Genetic variations in Tibetan populations and high-altitude adaptation at the Himalayas

Modern humans have occupied almost all possible environments globally since exiting Africa about 100,000 years ago. Both behavioral and biological adaptations have contributed to their success in surviving the rigors of climatic extremes, including cold, strong ultraviolet radiation, and high altitude. Among these environmental stresses, high-altitude hypoxia is the only condition in which traditional technology is incapable of mediating its effects. Inhabiting at >3,000-m high plateau, the Tibetan population provides a widely studied example of high-altitude adaptation. Yet, the genetic mechanisms underpinning long-term survival in this environmental extreme remain unknown. We performed an analysis of genome-wide sequence variations in Tibetans. In combination with the reported data, we identified strong signals of selective sweep in two hypoxia-related genes, EPAS1 and EGLN1. For these two genes, Tibetans show unusually high divergence from the non-Tibetan lowlanders (Han Chinese and Japanese) and possess high frequencies of many linked sequence variations as reflected by the Tibetan-specific haplotypes. Further analysis in seven Tibetan populations (1,334 individuals) indicates the prevalence of selective sweep across the Himalayan region. The observed indicators of natural selection on EPAS1 and EGLN1 suggest that during the long-term occupation of high-altitude areas, the functional sequence variations for acquiring biological adaptation to high-altitude hypoxia have been enriched in Tibetan populations.     

Tibetan and Andean patterns of adaptation to high-altitude hypoxia

Understanding the workings of the evolutionary process in contemporary humans requires linking the evolutionary history of traits with their current genetics and biology. Unusual environments provide natural experimental settings to investigate evolution and adaptation. The example of high-altitude hypoxia illustrates some of the progress and many of the remaining challenges for studies of evolution in contemporary populations. Current studies exemplify the frequently encountered problem of determining whether large, consistent population differences in mean values of a trait reflect genetic differences. In this review I describe 4 quantitative traits that provide evidence that indigenous populations of the Tibetan and Andean plateaus differ in their phenotypic adaptive responses to high-altitude hypoxia. These 4 traits are resting ventilation, hypoxic ventilatory response, oxygen saturation, and hemoglobin concentration. The Tibetan means of the first 2 traits were more than 0.5 standard deviation higher than the Aymara means, whereas the Tibetan means were more than 1 standard deviation lower than the Aymara means for the last 2 traits. Quantitative genetic analyses of within-population variance revealed significant genetic variance in all 4 traits in the Tibetan population but only in hypoxic ventilatory response and hemoglobin concentration in the Aymara population. A major gene for oxygen saturation was detected among the Tibetans. These findings are interpreted as indirect evidence of population genetic differences. It appears that the biological characteristics of sea-level humans did not constrain high-altitude colonists of the 2 plateaus to a single adaptive response. Instead, microevolutionary processes may have operated differently in the geographically separated Tibetan and Andean populations exposed to the same environmental stress. Knowledge of the genetic bases of these traits will be necessary to evaluate these inferences. Future research will likely be directed toward determining whether the population means reflect differences identified at the chromosomal level. Future research will also likely consider the biological pathways and environmental influences linking genotypes to phenotypes, the costs and benefits of the Tibetan and Andean patterns of adaptation, and the question of whether the observed phenotypes are indeed adaptations that enhance Darwinian fitness.     

Identifying Signatures of Natural Selection in Tibetan and Andean Populations Using Dense Genome Scan Data

High-altitude hypoxia (reduced inspired oxygen tension due to decreased barometric pressure) exerts severe physiological stress on the human body. Two high-altitude regions where humans have lived for millennia are the Andean Altiplano and the Tibetan Plateau. Populations living in these regions exhibit unique circulatory, respiratory, and hematological adaptations to life at high altitude. Although these responses have been well characterized physiologically, their underlying genetic basis remains unknown. We performed a genome scan to identify genes showing evidence of adaptation to hypoxia. We looked across each chromosome to identify genomic regions with previously unknown function with respect to altitude phenotypes. In addition, groups of genes functioning in oxygen metabolism and sensing were examined to test the hypothesis that particular pathways have been involved in genetic adaptation to altitude. Applying four population genetic statistics commonly used for detecting signatures of natural selection, we identified selection-nominated candidate genes and gene regions in these two populations (Andeans and Tibetans) separately. The Tibetan and Andean patterns of genetic adaptation are largely distinct from one another, with both populations showing evidence of positive natural selection in different genes or gene regions. Interestingly, one gene previously known to be important in cellular oxygen sensing, EGLN1 (also known as PHD2), shows evidence of positive selection in both Tibetans and Andeans. However, the pattern of variation for this gene differs between the two populations. Our results indicate that several key HIF-regulatory and targeted genes are responsible for adaptation to high altitude in Andeans and Tibetans, and several different chromosomal regions are implicated in the putative response to selection. These data suggest a genetic role in high-altitude adaption and provide a basis for future genotype/phenotype association studies necessary to confirm the role of selection-nominated candidate genes and gene regions in adaptation to altitude.     

Andean, Tibetan, and Ethiopian patterns of adaptation to high-altitude hypoxia

Research on humans at high-altitudes contributes to understandingthe processes of human adaptation to the environment and evolution.The unique stress at high altitude is hypobaric hypoxia causedby the fall in barometric pressure with increasing altitudeand the consequently fewer oxygen molecules in a breath of air,as compared with sea level. The natural experiment of humancolonization of high-altitude plateaus on three continents hasresulted in two—perhaps three—quantitatively differentarterial-oxygen-content phenotypes among indigenous Andean,Tibetan and Ethiopian high-altitude populations. This paperillustrates these contrasting phenotypes by presenting evidencefor higher hemoglobin concentration and percent of oxygen saturationof hemoglobin among Andean highlanders as compared with Tibetansat the same altitude and evidence that Ethiopian highlandersdo not differ from sea-level in these two traits. Evolutionaryprocesses may have acted differently on the colonizing populationsto cause the different patterns of adaptation. Hemoglobin concentrationhas significant heritability in Andean and Tibetan samples.Oxygen saturation has no heritability in the Andean sample,but does among Tibetans where an autosomal dominant major genefor higher oxygen saturation has been detected. Women estimatedwith high probability to have high oxygen saturation genotypeshave more surviving children than women estimated with highprobability to have the low oxygen saturation genotype. Thesefindings suggest the hypothesis that ongoing natural selectionis increasing the frequency of the high saturation allele atthis major gene locus.     

Hemoglobin concentration of high-altitude Tibetans and Bolivian Aymara

Elevated hemoglobin concentrations have been reported for high-altitude sojourners and Andean high-altitude natives since early in the 20th century. Thus, reports that have appeared since the 1970s describing relatively low hemoglobin concentration among Tibetan high-altitude natives were unexpected. These suggested a hypothesis of population differences in hematological response to high-altitude hypoxia. A case of quantitatively different responses to one environmental stress would offer an opportunity to study the broad evolutionary question of the origin of adaptations. However, many factors may confound population comparisons. The present study was designed to test the null hypothesis of no difference in mean hemoglobin concentration of Tibetan and Aymara native residents at 3,800–4,065 meters by using healthy samples that were screened for iron deficiency, abnormal hemoglobins, and thalassemias, recruited and assessed using the same techniques. The hypothesis was rejected, because Tibetan males had a significantly lower mean hemoglobin concentration of 15.6 gm/dl compared with 19.2 gm/dl for Aymara males, and Tibetan females had a mean hemoglobin concentration of 14.2 gm/dl compared with 17.8 gm/dl for Aymara females. The Tibetan hemoglobin distribution closely resembled that from a comparable, sea-level sample from the United States, whereas the Aymara distribution was shifted toward 3–4 gm/dl higher values. Genetic factors accounted for a very high proportion of the phenotypic variance in hemoglobin concentration in both samples (0.86 in the Tibetan sample and 0.87 in the Aymara sample). The presence of significant genetic variance means that there is the potential for natural selection and genetic adaptation of hemoglobin concentration in Tibetan and Aymara high-altitude populations. Am J Phys Anthropol 106:385–400, 1998. © 1998 Wiley-Liss, Inc.     

Correlation of DNA methylation patterns to the phenotypic features of Tibetan elite alpinists in extreme hypoxia

High altitude is an extreme environment that imposes hypoxic pressure on physiological processes, and natives living at high altitudes are more adaptive in certain physiological processes. So far, epigenetic modifications under extreme changes in hypoxic pressures are relatively less understood. Here, we recruit 32 Tibetan elite alpinists (TEAs), who have successfully mounted Everest (8848 m) at least five times. Blood samples and physiological phenotypes of TEAs and 32 matched non-alpinist Tibetan volunteers (non-TEAs) are collected for analysis. Genome-wide DNA methylation analysis identifies 23,202 differentially methylated CpGs (P_adj < 0.05, |β| > 0.1) between the two groups. Some differentially methylated CpGs are in hypoxia-related genes such as PPP1R13L, MAP3K7CL, SEPTI-9, and CUL2. In addition, Gene ontology enrichment analysis reveals several inflammation-related pathways. Phenotypic analysis indicates that 12 phenotypes are significantly different between the two groups. In particular, TEAs exhibit higher blood oxygen saturation levels and lower neutrophil count, platelet count, and heart rate. For DNA methylation association analysis, we find that two CpGs (cg16687447, cg06947206) upstream of PTEN were associated with platelet count. In conclusion, extreme hypoxia exposure leads to epigenetic modifications and phenotypic alterations of TEA, providing us clues for exploring the molecular mechanism underlying changes under extreme hypoxia conditions.     

Genetic association analysis highlights new loci that modulate hematological trait variation in Caucasians and African Americans

Red blood cell, white blood cell, and platelet measures, including their count, sub-type and volume, are important diagnostic and prognostic clinical parameters for several human diseases. To identify novel loci associated with hematological traits, and compare the architecture of these phenotypes between ethnic groups, the CARe Project genotyped 49,094 single nucleotide polymorphisms (SNPs) that capture variation in ~2,100 candidate genes in DNA of 23,439 Caucasians and 7,112 African Americans from five population-based cohorts. We found strong novel associations between erythrocyte phenotypes and the glucose-6 phosphate dehydrogenase (G6PD) A-allele in African Americans (rs1050828, P<2.0×10(-13), T-allele associated with lower red blood cell count, hemoglobin, and hematocrit, and higher mean corpuscular volume), and between platelet count and a SNP at the tropomyosin-4 (TPM4) locus (rs8109288, P=3.0×10(-7) in Caucasians; P=3.0×10(-7) in African Americans, T-allele associated with lower platelet count). We strongly replicated many genetic associations to blood cell phenotypes previously established in Caucasians. A common variant of the α-globin (HBA2-HBA1) locus was associated with red blood cell traits in African Americans, but not in Caucasians (rs1211375, P<7×10(-8), A-allele associated with lower hemoglobin, mean corpuscular hemoglobin, and mean corpuscular volume). Our results show similarities but also differences in the genetic regulation of hematological traits in European- and African-derived populations, and highlight the role of natural selection in shaping these differences.     

The Genetic Architecture of Adaptations to High Altitude in Ethiopia

Although hypoxia is a major stress on physiological processes, several human populations have survived for millennia at high altitudes, suggesting that they have adapted to hypoxic conditions. This hypothesis was recently corroborated by studies of Tibetan highlanders, which showed that polymorphisms in candidate genes show signatures of natural selection as well as well-replicated association signals for variation in hemoglobin levels. We extended genomic analysis to two Ethiopian ethnic groups: Amhara and Oromo. For each ethnic group, we sampled low and high altitude residents, thus allowing genetic and phenotypic comparisons across altitudes and across ethnic groups. Genome-wide SNP genotype data were collected in these samples by using Illumina arrays. We find that variants associated with hemoglobin variation among Tibetans or other variants at the same loci do not influence the trait in Ethiopians. However, in the Amhara, SNP rs10803083 is associated with hemoglobin levels at genome-wide levels of significance. No significant genotype association was observed for oxygen saturation levels in either ethnic group. Approaches based on allele frequency divergence did not detect outliers in candidate hypoxia genes, but the most differentiated variants between high- and lowlanders have a clear role in pathogen defense. Interestingly, a significant excess of allele frequency divergence was consistently detected for genes involved in cell cycle control and DNA damage and repair, thus pointing to new pathways for high altitude adaptations. Finally, a comparison of CpG methylation levels between high- and lowlanders found several significant signals at individual genes in the Oromo.     

Sequencing and alignment of mitochondrial genomes of Tibetan chicken and two lowland chicken breeds

Tibetan chicken lives in high-altitude area and has adapted well to hypoxia genetically. Shouguang chicken and Silky chicken are both lowland chicken breeds. In the present study, the complete mitochondrial genome sequences of the three chicken breeds were all sequenced. The results showed that the mitochondrial DNAs (mtDNAs) of Shouguang chicken and Silky chicken consist of 16784 bp and 16785 bp respectively, and Tibetan chicken mitochondrial genome varies from 16784 bp to 16786 bp. After sequence analysis, 120 mutations, including 4 single nucleotide polymorphisms (SNPs) in tRNA genes, 9 SNPs and 1 insertion in rRNA genes, 38 SNPs and 1 deletion in D-LOOP, 66 SNPs in protein-coding genes, were found. This work will provide clues for the future study on the association between mitochondrial genes and the adaptation to hypoxia.     

Sequencing and alignment of mitochondrial genomes of Tibetan chicken and two lowland chicken breeds

Tibetan chicken lives in high-altitude area and has adapted well to hypoxia genetically. Shouguang chicken and Silky chicken are both lowland chicken breeds. In the present study, the complete mito-chondrial genome sequences of the three chicken breeds were all sequenced. The results showed that the mitochondrial DNAs (mtDNAs) of Shouguang chicken and Silky chicken consist of 16784 bp and 16785 bp respectively, and Tibetan chicken mitochondrial genome varies from 16784 bp to 16786 bp. After sequence analysis, 120 mutations, including 4 single nucleotide polymorphisms (SNPs) in tRNA genes, 9 SNPs and 1 insertion in rRNA genes, 38 SNPs and 1 deletion in D-LOOP, 66 SNPs in pro-tein-coding genes, were found. This work will provide clues for the future study on the association between mitochondrial genes and the adaptation to hypoxia.Tibetan chicken, lowland chicken, mitochondrial genome, hypoxia.     

Elevation as a selective force on mitochondrial respiratory chain complexes of the Phrynocephalus lizards in the Tibetan plateau

Animals living in extremely high elevations have to adapt to low temperatures and low oxygen availability (hypoxia), but the underlying genetic mechanisms associated with these adaptations are still unclear. The mitochondrial respiratory chain can provide >95% of the ATP in animal cells, and its efficiency is influenced by temperature and oxygen availability. Therefore, the respiratory chain complexes (RCCs) could be important molecular targets for positive selection associated with respiratory adaptation in high-altitude environments. Here, we investigated positive selection in 5 RCCs and their assembly factors by analyzing sequences of 106 genes obtained through RNA-seq of all 15 Chinese Phrynocephalus lizard species, which are distributed from lowlands to the Tibetan plateau (average elevation >4,500 m). Our results indicate that evidence of positive selection on RCC genes is not significantly different from assembly factors, and we found no difference in selective pressures among the 5 complexes. We specifically looked for positive selection in lineages where changes in habitat elevation happened. The group of lineages evolving from low to high altitude show stronger signals of positive selection than lineages evolving from high to low elevations. Lineages evolving from low to high elevation also have more shared codons under positive selection, though the changes are not equivalent at the amino acid level. This study advances our understanding of the genetic basis of animal respiratory metabolism evolution in extreme high environments and provides candidate genes for further confirmation with functional analyses.     

Effect of altitude exposure on platelets.

Since decompression from depth is known to produce a fall in platelet count, the effect of altitude decompression and high-altitude exposure on platelets was investigated. Sixteen subjects decompressed without hypoxia to 20,000 ft simulated altitude for two hours showed a significant (P less than 0.01) drop in circulating platelet count of approximately 10% for three days following decompression. Four of five subjects similarly exposed had a shortened autologous platelet survival compared to that prior to exposure. Subjects exposed to 9,800 ft and then 17,600 ft in a mountain environment showed a significant mean decrease in platelet count on day 2 of 7% and 25% respectively, which had returned to control by day 5. Nonhypoxic and hypoxic decompressed rabbits which received homologous chromium-51-labeled platelets had an increase in lung radioactivity compared with sea-level controls. It is postulated that altitude decompression produces platelet reductions similar to these seen after decompression from depth, and that platelets sequester in the pulmonary vascular bed.     

GCH1 plays a role in the high-altitude adaptation of Tibetans

Tibetans are well adapted to high-altitude hypoxia.Previous genome-wide scans have reported many candidate genes for this adaptation,but only a few have been studied.Here we report on a hypoxia gene (GCH1,GTP-cyclohydrolase I),involved in maintaining nitric oxide synthetase (NOS) function and normal blood pressure,that harbors many potentially adaptive variants in Tibetans.We resequenced an 80.8 kb fragment covering the entire gene region of GCH1 in 50 unrelated Tibetans.Combined with previously published data,we demonstrated many GCH1 variants showing deep divergence between highlander Tibetans and lowlander Han Chinese.Neutrality tests confirmed a signal of positive Darwinian selection on GCH1 in Tibetans.Moreover,association analysis indicated that the Tibetan version of GCH1 was significantly associated with multiple physiological traits in Tibetans,including blood nitric oxide concentration,blood oxygen saturation,and hemoglobin concentration.Taken together,we propose that GCH1 plays a role in the genetic adaptation of Tibetans to high altitude hypoxia.     

Growth and nutritional status of Tibetan children at high altitude

Growth and development are clearly affected by high-altitude exposure to hypoxia, nutritional stress, cold or a combination of these factors. Very little research has been conducted on the growth and nutritional status of children living on the Tibetan Plateau. The present study evaluated the environmental impact on human growth by analyzing anthropometric characteristics of Tibetan children aged 8-14, born and raised above 4000 m altitude on the Himalayan massif in the prefecture of Shegar in Tibet Autonomous Region. Data on anthropometric traits, never measured in this population, were collected and the nutritional status was assessed. A reference data set is provided for this population. There was no evidence of wasting but stunting was detected (28.3%). Children permanently exposed to the high-altitude environment above 4000 m present a phenotypic form of adaptation and a moderate reduction in linear growth. However, it is also necessary to consider the effects of socioeconomic deprivation.     

C 蛋白基因多态性对脓毒血症患者血小板功能的影响

目的:探讨蛋白C基因突变及基因型频率分布和脓毒血症患者血小板功能及血清TXB2水平的相关性。方法:纳入112例脓毒血症患者,健康人群50例为对照组。采用PCR-RFLP法测定所有受试者蛋白C基因rs 17999808C/A位点和rs1799809位点基因型及突变率。入院24小时内测定脓毒血症患者血小板计数、最大聚集率及血清TXB2水平,并对其进行SOFA评分。结果:病例组和对照组rs17999808位点和rs1799809位点间基因型分布频率无统计学差异(P0.05)。rs 17999808基因型分布C/C占81.48%、C/A占12.96%、A/A占5.55%。rs1799809位点G/G占76.54%、G/C占15.43%、C/C占8.02%。rs17999808位点和rs1799809位点突变率分贝为12.03%、15.74%。病例死亡34例,死亡率30.35%。rs17999808位点突变纯合子患者(A/A)死亡率及SOFA评分明显增高,和野生纯合子及突变杂合子患者差异有统计学意义(P0.05)。rs17999808位点C/C野生纯合子患者血小板计数和TXB2浓度明显高于C/A和A/A患者,血小板聚集率低于后两者,差异有统计学意义(P0.05)。突变纯合子A/A患者较C/C、C/A患者两两相比,差异有统计学意义(P0.05)。rs17999809位点突变和TXB2浓度有相关性(P0.05)。结论:蛋白C基因rs17999808位点突变增加了脓毒血症患者死亡风险,这可能和其改变血小板功能有关。     

Platelet count and function at high altitude and in high-altitude pulmonary edema.

Platelet aggregation is the key process in primary hemostasis. Certain conditions such as hypoxia may induce platelet aggregation and lead to platelet sequestration primarily in the pulmonary microcirculation. We investigated the influence of high-altitude exposure on platelet function as part of a larger study on 30 subjects with a history of high-altitude pulmonary edema (HAPE) and 10 healthy controls. All participants were studied in the evening and the next morning at low altitude (450 m) and after an ascent to high altitude (4,559 m). Platelet count, platelet aggregation (platelet function analyzer PFA100; using epinephrine and ADP as activators), plasma soluble P (sP)-selectin, and the coagulation parameters prothrombin fragments 1+2 and thrombin-antithrombin complex were measured. High-altitude exposure decreased the platelet count, shortened the platelet function analyzer closure time by approximately 20%, indicating increased platelet aggregation, increased sP-selectin levels to approximately 250%, but left plasma coagulation unaffected. The HAPE-susceptible subjects were prophylactically treated with either tadalafil (a phosphodiesterase 5 inhibitor), dexamethasone, or placebo in a double-blind way. Subgroup analyses between these different treatments and comparisons of the seven placebo-treated individuals developing HAPE and controls revealed no differences in platelet count, platelet aggregation, or sP-selectin values. We conclude that exposure to high altitude activates platelets, which leads to platelet aggregation, platelet consumption, and decreased platelet count. These effects are, however, not more pronounced in individuals with a history of HAPE or actually suffering from HAPE than in controls and therefore may not be a pathophysiological mechanism of HAPE.     

Genetic adaptation to high altitude in the Ethiopian highlands

Background

Genomic analysis of high-altitude populations residing in the Andes and Tibet has revealed several candidate loci for involvement in high-altitude adaptation, a subset of which have also been shown to be associated with hemoglobin levels, including EPAS1, EGLN1, and PPARA, which play a role in the HIF-1 pathway. Here, we have extended this work to high- and low-altitude populations living in Ethiopia, for which we have measured hemoglobin levels. We genotyped the Illumina 1M SNP array and employed several genome-wide scans for selection and targeted association with hemoglobin levels to identify genes that play a role in adaptation to high altitude.

Results

We have identified a set of candidate genes for positive selection in our high-altitude population sample, demonstrated significantly different hemoglobin levels between high- and low-altitude Ethiopians and have identified a subset of candidate genes for selection, several of which also show suggestive associations with hemoglobin levels.

Conclusions

We highlight several candidate genes for involvement in high-altitude adaptation in Ethiopia, including CBARA1, VAV3, ARNT2 and THRB. Although most of these genes have not been identified in previous studies of high-altitude Tibetan or Andean population samples, two of these genes (THRB and ARNT2) play a role in the HIF-1 pathway, a pathway implicated in previous work reported in Tibetan and Andean studies. These combined results suggest that adaptation to high altitude arose independently due to convergent evolution in high-altitude Amhara populations in Ethiopia.     

HIF2A Variants Were Associated with Different Levels of High-Altitude Hypoxia among Native Tibetans

Hypoxia inducible factors, including HIF1A and HIF2A, play central roles in response to high-altitude hypoxia and genetic variants of HIF1A or HIF2A were associated with high-altitude sickness or adaptation. However, it remains to determine whether they are associated with tolerance to different levels of high-altitude selection pressure among native Tibetans. We recruited 189 Tibetan subjects living at 2,700 meters (Low level of high altitude, LHA), 197 at 3,200 meters (Middle level of high altitude of high altitude, MHA), 249 at 3,700 meters (High level of high altitude, HHA) and 269 at 4,700 meters (Very high level of high altitude, VHA) and performed association analysis of twelve tSNPs (tagging SNPs) in HIF1A and HIF2A with high-altitude. We found (1) a increasing trend of HIF2A rs5621780-C(18.4%, 15.9%, 32.8% and 31.1%, respectively, in LHA, MHA, HHA and VHA)(P = 3.56E-9); (2) increasing trends of HIF2A rs6756667-A(68.7%, 73.4%, 79.9% and 89.6%), rs7589621- G(74.6%, 77.9%, 83.7%, and 92.1%) and rs1868092-A(64.1%, 67.3%, 75.1% and 84.4%) (P = 3.56E-9, 4.68E-16, 1.17E-13 and 7.09E-14, respectively); (3) a increasing trend of haplotype AG (68.7%, 73.1%, 79.9% and 89.6%) (P = 2.22E-7) which was constructed by rs6756667 and rs7589621; (4) a strong linear correlation between major alleles of rs6756667-A (R ² = 0.997, P = 0.002), rs7589621-G (R ² = 0.994, P = 0.003), rs1868092-A (R ² = 0.985, P = 0.008) and altitude by linear correlation test. The associations between HIF2A variants and different level of high altitude support that extremely high-altitude hypoxia challenge imposes selective effects on HIF2A variants among native Tibetans.