High altitude polycythemia (HAPC) is a chronic plateau disease in which the organism lives for a long period of time on a plateau at an altitude of more than 2,500 m above sea level, and the lack of oxygen in the tissues leads to the compensatory overproliferation of erythrocytes (women’s hemoglobin ≥19 g/dL, men’s hemoglobin ≥ 21 g/dL), increased blood viscosity, and clinical symptoms such as dizziness, shortness of breath, and chest tightness (León-Velarde et al., 2005; Beall and Goldstein, 1987; Wu, 2005). HAPC is the most prevalent and harmful chronic high altitude disease among high altitude residents. It is estimated that up to 5%–10% of high altitude residents may develop HAPC(1). There are differences in the prevalence of HAPC by race, gender, age, altitude of residence, and duration of residence (Meyer et al., 2017). The risk of HAPC is associated with genetic, environmental and physiological factors. In the low oxygen environment of plateau, lowlanders are more likely to suffer from HAPC than highlanders (Garrido et al., 1996). It has been reported that the prevalence of HAPC is lower in Tibetan long-established populations at the same altitude compared to migrated Han Chinese populations (Wu et al., 2005). Genome-wide association studies (GWAS) have confirmed that the differences in genetic background between lowlanders and highlanders are the result of natural selection of genomic loci over hundreds of generations in highlanders. SNPs in genes such as EPAS1, PPARA, EGLN1, EDNRA, and PTEN were found to correlate with hemoglobin (Hb) concentrations in high altitude Tibetan populations, and they found that most of these genes play a role in the HIF-1 pathway (Simonson et al., 2010). In addition, Mallik N suggests that gain-of-function mutations in EPAS1 and loss-of-function mutations in EGLN1 lead to constitutive activation of EPO signaling and thus to HAPC. Strong and significant associations between Hb concentrations and haplotypic variation in PPARA and EGLN1 provide evidence of a genetic mechanism for the form of high-altitude acclimation that characterizes Tibetan populations (Simonson et al., 2010; Mallik et al., 2021). Polymorphisms in the CYP17A1 and CYP2E1 gene loci have been shown to play a role in mediating susceptibility to HAPC (Xu et al., 2015a).

Endothelial-type PAS structural domain protein 1 (EPAS1), also known as hypoxia-inducible factor 2 (HIF-2ɑ), is encoded by the EPAS1 gene located at 2p21, and is a protein composed of 870 amino acids (Karaghiannis et al., 2023). Numerous studies have shown that polymorphisms in EPAS1 are associated with the pathogenesis, pathologic staging, progression, and prognosis of cancers such as colorectal cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma, non-small cell lung cancer, and papillary thyroid carcinoma (Baba et al., 2010; Mohammed et al., 2011; Bangoura et al., 2004; Islam et al., 2020; Zhen et al., 2021; Zhang et al., 2023). EPAS1 rs7583392, rs6756667, and rs12467821 have been reported to be closely associated with plateau diseases and are thought to be related to high altitude acclimatization in Tibetans (Li et al., 2024; Basang et al., 2015; Bhandari et al., 2017; Pan et al., 2019). The hypoxia-inducible factor (HIF) oxygen signaling pathway plays an important role in hypoxia acclimatization in Tibetan populations (Simonson et al., 2010; Wang et al., 2011; Beall et al., 2010; Peng et al., 2011), but whether there is an association with HAPC is not yet known. The protein stability of HIF-2ɑ increases when the body is in a hypoxic state, and accumulated HIF-2ɑ continues to stimulate the HIF pathway, promoting transcription of downstream genes involved in erythropoiesis, hypoxic metabolism, inflammation, angiogenesis, and tumorigenesis (Wang et al., 2023; van Patot and Gassmann, 2011; Davis et al., 2022).

The peroxisome proliferator-activated receptor α gene (PPARA) is located on chromosome 22 and encodes peroxisome proliferator-activated receptor α (PPARα), a member of the nuclear receptor family of transcription factors, which regulates physiological processes, such as lipid metabolism, gluconeogenesis, and immune responses, particularly fatty acid β-oxidation in mitochondria and peroxisomes (Standage et al., 2017; Jay and Ren, 2007). Polymorphisms in the PPARA have been reported to be associated with the risk of liver disease, celiac disease, type 2 diabetes, and hyperlipidemia, among others (Jay and Ren, 2007; Li et al., 2014; Mostowy et al., 2016; Eurlings et al., 2002). A variant in the “C” allele of PPARA rs6520015 affects cardiac ejection function after acute plateau exposure (Standage et al., 2017; Yang et al., 2019). The PPARA promoter is associated with vascular function, target gene transcription and hemoglobin concentration during hypoxia in a Tibetan population (Shen et al., 2020).

In Tibetan populations at high altitude, EPAS1 and PPARA genes are involved in hypoxia adaptation by encoding transcription factors, and they play important roles in hypoxia-responsive pathways and oxidative stress (Simonson et al., 2010). However, whether the polymorphism of these genes is related to the incidence rate of HAPC in Chinese Tibetan population still needs to be investigated. Therefore, the aim of this study was to investigate the genetic relationship between polymorphisms in the EPAS1 and PPARA loci and HAPC in the Chinese Tibetan population.

This study was approved by the Research Ethics Committee of the First Affiliated Hospital of Dali University (approval number: DFY20171210002, date: 10 December 2017). We selected 162 participants from the Tibetan population in the Lhasa area of Tibet, all of whom were residents of high altitude areas. Among them, 78 cases were HAPC patients in the experimental group and 84 cases were healthy people in the control group. The inclusion criteria of the experimental group were: (León-Velarde et al., 2005): Hb ≥21 g/dL for men and ≥19 g/dL for women; (Beall and Goldstein, 1987); long-term residence in the plateau area with an elevation of more than 3000 m above sea level; and (Wu, 2005) having three or more of the following symptoms: headache, dizziness, fatigue, cyanosis, sleep disturbance, conjunctival congestion, and purplish skin. Patients with true cytosis and other secondary erythrocytosis; cardiovascular and cerebrovascular diseases, autoimmune diseases, malignant tumors, immune system diseases, and neurological diseases; and incomplete relevant data were excluded from the study. Clinical data: red blood cell count, hemoglobin, and hematocrit were also collected from the 2 groups.

More and more studies have suggested that genetic factors are one of the important factors contributing to the development of HAPC in Tibetan populations. EPAS1 rs12619696, rs13419896, and rs4953354 were significantly associated with the risk of HAPC disease in Tibetan populations on the Tibetan Plateau (Xu et al., 2015b). HAPC is an adverse consequence of adaptation to the high altitude environment and is closely related to the low-pressure environment of plateau hypoxia. The increasing number of erythrocytes and hemoglobin concentration poses a great threat to the health status of the population at high altitude (Wang et al., 2019). Therefore, it is important to explore the risk of HAPC genetically to improve the knowledge and diagnosis of this disease.

One of the hotspots of previous studies on the correlation between hypoxia adaptation and genetic polymorphisms is the EPAS1 gene, which encodes HIF-2α. Hypoxia-inducible factor (HIF) is a transcription factor that activates the adaptive hypoxia response at low levels of oxygen, and the two proteins, HIF-1 and HIF-2, consist of an unstable ɑ-chain and a stabilized β-chain (Jaśkiewicz et al., 2022; Yi et al., 2010); under normoxic conditions, the hydroxylation of HIF-1ɑ and HIF-2ɑ are post-translationally regulated by hydroxylation of specific proline residues within the oxygen-dependent degradation structural domain, which is carried out by prolyl hydrolase structural domain (PHD) proteins; hydroxylated HIF-α is recognized by von Hippel-Lindau (VHL) proteins, whose target HIFs are degraded via the ubiquitin-proteasomal pathway; in a hypoxic state, HIF-2ɑ hydroxylation is reduced, preventing the binding of HIF-2ɑ to VHL proteins, and the accumulated HIF-2ɑ continuously activates the HIF pathway, leading to a possible impact on the expression of downstream genes (Pamenter et al., 2020; Semenza, 2012; Sergi, 2019). Beall et al. found that EPAS1 variants downregulate its transcript levels, while its encoded transcription factor HIF-2α stimulates erythropoiesis, resulting in an increase in blood Hb concentration (Beall et al., 2010). Gruber et al. find that postnatal EPAS1 deficiency in mice causes anemia (Gruber et al., 2007). Tan et al. found that the EPAS1 G536W missense mutation causes erythrocytosis (Tan et al., 2013). In this study, we found that the rs6735530, rs6756667, rs7583392, and rs12467821 polymorphisms of the EPAS1 gene were significantly associated with HAPC in the Chinese Tibetan population by analysis. Among them, allele “G” in EPAS1 rs6756667 and allele “T” in rs12467821 were both significantly associated with increased risk of HAPC, and the analysis of codominant, dominant, recessive and overdominant models for rs6756667, rs12467821 showed an increased risk of HAPC. The allele “T” in EPAS1 rs6735530 is significantly associated with an increased risk of HAPC, and its codominant and recessive model analyses show an increased risk of HAPC. The allele “A” in EPAS1 rs7583392 is significantly associated with an increased risk of HAPC, and its codominant, dominant, and recessive models also show an increased risk of HAPC.

Under hypoxia, PPARα encoded by the PPARA gene enhances the regulation of anaerobic glycolysis and lactate accumulation in the venous blood, thereby increasing oxygen utilization and obtaining sufficient ATP for the organism (Kinota et al., 2023). In Tibetans, the expression of the putative favorable PPARA haplotype and PPARα was significantly and positively correlated with fatty acid oxidative capacity (FAO), and reduced expression of PPARα could enhance the body’s efficiency in utilizing oxygen, suggesting that PPARA is associated with hypoxic metabolic adaptation (Murray et al., 2018; Horscroft et al., 2017). We found no correlation between PPARA rs6520015 and the risk of HAPC in the Chinese Tibetan population. This is consistent with the results of previous studies. It has been reported that HIF can regulate PPARA by repressing its promoter during hypoxia, which reduces hemoglobin concentration in Tibetan population (Bhandari and Cavalleri, 2019; Bai et al., 2012).

In addition, the results of haplotype testing in this study showed that the TCAGC haplotype at the rs6520015, rs6735530, rs6756667, rs7583392, and rs12467821 loci decreases the risk of HAPC in Tibetans, and the TTGAT haplotype increases the risk of HAPC in Tibetans. And rs7583392 is in complete linkage disequilibrium with rs12467821. It indicates that rs7583392 and rs12467821 are highly interlinked, and the chance of their alleles appearing together on the same chromosome is higher than the frequency of random appearance. We also predicted the interactions between these SNPs by MDR analysis and found that there was no effect on the development of HAPC in the EPAS1 rs6756667 model and the PPARA rs6520015, EPAS1 rs6735530, EPAS1 rs6756667 model, although there was a strong interaction. This may have occurred because the MDR analysis could not adjust for covariates; and it could only analyze dichotomous phenotypes. It can only find interactions but does not identify the main effectors among the many factors. When we study fewer factors, there are limitations in using MDR analysis to accurately detect the effect of interactions on disease due to reduced dimensionality.

In this experiment, there are still some limitations. First, the sample size was small and limited to Tibetan patients in the Lhasa region of Tibet, so it is not possible to extrapolate the significance of the findings to other populations, and therefore the practical significance of the genetic polymorphisms needs to be investigated with larger sample sizes and different populations; second, we studied fewer loci, and in the future, we should investigate the correlation between other polymorphic loci in the EPAS1 and PPARA gene and the prevalence of HAPC.

In the Chinese Tibetan population, EPAS1 rs6735530, rs6756667, rs7583392, and rs12467821 polymorphisms were associated with the risk of developing HAPC, while PPARA rs6520015 was not significantly associated with HAPC.