The clinical manifestations of chronic obstructive pulmonary disease (COPD) are chronic respiratory symptoms (dyspnea, cough, sputum, acute exacerbations) due to airway (bronchitis, bronchiectasis) and/or alveolar anomalies (emphysema) that cause persistent and progressive exacerbation of airflow limitation [1]. Early COPD is asymptomatic or mildly symptomatic, meaning that diagnosis and treatment are easily delayed. Lung function damage such as fine bronchitis and destruction of the lung parenchyma (emphysema) has often already occurred when the diagnosis is confirmed [2, 3]. COPD is therefore a significant public health problem worldwide [4] and a major cause of mortality [5]. It presents a serious threat to patient survival and quality of life, as well as being a great burden to society [2].

COPD is the result of interactions between genes and the environment over time [6]. Epidemiologic investigations have shown a clear familial aggregation in some COPD patients [7, 8], thus revealing the importance of genetic factors. Genome-wide association studies (GWAS) have identified many candidate genes related to COPD pathogenesis [9], reinforcing the notion that genetic factors may play an important role in the development of COPD. Raguso et al. [10] found some similarities between chronic exposure to high altitude hypoxia in healthy people and chronic hypoxia in COPD patients, again suggesting that hypoxia has an important role in the development of COPD [11]. However, most of the current studies on candidate genes for COPD have focused on inflammation-related genes [12], with oxygen-sensitive genes deserving more in-depth investigation.

Egl-9 family hypoxia-inducible factor (EGLN1) acts as an oxygen sensor and catalyzes prolyl hydroxylation of the transcription factor hypoxia-inducible factor-1$\alpha$ under normoxic conditions, leading to its proteasomal degradation. EGLN1 therefore has a central role in the hypoxia-inducible factor-mediated hypoxia signaling pathway [13]. Tibetans have lived at high altitudes for generations and can adapt to environmental hypoxia. The gene products from some regions of their genome may be associated with high altitude adaptation, and EGLN1 is significantly associated with a reduced hemoglobin phenotype that is characteristic of plateau populations [14, 15, 16]. At high altitudes especially, chronic hypoxia causes EGLN1 to play an important regulatory role in the development of COPD [17, 18, 19]. However, it is not known whether genetic variations in EGLN1 are associated with the risk of developing COPD.

The Gannan Tibetan Autonomous Prefecture is located on the northeastern edge of the Qinghai-Tibetan Plateau [20] and is in a high elevation region [21]. A previous study by our group found that the prevalence of COPD in Gannan was 23.4% (20.7%–26.4%) [22]. Here, we conducted a case-control study in this Prefecture to investigate possible associations between EGLN1 single nucleotide polymorphisms (SNPs) (rs41303095 A$>$G, rs480902 C$>$T, rs12097901 C$>$G, and rs2153364 G$>$A) and COPD susceptibility, and to validate the effect of EGLN1 polymorphisms on lung function.

The development of GWAS in recent years has led to many studies showing that genetic polymorphisms are associated with the development of various diseases. This includes an association with COPD, thus providing a new direction for the study of COPD susceptibility. To study whether EGLN1 polymorphisms are involved in COPD, we investigated the association of four SNP loci in EGLN1 (rs41303095 A$>$G, rs480902 C$>$T, rs12097901 C$>$G, and rs2153364 G$>$A) with the risk of COPD in Gannan Tibetans. No significant association was found between any of the above SNPs and the risk of developing COPD in this population.

Positive selection genome-wide scans of Tibetan populations revealed the EGLN1 genetic locus encodes prolyl hydroxylase 2 (PHD2), which may allow for adaptive biological changes in humans in response to the low oxygen environment found in high plateaus [24]. Alterations in the hypoxia-inducible factor (HIF) pathway were shown to be the mechanism underlying this adaptive change [25, 26, 27]. Moreover, the genomic region containing EGLN1 was shown to be one of the strongest selection signals in Tibetans [14]. The EGLN family is itself a member of the larger 2-oxoglutarate and ferrous iron-dependent oxygenase family [28], which has a role in maintaining oxygen homeostasis in oxygen-organized metabolism in biological cellular tissues. The EGLN1 gene is located on human chromosome 1 (1q42.2) [29], which is the actual oxygen receptor in mammals [30]. Several studies have shown that under hypoxic stimulation, EGLN1 reduces its own activity [31, 32, 33], which means that HIF-1$\alpha$ is ineffectively degraded and continues to accumulate [34] and overexpress, thereby regulating related transcription factors. These factors mediate the hypoxic response and include genes that promote glucose uptake, facilitate glycolysis, stimulate angiogenesis, or regulate apoptosis and erythropoiesis [35]. They are particularly important in mediating erythropoiesis, angiogenesis, and lung development, thus contributing to the pathogenesis of diseases such as congenital erythrocytosis, pulmonary hypertension, and COPD [36]. The hypothesis that EGLN1 is involved in COPD-related responses by regulating HIF-1$\alpha$ in a hypoxic environment is therefore tenable.

Studies conducted by Mishra et al. [34] and Sharma et al. [37] on an Indo-Aryan population from the Tibetan plateau region found a significant difference in expression of the EGLN1 rs480902 variant between patients with high altitude pulmonary edema (HAPE) and healthy controls (p 0.05). This is consistent with the findings by Wu et al. [38] on Han Chinese individuals. The study by Mishra et al. [34] also found that arterial oxygen saturation (SaO${}_2$) levels were significantly higher in non-high-altitude pulmonary edema patients and healthy controls compared to high-altitude pulmonary edema patients. Bhandari et al. [39] found that Sherpas carrying EGLN1 rs12097901 and rs186996510 variant alleles had lower hemoglobin levels compared to wild-type allele carriers. The above finding may be attributed to the high expression of the EGLN1 gene. This would enhance the blocking effect of hydroxylated proline-catalyzed HIF-1$\alpha$ translational modification at positions 402 and 564 in the structural domain of HIF-1$\alpha$ oxygen-dependent degradation (ODD). This destabilizes HIF-1$\alpha$, preventing its downstream genes from maintaining cellular oxygen homeostasis and thereby hindering oxygen signaling. The hemoglobin concentration then increases to compensate for the losses associated with the low oxygen environment of the high-altitude plateau.

Online web tools such as NCBI and Ensembl were used in the present study to find the SNP location and functional information for the 4 SNPs investigated here. EGLN1 rs41303095 A$>$G is located at the 3${}^{\prime }$UTR end of EGLN1 (Chr: 231364492), with no studies so far having reported an association of this polymorphism with any disease. rs480902 C$>$T is located in the first intronic region of EGLN1 (Chr: 231395881), which prevents the translocation of HIF-1$\alpha$ to the nucleus and thus hinders oxygen signaling [40, 41]. Researchers have reported associations of this locus with diseases such as plateau pulmonary edema, altitude sickness, and chronic mountain sickness [34, 37, 38, 42, 43, 44, 45]. rs12097901 C$>$G is located in exon 1 of EGLN1 and is a missense mutation (Chr: 231421509) that causes HIF activation [46]. This SNP has been associated with high-altitude acclimatization, acute low-pressure hypoxia, and high-altitude erythropoiesis [14, 18, 39, 46]. rs2153364 G$>$A is located within the binding site for the EGLN1 5${}^{\prime }$UTR transcription factor c-Ets (Chr: 231424474). This site may affect the expression of EGLN1, and therefore its regulation of HIF-1$\alpha$, by influencing the binding efficiency of the c-Ets transcription factor [47]. Several studies have reported associations of this polymorphism with acute altitude sickness and hemoglobinopenia [47, 48, 49].

The prevalence of COPD in individuals aged 40 years or more in the Gannan region was previously found by our group to be 23.4% (20.7%–26.4%) [22]. This is considerably higher than the national incidence of 13.7% (12.1%–15.5%) [50]. Moreover, the current study found that EGLN1 rs12097901 C$>$G was associated with the level of pre-medication lung function indexes. These may be related to overexpression of the hypoxia gene EGLN1 in the hypoxic environment of the high plateau. Another study by our group found that the hematopoietic cytokine EPO rs1617640 A$>$C SNP was associated with COPD susceptibility in a southern Chinese (Guangzhou) population [51], with the C allele being associated with pre-medication lung function. We also found that the hypoxia-inducible factor EPAS1 rs13419896 G$>$A SNP could reduce susceptibility to COPD [52]. EPAS1 and EGLN1 are two of the preferred candidate genes previously reported in high altitude acclimatization studies in Tibetans [53, 54, 55].

In summary, it is reasonable to assume that EGLN1 may be involved in COPD pathogenesis. However, this study failed to show a statistically significant association between EGLN1 genetic variants and COPD. This may be due to the screening of only a few loci, insufficient sample size, recall bias in the case-control studies, and lack of quality control in the field work. Further studies will be conducted to overcome these shortcomings and to identify more relevant loci.

The present study found that the EGLN1 rs41303095 A$>$G, rs480902 C$>$T, rs12097901 C$>$G, and rs2153364 G$>$A SNPs were not significantly associated with the susceptibility to COPD in the Gannan Tibetan population.

COPD, chronic obstructive pulmonary disease; EGLN1, Egl-9 family of hypoxia-inducible factors; GWAS, genome-wide association analysis; SNPs, single nucleotide polymorphisms; FEV1, forceful expiratory volume in 1 second; FVC, forceful lung capacity; MAFs, minor allele frequencies; PCR, TaqMan real-time polymerase chain reaction.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

XZ designed the research study. XL wrote the main manuscript and revised it with JY, PZ. Statistical analysis of the results was done mainly by JY with the participation of XL. CZ, YS, XuW, HL and AL contributed to sample collection for the case-control and prevalence studies, and for electronic medical record entry. ZY provided assistance with experimental design and conduct. PZ has given a lot in the integration and utilization of literature searches. XiW and YW were responsible for the overall study design and quality control as well as directing the study methodology. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Boards of Xi’an Jiaotong University, Faculty of Medicine (Approval No.: XJTU 2016-411) and Guangzhou Medical University (Approval No.: GZMC2007-07-0676). In Furthermore, we interpreted the purpose of the study to all participants at the beginning of the study and obtained their signed informed consent.

We thank the Institute of Public Health, Guangzhou Medical University for their help. We thank the researchers in our laboratory for their guidance on experimental techniques.

This research was funded by the following grants: Fund project of Gansu Provincial Department of Education 2022CYZC-53 (Xinhua Wang); National Key Research and Development Program Project of Gansu Urban and Rural Natural Population Cohort Construction and Tumor Follow-up Research 2017YFC0907202 (Xinhua Wang).

The authors declare no conflict of interest.