Response to Letter on “Downregulation of EPAS1 and EGLN1 mRNA Expression Associated With High‐Altitude Adaptive Genetic Variants in Sherpa Highlanders”

1.

To the Editor,

We sincerely thank Malik for the thoughtful comments regarding our recent article examining the relationship between high‐altitude adaptive genetic variants and EPAS1/EGLN1 mRNA expression in Sherpa highlanders. We appreciate the opportunity to clarify several aspects of our study and agree that further investigations are needed to elucidate the mechanisms underlying high‐altitude adaptation.

First, regarding the concern that whole‐blood mRNA expression may be influenced by differences in leukocyte composition, we acknowledge that bulk blood transcriptomic measurements can be affected by cellular heterogeneity. Our study utilized whole‐blood RNA because of the logistical challenges associated with sample collection and processing in remote high‐altitude regions. We did not obtain differential leukocyte counts and therefore could not adjust for cell‐type composition. We agree that future studies incorporating complete blood counts and flow cytometric characterization would provide a more refined assessment of cell‐specific gene expression. Nevertheless, the substantial reductions in EPAS1 and EGLN1 transcript levels observed in Sherpas were consistent across both genes and paralleled findings previously reported in Tibetan highlanders, suggesting that the observed differences are unlikely to be explained solely by variations in leukocyte proportions.

Second, Sherpas are an Indigenous ethnic population of the Himalayan region who have resided at high altitude for centuries. The ancestors of modern Sherpas are believed to have migrated from eastern Tibet approximately 500 years ago. Through long‐term adaptation, Sherpas have developed remarkable tolerance to high‐altitude hypoxia and are rarely affected by acute or chronic mountain sickness (CMS). The primary objective of our study was to investigate the genetic mechanisms underlying this exceptional adaptation to high‐altitude environments.

To address this question, we compared Sherpa highlanders in Nepal with Nepalese lowlanders residing in Kathmandu (1330 m), who served as the reference lowland population. As Sherpas are Nepalese citizens, this comparison minimized potential confounding by environmental factors (other than altitude), socioeconomic conditions, and government welfare policies, thereby facilitating the investigation of genetic factors associated with high‐altitude adaptation. We referred to the Kathmandu residents as “non‐Sherpa” lowlanders to distinguish them from the Sherpa population and to avoid unnecessary complexity in terminology. Furthermore, recruiting large cohorts of lowlanders permanently residing at high altitude or highlanders permanently residing at low altitude is generally impractical because of substantial logistical challenges and the limited availability of such populations.

Moreover, EPAS1 and EGLN1 mRNA expression levels were not significantly correlated with oxygen saturation (SpO₂) among Sherpa highlanders (fig. 2B,C in Droma et al. ²⁰²⁶), suggesting that the transcriptional responses of EPAS1 and EGLN1 to hypoxia may be attenuated in Sherpas. This finding is consistent with the well‐established physiological adaptation of Sherpas to high‐altitude environments, characterized by a remarkable tolerance to chronic hypoxic stress.

Third, Malik correctly noted that the genotype‐stratified expression analyses involved relatively small subgroup sample sizes. We fully acknowledge that these analyses should be interpreted as exploratory because the limited sample size precluded adequately powered within‐stratum statistical testing and resulted in relatively large standard errors. Nevertheless, the primary objective of these analyses was to determine whether the direction of the expression differences was consistent with the population‐level observations. We agree that larger cohorts are required to provide adequately powered genotype–expression association analyses and to establish robust estimates of effect size.

Fourth, the two populations in Nepal are not only geographically proximate but have also shared extensive political, economic, and cultural interactions over the past five centuries within Nepal. Regarding population stratification, we acknowledge the possibility of genetic heterogeneity arising from the inclusion of multiple ethnic groups within both the Sherpa and non‐Sherpa populations in Nepal. However, previous genetic studies have demonstrated that human genetic variation is generally geographically structured, tends to be distributed continuously across populations, and rarely exhibits sharp geographic discontinuities (Jorde and Wooding 2004).

Nevertheless, the principal objective of this study was to compare allele frequencies and gene expression profiles between indigenous highlanders and lowlanders. We agree, however, that the inclusion of ancestry‐informative markers and principal component‐based adjustment would strengthen future studies investigating genotype–expression relationships and further reduce the potential for confounding due to population stratification.

Fifth, regarding serum erythropoietin (EPO), it has been demonstrated that excessive EPO production in response to high‐altitude hypoxia contributes to polycythemia in CMS. Figs. 5 and 6 (Droma et al. 2026) were not intended to demonstrate direct associations between circulating EPO concentrations and EPAS1 or EGLN1 genotypes, particularly given the limited statistical power resulting from the relatively small sample size. Rather, we proposed a biologically plausible framework based on the established roles of EPAS1 and EGLN1 in oxygen sensing and erythropoietic regulation. Indeed, EPO production is influenced by multiple regulatory mechanisms, and circulating EPO concentrations represent only one downstream component of the HIF pathway. We therefore agree that additional investigations incorporating other downstream molecular markers and functional assays are required to clarify the physiological consequences of the observed transcriptional differences.

Importantly, our study was intended as an observational investigation of naturally occurring genetic variation and gene expression patterns in the well‐characterized, high‐altitude‐adapted Sherpa population. We believe that our findings provide additional evidence supporting a role for the HIF pathway in human high‐altitude adaptation while recognizing the limitations of the present study.

We appreciate Malik's constructive comments, which highlight important directions for future research. We hope that larger studies incorporating transcriptomic, genomic, and functional approaches will further advance our understanding of the molecular mechanisms underlying human adaptation to chronic hypoxia.

Data Availability Statement

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