Globally, more than 140 million people live at altitudes above 2,500 m, mainly in the Tibetan Plateau, the Andes and the Ethiopian Plateau (1). When humans live at high altitude, they may develop high altitude polycythemia (HAPC) due to overcompensated proliferation of red blood cells caused by hypoxia, which is one of the typical chronic alpine diseases and has been a serious public problem at high altitudes (2). HAPC leads to a significant increase in blood viscosity, which results in microcirculatory damage and immune response disturbances such as vascular thrombosis, widespread organ damage and sleep disorders (3, 4). Central to the development of HAPC is the over compensatory proliferation of erythrocytes, and erythropoiesis is largely dependent on erythropoietin (EPO), a glycoprotein hormone regulated by hypoxia-inducible factor (HIF) (5). It is noteworthy that the risk of developing HAPC varies significantly among different indigenous high-altitude populations. For instance, compared to residents of the South American Andes, the indigenous Tibetan population of the Qinghai-Tibet Plateau exhibits superior hypoxic adaptation, with a relatively lower incidence of HAPC (6–8). This disparity suggests that different populations may have evolved distinct genetic adaptation mechanisms to high-altitude environments. It has been shown that EPAS1, ATP6V1E2 and other genes regulate the HIF pathway, and mutations in these loci have been shown to correlate with genetic susceptibility to HAPC in Tibetan populations (9–11).

Yi X et al. found population-specific variations in allele frequencies of several genes by whole exome sequencing of 50 Tibetan individuals, with the endothelial PAS structural domain protein 1 (EPAS1) gene showing the strongest signal of natural selection (12). The gene is located on chromosome 2p16-21 and is expressed mainly in tissues and organs involved in metabolism and oxygen supply, such as the placenta, vascular endothelium, and kidney (13–15). Previous studies have demonstrated that multiple single nucleotide polymorphisms (SNPs) in the EPAS1 gene—including rs1868092, rs4953396, and rs4953354—are significantly associated with hypoxic adaptation in the Tibetan population. However, the mechanisms underlying their association with high-altitude hypoxic anemia (HAPC) require further elucidation (16, 17).

ATP6V1E2 (also known as ATP6E1 or VMA4) is located on chromosome 2p21,and encodes the E subunit of the V-ATPase complex, which is a proton pump that regulates intracellular acid–base homeostasis (18). There are fewer direct studies on ATP6V1E2 and hypoxia, but several lines of evidence suggest that this gene may be involved in high altitude hypoxia acclimatization. Whole-exome sequencing of the Tibetan population revealed significant racial differences in allele frequencies of ATP6V1E2 (12),and significant correlations between the ATP6V1E2 locus and levels of red blood cell counts (RBC), hemoglobin (HGB), and hematocrit (HTC), which are important features of hypoxic acclimatization in high altitude populations (9). Notably, ATP6V1E2 is located in close proximity to the EPAS1 gene, which is a known key gene for high altitude acclimatization, suggesting a possible synergistic regulatory relationship.

Genetic studies of Tibetans at high altitude in China have shown that the EPAS1 and ATP6V1E2 genes exhibit significant adaptive evolution under the selective pressure of prolonged high altitude. Although existing studies have confirmed the association of polymorphisms in these two genes with the risk of HAPC in Tibetan populations, the relevant loci are still understudied. In this study, we analyzed the genetic polymorphisms of EPAS1 rs1868092, rs4953396, rs4953354 and ATP6V1E2 rs896210 in the Chinese Tibetan population and their associations with the susceptibility to HAPC, which will provide more theoretical basis for early screening and individualized prevention and treatment of HAPC in the Tibetan population in this region.

At present, HAPC remains a serious threat to the health of highlanders. In China, its main treatments include phlebotomy and therapeutic erythrocyte dialysis, and there is still a lack of effective means of curing the disease (23). Several studies have shown that Chinese Tibetans have a lower incidence of HAPC compared to other highlanders and lowlanders living at the same altitude, a phenomenon closely related to genetic factors (8, 17, 24). Therefore, an in-depth study of HAPC from a genetic perspective is important for the prediction, diagnosis, treatment, and prevention of this disease.

Low-pressure hypoxia due to altitude is the main cause of HAPC. Although the pathogenesis of HAPC remains unclear, the widely accepted hypothesis is that HIF-1 upregulates EPO secretion during exposure to hypoxia at high altitude, leading to increased erythrocyte (25). HIF-1 belongs to the PAS family of hypoxia-regulated transcription factors and consists of an oxygen-sensitive α-subunit (HIF-α) and a constitutively expressed β-subunit (HIF-β, also known as ARNT) (26). Under normoxic conditions, prolyl hydroxylases (PHDs) hydroxylate key proline residues of HIF-1α and HIF-2α using oxygen molecules and α-ketoglutarate as substrates (27). Hydroxylated HIF-α is recognized by von Hippel–Lindau to form the E3 ubiquitin ligase complex, which is then degraded via the proteasome pathway (28, 29). In contrast, under hypoxic conditions, this oxygen-dependent pathway ceases, resulting in the intracellular stabilization and accumulation of HIF-1α and HIF-2α and the formation of a heterodimer with ARNT, which then binds to the hypoxia-responsive element, locating within the regulatory element of the HIF target gene (30, 31). HRE activates the expression of hypoxia-related genes, such as target genes of EPO, vascular endothelial growth factor, and lactate dehydrogenase A (18). In particular, upregulation of EPO production promotes increased erythropoiesis, a molecular mechanism that links tissue hypoxia to a compensatory erythropoietic response. It has been found that treatment of HAPC rats with the HIF-2α inhibitor PT2385 resulted in significant reductions in the levels of EPO, HGB, RBC, and HCT compared with the untreated group (32). This result suggests that PT2385 may directly regulate red lineage hematopoiesis by inhibiting the HIF-2α-EPO pathway, thereby reducing the abnormal elevation of HGB and RBCs (32).

Oxygen-dependent regulation of HIF-2α is a central mechanism for cellular adaptation to chronic hypoxia, and it plays a key role in the transcriptional regulation of EPO (33). HIF-2α is encoded by EPAS1 gene, whose function is critical for hypoxic adaptation. Our functional studies of EPAS1 gene also indicate that EPAS1, as a core transcriptional regulator of the hypoxic response, plays a key role in hypoxic adaptation by interacting with proteins such as ARNT, HIF-1α, and TP53, and by integrating regulatory mechanisms such as hypoxic signaling (HIF-1) and other mediators. Specific mutations in EPAS1 (e.g., Tibetan-adapted mutations) reduce the incidence of HAPC, whereas certain mutations may increase HAPC susceptibility. Using population genetic analysis, we found that polymorphisms at EPAS1 rs1868092, rs4953396 and rs4953354 were significantly associated with HAPC in the Chinese Tibetan population. Among them, rs1868092-G, rs4953396-C and rs4953354-A were risk alleles for HAPC, and genetic modeling analysis showed that the mutant genotypes of these three SNPs significantly increased the susceptibility to HAPC in codominant, dominant and recessive models. In addition, the rs4953354 locus showed a super dominant effect. This finding is consistent with previous conclusions, both of which indicate that there is a significant correlation between EPAS1 gene polymorphisms and susceptibility to HAPC (34, 35).

This study identified a significant association between the ATP6V1E2 rs896210 polymorphism and the risk of HAPC. This gene encodes a key subunit of the V-ATPase, which is essential for maintaining lysosomal acidification. An unexpected study on Hif-2a conditional knockout mice provided key insights into its function: in this mouse model, downregulation of ATP6V1E2 expression directly led to impaired lysosomal acidification (36). This suggests that one potential mechanism of ATP6V1E2 in high-altitude adaptation may be: its variants influence lysosomal acidification and function (e.g., protein degradation, autophagy) by regulating V-ATPase activity, thereby indirectly modulating the stability and turnover of hypoxia-responsive proteins like HIF-α. Ultimately, this forms a synergistic regulatory network with EPAS1. Concurrently, this study confirmed through KEGG pathway analysis that both EPAS1 and ATP6V1E2 are enriched in the HIF-1 signaling pathway, indicating that they synergistically participate in the development of HAPC via this critical hypoxia response pathway. Although direct functional studies of ATP6V1E2 remain limited, its significance has been validated through multiple lines of evidence: not only is it located adjacent to the EPAS1 gene and shares a selective signal (9), but this study further directly confirmed their presence within a tightly integrated functional network through PPI network analysis. Collectively, these findings establish ATP6V1E2 as a key player in the hypoxic adaptation regulatory network.

This study also revealed the synergistic contribution of ATP6V1E2 and EPAS1 loci to Tibetan high-altitude adaptation through haplotype and linkage disequilibrium analysis. We identified two functionally distinct haplotypes: AGCA and GGCA as risk haplotypes for HAPC, while GAAG exhibits protective effects. Further linkage analysis confirmed strong linkage disequilibrium between rs896210 and rs4953396. Crucially, multiple interaction model analysis demonstrated robust statistical interactions among the four SNP loci, collectively determining HAPC risk. This suggests polygenic interactions are more critical than single-gene variants. This finding aligns with genomic evidence implicating polygenic regions (Genes such as ATP6V1E2, TMEM247, RHOQ, PIGF, and CRIPT) in high-altitude adaptation (9, 37), offering new insights into the functional role of this gene cluster. Consequently, future research may explore genetic testing for screening high-risk populations and investigate potential drug targets based on these genetic variations, providing novel strategies for HAPC prevention and precision treatment.

However, this study has several limitations. First, the subjects were exclusively Tibetan individuals from the Lhasa region of Tibet, potentially limiting the generalizability of the findings. Future studies should include populations from different geographic regions and ethnic groups to validate broader applicability. Second, the limited sample size, but post-hoc power analysis indicates sufficient detection capability for strong genetic effects (OR > 4.0), with some loci exhibiting >90% power, ensuring the reliability of major positive findings. However, the power for detecting weak effects (OR < 1.5) was low (most < 20%), requiring cautious interpretation of negative results. Future studies should expand sample sizes to enhance statistical power for detecting subtle genetic effects. Finally, this study focused solely on limited sites within the ATP6V1E2 and EPAS1 genes, which to some extent limited the comprehensive assessment of polygenic interactions. Future research should conduct deep saturation mutation screening of these two genes and subsequently extend the analysis to other members of the EPAS1 gene cluster to elucidate the polygenic inheritance architecture of HAPC at the systems level.

This study found that specific SNPs at the ATP6V1E2 and EPAS1 loci are not only independently associated with HAPC susceptibility but also exhibit significant genetic interactions, thereby providing new evidence for the polygenic interaction hypothesis. More importantly, establishing risk assessment models based on these SNPs holds promise for providing a theoretical basis for early screening and personalized prevention and treatment of HAPC among the Tibetan population in this region.