WILDERNESS & ENVIRONMENTAL MEDICINE, ], ]]]–]]] (2015)ORIGINAL RESEARCHEPAS1 Gene Polymorphisms Are Associated With High Altitude Polycythemia in Tibetans at the Qinghai-Tibetan Jin Xu, MD; Ying-Zhong Yang, PhD; Feng Tang, PhD; Qin Ga, MD; Wuren Tana, MD; Ri-Li Ge, MD, PhDFrom the Research Center for High Altitude Medical Sciences, Qinghai University School of Medicine, Qinghai, China (Drs Xu, Yang, Tang, Ga, Tana, and Ge); and the Department of Clinical Medicine, Qinghai University School of Medicine, Qinghai, China (Dr Xu).Objective.—To test the hypothesis that the polymorphisms in the EPAS1 gene are associated withthe susceptibility to high altitude polycythemia (HAPC) in Tibetans at the Qinghai-Tibetan Plateau.Methods.—We enrolled 63 Tibetan HAPC patients and 131 matched healthy Tibetans as a control group, from the Yushu area in Qinghai where the altitude is greater than 3500 m. Eight single- nucleotide polymorphisms (SNPs) of the EPAS1 gene, including rs12619696, rs13420857, rs2881504, rs4953388, rs13419896, rs4953354, rs10187368, and rs7587138, were genotyped by the Sequenom MassARRAY SNP assay.Results.—The frequencies of the G allele of EPAS1 SNP rs13419896rs13419896 were significantly higher in the HAPC group than in the control group (P o .05). Moreover, the A alleles of rs12619696 and rs4953354 were prevalent in the HAPC group, and their counterpart homozygotes were prevalent in the normal Tibetan group (P o .05).Conclusions.—Compared with normal Tibetans, Tibetans with HAPC are maladapted and have adifferent haplotype in EPAS1 SNPs rs12619696, rs13419896, and rs4953354.Key words: EPAS1, polymorphism, high altitude polycythemia, TibetansIntroductionIncreased numbers of circulating erythrocytes develop in high altitude dwellers to compensate for the hypoxia associated with high altitude. This phenomenon is termed high altitude polycythemia (HAPC), which is characterized by excessive erythrocytosis (males, hemo- globin [Hb] Z 21 g/dL; females, Hb Z 19 g/dL). HAPC is prevalent in 5% to 18% of the population on the Qinghai-Tibetan Plateau.1,2 HAPC leads to significant increase in blood viscosity, microcirculation disturbance, or even extensive organ damage.3,4 Although hypobaric hypoxia is likely to be a cause of HAPC at high altitude, the precise mechanisms underlying the pathogenesis of HAPC are not well understood.The Tibetans on the Qinghai-Tibetan plateau live permanently at an altitude up to 3000 to 4500 m; they adaptations Corresponding author: Ri-Li Ge, MD, PhD, Department of Internal Medicine, Research Center for High Altitude Medical Sciences, Qinghai University School of Medicine, 16 Kunlun Road, Xining 810001, Qinghai, China (e-mail: geriligao@hotmail.com).environment, as indicated by lower hemoglobin levels, lower hematocrits, higher oxygen saturation of blood in infants, and high work performance.5–7 However, some Tibetans living at high altitude still show elevated hemoglobin concentration and even exhibit HAPC.8–11 Growing evidence suggests that the hypoxia-inducible factor (HIF) oxygen-signaling pathway plays an impor- tant role in the adaptation of Tibetans.12–17 The human EPAS1 gene is located on chromosome 2p21–p16 and encodes the oxygen-sensitive alpha subunit of HIF-2, which is a key regulator of chronic hypoxia by regulat- ing a large number of genes involved in the cellular and systemic responses to hypoxia. These responses include erythropoiesis, iron homeostasis, pulmonary hyperten- sion and remodeling, vascular permeability, and lung and placental development.18 Our recent study showed in the EPAS1 gene are associated with the susceptibility to high altitude in the Han Chinese.19 pulmonary edema (HAPE) association of EPAS1 gene poly- morphisms with HAPC in the Tibetan population remains unclear.the polymorphismsrs4953354,rs13419896,Urs13419896,Up to now, 8 EPAS1 single-nucleotide polymorphisms (SNPs), including rs12619696, rs13420857, rs2881504, rs4953388, rs10187368, and rs7587138, have been shown to be related to the adapta- tion to high altitude.13,20,21 To explore the potential role of EPAS1 polymorphisms in the pathogenesis of HAPC in Tibetans, we examined these 8 SNPs of the EPAS1 gene in 63 subjects with HAPC and 129 healthy controls, all Tibetans from the Yushu area in Qinghai province, where the altitude is greater than 3500 m above sea level.A total of 63 patients with HAPC (mean age, 45.51 (cid:1) 10.07 years) and 131 control subjects (mean age, 45.14 (cid:1) 11.78 years) participated in this study. All participants lived in the Yushu area in southwest Qinghai province (altitude 3760 m), and they were all Kangba Tibetans. All HAPC patients were diagnosed at Yushu People’s Hospital between March 2011 and June 2013. The inclusion criteria were 1) an Hb concentration of at least 21 g/dL for men and at least 19 g/dL for women, and 2) that HAPC patients were local Tibetans normally living at 3600 to 4400 m. Patients with other diseases having similar clinical manifestations were excluded. Healthy Tibetans matching the patients in age, sex, and working conditions were randomly selected from a physical exami- nation at an outpatient clinic to serve as control subjects. None of the participants had a history of respiratory or cardiovascular disease, such as chronic obstructive pulmo- nary disease, pulmonary infection, asthma, shunt, valvular disease, congenital heart disease, or hypertensive heart disease. The research protocol was approved by the ethics committee at the Qinghai University School of Medicine (Xining, China). All participants in this study signed informed consent. Hemoglobin concentration and hematocrit (HCT) were determined from venous blood samples using the Mindray Hematology Analyzer (BC-2300; Mindray, Shenzhen, China), and oxygen saturation (SaO2) levels were tested by pulse oximeter (Ohmeda 3700 Pulse Oximeter; Datex-Ohmeda, Boulder, CO, USA).DNA EXTRACTION AND GENOTYPING ASSAYSGenomic DNA was extracted from venous blood by Gentra Puregene Blood Kit (Qiagen, Hilden, Germany) according to standard procedures. All selected SNPs were genotyped by the Sequenom MassARRAY SNP assay (Capital Bio Corporation, Beijing, China). SNP loci–tested polymerase chain reaction (PCR) primers and single base extension primers were designed by using the Sequenom MassAR- RAY Assay Design Genotyping Software and Tools(Sequenom, San Diego, CA, USA). The PCR reaction was performed under the following thermal cycling con- ditions: 941C for 4 minutes, then 941C for 20 seconds, 561C for 30 seconds, and 721C for 1 minute for 45 cycles, and 721C for 4 minutes. PCR products were treated with shrimp alkaline phosphataseshrimp alkaline phosphatase to remove free deoxyribonucleoside triphosphates, and single base extension reaction was performed, which consisted of 2.0 mL of EXTEND MIX, 0.619 mL of ddH2O, 0.94 mL of Extend primer mix, 0.2 mL of iPLEX buffer plus, 0.2 mL of iPLEX terminator, and 0.041 mL of iPLEX enzyme (Sequenom, San Diego, CA, USA). The thermal cycling conditions were as follows: 941C for 30 seconds, then 941C for 5 seconds, 521C for 5 seconds, and 801C for 5 seconds for 40 cycles, and 721C for 3 minutes. The MassARRAY Nanodispenser RS1000 (Capital Bio Corporation, Beijing, China) was used for dispensing the purified extension products onto a 384-element Spec- troCHIP bioarray (Sequenom, San Diego, CA, USA), and mass spectrometric analysis was performed using the MALDI-TOF (matrix-assisted laser desorption/ionization– time of flight) (Sequenom, San Diego, CA, USA). The results were analyzed using TYPER 4.0 software (Seque- nom, San Diego, CA, USA).STATISTICAL ANALYSISSPSS software (version 17.0; SPSS, Inc, Chicago, IL, USA) was used for statistical analysis. Allele frequencies were calculated based on genotype frequencies in HAPC and control groups, and the intergroup difference was estimated with the χ2 test. A probability value of less than .05 was considered significant. Deviations from the Hardy-Weinberg equilibrium (HWE) were assessed by processing the χ2 test for genotype frequency. Population genetic data were analyzed using Arlequin (version (http://cmpg.unibe.ch/software/arle quin35/). Genetic distances were used to measure the divergence between study groups. The genetic distance ranged between 0 and 1, where 1 was complete divergence and 0 indicated no divergence. The smaller the genetic distance, the closer the genetic relationship. A variable used in association with genetic distance to express the proportion of genetic diversity attributable to genotype frequency differences among study groups is FST.22 The odds ratio (OR), CIs, and probability values between the HAPC groups and control groups were calculated for alleles of polymorphic loci.CHARACTERISTICS OF SUBJECTSThe data for the sex, average age, SaO2, Hb, and HCT for the HAPC and control groups are reported in Table 1.EPAS1 and High Altitude PolycythemiaTable 1. Characteristics of the study groupsStudy groupAge (year)47.7 (cid:1) 1.7 42.8 (cid:1) 1.8 46.4 (cid:1) 1.5 43.2 (cid:1) 1.483.4 (cid:1) 0.9a 85.1 (cid:1) 0.9a 88.8 (cid:1) 0.4 87.3 (cid:1) 0.623.0 (cid:1) 0.3a 20.1 (cid:1) 0.2a 16.2 (cid:1) 0.1 15.8 (cid:1) 0.266.6 (cid:1) 2.1a 59.9 (cid:1) 0.8a 46.6 (cid:1) 0.6 41.6 (cid:1) 2.1The data represent the mean and SE of HAPC patients and healthy control subjects. HAPC, high altitude polycythemia; SaO2, arterial oxygen saturation; Hb, hemoglobin; HCT, hematocrit. a P o .05 vs control group.The incidence of HAPC was much higher in men than in women, consistent with the preponderance of men with HAPC. The SaO2 was significantly lower, whereas Hb and HCT were significantly higher in the HAPC group compared with the control group (P o .05).GENOTYPE AND ALLELE DISTRIBUTIONThe genotypic distributions and allelic frequencies of 8 EPAS1 SNPs (rs12619696, rs13420857, rs2881504, rs4953388, rs113419896, rs4953354, rs10187368, and rs7587138) in HAPC and control study groups are shown in Tables 2 and 3.All these polymorphisms were found to be in HWE in both study groups (Table 3). The AA genotypes of rs12619696 and rs4953354 were significantly more prevalent among the HAPC group (8.2% and 12.9%) than the control group (2.3% and 3.9%) with an OR of 0.227 (95% CI, 0.052 to 1.001; P ¼ .035) and 0.199 (95% CI, 0.060 to 0.658; P ¼ .004), respectively. Furthermore, the genotype GG rs13419896 differed significantly between HAPC and control groups with an OR of 0.062 (95% CI, 0.007 to 0.530; P ¼ .001; Furthermore, we found thatthere were significant differences in the allele frequency of the rs12619696rs12619696 SNP between the 2 groups (P ¼ .014; Table 2); the A allele was much more prevalent among the HAPC group (24.6%) than the control group (14.3%), with an OR of 0.513 (95% CI, 0.299 to 0.881). The difference was significant when applying the FST statistics on the genetic distance (.121; Table 3). The G allele for the significantly more prevalent rs13419896 SNP was among the HAPC group (27%) than the control group (14.7%; P ¼ .004; Table 2), with an OR of 0.466 (95% CI, 0.275 to 0.789), and the difference was significant when applying the FST statistics on the genetic distance (.203; Table 3). We observed a significantly higher incidence of the A allele of the rs4953354rs4953354 SNP in the HAPC group (34.7%) than in the control group (19.0%), with an OR of 0.442 (95% CI, 0.272 to 0.716; P ¼.001;Table 2), and the difference was significant when applying the FST statistics on the genetic distance (.410; Table 3). We also found a significant difference for the rs2881504 SNP (A/G) between the HAPC and control groups (P o .001; Table 2).We showed thatthe rs12619696 SNP was signifi- cantly associated with HAPC risk under the dominant model of inheritance (OR, 0.515; 95% CI, 0.271 to 0.981; P ¼ .042). The rs13419896 SNP was significantly associated with HAPC risk under the dominant model (OR, 0.506; 95% CI, 0.269 to 0.964; P ¼ .034) as well as the recessive model of inheritance (OR, 13.964; 95% CI, 1.642 to 118.737; P ¼ .002; Table 2). In addition, the rs4953354 SNP was significantly associated with HAPC risk both under the dominant (OR, 0.399; 95% CI, 0.215 to 0.742; P ¼ .003) and recessive models of inheritance (OR, 3.674; 95% CI, 1.149 to 11.745; P ¼ .020; Table 2).DiscussionIn a previous study we genotyped 207 SNPs of the EPAS1 gene in Chr2: 46304028–46851921 in a sample of 31 healthy Tibetans. The results suggested that the polymorphism in the EPAS1 gene is associated with adaptation to high altitude in Tibetans.15 Thus we hypothesized that Tibetans with HAPC may carry a different genotype and alleles, and we genotyped 8 SNPs of the EPAS1 gene, 7 of them located in the same region of Chr2: 46304028–46851921. We genotyped 8 SNPs of the EPAS1 gene by the SNP assay and analyzed the haplotypes in HAPC and control groups. Collectively, our results indicate that there are significant differences in 3 SNPs (rs12619696, rs13419896, and rs4953354) between the 2 groups.A previous study reported thatrs12619696 was associated with different patterns of adaptation to high altitude between Tibetans and Andeans.21 In this study, we found that both the AA genotype and A allele of rs12619696 were significantly more prevalent among the HAPC group (8.2% and 24.6%) than the control groupTable 2. Comparison of genotype distributions, allele frequencies, and association with HAPC risk in HAPC and control groupsGenotype or allele associated with SNPHAPC (n %)Control (n %)OR (95% CI)rs12619696Dominant modelRecessive modelrs13420857 Dominant modelRecessive modelDominant modelRecessive modelDominant modelRecessive modelrs13419696 Dominant modelRecessive model110 (91.7) 221 (85.7) 126 (97.7)107 (82.9) 235 (91.1) 128 (99.2) 107 (82.9) 200 (77.5) 122 (94.6) 174 (67.4) 117 (90.7)220 (85.3) 128 (99.2)0.587 (0.297–1.160) 0.227 (0.052–1.001)0.513 (0.299–0.881)0.515 (0.271–0.981)3.750 (0.886–16.237)2.625 (0.146–47.183) 2.098 (0.129–34.220) 0.929 (0.427–2.019)2.169 (0.133–35.283) 1.165 (0.501–2.710)1.048 (0.243–4.509)0.796 (0.196–3.239)2.840 (1.883–4.283)0.871 (0.218–3.489)1.308 (0.695–2.459)0.762 (0.391–1.487) 0.402 (0.154–1.049)0.672 (0.431–1.048)0.663 (0.352–1.248)2.145 (0.887–5.185)0.634 (0.325–1.234) 0.062 (0.007–0.530)0.466 (0.275–0.789)0.506 (0.269–0.954)13.964 (1.642–118.737)0.459 (0.238–0.883)EPAS1 and High Altitude PolycythemiaGenotype or allele associated with SNPHAPC (n %)Control (n %)OR (95% CI)Table 2 (continued )Dominant modelRecessive modelrs10187368 Dominant modelRecessive modelDominant modelRecessive model115 (95.8) 102 (85.0) 209 (81.0) 124 (96.1)124 (96.1) 253 (98.1) 124 (96.1) 217 (84.1) 124 (96.1) 0.199 (0.060–0.658)0.442 (0.272–0.716)0.399 (0.215–0.742)3.674 (1.149–11.745)0.444 (0.123–1.595)0.455 (0.129–1.601)0.444 (0.123–1.595)0.388 (0.042–3.604) 0.433 (0.049–3.816)0.934 (0.512–1.705)0.420 (0.048–3.679)0.979 (0.496–1.934)HAPC, high altitude polycythemia; OR, odds ratio, SNP, single-nucleotide polymorphism. a P o .05 vs control.(2.3% and 14.3%), with an OR of 0.227 (95% CI, 0.052 to 1.001; P o .05) and 0.513 (95% CI, 0.299 to 0.881; P o .05). In addition, rs12619696 was significantly associated with HAPC risk under the dominant model of inheritance (OR, 0.515; 95% CI, 0.271 to 0.981; For the SNP rs13419896, the A allele is proposed as being advantageous for Tibetans.14,22 In this study, the A allele was reported as 73% in Tibetans with HAPC and 85.3% in a control group who were Tibetans adapted to high altitude. The genotype AA differed significantly between the HAPC (55.7%) and control groups (71.3%). Therefore, the A allele of rs13419896 is the allele that is advantageous for Tibetans to adapt to hypoxia at high altitude. A high incidence of the G allele is associated with HAPC in Tibetans. the G allele of rs13419896 was significantly more prevalent among the HAPC group (27%) than the control group (14.7%; P o .05). The SNP rs13419896 was also significantlyassociated with HAPC risk under the dominant as well as the recessive model of inheritance.For the SNP rs4953354,the G allele had a high incidence in healthy Tibetans.14,22 Interestingly, we observed a significantly lower incidence of the G allele in the HAPC group (65.3%) compared with the control group (81.0%). However, the AA genotype was signifi- cantly more prevalent among the HAPC group (12.9%) than the control group (3.9%). The rs13419896 SNP was significantly associated with HAPC risk both under the dominant and recessive models of inheritance. Therefore, our results indicated that a high incidence of the A allele and AA genotype of rs4953354 is associated with the risk of HAPC in Tibetans.In addition, we found significant differences between 2 groups as measured by FST on the genetic distances for the 3 SNPs: rs12619696, rs13419896, and rs4953354. The divergences resulted from different genotypes of these SNPs in the 2 groups.Table 3. Comparison of Hardy-Weinberg Equilibrium (HWE) and genetic distance in HAPC and control groupsReferencesN HWE P value Genetic distanceMed Clin North Am. 2004;22:329–355, viii.1. Gallagher SA, Hackett PH. High-altitude illness. Emergrs12619696 HAPCrs13420857 HAPCrs2881504 HAPCrs4953388 HAPCrs13419896 HAPCrs4953354 HAPCrs10187368 HAPCrs7587138 HAPCControl 129 .802 Control 129 .802 Control 129 .808 Control 129 .502 Control 129 .207 Control 129 .843 Control 129 .822 Control 129 .251HAPC, high altitude polycythemia; SNP, single-nucleotide poly- morphism; SG, study group; FST, proportion of genetic diversity attributable to genotype frequency differences among study groups.a P o .05 computed from FST statistics.However, we should note several limitations of this study. First, we did not measure other clinical parameters of the subjects, such as systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulmonary artery systolic pressure, which would provide more information on the significance of EPAS1 SNPs in Tibetans with HAPC. Second, we only focused on 8 SNPs of EPAS1 and did not screen other genes. Third, we examined a modest number of subjects in the study. More subjects will be included in the 2 groups to increase the power to detect the association of EPAS1 SNPs with HAPC in Tibetans.AcknowledgmentsThis study was supported by grants from the National Basic Research Program of China (No. 2012CB518200), Program of International S&T Cooperation of China (No. 2011DFA32720), Natural Science Foundation of China (No. 31160219), The High Altitude Medical Sciences Key Laboratory of Qinghai (2013-Z-Y-05), and The Key Laboratory Development Foundation of Qinghai (No. 2014-Z-Y-07 & 2014-Z-Y-30).2. Windsor JS, Rodway GW. 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