Association Study Between αENaC And β₂-AR Genes Polymorphisms And Hypertension In Kazak And Uygur Populations In Xinjiang

Objective:Essential hypertension is the most common cardiovascular diseases that threaten human health and may lead to a series severe results such as cardiac and renal failure and shock.Hypertension is a complex disease resulted from the interaction of the cumulative effect of multiple genetic and environmental factors and genetic factors play an important role.Along with the completion of human genome project,it has been a key aim to study the sequence variations that correlate with certain diseases.As the third generation of genetic markers,Single Nucleotide Polymorphism(SNP)is characterized as large number and wide distribution.SNP or haplotype that are related to diseases may be obtained through systematically identifying SNPs especially those in coding and regulating region and the case-control study.This will play an essential role in searching the genetic mechanisms of some complex polygenic diseases.We investigated three epidemiology research in the pastoral area of balikun county and the rural area of Tuluafan to report the epidemiological characteristics of hypertension and assessment of the risk factors relevant to the high prevalence of hypertension.And we referring to the Chinese data in HapMap project and study SNPs of Epithelium Natrium Channel geneαsubunit(αENaC)andβ₂-adrenergic receptor gene(β₂-AR)that are important candidate genes of essential hypertension in Kazak and Uygur populations in Xinjiang province, Systematically screen for and examine SNPs in two ethnic groups and the pattern of intragenic linkage disequilibrium and the haplotype structure were estimated,Then investigate the relation between these polymorphisms and haplotypes and hypertension; Compare with every SNP genotype and allele gene frequency and distribution in two ethnic groups,we sought to demonstrate effect of candidate genes in hypertension pathogenesis from genetic ways and further search the mechanisms in more polulations. Methods:(1)Three epidemiology research was carried out in a natural population by cluster sampling and collected blood samples of 1141 kazak subjects who come from the pastoral area of balikun county,including 703 patients and 438 controls subjects,894 Uygur subjects who come from the rural area of Tuluafan,including 424 patients and 470 controls subjects.(2)All the sequence variants located promoter,exon,intron regions ofαENaC gene were identified by directly sequencing of PCR product derived from 40 randomly selected samples.The pattern of pairwise LD between the common(minor allele frequency＞5%)SNPs was measured by D¹ andγ²,and the tSNP were selected according to Haploview software and the papers;All tSNPs ofαENaC gene were futher genotyped in all subjects by TaqMan system and polymerase chain reaction -restriction fragment length polymorphism(PCR-RFLP).(3)On the basic of previous research,We chose theβ₂-AR gene to study the two high frequency polymorphisms Arg16Gly and Gln27Glu located encode region,then the TaqMan assay is used for the detectionβ₂-AR genotype.(4)The case-control study in a large group was carried out separately for each of the tSNP and followed up by haplotype analyses to determine the relation between SNPs and hypertension in two ethnic groups in Xinjiang and investigate the distribution of candidate genes polymorphisms in two ethnic groups.(5)Spss software version 13.0 for windows was used to analyze the statistics data, association for dichotomous variables and the Hardy-Weinberg equilibrium for the polymorphisms were tested by chi-square analysis.Association for 2 groups was tested by Student' s t test for continuous variables,Analysis of covariance(ANCOVA)was used to ajuSted mean;Ajusted Odds ratios(ORs)with 95%confidence intervals from logistic regression analysis was used to estimate the relative risk of hypertension associated with genotype:linkage disequilibrium(LD)analysis was carried out in a pair-wise fashion by SHESsis software online,haplotype block was determined by haploview software and haplotypes for HT and NT were inferred by the statistical software package PHASE version 2.0.Results:(1)Epidemiology research indicate:The prevalence of hypertension is 61.6%and 47.4%respectively in Kazak and Uygur polulations,the average levels of systolic blood pressure and diastolic blood pressure in Kazak is significantly higher than those in Uygur and the prevalence of hypertension increases with age increasing in both populations, body mass index(BMI),serum total cholesterol(TC),triglycerides(TG)and the rate of cigarette-smoking in the male in EH group are significantly higher than NT group.(2)In a total length of 7 kb ofαENaC gene explored,we identified 9 SNPs named G(-947)A,G(-321)A,A(+6)G,G(+49)C,C(+866)T,A334T,C(+20844)T,A(+26622)G and T663A that located promoter,exon2,6,13 and intron 1,7,11,All 9 polymorphisms were previously reported in the dbSNP at NCBI.(3)From 3 tSNPs selected from 9 polymorphsims we can predict common haplotype and other SNPs.They are G(-947)A that located promoter,A(334)T located exon 6 and A(663)T located exon 13,then all the genotypes of 3 SNPs were detected.(4)The genotypes of theαENaC gene variants were found to be in Hardy-Weinberg equilibrium in two ethnic groups;Single-locus association study indicated:Frequencies of genotype and allele of 3 SNPs in EH group were not significantly different from NT group respectively in Kazak and uygur populations(P＞0.05).(5)Pairwise LD was carried out for SNP respectively,Only G(-947)A and G(+49)C,A(+26622)G and T663A polymorphisms were in LD and most of the pairwise LD values of the polymorphisms were in weak or no disequilibrium.Hyplotype based association study in the two populations indicate:8 haplotypes(H1～8)was reconsreucted by 3 tSNPs and H2,H3,H6 and H7 haplotype frequency in EH is different from HT group in Kazak population and most haplotypes frequency except for H1 and H3 in EH is also different from HT group in uygur population.(6)Compare with hylotype frequency distribution in two ethnic and there were significantly different in Kazak and uygur populations(P＜0.05).(7)β₂-AR gene Arg16Gly and Gln27Glu genotype are detected by TaqMan assay in arge cohort,The genotypes of theβ₂-AR gene variants were found to be in Hardy-Weinberg equilibrium in two ethnic groups,We did not observe any prevalent genotypes of theαENaC polymorphisms between controls and hypertension patients in two ethnic groups(P＞0.05).(8)Pairwise LD was carried out for SNP,D¹ = 0.8 and 0.7 in Kazak and uygur populations respectively and two loci were in weak disequilibrium;Four common haplotypes were inferred after haplotype reconsreuction and haplotype frequencies has no statistic significant in case and in control in the two ethnic groups(P＞0.05). Conclusions:(1)The prevalence of hypertension in kazak is higher than this in Uygur,the average levels of systolic blood pressure and diastolic blood pressure is significantly different between Kazak and Uygur groups,and the group of high blood pressure is associated with higher levels of serum TC,TG,BMI and rate-smoking.(2)Our results suggest that single polymorphism of theαENaC gene might be not associated with hypertension in Kazak and Uygur populations in Xinjiang,In multilocus haplotype analysis,theαENaC gene haplotype profile is significantly different between cases and controls in both ethnic groups,because multilocus haplotype analysis has more detection power than single polymorphism locus,we speculate there is ethnic-specific disease predisposing or protecting haplotypes in the hypertensives in Kazak and Uyghur populations and our data support the presence of gene-dose effect for some haplotypes but not the single polymorphism locus.(3)Frequecy of haplotype is significantly different in Kazak and uygur populations, we indicateαENaC gene polymorphisms have ethnic specificity and maybe association with EH trait in two populations,(4)Both single polymorphism locus of theβ₂-AR gene and haplotype that are reconstructed by SNPs might be also not associated with hypertension in Kazak and Uygur populations in Xinjiang.