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Natural selection on EPAS1 (HIF2α) associated withlow hemoglobin concentration in Tibetan highlandersCynthia M. Bealla,1, Gianpiero L. Cavallerib,1, Libin Dengc,2, Robert C. Elstond, Yang Gaoc, Jo Knighte,f, Chaohua Lic,Jiang Chuan Lig, Yu Liangh, Mark McCormackb, Hugh E. Montgomeryi,1, Hao Panc, Peter A. Robbinsj,1,3,Kevin V. Shiannak, Siu Cheung Taml, Ngodrop Tseringm, Krishna R. Veeramahn, Wei Wangh, Puchung Wangduim,Michael E. Wealee,1, Yaomin Xuo, Zhe Xuc, Ling Yangh, M. Justin Zamanp, Changqing Zengc,1,3, Li Zhango,1,Xianglong Zhangc, Pingcuo Zhaxih,1,4, and Yong Tang Zhengq

aDepartment of Anthropology, Case Western Reserve University, Cleveland, OH 44106-7125; bMolecular and Cellular Therapeutics, The Royal College ofSurgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland; cBeijing Institute of Genomics, Key Laboratory of Genome Sciencesand Information, Chinese Academy of Sciences, Beijing 100029, China; dDepartment of Epidemiology and Biostatistics, Case Western Reserve University,Cleveland, OH 44106-7281; eDepartment of Medical and Molecular Genetics, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom;fNational Institute for Health Research, Biomedical Research Centre, Guy’s and St. Thomas’ National Health Service Foundation Trust and King’s CollegeLondon, London SE1 7EH, United Kingdom; gYunnan Institute of Population and Family Planning Research, Kunming 650021, China; hBeijing GenomicsInstitute at Shenzhen, Shenzhen 518000, China; IInstitute for Human Health and Performance, University College London, London N19 5LW, United Kingdom;jDepartment of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom; kInstitute for Genome Sciences and Policy, Centerfor Human Genome Variation, Duke University, Durham, NC 27708; lSchool of Biomedical Sciences, Chinese University of Hong Kong, Shatin NT, Hong Kong,China; mTibet Academy of Social Sciences, Lhasa 850000, Tibet Autonomous Region, China; nDepartment of History, The Centre for Society and Genetics andthe Novembre Laboratory, University of California, Los Angeles, CA 90095-7221; oDepartment of Quantitative Health Sciences, Cleveland Clinic, Cleveland, OH44195; pDepartment of Epidemiology and Public Health, University College London, London WC1E 6BT, United Kingdom; and qKunming Institute of Zoology,Chinese Academy of Sciences, Kunming 650223, China

Edited by Peter T. Ellison, Harvard University, Cambridge, MA, and approved May 17, 2010 (received for review February 26, 2010)

By impairing both function and survival, the severe reduction inoxygen availability associated with high-altitude environments islikely to act as an agent of natural selection. We used genomic andcandidate gene approaches to search for evidence of such geneticselection. First, a genome-wide allelic differentiation scan (GWADS)comparing indigenous highlanders of the Tibetan Plateau (3,200–3,500 m) with closely related lowland Han revealed a genome-widesignificantdivergenceacrosseightSNPs locatednearEPAS1. Thisgeneencodes the transcription factor HIF2α, which stimulates productionof red blood cells and thus increases the concentration of hemoglobinin blood. Second, in a separate cohort of Tibetans residing at 4,200m,we identified 31 EPAS1 SNPs in high linkage disequilibrium thatcorrelated significantly with hemoglobin concentration. The sex-ad-justed hemoglobin concentration was, on average, 0.8 g/dL lower inthe major allele homozygotes compared with the heterozygotes.These findings were replicated in a third cohort of Tibetans residingat 4,300m. The alleles associatingwith lower hemoglobin concentra-tions were correlated with the signal from the GWADS study andwere observed at greatly elevated frequencies in the Tibetan cohortscomparedwith the Han. High hemoglobin concentrations are a cardi-nal feature of chronicmountain sickness offering oneplausiblemech-anism for selection. Alternatively, as EPAS1 is pleiotropic in its effects,selectionmay have operated on someother aspect of the phenotype.Whichever of these explanations is correct, the evidence for geneticselection at the EPAS1 locus from the GWADS study is supported bythe replicated studies associating function with the allelic variants.

chronic mountain sickness | high altitude | human genome variation |hypoxia | hypoxia-inducible factor

The high plateaus of Central Asia and the Andes were amongthe last areas occupied as Homo sapiens spread across the

globe during the past 100,000–200,000 y. In the case of the Ti-betan plateau, early visitors appearedmore than 30,000 y ago, andthe plateau has been colonized for more than 10,000 y (1, 2). Thelow partial pressure of oxygen resulting from the extreme altitudewould have presented a formidable biological challenge to suchcolonists. Individuals from low-altitude populations (Europeanand Han) who move to live at high altitude suffer from a numberof potentially lethal diseases specifically related to the low levelsof oxygen (3–5) and struggle to reproduce at these altitudes (6–9).The hypoxia of altitude (hypobaric hypoxia) would thus haveexerted substantial evolutionary selection pressure.

The classic disease associated with long term residence at highaltitude is chronic mountain sickness, or Monge’s disease, afterCarlos Monge-Medrano who first identified the condition amongAndean highlanders (10). The cardinal feature is a very highconcentration of the oxygen-carrying pigment, hemoglobin, in theblood, caused by an overproduction of red blood cells (excessiveerythrocytosis). Tibetan highlanders are particularly resistant todeveloping chronic mountain sickness (4, 11), and exhibit little orno increase in hemoglobin concentration with increasing altitude,even at 4,000 m (13,200 ft) and only moderate increases beyond(12, 13). Typically, Tibetans average at least 1 g/dL and as muchas approximately 3.5 gm/dL (i.e. approximately 10–20%) lowerhemoglobin concentration in comparison with their Andeancounterparts (14–16) or acclimatized lowlanders, such as the Hanwho have moved to altitudes above 2,500 m (4, 17–23). This sug-gests that Tibetans have evolved a blunted erythropoietic responseto high-altitude hypoxia. The induction of erythrocytosis by hyp-oxia involves the hypoxia-inducible factor (HIF) family of tran-scription factors and, in particular, EPAS1 (or HIF2α) (24, 25).Three independent studies, but with mutually reinforcing

results, were performedby groups that have since come together toform a consortium with the aim of reporting on the findings. Thefirst study was a genome-wide allelic differentiation scan thatcompared SNP frequencies of a Yunnan Tibetan population re-siding at 3,200–3,500 m with the HapMap Phase III Han sample.

Author contributions: C.M.B., G.L.C., J.C.L., Y.L., H.E.M., P.A.R., S.C.T., N.T., W.W., P.W.,L.Y., C.Z., P.Z., and Y.T.Z. designed research; C.M.B., G.L.C., Y.G., C.L., J.C.L., Y.L., M.M.,H.P., K.V.S., S.C.T., N.T., W.W., P.W., Z.X., L.Y., C.Z., X.Z., P.Z., and Y.T.Z. performed re-search; G.L.C., L.D., R.C.E., Y.G., J.K., K.R.V., M.E.W., Y.X., Z.X., M.J.Z., and L.Z. analyzeddata; and C.M.B., G.L.C., H.E.M., P.A.R., M.E.W., and C.Z. wrote the paper.

The authors are listed alphabetically and declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1C.M.B., G.L.C., H.E.M., P.A.R., M.E.W., C.Z., L.Z., and P.Z. contributed equally to this work.2Present address: Faculty of Basic Medical Science, Nanchang University, Nanchang330006, China.

3To whom correspondence may be addressed. E-mail: peter.robbins@dpag.ox.ac.uk orczeng@big.ac.cn.

4Present address: The People’s Hospital of the Tibet Autonomous Region, Lhasa, 850000Tibet Autonomous Region, China.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1002443107/-/DCSupplemental.

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Mitochondrial, Y chromosome, and autosomal DNA evidence allsuggest a northor eastAsianorigin formodernTibetans (1, 26–28).Thus, theHan comprise a lowlandpopulation that is closely relatedto the Tibetans but which has not undergone selection for high-altitude adaptation. From this study, a signal of selection close toEPAS1 was identified at a genome-wide level of significance. Thesecond study comprised a candidate gene analysis of EPAS1 ina separate sample of Tibetans from 4,200m on theTibetan plateauand identified a significant association between genotype and he-moglobin concentration, with the major alleles associating withthe lower hemoglobin levels. These alleles were present at lowfrequency in the Han. The third study replicated the hemoglobinassociation in an independent sample of Tibetans from 4,300 m.

ResultsGenome-Wide Allelic Differentiation Scan. A genome-wide allelicdifferentiation scan (GWADS) was used to compare a cohort ofTibetan residents (n=35) sampled from four townships at altitudesof 3,200–3,500 m in Yunnan Province, China, with HapMap PhaseIII Han individuals (n = 84). We postulated that any markeddifferences in SNP frequencies between the Yunnan Tibetan andthe HapMap Han populations could reflect a history of divergentselection on functional variation that contributes to increased sur-vival at high altitude. (See SI Materials and Methods for detailedmethodology.) Of the 502,722 SNPs that were included in theanalysis, eight SNPs emerged as having genome-wide significance(P values ranging from 2.81× 10−7 to 1.49× 10−9), all locatedwithin235 kb on chromosome 2 (Fig. 1 and Table S1).All eight GWADS significant SNPs were in high pairwise

linkage disequilibrium in the Yunnan sample (0.23< r2 < 0.82),forming an extended haplotype with a frequency of 46% in theYunnan Tibetan sample but only 2% in the Han sample [esti-mated via an expectation-maximization algorithm using Haplo-view software (29, 30)]. The SNPs lie between 366 bp and 235 kbdownstream of EPAS1 but, as we show below, the region of highlinkage disequilibrium extends into EPAS1 itself. In addition tothis genome-wide significant finding relating to EPAS1, evidencefor other signals of selection was also found. Regions of sub-genome wide significance were in close proximity to other genesof the HIF pathway and present intriguing targets for follow-upstudies (see SI Text for further details).

Candidate Gene Study for EPAS1. Independent of the GWADSstudy, a candidate gene study (based on the pathway linkinghypoxia, EPAS1, and erythropoietin) addressed the functionalconsequence of EPAS1 variants by testing for association with

hemoglobin concentration in a sample of 70 Tibetans residing at4,200 m in Mag Xiang, Xigatse Prefecture in the Tibet Autono-mous Region, China (Table S2). One hundred and three non-coding SNPs across theEPAS1 gene were selected for genotyping.Of these, 49 had a minor allele frequency ≥5%, and were thusamenable to regression analysis (Materials and Methods) thatidentified 31 SNP sites significantly associated with hemoglobinconcentration (Fig. 2 and Table S3). The major (most frequent)allele of every significant SNP was associated with lower hemo-globin concentration. After adjusting for sex differences, indi-viduals who were homozygous for the major allele had an averagehemoglobin concentration that was 0.8 ± 0.15 g/dL (range from0.3 to 1.0 g/dL) lower than individuals who were heterozygous forthe major allele. Conditional linear regression analyses showedthat once the most significant SNP (rs4953354) was included, nosignificant improvement in fit was obtained after Bonferronicorrection by adding any other SNP, consistent with a singlecausal variant model. Many of the SNP sites were in high linkagedisequilibrium (Fig. 2). Genotypes for the eight GWADS signif-icant SNPs were available on 29 of the 70 individuals in the MagXiang cohort, too few to show statistical association with hemo-globin concentration. However, all eight GWADS SNPs werehighly correlated (0.54 < r2 ≤ 1) with variants associating withhemoglobin concentration in the complete Mag Xiang cohort(Table S4). Thus, the genome-wide and the candidate-geneanalyses can be linked, with the latter study demonstrating thatthere is a phenotype associated with the signal of selection.

Replication of Candidate Gene Study for EPAS1. We replicated theassociation of EPAS1 SNPs with hemoglobin concentration inanother sample of 91 Tibetans residing at 4,300 m in ZhaxizongXiang, Xigatse Prefecture, China (Table S2). Of the 49 MagXiang SNPs with a minor allele frequency ≥5%, 48 were suc-cessfully genotyped in the Zhaxizong Xiang sample. Of these,45 sites had a minor allele frequency ≥5% and 32 sites weresignificantly associated with hemoglobin concentration. Afteradjusting for sex differences, individuals who were homozygousfor the major allele had an average hemoglobin concentrationthat was 1.0 ± 0.14 g/dL (range from 0.5 to 1.2 g/dL) lower thanindividuals who were heterozygous for the major allele (Fig. 3 andTable S3). Twenty-six SNPs were associated with hemoglobinconcentration in both samples and the direction of the effect wasthe same. Conditional linear regression again found that, afterincluding the most significant SNP (rs13419896), no further SNPswere significant after Bonferroni correction. This was also thecase if the most significant SNP from the Mag Xiang sample

Fig. 1. A genome-wide allelic differentiation scan that compares Tibetan residents at 3,200–3,500 m in Yunnan Province, China with HapMap Han samples.Eight SNPs near one another and EPAS1 have genome-wide significance. The horizontal axis is genomic position with colors indicating chromosomes. Thevertical axis is the negative log of SNP-by-SNP P values generated from the Yunnan Tibetan vs. HapMap Han comparison. The red line indicates the thresholdfor genome-wide significance used (P = 5 × 10−7). Values are shown after correction for background population stratification using genomic control.

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(rs4953354) was used instead of rs13419896. Genotypes for theeight GWADS significant SNPs were available on 89 samplesfrom the Zhaxizong Xiang cohort. Three of these SNPs correlatedsignificantly with hemoglobin concentration (Table S4), thus sup-porting the association of a phenotype with the signal of selectionthat has been obtained from this area of the genome.Comparing allelic frequencies between the two Tibetan samples

and the HapMap Han sample, we note that the largest allelefrequency differences occur at the EPAS1 SNP sites that are as-sociated with low hemoglobin concentration (Fig. 4). Linkage dis-equilibrium (LD) among these 26 SNP sites was also elevated inthe twoTibetan cohorts compared with theHapMapHan (SI Text).

DiscussionThe results from the GWADS study revealed a level of allelefrequency differentiation near EPAS1 that far exceeds the ge-nome-wide average (Fig. 1). The association studies demonstratedthat the SNP variants that were at higher frequencies within theTibetan population were associated with lower hemoglobin con-centrations. As large genome-wide association studies of thedeterminants of hemoglobin concentration in other populations atlow altitude have failed to detect a signal associated with EPAS1(31–34), our results suggest either that there is a genetic variantthat is quite specific to the Tibetan population or that the variant isquite specific to moderating hemoglobin concentration only under

conditions of high altitude. Such specificity of effect in relation toTibetan highlanders is in keeping with a model of selection pres-sure on EPAS1 under the stress of high-altitude hypoxia. In-terestingly, a comparison between the HapMap Han and Andeanhighlanders—both of whom have a vigorous erythropoietic re-sponse (15)—did not detect selection at the EPAS1 locus (35). Itshould be noted, however, that the Andean study (35) applieda different array of methodologies to detect selection and over-lapping results are not necessarily expected, given the differingnature of the selection signals that particular techniques arepowered to detect. It is also possible that the Andean and Tibetanpopulations have developed different genetic adaptations to thehypoxia of high altitude given the differences in physiology thatare known to exist between these populations (13).The association studies revealed that genetic variation across

EPAS1 accounts for a large proportion of the variation in he-moglobin concentration in these populations. After controllingfor sex, the average difference in hemoglobin concentration be-tween major allele homozygotes and heterozygotes was 53% ofone SD in theMagXiang sample and 50% in the ZhaxizongXiangsample. In absolute terms, these differences were several foldlarger than for any of the determinants of hemoglobin concen-tration in populations residing at low altitude (31–34). Our find-ings are, however, consistent with previous high estimates ofheritability (h2) for hemoglobin concentration of 0.66 among

Fig. 2. Sex-adjusted hemoglobin concentrations and allelic variation in EPAS1 SNPs in a Tibetan sample from Mag Xiang (4,200 m), Tibet AutonomousRegion, China. Values were, on average, 0.8 g/dL lower for individuals who were homozygous for the major alleles compared with those who were het-erozygous. (Top) The results of testing variants at 49 SNPs with a minor allele frequency ≥5% for genotypic association with sex-adjusted hemoglobinconcentration. (Middle) The estimated hemoglobin concentration difference (mean ± 95% confidence interval) between the major allele homozygote andheterozygote genotypes at each SNP. Filled circles represent SNPs detected as having a significant association with hemoglobin concentration, with the falsediscovery rate controlled at <0.05 across the EPAS1 locus. Open diamonds represent SNPs without significant association. (Bottom) The pairwise linkagedisequilibrium measured as r2 between SNPs.

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Tibetans at 4,850–5,450 m (36) and of 0.86 among Tibetans at3,800–4,065 m (15). These values estimate the proportion ofadditive genetic variation relative to total phenotypic variance.The combined findings of our association and conditional linearregression analysis are consistent with a model in which a singlecausal variant at the EPAS1 locus accounts for a substantialproportion of the heritability. Under this model, hemoglobin-associating SNPs should be interpreted as markers and are pre-sumed to have differentiated because they are closely linked to anas yet to be identified causal variant. Functional studies will berequired to identify how this variation works to restrain thehematopoietic response.We have described a signal of natural selection on or near

EPAS1 that is associated with a blunting of the normal erythro-poietic response to hypoxia. As EPAS1 is pleiotropic, other res-ponses to hypoxia may be similarly affected. Some insight intothese may be given by studies of a few individuals/families, livingat low altitudes, who have been reported to have gain of functionmutations inEPAS1 (37–40). As expected, these individuals exhibitexcessive erythrocytosis, but they also appear to be particularlysusceptible to thrombotic events and to developing pulmonaryhypertension—although the total number of cases reported issmall. A larger number of cases have been reported for the slightlyless specific genetic disorder of Chuvash Polycythemia, where ho-mozygosity for hypomorphic alleles for VHL results in elevated

levels of both HIF1α and EPAS1/HIF2α (41). The phenotype forChuvash Polycythemia appears very similar to that for the specificEPAS1 gain of function mutations, with excessive erythrocytosis,an excess risk of thrombotic events at a young age, and pulmonaryhypertension (42–45). In both conditions, the excessive eryth-rocytosis is generally managed by venesection in the belief that thismay reduce the incidence of thrombotic events.The clinical similarity between the phenotypes of these genetic

disorders and chronic mountain sickness is striking. Indeed, itcaused one group of investigators to observe in respect of theEPAS1 gain of function mutations that “it raises the possibilitythat polymorphic variation in HIF2α [EPAS1] contributes to themarked differential susceptibility to erythrocytosis, reducedplasma volume and pulmonary hypertension in humans at highaltitude” (39). Chronic mountain sickness occurs among Tibetansat a lower prevalence than Han lowlanders (1.2% compared with5.6%) living in the Tibet Autonomous Region (4, 46). Chronicmountain sickness remains at that low level throughout adulthoodamong Tibetans but, in Peruvians, prevalence increases with agefrom approximately 13% in 20 to 39 y olds to approximately 36%in 55 to 69 y olds at 4,300 m (47). In Andeans, excessive eryth-rocytosis at high altitude has been associated with significantpulmonary hypertension (48), an increased risk of stroke (49), andalso an increased risk of poor outcome in pregnancy (stillbirth,preterm birth, or small for gestational age at birth) (50). These

Fig. 3. Sex-adjusted hemoglobin concentrations and allelic variation in EPAS1 SNPs in a Tibetan sample from Zhaxizong Xiang (4,300 m), Tibet AutonomousRegion, China. Values were, on average, 1.0 g/dL lower for individuals who were homozygous for the major alleles compared with those who were het-erozygous (Top) The results of testing variants at 45 SNPs with a minor allele frequency ≥5% for genotypic association with sex-adjusted hemoglobinconcentration. (Middle) The estimated hemoglobin concentration difference (mean ± 95% confidence interval) between the major allele homozygote andheterozygote genotypes at each SNP. Filled circles represent SNP detected as having a significant association with hemoglobin concentration with the falsediscovery rate controlled at <0.05 across the EPAS1 locus. Open diamonds represent SNP without significant association. (Bottom) The pairwise linkagedisequilibrium measured as r2 between SNPs.

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findings provide insight into some of the sources of elevatedmorbidity and mortality on which selection may have operated toinfluence allelic frequencies for EPAS1 among the early colo-nizers of the Tibetan plateau.Although the similarity between chronic mountain sickness

and the EPAS1 gain of function phenotype in lowlanders isstriking, there nevertheless may be other aspects to the pheno-type that are not revealed at low altitude but are only revealed athigh altitude, when oxygen availability is also limited. In partic-ular, EPAS1 plays very important, if still poorly understood, rolesin both placental and embryonic development (51–54) and pos-sibly also in the pathogenesis of fetal growth restriction (55). It iswell recognized that reproductive success is more difficult at highaltitude than at low altitude, and more difficult for nonnativesthan natives (6). For example, pre- and postnatal mortality arethreefold higher in the Han than in the Tibetans, and birthweight is significantly lower (56). This may relate in part to thepresence of greater uterine arterial blood flow and lower he-moglobin concentration in the Tibetans (9, 57). As such, naturalselection on EPAS1 may also have operated via effects duringpregnancy that affect both pre- and postnatal mortality.In conclusion, this study provides evidence for natural selection

in Tibetan highlanders at a specific human gene locus. The findingis further supported by a demonstration, in two independentsamples, that genetic variation at this locus has an associatedphenotype. The known physiological roles associated with thisgene locus provide insight into some of the factors that are likelyto have influenced human adaptation and survival following col-onization of the Tibetan Plateau.

Materials and MethodsHuman Volunteers. Ethics and consent. This study was approved by the ethicscommittees of the Yunnan Population and Family Planning Institute(Kunming, China); the Beijing Genomics Institute at Shenzhen (Shenzhen,China); the Beijing Institute of Genomics, Chinese Academy of Sciences (Bei-jing, China)and Case Western University (Cleveland, OH). All work was con-ducted in accordance with the principles of the Declaration of Helsinki. Allparticipants were recruited after obtaining informed consent.Sample collection. SamplingwasconductedinthreegeographicregionsofChinaapproximately 2,400 kilometers apart. Theywere (i) theNorthWestern regionof Yunnan province (28°26’N 98°52’E), (ii) Mag Xiang, Xigatse Prefecture,Tibet Autonomous Region (29°15’N 88°53’E), and (iii) Zhaxizong Xiang,

Xigatse Prefecture, Tibet Autonomous Region (28°34’N 86°38’E). Genotypicdata from the HapMap Phase III Han population were also included in thisanalysis. Further details on sample collection are given in the SI Materialsand Methods.Genotyping. All genotyping was conducted at the Beijing Institute of Geno-mics. The whole genome genotyping was conducted using the IlluminaVeracode platform and 610-Quad high throughput genotyping chips. Gen-otyping within EPAS1 was conducted using a customer-designed IlluminaGoldenGate assay (384 SNP plex) for all samples from Mag Xiang and someof the samples from Zhaxizong Xiang. The remainder of the samples fromZhaxizong Xiang were genotyped using MassARRAY assays. Further detailsof these and the quality control procedures are given in the SI Materialsand Methods.Phenotyping. Hemoglobin concentration was measured in duplicate imme-diately after provision of a venipuncture blood sample by individuals in theMag Xiang sample (58). Individuals were screened with the aim of includingonly healthy, native residents. Excluded were individuals who had anemia(men and women with hemoglobin concentrations below 13.7 g/dL and 12g/dL, respectively), hypertension, fever, poor lung function, extreme hyp-oxemia, or who were currently or recently pregnant, or who had symptomsor medication indicative of heart or lung disease. The Zhaxizong Xiangsample was obtained in the course of a health survey and included all vol-unteers who were native residents.

Statistical Analysis. GWADS. To identify variation between the Yunnan Ti-betan and the HapMap Han populations, we calculated SNP-by-SNP χ2 sta-tistics for allele frequencies and corrected for background populationstratification through a genomic control procedure (30). This approachallows genome-wide significant signals of allele frequency differentiation tobe readily declared by examining genomic distributions of χ2 values in thesample of approximately 500,000 SNPs. A threshold of genome-wide sig-nificance was set at 5 × 10−7 (59). A full description of the method, includinga simulation for two populations with a degree of genomic divergenceequal to that between the Yunnan Tibetan and HapMap Han populations, isgiven in the SI Text.Candidate gene studies. Candidate gene association analysis of EPAS1 SNPgenotype with hemoglobin concentration phenotype was performed sepa-rately in the two Tibet Autonomous Region samples. Mean characteristics forthese populations are given in Table S2. For each SNP, a linear additive geneticmodel was fitted with hemoglobin concentration as the response variable,the SNP as the predictive variable (entered as a numerical variable—1, 2, 3—corresponding to the three genotypes sorted by descending allelic frequency)and with gender as a covariate. The P values of the likelihood ratio test wereobtained from a comparison with the null model (i.e., only gender in themodel). The estimated difference stands for the increase in the sex-adjustedmean with the addition of one copy of the minor allele taking the mostfrequent homozygous genotypes as the reference. Unless otherwise stated,an adjustment for multiple comparisons was implemented by controlling thefalse discovery rate at less than 0.05 across the EPAS1 gene. The R languageand environment (R Project for Statistical Computing, http://www.r-project.org) was used for all related analysis and graphics. Conditional linear analyseswere undertaken by including a specified SNP as an additional covariate inthe model and were implemented using plink (http://pngu.mgh.harvard.edu/~purcell/plink/).

ACKNOWLEDGMENTS. We thank Wei Chen, Jian Bai, and Feng Cheng ofBeijing Institute of Genomics for their contribution in genotyping and dataprocessing and three anonymous reviewers for their critical and constructivecomments. We also thank the people of Shangri-La and De Qin Xians,Yunnan Province; Mag Xiang and Zhaxizong Xiang, Tibet AutonomousRegion, for their cooperation and hospitality during data collection. We aregrateful to the Tibet Academy of Social Sciences for their collaboration andenabling permission to collect data in Mag Xiang. This work was supportedby the National Science Foundation; National Institutes of Health NationalCenter for Research Resources, National Institute of General Medical Scien-ces, National Cancer Institute, National Heart, Lung, and Blood Institute;National Natural Science Foundation of China Grant 30890031 and Ministryof Science and Technology Grant 2006DFA31850 (to C.Z.); Chinese Academyof Sciences Grant KSCX2-YW-R-76 and Science and Technology Plan ofthe Tibet Autonomous Region Grant 2007-2-18 to Beijing Genomics Instituteat Shenzhen; and an International Joint Project award from the Royal Soci-ety. This consortium grew from a catalysis meeting sponsored by the Na-tional Science Foundation-supported National Evolutionary Synthesis Center(http://www.NESCent.org).

Fig. 4. Differences in allelic frequency at SNPs within EPAS1 between theHapMap Han, Mag Xiang and Zhaxizong Xiang cohorts. The horizontal axisis SNP position according to build 36.1. The vertical axis is allelic frequency,with the allele selected for display as the one occurring most frequently inthe Mag Xiang cohort. Squares denote data for HapMap Han; circles denotedata for Mag Xiang Tibetans; triangles denote data for Zhaxizong XiangTibetans. Filled symbols denote those SNPs having significant associationswith hemoglobin in both Mag Xiang and Zhaxizong Xiang cohorts; opensymbols denote those SNPs without both such associations.

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1. ZhaoM, et al. (2009)Mitochondrial genome evidence reveals successful Late Paleolithicsettlement on the Tibetan Plateau. Proc Natl Acad Sci USA 106:21230–21235.

2. Aldenderfer M (2003) Moving up in the world: Archaeologists seek to understandhow and when people came to occupy the Andean and Tibetan plateaus. Am Sci 91:542–549.

3. Wu T, Miao C (2002) High altitude heart disease in children in Tibet. High Alt Med Biol3:323–325.

4. León-Velarde F, et al. (2005) Consensus statement on chronic and subacute highaltitude diseases. High Alt Med Biol 6:147–157.

5. Wu T (2004) Life on the high Tibetan plateau. High Alt Med Biol 5:1–2.6. Julian CG, Wilson MJ, Moore LG (2009) Evolutionary adaptation to high altitude: A

view from in utero. Am J Hum Biol 21:614–622.7. Monge C (1973)Acclimatization in the Andes (Johns Hopkins University Press, Baltimore).8. Moore LG, Young D, McCullough RE, Droma T, Zamudio S (2001) Tibetan protection

from intrauterine growth restriction (IUGR) and reproductive loss at high altitude. AmJ Hum Biol 13:635–644.

9. Moore LG, Zamudio S, Zhuang J, Sun S, Droma T (2001) Oxygen transport in tibetanwomen during pregnancy at 3,658 m. Am J Phys Anthropol 114:42–53.

10. Monge-M C, Encinas E, Heraud C, Hurtado A (1928) La Enfermedad de los Andes(Sindromes Eritemicos) (Imprenta Americana, Lima).

11. Pei SX, et al. (1989) Chronic mountain sickness in Tibet. Q J Med 71:555–574.12. Beall CM (2007) Two routes to functional adaptation: Tibetan and Andean high-

altitude natives. Proc Natl Acad Sci USA 104 (Suppl 1):8655–8660.13. Beall CM (2002) Biodiversity of Human Populations in Mountain Environments.

Mountain Biodiversity: A Global Assessment, eds Korner C, Spehn EM (The ParthenonPublishing Group, New York), pp 199–210.

14. Winslow RM, et al. (1989) Different hematologic responses to hypoxia in Sherpas andQuechua Indians. J Appl Physiol 66:1561–1569.

15. Beall CM, et al. (1998) Hemoglobin concentration of high-altitude Tibetans andBolivian Aymara. Am J Phys Anthropol 106:385–400.

16. Beall CM (2002) Biodiversity of Human Populations in Moutain Environments.Mountain Biodiversity: A Global Assessment, eds Korner C, Spehn EM (The ParthenonPublishing Group, New York), pp 199–210.

17. Garruto RM, et al. (2003) Hematological differences during growth among Tibetansand Han Chinese born and raised at high altitude in Qinghai, China. Am J PhysAnthropol 122:171–183.

18. Ward MP, Milledge JS, West JB (1995) High Altitude Medicine and Physiology(Chapman and Hall Medical, London).

19. Zhuang J, et al. (1996) Smaller alveolar-arterial O2 gradients in Tibetan than Hanresidents of Lhasa (3658 m). Respir Physiol 103:75–82.

20. Moore LG, et al. (1992) Are Tibetans better adapted? Int J Sports Med 13(Suppl 1):S86–S88.

21. Droma T, et al. (1991) Increased vital and total lung capacities in Tibetan compared toHan residents of Lhasa (3,658 m). Am J Phys Anthropol 86:341–351.

22. Sun SF, et al. (1990) Greater maximal O2 uptakes and vital capacities in Tibetan thanHan residents of Lhasa. Respir Physiol 79:151–161.

23. Zhuang J, et al. (1993) Hypoxic ventilatory responsiveness in Tibetan compared withHan residents of 3,658 m. J Appl Physiol 74:303–311.

24. Chi JT, et al. (2006) Gene expression programs in response to hypoxia: Cell typespecificity and prognostic significance in human cancers. PLoS Med 3:e47.

25. Rankin EB, et al. (2007) Hypoxia-inducible factor-2 (HIF-2) regulates hepaticerythropoietin in vivo. J Clin Invest 117:1068–1077.

26. Su B, et al. (2000) Y chromosome haplotypes reveal prehistorical migrations to theHimalayas. Hum Genet 107:582–590.

27. Qian Y, et al. (2000) Multiple origins of Tibetan Y chromosomes. Hum Genet 106:453–454.

28. Gayden T, et al. (2009) Genetic insights into the origins of Tibeto-Burman populationsin the Himalayas. J Hum Genet 54:216–223.

29. Barrett JC, Fry B, Maller J, Daly MJ (2005) Haploview: Analysis and visualization of LDand haplotype maps. Bioinformatics 21:263–265.

30. Devlin B, Roeder K, Wasserman L (2001) Genomic control, a new approach to genetic-based association studies. Theor Popul Biol 60:155–166.

31. Benyamin B, et al. (2009) Common variants in TMPRSS6 are associated with iron statusand erythrocyte volume. Nat Genet 41:1173–1175.

32. Chambers JC, et al. (2009) Genome-wide association study identifies variants inTMPRSS6 associated with hemoglobin levels. Nat Genet 41:1170–1172.

33. Ganesh SK, et al. (2009) Multiple loci influence erythrocyte phenotypes in theCHARGE Consortium. Nat Genet 41:1191–1198.

34. Soranzo N, et al. (2009) A genome-wide meta-analysis identifies 22 loci associatedwith eight hematological parameters in the HaemGen consortium. Nat Genet 41:1182–1190.

35. Bigham AW, et al. (2009) Identifying positive selection candidate loci for high-altitudeadaptation in Andean populations. Hum Genomics 4:79–90.

36. Beall CM, Blangero J, Williams-Blangero S, Goldstein MC (1994) Major gene forpercent of oxygen saturation of arterial hemoglobin in Tibetan highlanders. Am JPhys Anthropol 95:271–276.

37. Percy MJ, et al. (2008) A gain-of-function mutation in the HIF2A gene in familialerythrocytosis. N Engl J Med 358:162–168.

38. Percy MJ, et al. (2008) Novel exon 12 mutations in the HIF2A gene associated witherythrocytosis. Blood 111:5400–5402.

39. Gale DP, Harten SK, Reid CDL, Tuddenham EGD, Maxwell PH (2008) Autosomaldominant erythrocytosis and pulmonary arterial hypertension associated with anactivating HIF2 α mutation. Blood 112:919–921.

40. Martini M, et al. (2008) A novel heterozygous HIF2AM535I mutation reinforces the roleof oxygen sensing pathway disturbances in the pathogenesis of familial erythrocytosis.Haematologica 93:1068–1071.

41. Ang SO, et al. (2002) Disruption of oxygen homeostasis underlies congenital Chuvashpolycythemia. Nat Genet 32:614–621.

42. Gordeuk VR, et al. (2004) Congenital disorder of oxygen sensing: Association of thehomozygous Chuvash polycythemia VHL mutation with thrombosis and vascularabnormalities but not tumors. Blood 103:3924–3932.

43. Sergeyeva A, et al. (1997) Congenital polycythemia in Chuvashia. Blood 89:2148–2154.44. Smith TG, et al. (2006) Mutation of von Hippel-Lindau tumour suppressor and human

cardiopulmonary physiology. PLoS Med 3:e290.45. Bushuev VI, et al. (2006) Endothelin-1, vascular endothelial growth factor and systolic

pulmonary artery pressure in patients with Chuvash polycythemia. Haematologica 91:744–749.

46. Ren YH, et al. (2010) Incidence of high altitude illnesses among unacclimatizedpersons who acutely ascended to Tibet. High Alt Med Biol 11:39–42.

47. Monge-C C, Arregui A, León-Velarde F (1992) Pathophysiology and epidemiology ofchronic mountain sickness. Int J Sports Med 13 (Suppl 1):S79–S81.

48. Penaloza D, Arias-Stella J (2007) The heart and pulmonary circulation at highaltitudes: Healthy highlanders and chronic mountain sickness. Circulation 115:1132–1146.

49. Jaillard AS, Hommel M, Mazetti P (1995) Prevalence of stroke at high altitude (3380m) in Cuzco, a town of Peru. A population-based study. Stroke 26:562–568.

50. Gonzales GF, Steenland K, Tapia V (2009) Maternal hemoglobin level and fetaloutcome at low and high altitudes. Am J Physiol Regul Integr Comp Physiol 297:R1477–R1485.

51. Pringle KG, Kind KL, Sferruzzi-Perri AN, Thompson JG, Roberts CT (2009) Beyondoxygen: Complex regulation and activity of hypoxia inducible factors in pregnancy.Hum Reprod Update, 10.1093/humupd/dmp046.

52. Dunwoodie SL (2009) The role of hypoxia in development of the Mammalian embryo.Dev Cell 17:755–773.

53. Meade ES, Ma YY, Guller S (2007) Role of hypoxia-inducible transcription factors1alpha and 2alpha in the regulation of plasminogen activator inhibitor-1 expressionin a human trophoblast cell line. Placenta 28:1012–1019.

54. Cowden Dahl KD, et al. (2005) Hypoxia-inducible factors 1alpha and 2alpha regulatetrophoblast differentiation. Mol Cell Biol 25:10479–10491.

55. Dai SY, Kanenishi K, Ueno M, Sakamoto H, Hata T (2004) Hypoxia-inducible factor-2alpha is involved in enhanced apoptosis in the placenta from pregnancies with fetalgrowth restriction. Pathol Int 54:843–849.

56. Moore LG, Young D, McCullough RE, Droma T, Zamudio S (2001) Tibetan protectionfrom intrauterine growth restriction (IUGR) and reproductive loss at high altitude. AmJ Hum Biol 13:635–644.

57. Xing Y, et al. (2009) Hemoglobin levels and anemia evaluation during pregnancy inthe highlands of Tibet: A hospital-based study. BMC Public Health 9:336.

58. Hoit BD, et al. (2005) Nitric oxide and cardiopulmonary hemodynamics in Tibetanhighlanders. J Appl Physiol 99:1796–1801.

59. Wellcome Trust Case Control Consortium (2007) Genome-wide association study of14,000 cases of seven common diseases and 3,000 shared controls. Nature 447:661–678.

11464 | www.pnas.org/cgi/doi/10.1073/pnas.1002443107 Beall et al.

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