Genetic Basis Of Essential Hypertension In Tibetan

PartⅠBackground:Human essential hypertension is a multi-factorial trait and thought to be resulted from the interaction of genetic and environment factors. Components of the renin-angiotensin system have been repeatedly investigated as a cardinal source of candidate genes for essential hypertension. In particular, suggestive or significant association has been detected in some studies between the angiotensinogen(AGT), angiotensinⅠconverting enzyme(ACE), and CYP11B2 gene with hypertension. Although variants in these genes have shown association with hypertension in some studies, these associations are often not reproducible in studies of other populations. Based on this, reproduction of the finding in a different ethnic population is considered to be important.Methods:In this context, we here conducted the case-control study to explore the association between multigenic polymorphisms and the development of EH in Tibetan population from Lhasa. In this study, we analyzed 7 gene polymorphisms evenly interspersed in 3 candidate genes of RAAS. Using polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) method, polymerase chain reaction based single strand conformation polymorphism (PCR-SSCP) method, and polymerase chain reaction products direct sequencing method, we systematically screened polymorphisms and genotyped. Because our genetic data were multi locus, the interactions between these polymorphisms should be evaluated and addressed. Traditionally, one approach to modeling gene-gene interactions in complex diseases is parametric-based logistic regression. The MDR method has been successfully applied to detecting gene-gene and gene-environment interactions for a variety of clinical endpoints. Accordingly, we have adopted the MDR method in the present study aiming to explore the synergistic effects of these polymorphisms on the development of EH in Tibetan population.Results:Singe-locus analysis:In the RAAS, single-locus analysis showed that the differences of CYP11B2 C-344T locus genotypes and alleles frequency distributions between Essential Hypertension and healthy.controls were statistically significant in Tibetan population (P=0.002和P＜0.01) and CC genotype is more susceptibile to hypertension than TT genotype;Linkage Disequilibrium (LD) Analysis:The LD analysis constructed a linkage block between ACE I/D and A2350G polymorphisms, while the linkage is not complete. Gene-Gene Interaction Analysis and Haplotype Analysis:The gene-gene interaction analysis indicated the overall best model including I/D and A2350G polymorphisms with strong synergistic effects. Haplotypes D-A (in order of I/D and A2350G polymorphisms) conferred high genetic susceptibility to EH (9.2% vs.2.1%, P＜0.001)Conclusions:.The gene polymorphism study showed that CYP11B2 gene-344C/T polymorphism was significantly associated with essential hypertension in Tibetan population, and C allele is more susceptibile to hypertension than T allele. The gene-gene interaction analysis indicated haplotypes I-G (in order of I/D and A2350G polymorphisms) is associated with essential hypertension in Tibetan population, and it conferre high genetic susceptibility to EH.Part IIBackground:In order to explain the results of population-based genetic study, we constructed the expression plasmid containing the luciferase reporter gene inserted with different alleles of CYP11B2 gene C-344T locus. To study whether the certain polymorphisms in the regulation region alter CYP11B2 gene transcription, the above plasmids and an intra-control were cotransfected into 293T cell by eukaryote gene transfection techniques.Methods:The insertion fragment contained different alleles of CYP11B2 gene C-344T locus were obtained by PCR amplification from genome DNA and subcloned in the pMD-18T vector. The reporter expression plasmids were constructed by ligating the cohesive-end fragment produced by restriction endonuclease Kpn I and Xho I in pGL3-Basic/Enhancer vector and subcloned pMD-18T vector. To study whether certain polymorphisms in the regulation region alter CYP11B2 gene transcription, the above plasmids and pRL-TK as an intra-control were cotransfected into 293T cell by eukaryote gene transfection techniques.Results:The results of transfecting human 293T cell in proliferation phase showed that the transcriptional activity had significant differences between C allele and T allele, and the transcriptional activity of the reporter expression plasmids with C allele was significantly higher than that of the reporter expression plasmids with T allele. Conclusions:we studied the function of relevant allele based on the results of association analysis; the results showed that the polymorphisms of the CYP11B2 gene C-344T locus might be involved in the regulation of transcription of the gene.PartⅢBackground:Genomewide association study (GWAS) is a novel strategy for discovering genetic basis of human complex diseases, through using millions of single nucleotide polymorphism(SNPs) as marks to conduct case-control association studies. In recent years,following the implementation of Human Genome Project and development of Genome Chips, large number of human complex diseases associated genetic variants has been identified through GWAS method,which provides important clues for understanding the mechanisms of related diseases. Attempts by researchers to identify genetic variants associated with hypertension have been challenging and of relatively low yield.Methods:Using a novel approach we call SNP-MaP (SNP Microarrays and Pooling), we conducted a GWAS to explore the association between multigenic polymorphisms and the development of EH in 200 patients with essential hypertension and 200 health controls in Tibetan population from Lhasa. In this study, equimolar amounts of DNA from patients or controls were pooled and genotyped using an Affymetrix GeneChip(?) Array 6.0. After detected the SNP locus, find the SNP frequencies in 300 patients and 300 controls respectively by sequencing or PCR-RFLP. The relationship between SNPs and EH was analyzed.Results:5 sequence tagged sites met the genomic criteria of P＜9.2×10^-8 after Bonferroni correction among whole-genome genetic markers. One SNP locus (rs9865108) on chromosome 3 were located within 20 kb of the gene SUCNR1 and rs17136827 on chromosome 10 were located in the intron 1 of the gene CAMKID. While no gene was found within 50 kb region of the SNPs(rs1866525, rs12541835 and rs4547758). The differences of genotypes and alleles frequency distributions of the 5 SNP locus was detected by sequencing or PCR-RFLP. The results show that differences of rs17136827 and rs1866525 genotypes and alleles frequency distributions between essential hypertension and healthy controls were not statistically significant, and differences of rs9865108, rs12541835 and rs4547758 genotypes and alleles frequency distributions between essential hypertension and healthy controls were statistically significant in population.Conclusions:Our results demonstrate that Affymetrix Human SNP Microarrays and Pooling provide a very effective platform for genome-wide analysis of SNPs which are susceptible to essential hypertension. Polymorphisms of SNPs(rs9865108, rs12541835 and rs4547758) are associated with essential hypertension in Tibetan population.