and evaluate of EH Correlation factor evaluate sex man=1，woman=2nation Korea=1，Han=2educational background below middle school=1，above middle school=2income 1000=2smoking no=0，yes=1drinking no=0，yes=1stress incident no=0，yes=1life tension no=0，yes=1WHR 0.85=0,mam0.9=1，woman0.85=1intake of Salt 8=0，8=1intake of Meat 1-2order/week=0，3order/week=1intake of Hot same as aboveintake of Vegetables same as aboveintake of Egg same as aboveintake of Sweet same as aboveintake of Fish same as aboveintake of Milk same as aboveeHypertension FMH no=0，yes=1DM history no=0，yes=1Hyperlipemia history no=0，yes=1Heart disease history no=0，yes=1Cerebrovascular disease history no=0，yes=1 Table 2Analysis result of multiple factor logistic regression for Essential hypertension in Yanbian Korean and Han Chinese populations EXP(B) 95% Variable S.E. Wald P OR Lower Upper Drinking 1.246 0.559 4.969 0.026 3.477 1.162 10.400Salt intake 1.138 0.476 5.723 0.017 3.119 1.228 7.923Hypertension FMH 2.660 1.263 4.436 0.035 14.302 1.203 170.028 Table 3Clinical Characteristics of the study population Yanbian Korean Chinese Yanbian Han Chinese hypertension healthy group P hypertension healthy group PAge(years) 53.9210.49 49.8410.32 0.276 52.399.60 49.2510.84 0.172SBP(mmHg) 148.5416.51 117.3912.77 0.001 * 155.4017.63 118.6210.41 0.001*DBP(mmHg) 94.1310.02 77.767.13 0.001* 92.289.32 78.287.53 0.001*BMI(kg/m2) 25.593.32 22.685.24 0.001* 25.923.10 23.073.77 0.001*Body fat (%) 33.696.10 29.196.69 0.001* 32.407.13 29.336.67 0.002*FPG(mmol/L) 5.812.01 5.281.48 0.061 5.832.10 5.482.02 0.054 TC(mmol/L) 5.281.04 4.911.03 0.080 5.091.27 4.880.91 0.055TG(mmol/L) 2.581.97 2.211.34 0.183 2.301.51 1.981.73 0.143HDL-C (mmol/L) 1.520.33 1.450.31 0.162 1.420.43 1.450.27 0.588LDL-C (mmol/L) 2.860.77 2.640.68 0.173 2.860.91 2.420.65 0.162Insulin secretion(ng/mL) 21.1313.31 19.4610.75 0.712 22.2716.28 20.0214.10 0.053APM-1(g/mL) 2.643.39 5.014.60 G AA AG GG A G AA AG GG A GHypertension 76(67.85) 33(