DNA methylation in adaptation to high-altitude environments and pathogenesis of related diseases

Abstract

High-altitude environments, characterized by hypoxia, low temperatures, and intense ultraviolet radiation, pose significant challenges to human physiology and health. DNA methylation, as a key epigenetic regulatory mechanism, plays a central role in human adaptation to high-altitude environments and in disease pathogenesis. Current research indicates that high-altitude native populations (such as Tibetans and Andeans) modulate the methylation of hypoxia-responsive genes like EPAS1 and EGLN1 to enhance oxygen transport efficiency and energy metabolism patterns, while simultaneously suppressing excessive erythropoiesis and oxidative stress damage. This epigenetic regulation not only compensates for the lag in genetic adaptation over time but also forms synergistic networks with genetic variations. For instance, the functional SNPs of the EPAS1 gene are co-localized with its differentially methylated regions, revealing a delicate balance between genetic and epigenetic interactions under environmental stress. On the other hand, aberrant methylation patterns may disrupt the homeostasis of the HIF pathway, leading to acute and chronic high-altitude illnesses. This article provides a review of the recent research progress in plateau medicine and DNA methylation (up to 2025), including human clinical studies and animal model research. This includes research on high-altitude adaptation/acclimatization, as well as studies on inadequate adaptation to high altitude in relation to acute and chronic high-altitude-related diseases, cognitive decline, and pregnancy risks. By elucidating the core mechanisms underlying the “environmen – epigenetics – phenotype” axis, this work aims to provide a theoretical foundation for precision health interventions in high-altitude regions.

Introduction

High-altitude environments, characterized by unique geographical and climatic conditions, present significant challenges to human physiology and health [1]. Typical high-altitude regions, including the Himalayas, Andes, and Tibetan Plateau [2], are situated at elevations above 2,500 m and are marked by extreme conditions such as hypoxia, low temperatures, intense ultraviolet radiation, and nutritional constraints [3]. These harsh environmental factors compel populations and animals in high-altitude areas to adapt through various physiological and molecular mechanisms to maintain normal bodily functions and life activities [4]. Hypobaric hypoxia is the most prominent stressor in high-altitude environments [5]. Prolonged exposure to hypoxic conditions triggers a series of physiological responses, including increased red blood cell production [6], enhanced lung capacity [7], and altered vascular regulation [8], all aimed at improving oxygen transport and utilization efficiency. However, the hypoxic environment also increases the risk of acute mountain sickness and chronic high-altitude diseases [9].

With advancements in molecular biology and genomics, researchers have increasingly recognized the crucial role of genetic factors in high-altitude adaptation. Over evolutionary time, indigenous highland populations have developed unique physiological adaptations through genetic regulation, including enhanced adaptation to hypoxia, optimized energy metabolism, and stable vascular regulation [10–12]. The Tibetan population’s exceptional adaptation to high-altitude hypoxia has become a classic model for studying human environmental adaptation [13]. Genome-wide association studies (GWAS) have identified positive selection of genes involved in hypoxia-inducible factor (HIF) pathways, such as EPAS1 and EGLN1, as a core genetic basis for Tibetan adaptation to high altitudes [14–17].

However, genetic variation accounts for only part of the phenotypic differences. Growing evidence suggests that epigenetic regulation, serves as an “environmental sensor,” dynamically mediating gene expression plasticity in high-altitude adaptation (Fig. 1) [18, 19]. Epigenetic research focuses on regulatory mechanisms that affect gene expression. These mechanisms operate not through changes in DNA sequence but through DNA methylation, histone modification, chromatin remodeling, and non-coding RNA expression.

Fig. 1.

The interactive model of adaptation to high-altitude environments. The inner circle represents traditional adaptation models as independent entities, categorized into physiological, developmental, transgenerational, and genetic aspects, all of which can interact with one another. The epigenome process can be viewed as the central hub connecting the environmental, physiological, and genomic elements of the adaptation process. These processes are further manifested in specific adaptation mechanisms in the outer circle, such as epigenetic regulation, genetic variation, physiological plasticity, behavioral adaptation, the synergistic role of the gut microbiome, and others. Moreover, these specific mechanisms can also influence each other

DNA methylation, one of the most studied epigenetic modifications, involves the addition of a methyl group to the cytosine in cytosine-phosphate-guanine dinucleotide (CpG). This covalent modification at the fifth carbon atom of cytosine (5mC) regulates chromatin structure and gene activity, with patterns influenced by environmental stress and nutritional status [20, 21]. Recent breakthroughs in epigenomics have shown that DNA methylation is not merely a complementary mechanism to genetic variation, but also a key mediator that links environmental signals to phenotypic outcomes [22, 23]. Studies indicate that, in the context of increasing urbanization, early-life stress can elevate glucocorticoid levels and alter adult phenotypes, with DNA methylation potentially playing a crucial role in mediating these environmental effects [24]. Additionally, DNA methylation is likely pivotal in responding to environmental changes, as it can remain stable throughout an individual’s lifetime and exhibit varying degrees of heritability [25]. For instance, a study by Peng et al. used surrounding road conditions as a measure of environmental exposure and identified four highly methylated sites in the LAMB2 gene that were associated with lower cognitive scores on the KBIT-2 [26]. Furthermore, asbestos exposure has been linked to hypermethylation of the p16/CDKN2A promoter and hypomethylation of RARB and DAPK in lung cancer [27, 28]. These findings suggest that dynamic changes in DNA methylation may be a key driver of phenotypic changes induced by environmental factors.In studies related to adaptation to high-altitude environments, DNA methylation has been the main research model.

Although other epigenetic mechanisms have been relatively under-researched, they also play an important role in revealing the mechanisms of human adaptation to high-altitude environments and maladaptation. Histone modifications affect chromatin structure, which in turn regulates the binding of transcription factors to DNA and modulates gene expression. A previous study has shown allele differences in histone acetyltransferase p300 (EP300) between populations native to the Tibetan Plateau and those from low-altitude regions (including Asia, Europe, and Africa) [29]. The study hypothesizes that the adaptive allele of EP300 may be associated with reduced blood nitric oxide (NO) levels, which could serve as a protective mechanism to prevent excessive NO production in Tibetans. Non-coding RNAs (ncRNAs), which do not encode proteins, include microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). These RNA molecules have regulatory functions and have been shown in previous studies to be involved in the regulation of high-altitude adaptation [30–32]. Moreover, research has indicated that ncRNAs may be associated with the pathogenesis of altitude-related diseases such as acute mountain sickness (AMS) [33, 34], chronic mountain sickness (CMS) [35], high-altitude retinopathy [36], and high-altitude deep vein thrombosis (HADVT) [37].

This review aims to systematically integrate recent cross-disciplinary research findings in plateau medicine and epigenetics, with a focus on the role of DNA methylation in adaptation to high-altitude environments and the pathogenesis of high-altitude diseases. By analyzing the core mechanisms linking the “environment-epigenetics-phenotype,” this paper seeks to provide a theoretical foundation for precise interventions in high-altitude health. This article will focus on the corresponding DNA methylation patterns in humans (Table 1), but without relevant studies we will further use animal models to elucidate possible mechanisms.

Table 1.

DNA methylation changes in humans at high-altitude

Type	Authors	Method	Altitude (m)	Population	Sample size	Gene	Change	Pathways likely to be affected	DOI
1. High-altitude acclimatization
Acute	Childebayeva et al.	Quantitative pyrosequencing	1400 3440 4240 5160	European	21	LINE-1, EPO and RXRa	Decrease	Erythropoiesis	10.3389/fgene.2019.01062
						EPAS1 and PPARa,	Increase	Metabolic changes; HIF pathway activation
Acute	Childebayeva et al.	llumina Infinium MethylationEPIC BeadChip	1400 4240	European	15	ANGPT1, CREBBP, CUL2, HIF1A, HK1, HMOX1, PDK1, PIK3R3, PLCG1, PRKCG, RELA, STAT3, ABL1, etc.	DM	HIF pathway activation; RAS pathway activation; Inflammation related; Changes in cognitive function	10.3389/fphys.2021.660906
Long-term	Cheng et al.	Illumina HumanMethylation 450 BeadChips	0 4100	Chinese Han	761	SPATA13-AS1, KPNB1, IFT27, NMRAL1, FAM126A, PABPN1, BORCS6, NDUFS8, RAB5B, C12orf65, etc.	DM	HIF pathway activation; Steroid biosynthesis; Protein processing	10.1038/s41421-025-00795-z
2. High-altitude adaptation
	Cheng et al.	Illumina HumanMethylation 450 BeadChips	4100	Chinese Tibetan and Han	986	TMEM247, PF4V1, KLK8, EPAS1, GS1-259H13.2, INSM1, PRSS16, SMARCD1, MIR133A1HG, etc.	DM	HIF pathway activation; Immune-related diseases; Hematopoietic lineage;	10.1038/s41421-025-00795-z
	Lin et al.	Illumina Hiseq X Ten	NA	Chinese Han, Tibetan, Zhuang and Mongolian	32	ARNT, EPAS1, EGLN1, etc.	DM	HIF pathway activation	10.1007/s11427-022-2284-8
	Childebayeva et al.	Illumina Infinium MethylationEPIC BeadChip	150 4300	Peruvian Quechua	113	LOXL3, P4HA1, RUNX1, MEIS, BHMT, LHDC, etc.	DM	HIF pathway activation; Hematopoietic stem cell oxidative defense; Sugar metabolic pathway	10.1093/gbe/evaa239
	Childebayeva et al.	Quantitative pyrosequencing	0 4388	Peruvian Quechua	572	LINE-1	Increase	Assess the overall level of DNA methylation	10.1080/15592294.2018.1561117
						EPAS1	Decrease	Increased transcriptional activity of HIF
	Alkorta-Aranburu et al.	Infinium HumanMethylation 27	1200 3700 1560 4000	Christian Amhara, Muslim Oromo	68	APOBEC3G, PITX2, MT1G, OR2K2, TLR6, SUMO1, PDE4A, HIVEP2, etc.	DM	Angiogenesis and regulation	10.1371/journal.pgen.1003110
In terms of pregnancy	William E. Gundling et al.	Illumina HumanMethylation 450 BeadChips	3600	Andean and European	70	LYVE1, STC1, FOS, ALOX5AP, NAPRT1, PLA2G2A, CCL2, CCL3L3, etc.	DM	Involved in the response to oxidative stress and inflammation	10.1101/261974
3. Repeated exposure to extreme hypoxia
	Basang et al.	Illumina Methylation EPIC array (850 K)	4700  > 8000	Chinese Tibetan	64	ANKMY1, PPP1R13L, NRIP2, MAP3K7CL, SEPTI-9, KCNJ15, CUL2, PTEN, etc.	DM	Immune cell activation and differentiation; Inflammation related; HIF pathway activation	10.1016/j.jgg.2021.05.015
	S. Zhang et al.	Illumina Methylation EPIC array (850 K)	4700  > 8000	Chinese Tibetan	64	LIPN, PLCH1, EMR1, STX5, etc.	DM	Oxyhemoglobin saturation Lipase activity and skin keratosis	10.3390/ijms252312652
4. High-altitude diseases
HAPE	Sharma et al.	Bisulfite-converted DNA sequencing	3500	Indian	96	EGLN1	Decrease	Regulation of PHD2 protein expression	10.1186/s13148-022-01338-z
HAPE	Mishra et al.	Bisulfite-converted DNA sequencing	3500	Indian	200	Apelin	Increase	Regulates blood vessels and oxygen homeostasis	10.1073/pnas.1422759112
HAPE	Jin et al.	MALDI-TOF	> 4000	Chinese Han	106	CYP2S1	DM	Inflammation and cell proliferation	10.1016/j.gene.2022.146590
HAPE	P. Wang et al.	MALDI-TOF	> 4000	Chinese Han	106	CYP39A1	DM	Inflammation and cell proliferation	10.2147/PGPM.S397862
HAPC	Zhaxi et al.	Quantitative methylation-specific real-time PCR	> 4500	Chinese Tibetan	130	BMPR2	Increase	Erythropoiesis and vascular remodeling	10.1007/s10528-024-10798-2
						TGF-β	Increase	Inflammation, angiogenesis, wound healing, and immune cell function
HAPC	Ji et al.	Bisulfite-converted DNA sequencing	NA	Chinese Tibetan	20	MCCC2, RP3-399L15.3, etc.	DM	Serotonin synapses	10.19746/j.cnki.issn.1009-2137.2025.02.041
				Chinese Han	20	NAA25, CORO2B, etc.	DM	Gutamatergic synapse, Rap1 signaling pathway and cholinergic synapse
Iron overload	Q. Zhao et al.	Illumina HiSeq 2500 platform	> 2500	Chinese Tibetan	6	FLVCR1, HMOX2 and HFE	Increase	Iron absorption and transport Hemoglobin production and metabolism	10.1266/ggs.21 − 00006
						ACO1, IREB2, SLC40A1 and TFRC	Decrease
						SLC11A2, CYBRD1 and NEDD8	DM
HADVT	Vijay et al.	Bisulfite sequencing PCR	> 3500	Indian	20	Thrombomodulin	Increase	Modulating thrombin activity	10.1016/j.thromres.2022.04.018

Abbreviation: HAPE, high-altitude pulmonary edema; HAPC, high-altitude polycythemia; HADVT, high-altitude deep vein thrombosis; NA, not available; DM, differential methylation

Biological basis of adaptation to high-altitude environments and high-altitude diseases

Physiological mechanisms of adaptation to high-altitude environments

Adaptation to high-altitude environments is a complex physiological process that can be divided into two categories: the acclimatization of lowland populations to high-altitude conditions and the long-term adaptation of highland populations (Fig. 2). The acclimatization of lowland populations refers to the rapid physiological response of individuals from lowland areas when ascending to high-altitude regions, commonly known as “high-altitude acclimatization.” This acclimatization occurs when individuals quickly ascend to high-altitude regions, resulting in physiological changes. These changes include adjustments in the cardiovascular system, such as modifications in heart rate and blood pressure, and in the respiratory system, such as increased ventilation and alterations in blood oxygen saturation [38, 39].

Fig. 2.

Adaptation to high-altitude environments and high-altitude diseases. BP, blood pressure; HR, heart rate; RBC, red blood cell count; Hb, Hemoglobin (by figdraw.com)

In contrast, the adaptation of highland populations refers to the genetic and physiological traits exhibited by populations that have inhabited high-altitude areas for generations, such as Tibetans and Sherpas. These populations demonstrate inherent adaptability to high-altitude environments, a phenomenon known as “high-altitude adaptation.” High-altitude populations typically exhibit unique adaptation patterns in blood indices such as hemoglobin concentration [40]. Additionally, highland populations typically possess larger lung volumes [7, 41], which improve the efficiency of oxygen intake and transport, thereby supporting normal physiological function in hypoxic environments. High-altitude adaptation also involves structural changes in the cardiovascular system, such as remodeling of the ventricular structures and functions to adapt to prolonged hypoxic conditions [42, 43]. In highland populations, the mechanical function of the heart may alter to enhance oxygen delivery efficiency while mitigating the effects of oxidative stress [44]. Furthermore, high-altitude environments also influence the composition of the gut microbiota in highland populations. The gut microbiota, closely associated with high-altitude conditions and dietary habits, plays a crucial role in mediating the adaptation of highland populations to their environment [45, 46].

Although high-altitude populations from different regions exhibit convergence in the process of adapting to hypoxic environments at high altitudes [47, 48], physiological differences still exist among different high-altitude populations (Fig. 2) [49, 50]. For instance, the hemoglobin concentration of Andean highlanders is higher than that of Ethiopian and Tibetan highlanders [49, 51]. Strong genetic evidence indicates that both Tibetan and Ethiopian populations have genetically adapted to high altitudes [14, 52, 53], leading to the speculation that the difference may suggest that the adaptation of Andean populations to high altitudes is still ongoing [54, 55].

Manifestations of high-altitude diseases

The pathological manifestations of maladaptation to high-altitude include acute high-altitude sickness, CMS, and cognitive impairments induced by high-altitude environments (Fig. 2). Acute high-altitude sickness is a prevalent health issue in high-altitude regions, typically occurring when individuals ascend rapidly to high altitudes without adequate acclimatization. It can be classified into three types, with AMS being the most common. AMS is characterized by symptoms such as headache, nausea, vomiting, loss of appetite, dizziness, fatigue, and sleep disturbances, as well as life-threatening conditions like high-altitude cerebral edema (HACE) and high-altitude pulmonary edema (HAPE) [56, 57]. HACE affects the brain, presenting with mental status changes and ataxia, along with other AMS symptoms. HAPE affects the lungs, causing progressive shortness of breath and exercise intolerance, accompanied by other symptoms, including chest tightness, orthopnea, and cough with pink frothy sputum. Studies have shown that the onset of acute high-altitude sickness is associated with a decrease in arterial oxygen saturation, and changes in oxygen saturation can serve as an important indicator for its occurrence [58]. Moreover, acute high-altitude sickness is also linked to changes in cerebral hemodynamics, particularly the increased blood flow velocity and reduced resistance in the posterior circulation area [59].

CMS is a clinical syndrome caused by the loss of the ability to adapt to high-altitude environments following prolonged residence in high-altitude areas or migration from lowland regions. It is characterized by excessive erythrocytosis, severe hypoxemia, and pulmonary hypertension [60]. Studies have shown that while exercise capacity in chronic mountain sickness patients is affected by pulmonary hypertension and relatively low ventilation, their aerobic exercise capacity is maintained due to increased oxygen-carrying capacity in the blood and enhanced pulmonary diffusion ability [60]. Additionally, maladaptation to high-altitude is associated with alterations in neurobehavioral functions. In high-altitude environments, reaction time and cognitive function may be impaired [61, 62]. Research has demonstrated that exposure to high-altitude environments leads to cognitive decline, particularly within the first 24 h, during which the cognitive processing speed of lowland residents significantly decreases [63]. Long-term residents of high-altitude regions may experience significant psychological and cognitive impairments, which are closely linked to reduced brain tissue oxygenation, lower sleep quality, and biochemical dysfunction [64]. Furthermore, some symptoms of chronic mountain sickness, such as cyanosis, sleep disorders, and excessive erythrocytosis, are associated with specific cognitive impairments, suggesting that these symptoms may serve as early indicators of cognitive decline [65]. High-altitude environments also pose considerable risks to pregnancy. Studies have shown that hypoxic conditions at high altitudes reduce uterine artery diameter and blood flow, adversely affecting fetal oxygen supply and leading to fetal growth restriction [66]. Additionally, high-altitude environments influence the expression of ion channel genes in the placenta, increasing the risk of pregnancy-induced hypertension and preeclampsia [67]. High-altitude environments may also increase maternal psychological and physiological stress, which could further impact pregnancy outcomes [68].

DNA methylation and adaptation to high-altitude environments

DNA methylation regulation of the core hypoxic response pathways

The HIF pathway serves as the core regulatory network for organisms’ responses to hypoxia [69]. Under normoxic conditions, prolyl hydroxylase domain proteins (PHD) hydroxylate the proline residues of the HIF-α subunit. This modification creates a specific recognition site for the von Hippel-Lindau (VHL) E3 ubiquitin ligase. VHL then mediates the ubiquitination of HIF-α, which leads to its subsequent degradation by the proteasome [70]. The transcription of PHD is regulated by the EGLN1 gene. Additionally, the inhibitory factor inhibiting HIF (FIH) can hydroxylate asparagine-803 in the C-terminal transactivation domain of HIF-2α, further guiding the VHL-mediated ubiquitination process [71] (Fig. 3).

Fig. 3.

Mechanism schematic of the HIF pathway. Under normoxic conditions, PHD and FIH hydroxylate HIF-α. This modification provides a site for VHL to ubiquitinate HIF-α. Ultimately, HIF-α is degraded by proteases. Under hypoxic conditions, the insufficient oxygen content reduces PHD hydroxylation, leading to an increase in the retention of HIF-α. Hypoxia also increases the methylation modification of the PHD encoding gene EGLN1, resulting in decreased PHD expression. In contrast, the methylation modification of the HIF-α encoding gene EPAS1 decreases, leading to an increase in HIF-α expression. HIF-α and HIF-β form a dimer in the nucleus, regulating downstream target genes by binding to HREs. (by figdraw.com)

Under hypoxic conditions, the activity of PHD is significantly reduced [72]. This reduction is due to the limitation of oxygen-dependent co-substrate supply. Additionally, the methylation level of its encoding gene, EGLN1, may increase. Together, these factors inhibit PHD protein expression. This is confirmed in Tibetan populations, where EGLN1 follows an upregulated DNA methylation pattern [72]. This dual regulatory mechanism effectively maintains the stability of HIF-α subunits, preventing their ubiquitination and degradation. At the same time, hypoxia can downregulate the methylation modification level of the HIF-α encoding gene EPAS1 [70], promoting HIF-α protein synthesis by enhancing gene transcription. EPAS1 is a protein subunit that contributes to the formation of HIF-2α and is a member of the transcription factor family [73]. HIF-2α forms a complex with HIF-β through its Per-ARNT-Sim domain, initiating the expression of genes related to oxygen supply and cellular adaptation. DNA methyltransferase 3a (DNMT3a) can methylate and silence EPAS1 in differentiated cells, leading to reduced expression of HIF-2α mRNA and downstream target molecules [74]. Compared to the Han population, EPAS1 in Tibetan populations is expressed in a downregulated DNA methylation pattern [72]. Accumulated HIF-α subunits then translocate to the nucleus, where they form a transcriptionally active heterodimer with constitutively expressed HIF-β subunits. This complex specifically recognizes and binds to hypoxia response elements (HRE) in the promoter regions of downstream target genes. It then initiates various adaptive physiological responses, such as angiogenesis and the reprogramming of glucose metabolism (Fig. 3). In Tibetan high-altitude adaptation, key genes in the HIF pathway, such as EGLN1 and EPAS1, have unique methylation patterns due to natural selection, enhancing tolerance to hypoxia compared to lowland populations (e.g., Chinese Han) [72, 75]. A similar methylation regulation pattern of the HIF pathway has also been observed in high-altitude adaptation studies in Andean populations [76, 77].

However, some studies have found that in lowland populations, EPAS1 methylation is upregulated under short-term high-altitude acclimatization. The researchers suggest that this upregulation may occur due to the short duration of exposure, during which EPAS1 is undergoing demethylation. The method used to detect methylation via bisulfite conversion does not distinguish between methylation and hydroxymethylation, and hydroxymethylation at CpG sites is an intermediate stage of demethylation [78–80]. Hydroxymethylation at CpG sites is mediated by the Ten-Eleven Translocation (TET) enzymes [81], which are part of the 2-oxoglutarate-dependent dioxygenase (2-OGD) superfamily. 2-OGD has been shown to be transcriptionally upregulated under hypoxic conditions [82], suggesting that DNA methylation and demethylation work in tandem to maintain environmental adaptation. PHD2 is also a member of the 2-OGD family, and it shows an upregulation trend under acute hypoxia [83], possibly serving as a negative feedback mediator for HIF-1 activity during hypoxic stress [84]. However, in the high-altitude Tibetan population, PHD2 is downregulated compared to the high-altitude Han population [40]. Additionally, both the genetic variations and epigenetic modifications of EGLN1 in Tibetans also demonstrate a downregulation pattern [40, 72], further suggesting that high-altitude Tibetans are better adapted to the hypoxic environment than their Han counterparts.

Previous research has primarily focused on the role of genetic variation patterns in the HIF-related pathways involved in high-altitude adaptation. In Tibetan populations, variations in the EPAS1 and EGLN1 genes are considered important factors in adapting to high-altitude hypoxic environments [17, 85]. These genetic variations help individuals better adapt to low-oxygen environments by regulating hemoglobin concentration and oxygen transport capacity [52, 86]. Research has also shown that the HIF pathway exhibits selective variation across different high-altitude populations. For example, in Andean highlanders, although the EGLN1 gene also shows selective signals, its specific adaptation mechanisms may differ from those in Tibetan populations [87]. Variations in hematocrit levels in Tibetans and Andeans are mediated by different variations and mechanisms involving the same gene (EPAS1) [88]. This difference may reflect the varying selective pressures and evolutionary paths experienced by different populations in adapting to high-altitude environments.

However, local genetic variations and SNPs in one-carbon metabolism can influence DNA methylation [89, 90], which is also evident in high-altitude adaptation. A recent large-scale population study on high-altitude adaptation revealed that genetic variation loci related to EGLN1, EPAS1, and HLA-DQB1 in the Tibetan population are associated with DNA methylation in high-altitude adaptation [75]. The study pointed out that the epigenetic changes at known adaptive loci are at least partially driven by genetic adaptation. These genetic signals influence the formation of DNA methylation, potentially controlling the regulation of gene expression levels associated with adaptive phenotypes. Another study on DNA methylation in the adaptation of Chinese populations to high-altitude environments also confirmed a relatively significant association between the high-altitude adaptation-related SNP rs78561501 and methylation changes on EGLN1 [72]. In Andean populations, local variations at two SNPs (rs2044456 and rs7579899) in the EPAS1 gene were also found to be related to EPAS1 methylation [76].

A whole-genome DNA methylation study of lowland populations undergoing short-term high-altitude acclimatization identified differential methylation genes such as ANGPT1, CREBBP, and CUL2, whose roles in high-altitude acclimatization remain unclear but are closely related to HIF-1α [91]. In a study involving repeated high-altitude exposures, it was found that methylation changes at the CUL2 locus might inhibit its expression [92]. CUL2 is involved in HIF-1α degradation and influences the hypoxia regulatory system in various cells [93]. The study also identified hypomethylated genes such as SEPTI-9 and PPP1R13L, where SEPTI-9 enhances HIF-1α transcriptional activity [94], and PPP1R13L gene expression may have a similar impact on focal adhesion kinase (FAK) as HIF-1α [95, 96].

DNA methylation regulation of oxygen transport in high-altitude environments

A central feature of high-altitude adaptation/acclimatization is the maintenance of oxygen supply under hypoxic conditions and the regulation of red blood cell production, with DNA methylation playing a key role in mediating the expression of relevant genes. Erythropoietin (EPO), the principal regulator of red blood cell production, increases rapidly under hypoxic conditions [97–99]. EPO contains a conserved HIF-1 binding site (HBS) CGTG in its 3’ enhancer, which includes a CpG site [100, 101]. Studies have shown that, under hypoxia, the hypomethylation of HBS is linked to EPO activation [100]. However, other research indicates that as altitude increases, the methylation level of HBS initially decreases but later begins to recover [78]. In high-altitude acclimatization studies, EPO has been shown to peak 1 to 3 days post-exposure, followed by a decrease in a highly altitude-dependent manner, consistent with the DNA methylation pattern of HBS [102, 103]. Furthermore, EPO levels in Tibetan populations are comparable to those in Han populations living at the same altitude [40].

Research suggests that red blood cell production homeostasis in Tibetans may be more closely related to the upstream regulator EPAS1 [72, 104]. Under hypoxic conditions, EPAS1 regulates the stability of HIF-2α, enhancing its transcription and forming a heterodimer with aryl hydrocarbon receptor nuclear translocator. This complex binds to HRE in the EPO gene promoter, thereby promoting EPO transcription. This process not only stimulates red blood cell production but also ensures an adequate iron supply for hemoglobin synthesis by regulating iron metabolism [105]. In addition, EPAS1 also influences iron absorption and utilization by regulating the expression of hepcidin, thereby further affecting erythropoiesis [106]. This may be the most crucial pathway through which the EPAS1 gene regulates hemoglobin and erythrocyte count during chronic hypoxia. In the previously mentioned acute high-altitude exposure study, a significant positive correlation between RXRa methylation and hemoglobin levels was also observed [78]. RXRa, a member of the retinoic acid receptor family, is crucial for normal hematopoiesis during development [107, 108]. As a negative regulator of EPAS1, EGLN1 has been shown to exhibit opposing methylation patterns compared to EPAS1 in Tibetan populations, potentially affecting related pathways [72]. Previous studies have indicated that genetic variation at the rs186996510 locus of EGLN1 is significantly associated with hemoglobin levels in Tibetans, contributing to the maintenance of lower hemoglobin levels in this population [109]. Recent studies have also shown that DNA methylation levels at the EGLN1 and EPAS1 loci are significantly correlated with hemoglobin levels [75].

Oxygen saturation, a key physiological parameter for assessing the efficiency of oxygen transport, reflects dynamic changes that provide insight into the body’s capacity for oxygen adaptation in high-altitude environments. The human body gradually adapts to reduced oxygen pressure through various mechanisms, with vascular function regulation being one of the core adaptive processes. In a study involving repeated extreme high-altitude exposure, 64 Tibetans living at 4700 m were selected, with 32 volunteers undergoing repeated extreme high-altitude exposure (up to 8848 m). The results showed that the repeated exposure group exhibited higher percutaneous oxygen saturation (SpO₂) levels and lower systolic blood pressure. Genome-wide differential methylation analysis revealed that methylation modifications at CpG sites in genes such as PLCH1 and LIPN may jointly regulate oxygen transport and vascular tone [110]. An acute high-altitude exposure study identified two CpG sites near the CLK2 and TOR4 genes that are associated with arterial oxygen saturation (SaO2) [91]. Additionally, important CpG sites linked to the renin-angiotensin system (RAS) pathway were found, including methylation changes in ANGPT1 and RASA3. ANGPT1 expression is linked to increased vascular density [111], while the RAS system is an important pathway for regulating blood pressure [112]. Previous studies have also found that during acute high-altitude acclimatization, methylation of EPO loci is negatively correlated with systolic blood pressure [78]. DNA methylation can influence the expression of genes in endothelial and vascular smooth muscle cells, affecting their proliferation, apoptosis, and function, thereby maintaining normal vascular function in high-altitude environments [113, 114]. A comparison of 27,578 CpG sites in a study of Ethiopian populations living between 4000 and 1500 m revealed differences in methylation of several genes, such as APOBEC3G, PITX2, MT1G and OR2K2, all of which may be involved in angiogenesis [115].

DNA methylation regulation of energy metabolism in high-altitude environments

DNA methylation plays a critical role in the adaptation of energy metabolism under hypoxic conditions at high altitudes. Previous studies have shown that, during acute high-altitude acclimatization, methylation of the CpG island in the RXRa promoter region decreases, while RXRa remains a transcription factor involved in fat metabolism and intracellular receptor signaling [78]. RXRa binds to PPARa to form an active transcription complex, whose activity is diminished under hypoxic conditions, thereby inhibiting fatty acid metabolism [116]. This study also observed that increased methylation in the PPARa promoter region is correlated with altitude [78], which aligns with previous findings of decreased PPARa expression under hypoxic conditions [117]. PPARa is a transcription factor involved in regulating fatty acid metabolism and oxidation. PPARs activate genes encoding acyl-CoA oxidase, a rate-limiting enzyme in the peroxisomal β-oxidation pathway, and promote fatty acid oxidation by regulating uncoupling protein 3 [118]. Reduced PPARa expression leads to restricted fatty acid breakdown, resulting in greater reliance on anaerobic glycolysis under hypoxic conditions [119, 120]. Previous research has also found that, compared to lowland populations, PPARa expression is lower in Himalayan Sherpas, and their skeletal muscle fatty acid oxidation capacity is reduced [121]. Notably, since RXRa and PPARa can form a transcription complex and their methylation patterns show opposite trends, this suggests a potential interaction between the two, although the specific mechanism remains unclear. In another study on high-altitude acclimatization, the methylation level of CLK2 was positively correlated with SpO₂ [91]. During acute hypoxia, decreased CLK2 methylation may enhance the transcriptional activity of gluconeogenesis genes regulated by PPARGC1A. This increase in hepatic gluconeogenesis and glucose output helps meet metabolic demands under low-oxygen conditions, as previously observed [122, 123]. In extreme high-altitude exposure studies [110], high methylation of the ACSL3 gene may suppress its expression, leading to increased fat storage [124], which is consistent with lipid accumulation under hypoxic conditions [125]. These findings highlight how DNA methylation-mediated epigenetic regulation enables the body to reduce reliance on oxygen and adapt to hypoxic environments, thereby maintaining energy metabolic homeostasis.

Other aspects of DNA methylation regulation in high-altitude environments

In high-altitude hypoxic stress environments, oxidative stress levels are significantly elevated, as hypoxia induces the production of reactive oxygen species (ROS), which can impact genomic stability [126]. ROS is often associated with a global decrease in DNA methylation [127, 128]. First, ROS directly attacks DNA, leading to oxidative DNA damage (such as 8-hydroxydeoxyguanosine), and these damages near CpG dinucleotides strongly inhibit the activity of DNA methyltransferases that methylate cytosine residues [129–131]. Second, under oxidative stress conditions, the activated TET hydroxylases catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) [132]. This 5hmC is further stabilized through base excision repair and replaced by unmethylated cytosine. However, some studies have found that ROS may induce hypermethylation at certain specific sites [133]. It is possible that ROS acts as a catalyst for DNA methylation [134], or ROS may induce site-specific hypermethylation by upregulating the expression of DNMTs or forming new DNMT-containing complexes [135].

Long interspersed nuclear element-1 (LINE-1), a highly repetitive DNA element in the genome, is often used as a marker for overall DNA methylation levels [136, 137]. Research has shown that excessive ROS production can reduce LINE-1 repeat sequence methylation, potentially activating transposition or regulating neighboring genes, leading to genomic instability [138, 139]. A cohort study of Andean populations found that individuals born at high altitudes or those who had lived at high altitudes for extended periods had higher LINE-1 methylation than lowland populations, indicating adaptation to hypoxic conditions in highland populations [77]. However, under acute high-altitude exposure, LINE-1 methylation levels decreased [78]. This change in methylation status may sensitively reflect the cumulative effects of chronic hypoxia on the epigenome, suggesting that LINE-1 could serve as a potential molecular marker for hypoxic adaptation. A significant decrease in methylation levels of the repeat element LINE-1 was also observed during long-term high-altitude acclimatization [75].

The effects of high-altitude environments on pregnancy and placental adaptation in highland populations may be influenced by DNA methylation mechanisms. Studies on placental tissues in Andean populations have revealed significant upregulation of several inflammatory chemokines (e.g., CCL2 and CCL3L3), whose expression is regulated by DNA methylation [140]. The high expression of CCL2 and CCL3L3 may promote the recruitment of placental immune cells and stimulate angiogenesis, thereby optimizing placental remodeling [141, 142]. These findings emphasize the crucial role of epigenetic regulation in immune tolerance and vascular remodeling at the maternal-fetal interface. In Andean Highland Populations, allele frequency shifts and methylation dynamics at the dysferlin (DYSF) gene rs10166384 locus highlight the evolutionary significance of epigenetic selection [143]. This locus is located within the TEAD4 transcription factor binding site, where increased DNA methylation limits TEAD4 binding, leading to reduced DYSF gene expression [144]. The study found that the Andean population exhibits the A allele at this locus [143], where CpG sites are typically methylated, and the G-to-A change reduces the likelihood of methylation because methylation rarely occurs at CpA sites [145]. This genetic and epigenetic dual effect may enhance dysferlin expression in the trophoblast, thereby improving placental cell fusion and repair functions [146, 147], a viewpoint supported by previous research [140]. This mechanism suggests that, at high altitudes, selective maintenance of advantageous alleles and regulation of their methylation levels may facilitate rapid adaptation to critical physiological processes.

DNA methylation and high-altitude diseases

DNA methylation in acute high-altitude sickness

Acute high-altitude sickness are multifactorial pathophysiological conditions triggered by acute hypoxic stress, involving inflammation, immune responses, cellular metabolism, and vascular regulation. HAPE, a type of acute high-altitude disease, is characterized by severe symptoms and, without timely intervention, can quickly progress to coma or even death [148]. Previous studies have shown that genetic polymorphisms in genes such as IL6, CYP4F2, and NR3C2 are significantly associated with the risk of HAPE [149–151]. However, environmental factors can dynamically modify DNA methylation of key hypoxic response genes through epigenetic regulation, contributing to the multifaceted pathophysiological responses of HAPE.

At the level of oxygen sensing and signal transduction, studies have found that, compared to high-altitude acclimatization lowland populations and high-altitude ethnic groups, HAPE patients exhibit reduced methylation of the EGLN1 promoter and gene body regions [152]. This reduction may be associated with enhanced transcriptional activity. This leads to overexpression of the PHD2 protein, which increases the degradation of HIF-1α, affecting vasoconstriction and oxygen transport, ultimately contributing to the development of HAPE [153]. The study also found that methylation regions of EGLN1 co-localize with functional SNPs (e.g., rs186996510), suggesting that genetic-epigenetic interactions may regulate the inhibition of the HIF signaling pathway by altering the binding efficiency of transcription factors such as FOS and POLR2A [152, 153].

Regarding vascular tone regulation, the apelin signaling pathway plays a crucial role in both adaptive and maladaptive physiological responses of endothelial and smooth muscle cells [154]. A study have shown that, in HAPE patients, methylation levels of the apelin gene promoter, 5′ UTR, and intron 1 regions are significantly elevated, especially in the 5′ UTR region [155]. This methylation is associated with the downregulation of apelin expression and reduced plasma apelin-13 levels. Methylation inhibits the binding of HIF to HRE, hindering the hypoxia-dependent transcriptional activation of apelin and leading to decreased apelin mRNA expression and plasma apelin-13 levels in HAPE patients. Consequently, endothelial-dependent vasodilation mediated by the apelin receptor is impaired, accompanied by decreased expression of endothelial nitric oxide synthase (NOS3) and reduced nitrite levels, ultimately weakening the AKT-NOS3 pathway-mediated vasodilation [156–158]. The study also identified synergistic effects of risk alleles for apelin, such as rs3761581G and rs2235312T, when combined with high methylation of its CpG islands [155]. These effects may exacerbate vascular tone imbalance by reducing transcriptional activity, further supporting the genetic-epigenetic interaction. Under hypoxic conditions, inflammation regulation is influenced by increased oxidative stress, which activates cytochrome P450 enzymes (CYP) [159, 160]. These enzymes metabolize endogenous substances, such as arachidonic acid, vitamins, and steroids, thereby modulating the body’s antioxidant capacity and inflammatory response. This process may impact the progression of HAPE [159, 160]. Studies analyzing the methylation of CYP2S1 and CYP39A1 in the HPEA population have found that methylation of these genes is associated with the risk of HPEA, although the specific pathways remain unclear [161, 162].

High-altitude headache is the main symptom of AMS. HACE, which progresses further from AMS, is characterized by changes in mental state, including cognitive impairment, drowsiness, rigidity, and ataxia [163]. Thus, the disease progression of AMS and HACE is consistent, with the main pathological factor being brain vasodilation caused by hypoxemia [164]. This may be related to the pulmonary vascular dysfunction of HPAE mentioned above [154]. However, the role of DNA methylation regulation in the pathogenesis of AMS and HACE is still unclear. Previous studies on acute high-altitude acclimatization have found changes in DNA methylation related to the RAS pathway and inflammatory factor pathways [91]. We hypothesize that abnormal DNA methylation regulating vascular modulation and inflammatory factors may be involved in the pathological development of AMS and HACE.

DNA methylation in CMS

CMS, also known as high-altitude polycythemia (HAPC), is characterized by excessive red blood cell proliferation. This condition leads to increased blood viscosity, microcirculatory obstruction, and thrombosis, which can result in widespread organ damage, sleep disturbances, and a heightened risk of stroke and myocardial infarction in early adulthood [165, 166]. Studies have shown that high methylation of the BMPR2 and TGF-β gene promoters is significantly associated with elevated hemoglobin levels in HAPC patients [167]. The transcriptional inhibition of BMPR2 may interfere with the BMP/SMAD signaling pathway, resulting in an imbalance in hepcidin expression and promoting abnormal iron metabolism and excessive red blood cell production [168, 169]. TGF-β regulates erythropoietin gene expression under hypoxia and negatively regulates red blood cell differentiation and maturation. High methylation modifications may reduce its transcriptional activity, promoting red blood cell proliferation [170, 171]. The study also found that low methylation of the ABCA1 gene is closely related to hemoglobin levels but not to HAPC. ABCA1 is a direct transcriptional target of HIF-1α in human proximal tubule cells [172]. In a separate study, researchers performed a genome-wide differential methylation analysis by comparing Han Chinese and Tibetan patients with HAPC to healthy controls of the same ethnicities [173]. The results of this study indicate that the five genes with the greatest methylation differences between HAPC patients and the control group are MCCC2, ARSJ, CTNNA3, SLC39A10, and SWAP70. The protein regulated by the MCCC2 gene is a subunit of 3-methylcrotonyl-CoA carboxylase, which plays a key role in the metabolism of leucine and isovaleric acid. Previous studies have found that MCCC2 plays an important role in energy metabolism and cellular functions in various cancers, influencing markers of glycolysis and glucose consumption, among other aspects [174, 175]. The zinc transporter protein encoded by SLC39A10 is a key zinc transporter in hematopoiesis and is closely related to zinc deficiency anemia. It plays a crucial role in embryonic hematopoiesis [176]. Mutations related to SWAP70 may lead to hematologic diseases. SWAP70 is a newly identified regulatory factor in integrin-mediated erythroid development and stress-induced erythropoiesis [177]. However, the sample size in this study was relatively small, and further mechanisms were not elaborated upon.

In CMS animal models, chronic hypoxia increased DNMT3a/b expression, leading to high methylation of the VHL gene promoter, inhibiting its transcription, and reducing VHL protein expression [178]. VHL is a core component of the E3 ubiquitin ligase complex for HIF factors. Loss of VHL function causes abnormal accumulation of HIF-α, which activates the EPO signaling pathway [178, 179]. This drives the proliferation and differentiation of erythroid progenitors and disrupts the balance between myeloid and erythroid cells [178, 179]. The study also showed that the DNMT inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC) can reverse the methylation of the VHL promoter, restore its expression, and effectively inhibit the HIF-2α/EPO pathway [178]. This prevents excessive bone marrow erythropoiesis and further confirms the critical role of DNA methylation regulation in chronic hypoxia maladaptation. Recent studies have also shown that DNA methylation levels at the EGLN1 and EPAS1 loci are significantly correlated with hemoglobin levels [75].

High-altitude pulmonary hypertension (HAPH) is a type of CMS and is classified as third-type pulmonary hypertension (PH). It is characterized by persistent constriction of small pulmonary arteries, smooth muscle cell proliferation, and vascular remodeling, which leads to elevated pulmonary arterial pressure in low-oxygen environments at high altitude [180]. Severe pulmonary hypertension exacerbates the burden on the right heart, resulting in right ventricular hypertrophy and contributing to the development of high-altitude heart disease. The role of epigenetic regulation in pulmonary arterial hypertension (PAH, first-type PH) has been widely recognized [181, 182]. However, research on epigenetic regulation in hypoxic pulmonary hypertension (HPH) remains scarce, and some animal models have revealed potential DNA methylation mechanisms.

Studies have found that low methylation of the Versican gene promoter leads to significant upregulation of its expression in HPH mouse models, which, through the regulation of the transcription factor Twist1, mediates endothelial-mesenchymal transition (EndMT) and drives pulmonary vascular remodeling and disease progression [183]. Additionally, glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway has been shown to influence the pathological process of HPH through DNA methylation [184, 185]. In mice, under hypoxic conditions, increased G6PD activity suppresses the expression of TET2, maintaining high methylation in the promoter region of the non-coding RNA lncPIN gene, which accelerates HPH pathological progression [184, 185]. Further study indicated that hypoxia-induced increased expression of DNMTs in rat pulmonary artery smooth muscle cells (PASMC) led to increased methylation of PTEN, reducing its expression [186]. Loss of PTEN triggers smooth muscle cell proliferation and vascular remodeling [187]. The study also found that inhibiting PTEN promoter methylation with 5-Aza-dC significantly suppressed pathological PASMC proliferation and migration, induced cell cycle arrest, and increased apoptosis. Previous research also indicated that pyroptosis is closely related to the pathological process of HPH [188]. In hypoxic rat pulmonary artery endothelial cells (HPAEC), DNA methylation and RNA methylation interaction mediates HPH-induced pyroptosis [189]. Hypoxia affects N6-methyladenosine (m6A) RNA modifications and downregulates lncRNA FENDRR [189]. This downregulation prevents the formation of an RNA-DNA triplex with the DRP1 promoter, increases DRP1 promoter methylation, and reduces DRP1 transcription. As a result, hypoxia-induced pyroptosis in HPAECs is promoted.

DNA methylation in high-altitude-related cognitive dysfunction

High-altitude hypoxic environments impair cognitive function, particularly with prolonged exposure [190, 191]. Hypoxia affects cerebral blood flow and oxygenation, which can contribute to cognitive decline [192]. Long-term exposure to hypoxic conditions at high altitudes may also reduce gray matter density and damage nerve fibers, both of which serve as structural bases for cognitive impairment [193, 194]. DNA methylation regulates the expression of genes involved in neural function, thereby influencing brain function and cognitive ability. In high-altitude rat models, sustained hypoxia significantly upregulated the expression of DNMT1 and DNMT3b in the hippocampus, leading to hypermethylation of the BDNF gene promoter region [195]. This abnormal methylation enhances the binding of methyl-CpG binding protein 2 (MeCP2) to the BDNF promoter, forming a transcriptional repressive complex with histone deacetylases on methylated DNA [196, 197], ultimately decreasing BDNF expression by 60-70%. BDNF is a neurotrophic factor with neuroprotective functions, involved in synaptic plasticity and memory formation [198]. Moreover, high-altitude environments decrease the phosphorylation of MeCP2 at the serine 421 site, impairing its dissociation from the BDNF promoter, which exacerbates transcriptional repression [195, 199]. The absence of BDNF expression weakens the TrkB/PI3K/Akt signaling pathway [200] and reduces the synthesis of synaptic proteins, such as synaptophysin and PSD-95 [201]. This leads to an increased rate of neuron apoptosis in the hippocampal CA1 region of high-altitude rats. This is accompanied by decreased target quadrant time in spatial memory tests and prolonged escape latency [195]. However, studies on the DNA methylation mechanisms associated with cognitive decline in humans at high altitudes remain scarce.

DNA methylation in high-altitude-related pregnancy risks

High-altitude environments, with their low oxygen, pressure, and temperature, can significantly affect pregnancy in women. Studies have shown that hypoxic conditions at high altitudes can reduce blood oxygen saturation in pregnant women, increasing the risk of complications such as hypertension and preeclampsia [202, 203]. In mammals like sheep, the uterine artery’s vascular structure and function are similar to humans [204], and their estrogen receptor-α (ERα) gene sequence and expression closely resemble those in humans [205]. A research team used sheep to simulate high-altitude pregnancy and explored the DNA methylation mechanisms involved in uterine artery changes [205–207]. They found that chronic hypoxia at high altitudes upregulated DNMT3b activity, significantly increasing the methylation of USF and Sp1 binding sites in the ERα gene promoter, leading to decreased binding of transcription factors USF1/2 and Sp1, ultimately suppressing ERα transcriptional activity [205]. Previous studies also found that pregnancy significantly increases ERα mRNA and protein abundance in the uterine artery, while chronic hypoxia inhibits this effect [208]. This epigenetic silencing weakens estrogen-mediated uterine artery dilation while enhancing protein kinase C signaling, which increases vascular tone [209–211].

The research team also discovered that promoter methylation of KCNMB1, which encodes the β1 subunit of the large-conductance calcium-activated potassium (BKCa) channel, was significantly increased under hypoxic conditions. This blocks the binding of Sp1 or ERα to DNA, inhibiting β1 subunit expression [206, 207]. This mechanism reduces the BKCa channel’s sensitivity to intracellular calcium, lowering the membrane depolarization threshold, thereby weakening smooth muscle hyperpolarization and increasing pressure-dependent myogenic tone [208, 212]. Notably, ex vivo experiments confirmed that the DNMT inhibitor 5-Aza-dC could specifically reverse hypoxia-induced CpG hypermethylation, restoring Sp1/ERα binding and downstream gene expression, providing direct evidence for epigenetic intervention [207]. A comparison between the human offspring of high-altitude hypertensive pregnancies and other offspring of normal high-altitude pregnancies revealed that the offspring of hypertensive pregnancies had higher systolic pulmonary artery pressure. Furthermore, a whole-genome differential methylation analysis showed significant differential methylation in several vascular-related genes, which may reveal the role of the environment in transgenerational disease inheritance through epigenetics [213].

DNA methylation in iron overload pathogenesis among highland populations

In the low-pressure hypoxic environment of high altitudes, the body requires substantial amounts of iron for hemoglobin synthesis, thereby facilitating oxygen transport. The prevalence of iron overload is higher in Tibetan men compared to Han Chinese [214]. A whole genome bisulfite sequencing study suggests that abnormal iron metabolism in Tibetans is significantly associated with dynamic DNA methylation imbalances [215]. The diagnostic criteria for iron overload in this study follow the European and American standards. After eliminating factors such as active inflammation, liver disease, tumors, hemolysis, and alcohol, a serum ferritin level > 1,000 ng/ml can be diagnosed as iron overload [216]. This study compared normal Tibetan male individuals from high-altitude areas (> 2500 m) with Tibetan males who had iron metabolism abnormalities. The study included groups based on the criteria of the iron overload group: serum ferritin > 1,000 ng/ml or the normal iron group: serum ferritin < 200 ng/ml. And individuals with hematological diseases, liver diseases, and those using statin medications were excluded from the study.

In the iron overload group, overall methylation levels at CG sites are significantly higher than those in the normal iron group. The differentially methylated regions are primarily enriched in functional regulatory elements of genes related to iron metabolism. Among these, FLVCR1, HMOX2, and HFE genes exhibit increased methylation, while ACO1, IREB2, SLC40A1, and TFRC genes show hypomethylation. The methylation levels of CpG sites in SLC11A2, CYBRD1, and NEDD8 are inconsistent. Hypomethylation of IREB2 and TFRC coordinately increases transferrin receptor expression, promoting cellular iron uptake and leading to elevated intracellular iron levels [217]. High methylation of FLVCR1 reduces its protein expression, impairing its ability to transport heme from the cytoplasm to the extracellular space, potentially causing heme deposition and oxidative damage. HFE can bind to transferrin receptor (TFR) and reduce its affinity for iron-loaded transferrin [218]. The HFE gene also plays a role in sensing extracellular and intracellular iron levels, and may regulate hepcidin expression under both intracellular and extracellular iron overload conditions [219]. In animal models, HFE knockout mice have higher iron levels than normal mice [220]. Abnormal methylation of HFE weakens the negative feedback regulation of iron absorption. Previous studies also suggest that SLC40A1, HFE, and TFR2 genes are associated with hemochromatosis and iron overload phenotypes [221]. In iron overload populations, hypomethylation of SLC40A1 and ACO1 genes may reflect adaptive changes to iron overload [215]. SLC40A1 encodes the metal transporter ferroportin 1 (FPN1), which is located on the basolateral membrane of enterocytes. It mediates the entry of ferrous iron into the portal vein and is considered a key coordinator of iron balance between intracellular and systemic iron homeostasis [222, 223]. Low methylation of SLC40A1 promotes FPN1 expression, enhancing iron efflux and preventing intracellular iron accumulation [223, 224]. ACO1, which binds to the 4Fe-4 S cluster and acts as aconitase, lowers cellular iron levels by isomerizing citrate into isocitrate [225, 226]. These epigenetic changes contribute to the development of a complex regulatory network, which may play a role in the pathological phenotype of iron overload in Tibetans. This offers new insights into the molecular mechanisms behind high-altitude adaptive iron metabolism disorders.

DNA methylation in other aspects of high-altitude injury

Hypoxic conditions at high altitudes significantly affect the expression pathways involved in epidermal barrier repair [227]. Studies have shown that methylation levels of the LIPN gene increase in individuals with repeated high-altitude exposure, potentially downregulating its expression [110], which affects triglyceride hydrolysis and keratinocyte differentiation [228]. LIPN-mediated lipid metabolism influences skin permeability and protection, with its high expression in both the skin and lungs highlighting its crucial role in maintaining epidermal barrier and lung health [229–231]. Under hypoxic conditions, fatty acid synthesis and barrier repair are impaired, and increased LIPN methylation may exacerbate these processes, leading to skin and lung damage [232, 233].

Prolonged exposure to high altitudes may accelerate biological aging and lead to the onset of ageing-related diseases [234]. As an important mechanism of epigenetic regulation, DNA methylation has been shown to be closely associated with the aging process through its dynamic changes [235]. A study employing the Best Linear Unbiased Prediction (BLUP) model quantified the deviation between individuals’ epigenetic age and chronological age using the age acceleration residual (AAR) [75]. The results indicated that the AAR of Han individuals who had migrated to high-altitude areas increased significantly by 1.3 years compared to both indigenous Tibetan populations and low-altitude Han individuals, while no significant difference was observed between the Tibetan and low-altitude Han groups. This suggests that acclimatization to high-altitude environments may accelerate the aging process. Moreover, the study found no significant correlation between AAR and the duration of high-altitude exposure, implying that the epigenetic reprogramming induced by high-altitude acclimatization may reach a stable state early after initial exposure. The phenomenon of high-altitude accelerating epigenetic aging has also been observed in the Andean population [76].

Exposure to high altitudes and hypoxia is a recognized risk factor for venous thromboembolism [236, 237]. This involves multiple pathophysiological mechanisms, including increased red blood cell count and blood viscosity, platelet aggregation, coagulation activation, and endothelial cell damage induced by hypoxia [238, 239]. Whole-genome expression analysis also suggests that hypoxia-triggered hypercoagulability leads to high-altitude venous thromboembolism [240]. DNA methylation, as a key mechanism regulating the interaction between environmental factors and gene expression, plays a role in this process as well. A study have shown that in high-altitude deep vein thrombosis (HADVT) patients, the DNA methylation levels in the thrombospondin (TM) gene promoter region are significantly increased, accompanied by decreased TM mRNA and protein expression, indicating a direct association between high DNA methylation of the TM gene and its transcriptional repression [241]. As a key anticoagulant protein, reduced expression of TM disrupts vascular homeostasis, thereby increasing the risk of thrombosis [242, 243]. The study found that DNMT1 and DNMT3B mRNA were significantly upregulated in the HADVT group and hypoxic endothelial cells [241], suggesting that methylation may stabilize HIF-1α, activating DNMTs, particularly DNMT3b, and promoting methylation of the TM gene promoter, which suppresses its transcription and translation [244]. In vitro experiments further confirmed that the DNMT inhibitor decitabine reversed hypoxia-induced TM methylation, restoring TM expression [241].

Conclusions and future perspectives

DNA methylation serves as a central regulatory mechanism in adaptation to high-altitude environments and the development of associated diseases. It acts as an “environmental sensor” by dynamically modifying the expression of key genes in response to hypoxic challenges. Current research indicates that high-altitude populations, such as Tibetans and Andeans, enhance oxygen transport efficiency and energy metabolism by reprogramming the methylation patterns of hypoxia-related genes, including EPAS1 and EGLN1, while concurrently inhibiting excessive erythropoiesis and oxidative stress damage. This epigenetic regulation not only compensates for the time lag associated with genetic adaptation but also interacts synergistically with genetic variations. For instance, functional SNPs in the EPAS1 and EGLN1 gene co-localize with its differentially methylated regions, highlighting the fine balance of “gene-epigenetic” interactions under environmental pressures [76, 77]. Conversely, aberrant methylation patterns may contribute to the onset of both acute and chronic high-altitude diseases by disrupting the homeostasis of the HIF pathway, endothelial function, and neural plasticity.

While significant progress has been made in this field, several gaps remain. First, the DNA methylation mechanisms underlying long-term (ranging from several months to years) high-altitude acclimatization in lowland populations have been scarcely explored. To date, only one study has revealed mechanisms related to long-term acclimatization [75]. However, that study involved a wide range of acclimatization durations (from six months to 63 years) and substantial age differences among participants, which may introduce considerable biases. Longitudinal studies require interdisciplinary collaboration and substantial resources, especially given the technical challenges associated with sample collection, such as peripheral blood or tissue biopsies taken at multiple time points. Secondly, in terms of high-altitude adaptation, there is currently a lack of direct studies on the differences in DNA methylation between three classic high-altitude populations: the Andeans, Tibetans, and Ethiopians. There is also a lack of research on the DNA methylation differences during high-altitude acclimatization between different lowland populations, such as East Asian and European populations. This may require collaborative research efforts from teams across different countries. The role of gut microbiota in high-altitude adaptation [45] / acclimatization [245] has been proven, and previous studies have also shown that gut microbiota are related to epigenetics such as DNA methylation [246]. However, direct studies on the role of gut microbiota and DNA methylation in high-altitude adaptation/acclimatization are still lacking.

Research on the DNA methylation mechanisms of high-altitude diseases still heavily relies on animal models or in vitro cell simulations, with a lack of clinical evidence from humans. While animal models can partially simulate pathological phenotypes, their hypoxic exposure models (such as normobaric hypoxia chambers) fail to replicate the comprehensive pressures of real high-altitude environments (e.g., low temperature, UV radiation). Additionally, the conservation of epigenetic regulation networks across species is questionable, which limits the clinical translational value of the research findings. This may be due to the ethical and medical constraints on invasive tissue sampling (e.g., pulmonary vascular or placental tissue) in high-altitude regions, which further hinders progress in this area.

Existing studies have shown that the risks of common hypoxia-related diseases, such as ischemic heart disease, chronic obstructive pulmonary disease (COPD), and sleep-disordered breathing, differ between high-altitude and lowland regions. For instance, the prevalence of COPD is lower at high altitudes, which may be related to the potential protective effects of the hypoxic environment on lung function [247]. This phenomenon may also be associated with the vascular roles of factors such as PTEN in HAPH, as discussed in Sect. 4.2. However, other studies have reported a higher prevalence of COPD at high altitudes, possibly due to household air pollution, particularly in resource-limited highland environments where indoor particulate concentrations tend to be elevated [248]. In addition, the risk of ischemic heart disease may be reduced at moderate altitudes, potentially due to adaptive cardiovascular responses to chronic hypoxia [249, 250]. These adaptations may be closely linked to altitude-induced epigenetic changes, such as DNA methylation of genes involved in the RAS pathway. However, acute exposure to high-altitude hypoxia, especially in individuals with pre-existing cardiovascular conditions, may conversely increase the risk of cardiovascular events [251]. Regarding sleep disorders, the incidence of central sleep apnea (CSA) is higher at high altitudes, particularly among males, which may be associated with increased peripheral chemosensitivity in hypoxic environments [252]. Overall, the altitude-related differences in the risks of these common hypoxia-related diseases may be closely tied to epigenetic mechanisms such as DNA methylation. However, targeted studies investigating these epigenetic differences, particularly DNA methylation, remain lacking and warrant further exploration.

With the rapid development of epigenetics technologies, emerging DNA methylation research methods provide important tools for deeply analyzing the mechanisms of high-altitude adaptation/acclimatization and the pathology of related diseases. Among these methods, oxidative bisulfite sequencing (oxBS-Seq) selectively distinguishes between 5mC and 5hmC. This technique allows researchers to dynamically and precisely monitor methylation and demethylation in high-altitude hypoxic environments. This method lays the foundation for revealing the hierarchical complexity of epigenetic regulation [253]. Single-cell methylation analysis technology overcomes the limitations of traditional bulk sequencing. It enables the analysis of heterogeneous methylation patterns in different immune cells, endothelial cells, or neuronal cells under high-altitude stress. This allows for the precise identification of susceptible cell subpopulations and their epigenetic regulatory networks [254]. Furthermore, the CRISPR-dCas9 epigenome editing tool can specifically modify the methylation status of gene loci [255]. Researchers can use this approach to directly verify the epigenetic regulatory mechanisms of high-altitude adaptation/acclimatization related genes, such as EPAS1 and EGLN1. It also helps clarify their causal relationships with diseases like polycythemia and HACE. These new technologies will promote the mapping of the high-altitude adaptation/acclimatization epigenetic landscape from multiple dimensions and high-resolution perspectives.

Future research should establish a prospective cohort of lowland populations with long-term high-altitude exposure. Researchers can then apply multi-omics technologies to clarify how methylation evolves from acute stress responses to chronic acclimatization. Further exploration of the epigenetic mechanisms underlying the differences in the incidence of common hypoxia-related diseases between high-altitude and lowland regions is also warranted. Further advances in translational medicine should aim to develop early diagnostic biomarkers for high-altitude diseases based on plasma free DNA methylation and explore the therapeutic potential of inhibitors targeting DNMTs and TET enzymes to reverse pathological methylation. By integrating technological innovation with clinical practice, DNA methylation research has the potential to move from molecular mechanism discovery to precise medical interventions, ultimately facilitating disease prevention and health promotion through the guidance of the “high-altitude adaptation/acclimatization epigenetic map.” This approach will provide robust support for the development of high-altitude medicine and offer innovative strategies for humanity’s conquest of extreme environments.

Abbreviations

CpG

Cytosine-phosphate-guanine dinucleotide

HIF

Hypoxia-inducible factor

5mC

5-methylcytosine

5hmC

5-hydroxymethylcytosine

HACE

High-altitude cerebral edema

HAPE

High-altitude pulmonary edema

HAPC

High-altitude polycythemia

HADVT

High-altitude deep vein thrombosis

HAPH

High-altitude pulmonary hypertension

CMS

Chronic mountain sickness

AMS

Acute mountain sickness

PHD

Prolyl hydroxylase domain proteins

VHL

Von Hippel-Lindau

FIH

Factor inhibiting HIF

DNMT

DNA methyltransferase

HRE

Hypoxia response elements

SNP

Single nucleotide polymorphism

TET

Ten-eleven translocation

2-OGD

2-oxoglutarate-dependent dioxygenase

EPO

Erythropoietin

HBS

HIF-1 binding site

RAS

Renin-angiotensin system

ROS

Reactive oxygen species

G6PD

Glucose-6-phosphate dehydrogenase

5-Aza-dC

5-aza-2′-deoxycytidine

AAR

Age acceleration residual

Author contributions

X.Z. and Q.S. set up the topic; X.Z. and Y.Y. performed writing and editing; Q.S. conducted modification and gave suggestions; X.Z., Y.Y. and Q.S. performed manuscript revision. All authors have read and agreed to the submitted version of the manuscript.

Funding

This work was financially supported by [Central Government Guidance for Local Science and Technology Development Fund Projects] (Grant number ZYYD2025ZY14).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This review did not conduct any experiments. The human and animal studies included in this review all underwent the relevant ethical review.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

RRIDs

This review did not involve original biological resources requiring RRID reporting.