An Integrative Framework for EWAS in Challenging Environmental Contexts: Application to Chronic Hypobaric Hypoxia

Abstract Background: Over the past decade, Epigenome-Wide Association Studies (EWAS) have emerged as a powerful approach to link environmental exposures, gene regulation, and phenotypes beyond genetic variation, but their ability to explain complex adaptations remains limited, highlighting the need for integrative strategies. Aims: This Thesis aimed to investigate DNA methylation alterations associated with chronic hypobaric hypoxia at high altitude, considering environmental, lifestyle, genetic and socio-psychological influences, through the development of an integrative framework combining genetic, epigenetic, and clinical data to clarify the role of DNA methylation in the manifestation of adaptive phenotypes. Material and Methods: The study population was a subcohort of the HIGHCARE-LAPS project, including 192 subjects from Peru, equally divided between Lowlanders (Lima, sea level) and Highlanders (Cerro de Pasco, 4,400 m). Genomic and epigenomic data (array-based) were collected from blood, together with lifestyle, socio-psychological, and clinical variables. A replication cohort from a public dataset was used when available. The framework included all key steps (data import, quality control, preprocessing, and normalization) and introduced alternative approaches to traditional EWAS, including adapted differential methylation analysis, sensitivity checks, detection of stochastic epigenetic mutations, rare epivariation, exploration of altitude–methylation–phenotype links, and the use of epigenetic clocks. Results and Conclusions: The application of this alternative framework has proven effective in reducing noise and strengthening traditional analyses, enabling the identification of new sites and regions potentially involved in hypoxia-related biological processes. It has also emerged that some adaptive phenotypes are mediated by DNA methylation in genes already known for such phenotypes, but not previously associated with chronic high-altitude hypoxia. Other metrics, such as epigenetic age acceleration, have shown an increase without negative associations with phenotypes, opening up new avenues for research. Overall, these findings highlight the value of adopting refined analytical strategies to better capture the complexity of environmental adaptation.