Tibetans are well adapted to the hypoxic environments at high altitude, yet the molecular mechanism of this adaptation remains elusive. We reported comprehensive genetic and functional analyses of EPAS1, a gene encoding hypoxia inducible factor 2α (HIF-2α) with the strongest signal of selection in previous genome-wide scans of Tibetans. We showed that the Tibetan-enriched EPAS1 variants down-regulate expression in human umbilical endothelial cells and placentas. Heterozygous EPAS1 knockout mice display blunted physiological responses to chronic hypoxia, mirroring the situation in Tibetans. Furthermore, we found that the Tibetan version of EPAS1 is not only associated with the relatively low hemoglobin level as a polycythemia protectant, but also is associated with a low pulmonary vasoconstriction response in Tibetans. We propose that the down-regulation of EPAS1 contributes to the molecular basis of Tibetans’ adaption to high-altitude hypoxia.

Structural variants (SVs) may play important roles in human adaptation to extreme environments such as high altitude but have been under-investigated. Here, combining long-read sequencing with multiple scaffolding techniques, we assembled a high-quality Tibetan genome (ZF1), with a contig N50 length of 24.57 mega-base pairs (Mb) and a scaffold N50 length of 58.80 Mb. The ZF1 assembly filled 80 remaining N-gaps (0.25 Mb in total length) in the reference human genome (GRCh38). Markedly, we detected 17 900 SVs, among which the ZF1-specific SVs are enriched in GTPase activity that is required for activation of the hypoxic pathway. Further population analysis uncovered a 163-bp intronic deletion in the MKL1 gene showing large divergence between highland Tibetans and lowland Han Chinese. This deletion is significantly associated with lower systolic pulmonary arterial pressure, one of the key adaptive physiological traits in Tibetans. Moreover, with the use of the high-quality de novo assembly, we observed a much higher rate of genome-wide archaic hominid (Altai Neanderthal and Denisovan) shared non-reference sequences in ZF1 (1.32%–1.53%) compared to other East Asian genomes (0.70%–0.98%), reflecting a unique genomic composition of Tibetans. One such archaic hominid shared sequence—a 662-bp intronic insertion in the SCUBE2 gene—is enriched and associated with better lung function (the FEV1/FVC ratio) in Tibetans. Collectively, we generated the first high-resolution Tibetan reference genome, and the identified SVs may serve as valuable resources for future evolutionary and medical studies.

Tibetans are well adapted to high-altitude environments. Among the adaptive traits in Tibetans, the relatively low hemoglobin level is considered a blunted erythropoietic response to hypoxic challenge. Previously, EPAS1 and EGLN1, the major upstream regulators in the hypoxic pathway, were reportedly involved in the hemoglobin regulation in Tibetans. In this study, we report a downstream gene (HMOX2) involved in heme catabolism, which harbors potentially adaptive variants in Tibetans. We first resequenced the entire genomic region (45.6 kb) of HMOX2 in Tibetans, which confirmed the previously suspected signal of positive selection on HMOX2 in Tibetans. Subsequent association analyses of hemoglobin levels in two independent Tibetan populations (a total of 1,250 individuals) showed a male-specific association between the HMOX2 variants and hemoglobin levels. Tibetan males with the derived C allele at rs4786504:T>C displayed lower hemoglobin level as compared with the T allele carriers. Furthermore, our in vitro experiments indicated that the C allele of rs4786504 could increase the expression of HMOX2, presumably leading to a more efficient breakdown of heme that may help maintain a relatively low hemoglobin level at high altitude. Collectively, we propose that HMOX2 contributes to high-altitude adaptation in Tibetans by functioning as a modifier in the regulation of hemoglobin metabolism.

Nitric oxide (NO) is an important molecule for vasomotor tone, and elevated NO signaling was previously hypothesized as a unique and adaptive physiological change in highland Tibetans. However, there has been lack of NO data from Tibetans living at low altitude and lowlander immigrants living at high altitude, which is crucial to test this hypothesis. Here, through cross-altitude (1990–5018 m) and cross-population (Tibetans and Han Chinese) analyses of serum NO metabolites (NOx) of 2086 individuals, we demonstrate that although Tibetans have a higher serum NOx level compared to lowlanders, Han Chinese immigrants living at high altitude show an even higher level than Tibetans. Consequently, our data contradict the previous proposal of increased NO signaling as the unique adaptive strategy in Tibetans. Instead, Tibetans have a relatively lower circulating NOx level at high altitude. This observation is further supported by data from the hypoxic experiments using human umbilical vein endothelial cells and gene knockout mice. No difference is detected between Tibetans and Han Chinese for endothelial nitric oxide synthase (eNOS), the key enzyme for circulating NO synthesis, suggesting that eNOS itself is unlikely to be the cause. We show that other NO synthesis-related genes (e.g. GCH1) carry Tibetan-enriched mutations significantly associated with the level of circulating NOx in Tibetans. Furthermore, gene network analysis revealed that the downregulation and upregulation of NOx is possibly achieved through distinct pathways. Collectively, our findings provide novel insights into the physiological and genetic mechanisms of the evolutionary adaptation of Tibetans to high-altitude hypoxia.