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.     

Screening of Microorganisms Producing 5-Amino-4-imidazole-carboxamide and D-Ribose from 5-Amino-4-imidazole-carboxamide-riboside

Rice has evolved metabolic and morphological adaptations to low-oxygen stress to grow in submerged paddy fields. To characterize the molecular components that mediate the response to hypoxia in rice, we identified low-oxygen stress early response genes by microarray analysis. Among the highly responsive genes, five genes, OsHREF1 to OsHREF5, shared strong homology. They encoded small proteins harboring two EF-hands, typical Ca²⁺-binding motifs. Homologous genes were found in many land plants, including SlHREF in tomato, which is also strongly induced by hypoxia. SlHREF induction was detected in both roots and shoots of tomato plants under hypoxia. With the exception of OsHREF5, OsHREF expression was unaffected by drought, salinity, cold, or osmotic stress. Fluorescent signals of green fluorescent protein-fused OsHREFs were detected in the cytosol and nucleus. Ruthenium red, an inhibitor of intracellular Ca²⁺ release, repressed induction of OsHREF1-4 under hypoxia. The HREFs may be related to the Ca²⁺ response to hypoxia.     

Genomic Analysis of Natural Selection and Phenotypic Variation in High-Altitude Mongolians

Deedu (DU) Mongolians, who migrated from the Mongolian steppes to the Qinghai-Tibetan Plateau approximately 500 years ago, are challenged by environmental conditions similar to native Tibetan highlanders. Identification of adaptive genetic factors in this population could provide insight into coordinated physiological responses to this environment. Here we examine genomic and phenotypic variation in this unique population and present the first complete analysis of a Mongolian whole-genome sequence. High-density SNP array data demonstrate that DU Mongolians share genetic ancestry with other Mongolian as well as Tibetan populations, specifically in genomic regions related with adaptation to high altitude. Several selection candidate genes identified in DU Mongolians are shared with other Asian groups (e.g., EDAR), neighboring Tibetan populations (including high-altitude candidates EPAS1, PKLR, and CYP2E1), as well as genes previously hypothesized to be associated with metabolic adaptation (e.g., PPARG). Hemoglobin concentration, a trait associated with high-altitude adaptation in Tibetans, is at an intermediate level in DU Mongolians compared to Tibetans and Han Chinese at comparable altitude. Whole-genome sequence from a DU Mongolian (Tianjiao1) shows that about 2% of the genomic variants, including more than 300 protein-coding changes, are specific to this individual. Our analyses of DU Mongolians and the first Mongolian genome provide valuable insight into genetic adaptation to extreme environments.     

Genetic screening of new genes responsible for cellular adaptation to hypoxia using a genome-wide shRNA library

Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.     

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.     

Comparative respiratory strategies of subterranean and fossorial octodontid rodents to cope with hypoxic and hypercapnic atmospheres

Subterranean rodents construct large and complex burrows and spend most of their lives underground, while fossorial species construct simpler burrows and are more active above ground. An important constraint faced by subterranean mammals is the chronic hypoxia and hypercapnia of the burrow atmosphere. The traits, regarded as “adaptations of rodents to hypoxia and hypercapnia”, have been evaluated in only a few subterranean species. In addition, well-studied subterranean taxa are very divergent to their sister groups, making it difficult to assess the adaptive path leading to subterranean life. The closely related sister genera Octodon and Spalacopus of Neotropical rodents offer a unique opportunity to trace the evolution of physiological mechanisms. We studied the ventilatory responses of selected octodontid rodents to selective pressures imposed by the subterranean niche under the working hypothesis that life underground, in hypoxic and hypercapnic conditions, promotes convergent physiological changes. To perform this study we used the following species: Spalacopus cyanus (the subterranean coruros) and Octodon degus (the fossorial degus) from central Chile. Ventilatory tidal volume and respiratory frequency were measured in non-anaesthetized spontaneously breathing animals. Acute hypoxic challenges (O₂ 1–15%) and hypercapnia (CO₂ 10%) were induced to study respiratory strategies using non-invasive whole body pletismography techniques. Our results show that coruros have a larger ventilatory response to acute hypoxia as than degus. On the other hand, hypercapnic respiratory responses in coruros seem to be attenuated when compared to those in degus. Our results suggest that coruros and degus have different respiratory strategies to survive in the hypoxic and hypercapnic atmospheres present in their burrows.     

GENETIC VARIATION IN HIF SIGNALING UNDERLIES QUANTITATIVE VARIATION IN PHYSIOLOGICAL AND LIFE‐HISTORY TRAITS WITHIN LOWLAND BUTTERFLY POPULATIONS

Oxygen conductance to the tissues determines aerobic metabolic performance in most eukaryotes but has cost/benefit tradeoffs. Here we examine in lowland populations of a butterfly a genetic polymorphism affecting oxygen conductance via the hypoxia‐inducible factor (HIF) pathway, which senses intracellular oxygen and controls the development of oxygen delivery networks. Genetically distinct clades of Glanville fritillary (Melitaea cinxia) across a continental scale maintain, at intermediate frequencies, alleles in a metabolic enzyme (succinate dehydrogenase, SDH) that regulates HIF‐1α. One Sdhd allele was associated with reduced SDH activity rate, twofold greater cross‐sectional area of tracheoles in flight muscle, and better flight performance. Butterflies with less tracheal development had greater post‐flight hypoxia signaling, swollen & disrupted mitochondria, and accelerated aging of flight metabolic performance. Allelic associations with metabolic and aging phenotypes were replicated in samples from different clades. Experimentally elevated succinate in pupae increased the abundance of HIF‐1α and expression of genes responsive to HIF activation, including tracheal morphogenesis genes. These results indicate that the hypoxia inducible pathway, even in lowland populations, can be an important axis for genetic variation underlying intraspecific differences in oxygen delivery, physiological performance, and life history.     

Effects of hypoxia on aerobic metabolism and active electrosensory acquisition in the African weakly electric fish Marcusenius victoriae

Environmental hypoxia has effected numerous and well-documented anatomical, physiological and behavioural adaptations in fishes. Comparatively little is known about hypoxia's impacts on sensing because it is difficult to quantify sensory acquisition in vivo. Weakly electric fishes, however, rely heavily on an easily-measurable sensory modality—active electric sensing—whereby individuals emit and detect electric organ discharges (EODs). In this study, hypoxia tolerance of a mormyrid weakly electric fish, Marcusenius victoriae, was assessed by examining both its metabolic and EOD rates using a critical threshold (p_crit) paradigm. The routine metabolic rate was 1.42 mg O₂ h⁻¹, and the associated critical oxygen tension was 14.34 mmHg. Routine EOD rate was 5.68 Hz with an associated critical tension of 15.14 mmHg. These metabolic indicators of hypoxia tolerance measured in this study were consistent with those in previous studies on M. victoriae and other weakly electric fishes. Furthermore, our results suggest that some aerobic processes may be reduced in favour of maintaining the EOD rate under extreme hypoxia. These findings underscore the importance of the active electrosensory modality to these hypoxia-tolerant fish.     

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.     

藏族的高原适应——西藏藏族生物人类学研究回顾

藏族生活在具有世界屋脊之称的青藏高原, 特殊的生态环境和特殊的文化背景造就了藏族特殊的适应高原缺氧机制, 引起了国内外学者的广泛关注和浓厚的研究兴趣。本文根据国内外数据库的文献并结合我们的研究工作, 从高原适应的角度回顾了30多年藏族人类学研究。回顾显示, 藏族由于长期生活在高原缺氧的环境中, 不仅形态和机能发生了适应性变化, 而且体成分也表现出相应的变化, 体现了形态、机能和体成分的统一。这些变化是长期进化形成的, 与安第斯山人等有明显不同, 就是在同一高原生活的西藏、青海、四川、甘肃和云南的藏族乃至尼泊尔和印度藏族的体质也表现出地域差异, 这些差异的产生是多种因素所致, 两个关键性的基因是导致两大高原人口高原适应机制不同的最主要的原因。     

Hypoxia-induced expression of HIF-1alpha and its target genes in umbilical venous endothelial cells of Tibetans and immigrant Han

The better adaptation of native Tibetans to hypoxia is thought to be partly due to improved umbilical circulation, which results in reduced pre- and postnatal fatalities. We hypothesized that the difference in umbilical circulation between native Tibetans and other high-altitude inhabitants was due to differences in the expression of hypoxia-induced factor (HIF-1) and its target genes vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). We tested this hypothesis by examining the effect of hypoxia on the expression of HIF-1alpha, VEGF, and iNOS in cultured umbilical venous endothelial cells (UVECs) from native Tibetans and immigrant Hans. UVECs were collected and cultured under hypoxic (0.5% oxygen) or normoxic conditions for 2, 4, 12 and 24 h. The mRNA levels of HIF-1alpha, VEGF, endothelial nitric oxide synthase (eNOS) and iNOS and the protein level of HIF-1alpha were determined with RT-PCR and Western blot analyses, respectively. In both immigrant Han and Tibetans, HIF-1alpha mRNA was constitutively expressed under normoxic condition, and remained constant after hypoxic exposure. In contrast, HIF-1alpha protein was undetectable under normoxic condition, but underwent dynamic changes in response to hypoxia. It was induced at 4 h, peaked at 12 h, and remained elevated at 24 h. Concurrent with the induction of HIF-1alpha protein, the mRNA levels of VEGF and iNOS were also up-regulated whereas that of eNOS was down-regulated. The lack of a hypoxia-related difference in the expression of HIF-1alpha and its target genes suggests that HIF-1alpha does not play a critical role in high altitude adaptation. Alternative mechanisms may be responsible for the better adaptation of native Tibetans.