Correlation Of Single Nucleotide Polymorphisms In EPAS1with High Altitude Polycythemia In Male Han Population

Backgrounds and objectives:When people live at plain enter into high altitude hypoxia environment, red blood cellcan compensatory increase and improve the hypoxemia. However, in some individuals,RBCs present hyperplasia and hemoglobin concentration(Hb) shows a significant increase,eventually leading to high altitude polycythemia(HAPC), which results in increased bloodviscosity, slow blood flow and poor microcirculation, and worsen hypoxemia in humanbody. The clinical characteristics of this disease mainly present as absolutely increasedRBCs and Hb concentration as well as damage of multi-system and multi-organ caused byhypoxemia, especially the performances show in the respiratory system, the cardiovascularsystem and the nervous system.HAPC may occur in both native people at high altitude and plain settlers, while nativepeople of high altitude rarely suffered from this disease, and those who immigrate to highaltitude have a significantly increased incidence. The incidence in males is obviously higherthan that of females, and the incidence shows a significantly positive correlation withaltitude, which means the higher altitude, the higher incidence, and the hematologicalcharacteristics for male is Hb≥210g/L while female is Hb≥190g/L. This disease mainlymanifested as an increase in the number of RBCs, whereas the exact mechanisms have notbeen elucidated, and the regulating mechanisms in physiological characteristics of low Hbconcentration in native Tibetans provide clues for us to explore the pathogenesis of HAPCin immigrants of Han population.Tibetans are considered as the best nation adapts to high altitude environment aroundthe world, and low Hb concentration is one important symbol of adaptation. Compared withthe immigrants Han at the same altitude, the Hb concentration of native Tibetans wassignificantly lower, even immigrants Han who express well acclimatization still show a higher Hb concentration than that of Tibetans at the same altitude, nevertheless, themechanisms that Tibetans show lower Hb concentration have not been elucidated. The rapiddevelopment of high-throughput technologies as gene chip and exons sequencing providemore new technical methods for us to research genetic mechanisms that Tibetans adapt tohigh altitude. Using these advanced technologies and methods, researches about the geneticmechanisms that Tibetans adapt to high altitude have got great progress. Recent researchresults revealed that some sites belong to DNA sequences of EPAS1gene and EGLN1genein native Tibetans at high altitude showed remarkable frequencies differences with Hanpeople at plain, moreover, these sites were markedly associated with low Hb concentrationin native Tibetans at high altitude, and the EPAS1gene got more association. Initial studiesfound that exons mutation of EPAS1would cause excessive erythrocytopoiesis at plainenvironment, indicating that EPAS1gene plays an important role in regulating Hbconcentration. Thus we can infer that EPAS1gene is involved in the pathogenesis of HAPCin Han population. Therefore, in this study, we select single nucleotide polymorphisms(SNPs) in EPAS1gene which show great frequencies differences between Tibetan and Han,and detect the genotypes distribution of SNPs mentioned above in male HAPC patients andhealthy controls among immigrants Han at high altitude region, analyze theirpolymorphisms so as to explore the correlation between SNPs of EPAS1and HAPC in maleHan population.Materials and methodsBetween2009and2011, during the process of survey at areas above sea level4000meters at Qinghai-Tibet plateau, we have acquired318cases of male Han HAPC bycollecting epidemiological data and clinical data as well as detecting Hb concentration ofperipheral venous blood samples of subjects, and we chose316healthy controls matchedwith age, altitude of working place, time spent living at high altitude and birth place.Genomic extraction kit was used to extract DNA from whole blood, finally we selected fiveSNPs of EPAS1(rs13419896, rs1868092, rs4953354, rs6756667, rs7583392) combinedwith Tibetan data published and CHB data from HapMap. Genotyping was finished by themethod of polymerase chain reaction-high resolution melting (PCR-HRM) and10%of genotyping results were randomly selected to DNA sequencing to test the accuracy ofgenotyping results after genotyping. Chi-square test was used to analyze distributions ofgenotypes and alleles of five SNPs mentioned above between cases and controls,non-conditional logistic regression was applied to analyze the risk of each genotype, andcompared with the published data of Tibetan and HapMap database.Results1. The genotypes distribution of rs7583392in controls did not match theHardy-Weinberg Equilibrium (HWE), so the data of rs7583392was excluded in furtheranalysis.2. The distributions of genotypes and alleles in rs13419896, rs1868092and rs4953354between male HAPC and healthy controls in Han population did not show significantdifference.3. A significant difference in the genotypes distribution of rs6756667was foundbetween male HAPC and healthy controls in Han population (p=0.049), and allelesdistribution showed a remarkable difference between male HAPC and healthy controls inHan population (p=0.01). The frequency of GG genotype in male Han HAPC was higherthan that in controls, and distribution of AG and GG genotypes expressed significantdifference(p=0.025).Taking G allele as a reference, the A allele was a protective factor formale HAPC in Han population (OR=0.65,95%CI=0.46-0.91).4. Non-conditional logistic regression indicated that when taking GG genotype as areference in rs6756667, the AG genotype decrease the risk of male HAPC in Hanpopulation(OR=0.64,95%CI=0.43-0.95), and combined AA genotype and AG genotypetogether would decrease risk of male HAPC in Han population(OR=0.62,95%CI=0.43-0.91). When taking the AA genotype as a reference in rs13419896, rs1868092andrs6756667, the AG and GG genotypes did not increase the risk of HAPC; Taking GGgenotype as a reference in rs4953354, the AG and AA genotype did not increase the risk ofHAPC.5. The distribution of haplotype of G-G-G constructed by rs13419896, rs1868092andrs4953354showed a significant difference between HAPC and controls, and this haplotype was a risk factor for male HAPC in Han population (p=0.003).6. For the first time, genotypes of AA in rs1868092and GG in rs4953354not reportedin CHB in HapMap database were detected in this study.ConclusionGene susceptibility locis of Han HAPC were found in EPAS1gene, the SNP siters6756667of EPAS1gene was associated with HAPC in male Han population, the A alleleof rs6756667was a protective factor for HAPC; Haplotype of G-G-G constructed byrs13419896, rs1868092and rs4953354was a risk factor for HAPC in male Han population,indicating that EPAS1gene is associated with the occurrence of HAPC in Han population.