Nat Genet. 2014 Sep;46(9):951-6. doi: 10.1038/ng.3067. Epub 2014 Aug 17.
A genetic mechanism for Tibetan high-altitude adaptation.
Lorenzo FR1, Huff C2, Myllymäki M3, Olenchock B4, Swierczek S5, Tashi T5, Gordeuk V6, Wuren T7, Ri-Li G7, McClain DA5, Khan TM8, Koul PA9, Guchhait P10, Salama ME11, Xing J12, Semenza GL13, Liberzon E14, Wilson A15, Simonson TS16, Jorde LB17, Kaelin WG Jr14, Koivunen P3, Prchal JT18.
1] Department of Medicine, University of Utah School of Medicine and George E. Wahlin Veterans Administration Medical Center, Salt Lake City, Utah, USA. [2].
1] Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA. [2] Department of Epidemiology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA. [3].
1] Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, Oulu, Finland. [2].
Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Department of Medicine, University of Utah School of Medicine and George E. Wahlin Veterans Administration Medical Center, Salt Lake City, Utah, USA.
Sickle Cell Center, University of Illinois, Chicago, Illinois, USA.
Research Center for High-Altitude Medicine, Qinghai University, Xining, People's Republic of China.
Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Sher-i-Kashmir Institute of Medical Sciences, Srinagar, India.
Regional Centre for Biotechnology, Gurgaon, India.
1] Department of Pathology, University of Utah, Salt Lake City, Utah, USA. [2] ARUP Laboratories, Hematopathology, Salt Lake City, Utah, USA.
1] Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA. [2] Department of Genetics, Rutgers, State University of New Jersey, Piscataway, New Jersey, USA.
Vascular Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
1] Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
Departmant of Family and Preventive Medicine, University of Utah School of Medicine, Salt Lake City, Utah, USA.
1] Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA. [2] Division of Physiology, University of California San Diego School of Medicine, La Jolla, California, USA.
Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA.
1] Department of Medicine, University of Utah School of Medicine and George E. Wahlin Veterans Administration Medical Center, Salt Lake City, Utah, USA. [2] Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA. [3].
Tibetans do not exhibit increased hemoglobin concentration at high altitude. We describe a high-frequency missense mutation in the EGLN1 gene, which encodes prolyl hydroxylase 2 (PHD2), that contributes to this adaptive response. We show that a variant in EGLN1, c.[12C>G; 380G>C], contributes functionally to the Tibetan high-altitude phenotype. PHD2 triggers the degradation of hypoxia-inducible factors (HIFs), which mediate many physiological responses to hypoxia, including erythropoiesis. The PHD2 p.[Asp4Glu; Cys127Ser] variant exhibits a lower K(m) value for oxygen, suggesting that it promotes increased HIF degradation under hypoxic conditions. Whereas hypoxia stimulates the proliferation of wild-type erythroid progenitors, the proliferation of progenitors with the c.[12C>G; 380G>C] mutation in EGLN1 is significantly impaired under hypoxic culture conditions. We show that the c.[12C>G; 380G>C] mutation originated ∼8,000 years ago on the same haplotype previously associated with adaptation to high altitude. The c.[12C>G; 380G>C] mutation abrogates hypoxia-induced and HIF-mediated augmentation of erythropoiesis, which provides a molecular mechanism for the observed protection of Tibetans from polycythemia at high altitude.
10.1038/ng.3067
Genome-wide allele frequency differentiation between Tibetans, Mongolians and Europeans. To measure genome-wide differentiation in allele frequency between Tibetans and Mongolians, we calculated the PBS for the c.12C>G and c.380G>C variants in EGLN1 for each Affymetrix SNP. We included all unrelated Tibetan, Mongolian and European individuals in the analysis for whom we had Affymetrix 6.0, c.12C>G and c.380G>C genotype data. The final set included 16 Tibetans, 11 Mongolians and 14 Europeans. The PBS for c.12C>G was 1.02 (empirical P value = 9 × 10−6). The four SNPs with PBS greater than 1.02 were all located in the EGLN1 region.
A genetic mechanism for Tibetan high-altitude adaptation
Nat Genet. 2014 Sep;46(9):951-956.
The p.[Asp4Glu; Cys127Ser] PHD2 mutant shows gain of function under hypoxia. (a) The Km value for O2 for the p.[Asp4Glu; Cys127Ser] PHD2 mutant is lower than that for wild-type PHD2. Shown is the effect of O2 concentration on the reaction velocity of wild-type PHD2 (red line) and the p.[Asp4Glu; Cys127Ser] mutant (blue line), using the HIF-2α ODDD as a substrate. The inset shows the corresponding double-reciprocal plot. The data presented are the average ± s.e.m. of eight independent assays comparing wild-type PHD2 and the p.[Asp4Glu; Cys127Ser] mutant in the same assay. Data from independent assays were normalized relative to the disintegrations per minute (d.p.m.) obtained with wild-type PHD2 at an O2 concentration of 212 μM. (b) Decreased HIF-2α accumulation in hypoxic cells expressing p.[Asp4Glu; Cys127Ser] PHD2 in knockdown-rescue experiments. Shown is a representative immunoblot analysis of Hep3B cells expressing Venus fluorescent protein (V), wild-type (WT) PHD2 or p.[Asp4Glu; Cys127Ser] PHD2. Cells were stably coinfected with an shRNA targeting the 3′ UTR of endogenous EGLN1 (not present in the rescue constructs) or a negative control shRNA and were grown in the presence of 21% or 1% O2 for 12 h. Decreased HIF-2α levels in cells expressing the p.[Asp4Glu; Cys127Ser] variant were observed in three independent experiments. The electrophoretic mobility of the exogenous PHD2 protein was slightly decreased owing to the presence of the Flag epitope. Tubulin was used as a loading control. (c) RT-PCR demonstrates knockdown of endogenous EGLN1 in Hep3B cells by the EGLN1-targeted shRNA and equal levels of transgene transcripts in cells expressing the transgenes for wild-type and p.[Asp4Glu; Cys127Ser] PHD2. Shown are representative data from three independent experiments.
Erythroid colony (BFU-E) assays. Erythroid colonies were enumerated after stimulation with various concentrations of EPO. (a) BFU-Es homozygous for the c.[12C>G; 380G>C] variant in EGLN1 are hypersensitive to EPO under normoxia. BFU-E colony assays in Tibetans homozygous (n = 2 biological replicates) or heterozygous (n = 1) for the c.[12C>G; 380G>C] variant in comparison to normal controls (n = 10) at different EPO concentrations. Values are normalized to the number of BFU-Es grown at ambient oxygen tension at 3,000 mU/ml EPO, which was expressed as 100%. Designated error bars in controls and for cells homozygous for c.[12C>G; 380G>C] represent ±s.d. (b) Sensitivity of BFU-Es to low EPO concentration (15 mU/ml). Hypersensitive BFU-Es from Tibetans with the c.[12C>G; 380G>C] variant (n = 6) lose their hypersensitivity to EPO as the oxygen tension decreases to 5% and fail to form colonies at 1% O2, whereas control colonies (n = 3) become hypersensitive to EPO with increasing hypoxia. (c) Colony growth of progenitor cells from a Tibetan with the c.[12C>G; 380G>C] mutation and controls at 3,000 mU/ml EPO. Tibetan BFU-E colonies with the c.[12C>G; 380G>C] mutation have decreased colony formation with ambient O2 levels and 5% O2, and they fail to grow at 1% O2. In contrast, BFU-Es from control subjects (n = 3) form more colonies as the oxygen tension decreases. (d) Size of erythroid colonies increases in BFU-Es from controls but decreases for progenitor cells from a Tibetan with the c.[12C>G; 380G>C] mutation. Average BFU-E colony size represented in pixels (y axis) is greater in BFU-E colonies from the control subject under hypoxia (5% O2), whereas, in the Tibetan subject with the c.[12C>G; 380G>C] mutation, hypoxia decreases BFU-E colony size. Error bars in Tibetans and controls represent ±s.d. (Fig. 3a is an independent experiment performed with different individuals and at a different time than the experiments in Fig. 3b–d, which consist of 6 biological replicates (5 Tibetans homozygous and 1 Tibetan heterozygous for c.[12C>G; 380G>C]) together with 3 controls.)
Hypoxia increases the proliferation of control BFU-E colonies but decreases that of colonies with the c.[12C>G; 380G>C] mutation in EGLN1. BFU-Es were grown in 3,000 mU/ml EPO, and all images were acquired at 40× magnification (scale bars, 1 mm). (a,b) Representative colonies from a control (wild type) subject and (c,d) a Tibetan subject homozygous for the c.[12C>G; 380G>C] mutation. Note the larger colony sizes for the control BFU-E colonies under hypoxia (5% O2) (b) relative to ambient oxygen tension (a). BFU-Es with the c.[12C>G; 380G>C] mutation exhibit smaller colony sizes under normoxia (c) in comparison to control cells (a), and the colonies are paler than the controls, reflecting the decreased hemoglobinization with the c.[12C>G; 380G>C] erythroid progenitors in comparison to the controls. (d) The decrease in colony size and hemoglobinization for Tibetan BFU-Es with the c.[12C>G; 380G>C] mutation is even more pronounced under 5% O2 (in comparison to b).
Acclimatization/genetics*
Adaptation, Physiological/genetics*
Asian Continental Ancestry Group/genetics*
Erythropoiesis/genetics
Hypoxia/genetics
Hypoxia-Inducible Factor-Proline Dioxygenases/genetics
Polycythemia/genetics
Polymorphism, Single Nucleotide
EGLN1 protein, human
Hypoxia-Inducible Factor-Proline Dioxygenases
HG005846/HG/NHGRI NIH HHS/United States
P01CA108671/CA/NCI NIH HHS/United States
I01 BX001140/BX/BLRD VA/United States
K08 HL119355/HL/NHLBI NIH HHS/United States
R00 HL118215/HL/NHLBI NIH HHS/United States
HL119355-01/HL/NHLBI NIH HHS/United States
UL1 TR001067/TR/NCATS NIH HHS/United States
HL098062/HL/NHLBI NIH HHS/United States