Supplementary Figure 1 Genetic relationship between local Tibetans and other East Asian populations.
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1 Supplementary Figure 1 Genetic relationship between local Tibetans and other East Asian populations. 1
2 (A) Principal-components analysis. The first two principal components (PCs) are shown. Each individual is represented by one dot, and color corresponds to the population. The percentage of variance explained by each PC is shown on the axis. (b) Individual grouping inferred by ADMIXTURE. Results from k = 4 are shown. Each individual s genome is represented by a vertical bar composed of colored sections, where each section represents the proportion of an individual s ancestry derived from one of the k ancestral populations. Individuals are arrayed horizontally and grouped by population as indicated. JPT, Japanese; CHB, Chinese; Local Tibetan, Tibetans collected in this study. The two Local Tibetans who have the wild-type D4E variant (TU09 and TU12) are indicated in the plots. 2
3 Supplementary Figure 2 Analysis of purified wild-type PHD2 enzyme, the D4E and C127S single mutants, and the D4E and C127S double mutant. Recombinant enzymes were expressed in insect cells and were affinity purified exploiting their C- terminal Flag tags in anti-flag affinity gel columns. The near-to-homogeneity purified enzymes were analyzed by 10% SDS-PAGE followed by Coomassie Blue staining. Molecular weight markers (kda) are shown on the left. 3
4 Fig. S3A Fig. S3B Supplementary Figure 3 Expression study of hypoxia and several HIF target genes. (a) Expression of hypoxia-regulated genes in granulocytes from Tibetan (n = 4) versus control (n = 6) subjects. (b) BFU-E expression of selected HIF target genes in Tibetan (n = 1) versus control (n = 2) erythroid progenitors grown at optimal EPO levels (3,000 mu). Two independent experiments (technical replicates) from BFU-Es were pooled together and utilized the statistical randomization test 4
5 using the Relative Expression Software Tool (REST, Qiagen). Error bars represent s.d. 5
6 Sample ID Race D4E C127S CA01 Filipino Wild-Type Heterozygous CA02 Japanese Wild-Type Heterozygous CA03 Japanese Wild-Type Wild-Type CA19 Japanese Wild-Type Wild-Type CA20 Japanese Wild-Type Wild-Type CA04 orean Wild-Ty e Heterozygous CA05 Korean Wild-Type Wild-Type CA06 Korean Wild-Type Heterozygous CA07 Korean Wild-Type Wild-Type CA08 Korean Wild-Type Wild-Type CA09 Korean Wild-Type Wild-Type CC01 European Wild-Type Heterozygous CC02 European Wild-Type Wild-Type CC03 European Wild-Type Heterozygous CC04 European Wild-Type Wild-Type CC05 European Wild-Type Wild-Type CC06 European Wild-Type Wild-Type CC07 European Wild-Type Wild-Type CC08 European Wild-Type Wild-Type CC09 European Wild-Type Heterozygous CC19 European Wild-Type Wild-Type CC20 European Wild-Type Wild-Type CC21 European Wild-Type Wild-Type CC22 European Wild-Type Wild-Type CC23 European Wild-Type Wild-Type CC24 European Wild-Type Heterozygous CC25 European Wild-Type Wild-Type CC26 European Wild-Type Wild-Type CC27 European Wild-Type Wild-Type CC28 European Wild-Type Wild-Type CM01 Mongolian Wild-Type Wild-Type CM02 Mongolian Heterozygous Heterozygous CM03 Mongolian Wild-Type Heterozygous CM04 Mongolian Wild-Type Heterozygous CM05 Mongolian Wild-Type Heterozygous CM06 Mongolian Wild-Type Wild-Type CM07 Mongolian Wild-Type Heterozygous CM08 Mongolian Wild-Type Heterozygous CM09 Mongolian Wild-Type Heterozygous CM10 Mongolian Wild-Type Wild-Type 1
7 CM11 Mongolian Wild-Type Wild-Type CA10 Japanese Wild-Type Wild-Type CA11 Japanese Heterozygous Heterozygous CA12 Japanese Wild-Type Heterozygous CA13 Japanese Wild-Type Wild-Type CA14 Korean Wild-Type Wild-Type CA15 Korean Wild-Type Heterozygous CA16 Korean Wild-Type Wild-Type CA17 Japanese Wild-Type Wild-Type CA18 Japanese Wild-Type Heterozygous CCH01 Han Chinese Wild-Type Heterozygous CCH02 Han Chinese Wild-Type Wild-Type CCH03 Han Chinese Wild-Type Wild-Type CCH05 Han Chinese Wild-Type Heterozygous CCH06 Han Chinese Wild-Type Homozygous CCH07 Han Chinese Wild-Type Heterozygous CCH13 Han Chinese Wild-Type Wild-Type CCH16 Han Chinese Wild-Type Heterozygous CCH17 Han Chinese Wild-Type Heterozygous CCH18 Han Chinese Wild-Type Heterozygous CCH20 Han Chinese Wild-Type Homozygous CCH21 Han Chinese Wild-Type Heterozygous CCH23 Han Chinese Wild-Type Heterozygous CHN35 Han Chinese Wild-Type Heterozygous CHN37 Han Chinese Wild-Type Wild-Type CHN43 Han Chinese Wild-Type Heterozygous CAU01 European Wild-Type Heterozygous CAU02 European Wild-Type Heterozygous CAU03 European Wild-Type Wild-Type CAU04 European Wild-Type Wild-Type CAU05 European Wild-Type Wild-Type CAU06 European Wild-Type Heterozygous CAU07 European Wild-Type Wild-Type CAU08 European Wild-Type Wild-Type CAU09 European Wild-Type Heterozygous CAU10 European Wild-Type Wild-Type CAU11 European Wild-Type Wild-Type CAU12 European Wild-Type Wild-Type CAU13 European Wild-Type Wild-Type CAU14 European Wild-Type Wild-Type CAU15 European Wild-Type Wild-Type 2
8 CAU16 European Wild-Type Wild-Type CAU17 European Wild-Type Wild-Type CAU18 European Wild-Type Wild-Type CAU19 European Wild-Type Wild-Type CAU20 European Wild-Type Heterozygous SCAU01 European Wild-Type Wild-Type SCAU02 European Wild-Type Wild-Type SCAU03 European Wild-Type Wild-Type SCAU05 European Wild-Type Wild-Type SCAU06 European Wild-Type Wild-Type SCAU08 European Wild-Type Wild-Type SCAU09 European Wild-Type Wild-Type SCAU11 European Wild-Type Wild-Type SCAU10 European Wild-Type Wild-Type SCAU12 European Wild-Type Heterozygous SCAU13 European Wild-Type Wild-Type SCAU14 European Wild-Type Wild-Type SCAU15 European Wild-Type Wild-Type SCAU16 European Wild-Type Wild-Type SCAU17 European Wild-Type Wild-Type SCAU18 European Wild-Type Wild-Type SCAU19 European Wild-Type Wild-Type SCAU20 European Wild-Type Heterozygous CE28 European Wild-Type Wild-Type CE36 European Wild-Type Wild-Type CE30 European Wild-Type Wild-Type CE31 European Wild-Type Wild-Type CE32 European Wild-Type Wild-Type CE34 European Wild-Type Wild-Type CE35 European Wild-Type Wild-Type CE37 European Wild-Type Wild-Type CE38 European Wild-Type Wild-Type CE42 European Wild-Type Wild-Type CE43 European Wild-Type Wild-Type CE33 European Wild-Type Wild-Type CE39 European Wild-Type Wild-Type CE44 European Wild-Type Wild-Type CE51 European Wild-Type Wild-Type CE52 European Wild-Type Wild-Type CE54 European Wild-Type Wild-Type 3
9 Supplementary Table 1 List of the 121 controls of different ethnicity used in screening for EGLN1 mutation. 4
10 SampleID Origin PHD2- D4E PHD2- C127S MG03 India Heterozygous Heterozygous MG05 India Heterozygous Heterozygous MG07 India Heterozygous Homozygous MG08 India Heterozygous Wild- Type MG11 India Heterozygous Heterozygous MG12 India Heterozygous Heterozygous MG14 India Heterozygous Heterozygous MG15 India Homozygous Homozygous MG16 India Homozygous Homozygous MG17 India Heterozygous Heterozygous MG19 India Homozygous Homozygous MG20 India Heterozygous Heterozygous MG21 India Wild- Type Homozygous MG22 India Homozygous Homozygous MG24 India Heterozygous Heterozygous MG25 India Homozygous Homozygous MG27 India Heterozygous Heterozygous MG30 India Homozygous Homozygous MG32 India Homozygous Homozygous MG35 India Heterozygous Homozygous MG36 India Wild- Type Homozygous MG47 India Heterozygous Homozygous MG51 India Wild- Type Homozygous TCV5 India Heterozygous Homozygous TCV6 India Homozygous Homozygous TCV7 India Heterozygous Homozygous XT01 China Wild- Type Heterozygous XT02 China Wild- Type Wild- Type XT03 China Heterozygous Heterozygous XT04 China Homozygous Homozygous XT05 China Homozygous Homozygous XT06 China Homozygous Homozygous XT12 China Homozygous Homozygous XT13 China Wild- Type Homozygous XT16 China Homozygous Homozygous XT18 China Heterozygous Heterozygous XT20 China Heterozygous Heterozygous XT21 China Wild- Type Wild- Type 5
11 XT22 China Homozygous Homozygous ZT02 China Heterozygous Heterozygous ZT03 China Homozygous Homozygous ZT04 China Heterozygous Heterozygous ZT05 China Homozygous Homozygous ZT06 China Heterozygous Homozygous ZT07 China Homozygous Homozygous ZT11 China Heterozygous Homozygous ZT17 China Homozygous Homozygous ZT18 China Heterozygous Heterozygous ZT20 China Heterozygous Homozygous ZT23 China Heterozygous Heterozygous ZT24 China Heterozygous Heterozygous ZT26 China Heterozygous Heterozygous ZT27 China Heterozygous Heterozygous ZT29 China Homozygous Homozygous ZT30 China Homozygous Homozygous ZT31 China Homozygous Homozygous ZT32 China Heterozygous Homozygous ZT33 China Homozygous Homozygous ZT34 China Heterozygous Homozygous ZT36 China Homozygous Homozygous ZT37 China Heterozygous Homozygous ZT38 China Heterozygous Heterozygous ZT39 China Homozygous Homozygous ZT40 China Homozygous Homozygous ZT41 China Heterozygous Heterozygous Supplementary Table 2 List of the cohort Tibetan subjects (n=65) from India and China, screened for the EGLN1 variant. 6
12 EGLN1:c.12C>G EGLN1:c.380G>C Salt Lake & Virginia, USA (n=26) Mutation frequency 22 (85%) 26 (100%) Homozygous 20 (91%) 21 (81%) Heterozygous 2 (9%) 5 (19%) Allele frequency 42 / 52 (81%) 47 / 52 (90%) Himachal Pradesh, India (n=26) Mutation frequency 23 (89%) 25 (96%) Homozygous 8 (35%) 16 (64%) Heterozygous 15 (65%) 9 (36%) Allele frequency 31 / 52 (60%) 41 / 52 (79%) Qinghai province, China (n=39) Mutation frequency 35 (90%) 37 (95%) Homozygous 18 (51%) 24 (65%) Heterozygous 17 (49%) 13 (35%) Allele frequency 53 / 78 (68%) 61 / 78 (78%) Supplementary table 3 Tabulation of the mutation and allelic frequency from U.S. Tibetan and cohorts of Tibetans from India and China. 7
13 Observed number/proportion of recombinant chromosomes Number of PHD2 D4E chromosomes Recombinatio n rate (cm) Upstream SNPs Downstream SNPs Age (in generations) Age (in years, 25 year generation time) Log likelihood Maximum likelihood estimate % lower bound % upper bound SNPs* upstream of PHD2 D4E Chr. position (HG18) rs number Inferred ancestr al allele of EGLN1: c.12c>g Genetic distance to next SNP (in cm) Cumulative probability of observing a crossover (in cm) Cumulative proportion of EGLN1:c.12C>G Chr. without an observed crossover A A C A C C G A C G A G C G T A A C G C T T G
14 C T A C T G A G G C A G G A G G C G T G A G C A G C A A C T T T SNPs* downstream of PHD2 D4E Chr. position (HG18) rs number Inferred ancestral allele of EGLN1: c.12c>g Genetic distance to next SNP (in cm) Cumulative probability of observing a crossover (in cm) Cumulative proportion of EGLN1:c.12C>G Chr. without an observed crossover G G G C G A
15 T G C G A G T A C A C G A C G C C C C T T A C G C C C C Supplementary Table 4 Estimating the age of EGLN1:c.12C>G. *All SNPs were included within 0.25 cm of EGLN1:c.12C>G that had an allele frequency of the inferred ancestral allele of EGLN1:c.12C>G of less than 0.8 to ensure that only informative SNPs that could result observable recombination events were included in the analysis. 10
16 EGLN1/PHD2 primer set (NCBI Reference Sequence: NG_015865) Primers Sequence (5'- 3') Exon Tm ( O C) Expected size PHD2- X1F CCCCTATCTCTCTCCCCG bp PHD2- X1R CCTGTCCAGCACAAACCC 56.2 PHD2- X2F AACCTATCTTGTGTTCCCTA bp PHD2- X2R GAGAAAAAGACACCTGTAAG 46.3 PHD2_X3F TTTGGTTACTCCTGCTTACA bp PHD2_X3R CGTTTACTCTACAGATTCCC 49.1 PHD2- X4F AATACCACTTTTTCCCCGCA bp PHD2- X4R TTTCCTGAAAGCATCACCTG 56 PHD2- X5F TTGGAAGTACAAGCGTGAT bp PHD2- X5R TTCCTCCTGTAAGCAATCA 50.9 PPARA primer set (NCBI Reference Sequence: NG_012204) 5'- UTR Primers Sequence (5'- 3') Exo n Tm ( O C) Expected size PPARA X1F GAGGGGCGGTGCGTGTC bp PPARA X1R CCTCGCAAGAGTCCTCGGTG PPARA X2F TCCAGAGAACAACCGTAAT bp PPARA X2R GCATCACAAAGTAATAACCC PPARA v5x- F CAAAATACCGTAAACTGGG V bp PPARA v5x- R AGACACGATGCTCCTAAAC V Coding region Primers Sequence (5'- 3') Exo n Tm ( O C) Expected size PPARA X3F ATTGTTCCTCTTTCCTCCCA bp PPARA X3R GCACACTTACCCGTGATGAC PPARA X4F GCGAAATCACATCCTCATAG bp PPARA X4R TAAGTAGTTGATGGTGGCG PPARA X5F GAGCAACAAAAAAGGTGAG bp PPARA X5R AACTGAGGAACGAACTGG PPARA X6F ATGAAGGATGGGTCTGA bp PPARA X6R TTGCTTAACTGTGGGATA PPARA X7F TGGTGTCCTCCTTTGTAG bp PPARA X7R ACTGGATTTCCCATTGTC PPARA X8F AGCAGTTCTTGGGTGAT bp PPARA X8R AGGTTAAAGTGGTGGATAT v5 = PPARA variant 5 PPARA promoter region primer set 11
17 Primers Sequence (5'- 3') Exon Tm ( O C) Expected size PPRA_HNF4- REF TGCCAGGTAATGTCTTTGAG NA bp PPRA_HNF4- RER TCGGGAACGCAATGTG NA 53.6 EPAS1/HIF2A primer set (NCBI Reference Sequence: NG_016000) Primers Sequence (5'- 3') Exon Tm ( O C) Expected size HIF2A_gX1F GCGGGAGCTTTACACTCG bp HIF2A_gX1R GGAGAGAAGGGAACCCAAGT 57.1 HIF2A_gX2F AGGTATGATAGGCTGACAGTAAC bp HIF2A_gX2R GGATTCACGGTAAAGGTCA 52.8 HIF2A_gX3F GTCCCATCTGTTTTCACTCCA bp HIF2A_gX3R ACCTTCCACTCATTTCTACCC 55 HIF2A_gX4F AATCTGGAAGGTGGCTCA bp HIF2A_gX4R TCTCATCTCACAGGCTACATAC 51.9 HIF2A_gX5-6F CAATAGGCTGCCAAGAA bp HIF2A_gX5-6R TGGGAGACCACCAAATAC 50.1 HIF2A_gX7F TGTTTGATTTGCCTTCTG bp HIF2A_gX7R AGACTCAACCCACCCTC 48.2 HIF2A_gX8F CCTGGTCCTCACTGTCGT bp HIF2A_gX8R TCCCAACCCAGTATCCC 52.4 HIF2A_gX9F ATGCCTGGAGTCCTACC bp HIF2A_gX9R AGCAGATCCCTCACCTC 48.4 HIF2A_gX10F GTGAGCCCGATGGTTGTG bp HIF2A_gX10R GAACACTAAAAGGAAGAAAACAGC 57.1 HIF2A_gX11F GCTGAAGGGACATTTGG bp HIF2A_gX11R CACCAGACAGCAGGCAC 51.9 HIF2A_gX12F GGAATAGTGTTTGTGAGGTCGTA bp HIF2A_gX12R TAAATGGGGTATCAGATGGC 55 HIF2A_gX13F GCCAGGAAGCCCTAGCAT bp HIF2A_gX13R AGATGGAAAGGGGAAATGG 57.4 HIF2A_gX14-15F AGCCCATCCTGGTTCTTC bp HIF2A_gX14-15R CAGTATTCTAGCCTGAGTTTGAG 53.7 HIF2A_gX16F AGACACCACTGAAGGAGCA bp HIF2A_gX16R TTTGGCAACAACAATAACG 52.8 Sequence of primers used to generate baculoviruses coding for PHD2 D4E and C127S mutants Primers Forward Reverse PHD2:p.D4E GATCCATGGCCAATGAGAGCGGCG CGGGCCCGCCGCTCTCATTGGCCATGG 12
18 PHD2:p.C127S GGCCCGG CGGCCGCGTCGCCGTCTCGTGCGGC CGCC ATCC GCGGCCGCACGAGACGGCGACGCGGC CGC PHD2 primers for Real-Time Quantitative PCR Primers Sequence (5'- 3') Exon Tm ( O C) Expected size PHD2- qpcr- F TATCCGGGCAATGGAACGGGT TAT bp PHD2- qrcr- R GGTTCAATGTCAGCAAACTGG GCT PHD2- endogen- F TAAACCTTCAGATTCGGTCG bp PHD2- endogen- R CAGATTTGAAGTTGATCATGC A 3 - UTR 62.6 Hydrolysis probe use for expression assay Gene Probe GeneID Chr Name Cat# ALAS2 212 Xp11.21 aminolevulinate, delta-, synthase 2 Hs _m1 EGLN q42.1 egl nine homolog 1 (C. elegans) Hs _m1 EPAS p21- p16 endothelial PAS domain protein 1 / HIF2A Hs _m1 EPOR p13.3- p13.2 erythropoietin receptor Hs _m1 HIF1A q23.2 hypoxia inducible factor 1, alpha subunit Hs _m1 HK q22 hexokinase 1 Hs _m1 PDGFB q13.1 platelet- derived growth factor beta polypeptide Hs _m1 PDK q31.1 pyruvate dehydrogenase kinase, isozyme 1 Hs _m1 PPARA q13.31 peroxisome proliferator- activated receptor alpha Hs _m1 SLC2A p16.1 solute carrier family 2 (facilitated glucose Hs _m1 transporter) TFRC q29 transferrin receptor (p90, CD71) Hs _m1 TGFB q13.1 transforming growth factor, beta 1 Hs _m1 VEGFA p12 vascular endothelial growth factor A Hs _m1 VHL p25.3 von Hippel- Lindau tumor suppressor Hs _m1 13
19 Supplementary Table 5 List of sequence primers and hydrolysis probe sets use in the analysis. Supplementary Note USA Subjects of Tibetan ancestry residing in Utah and Virginia, USA were recruited and screened by FRL, TT, JP using IRB approved protocol. Each subject signed IRB approved Informed consent for blood donation. INDIA Subjects of Tibetan ancestry residing in Srinagar and Himachal Pradesh Indian provinces were recruited by our Indian collaborators, Dr. Parvaiz A. Koul (a co-author of this manuscript) from Jammu and Kashmir, and the Director of Tibetan Delek Hospital in Dharamshala Himachal Pradesh, Dr. Tsetan Dorji. One of us (TT) is a native Tibetan and he explained the project in Tibetan language and stressed again voluntary nature of our study. Each subject signed IRB approved Informed consent for blood donation. CHINA Subjects of Tibetan ancestry residing in Qinghai province were recruited by our Chinese collaborator Dr. Ge Ri-Li (a co-author of this manuscript). One of us (TT) is a native Tibetan and he explained the project in Tibetan language and stressed again voluntary nature of our study. Each subject signed IRB approved Informed consent for blood donation. 14
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