Multi‐Ancestry Epigenome‐Wide Association Study of Asthma Exacerbations

Asthma exacerbations (AEs) are a major concern in public health due to their impact on the quality of life, the risk of mortality of patients, as well as the associated economic burden. Therefore, understanding AEs' biological bases could facilitate the development of personalized prevention and management strategies and the identification of therapeutic targets. Recently, the first epigenome-wide association study (EWAS) of AEs revealed epigenetic biomarkers in Europeans [1]. To improve our understanding of AE genetics across different populations, we conducted EWAS of AEs in minoritized admixed populations from the United States. Blood DNA methylation (DNAm) profiles were obtained for 599 Latino Americans (Genes-Environment and Admixture in Latino Americans Study) [2] and 394 African Americans (Study of African Americans, Asthma, Genes and & Environments) [2] (Tables S1, S2) with the Infinium MethylationEPICv1 microarray (Illumina). Association results of DNAm at differentially methylated positions (CpGs) with AEs revealed a significant association of the CpG cg21608069 annotated to the makorin ring finger protein 2 gene (MKRN2) (coefficient = 0.09, p-value = 9.56 × 10−8) (Table 1, Figures S1, S2). MKRN2 encodes a ubiquitin E3 ligase that promotes the degradation of the p65 subunit of the proinflammatory transcription factor NF-κB, affecting NF-κB-mediated inflammatory responses [3]. Interestingly, epigenetic variation at the MKRN2 gene has been previously associated with fractional exhaled nitric oxide (FeNO) levels in nasal samples from children [4]. Among four CpGs previously suggestively associated with AEs in Europeans [1], three CpGs were validated and showed consistent effects in Latino and African American populations at false discovery rate (FDR) < 5% adjusted for the four CpGs tested (Table 1): cg15508223 (LOC100130691, FDR = 7.75 × 10−7), cg25345365 (ZBTB16, FDR = 1.54 × 10−5), and cg03546163 (FKBP5, FDR = 0.018); cg06710464 was not significant (BAIAP2, p = 0.061). A multi-ancestry meta-analysis including Latinos, African Americans, and Europeans identified the same three CpGs associated with AEs at FDR < 5% and revealed the novel association of the CpG cg17148519 (USP7) (p-value = 1.41 × 10−7) (Table 1, Figure S2). The deubiquitinating enzyme ubiquitin-specific protease 7 (USP7) participates in the differentiation of functional Th2 cells involved in chronic allergic inflammation, including asthma, by regulating signal transducers and activators of transcription [5]. Interestingly, nasal DNAm levels at this CpG have been previously associated with pediatric asthma and FeNO levels [4]. Furthermore, we identified 22 significant differentially methylated regions (DMRs) associated with AEs in minoritized admixed populations (adjusted p-value < 0.05, Table 2). From these, the genomic region of three CpGs annotated to WD repeat domain 4 (WDR4) gene was consistent using an independent method (adjusted p-value = 2.69 × 10−6) and showed a stronger association in the multi-ancestry meta-analysis (adjusted p-value = 2.65 × 10−9) (Table 2). WDR4 encodes a subunit of a methyltransferase complex, and its expression levels have been linked to lung cancer [6]. Moreover, DNAm in this gene has been associated with the body mass index (BMI) in children and with different environmental exposures (i.e., smoking and polychlorinated biphenyls exposure) [4], although the role in asthma is unknown. Finally, the integration of epigenetic and gene expression data from 450 Latinos and 254 African American asthma patients from the discovery phase through a cis-expression quantitative trait methylation analysis revealed that DNAm at the CpG cg03546163 was associated with lower FKBP5 expression (FDR = 0.025) (Table S3). We supported previous findings of FKBP5 expression associated with severe asthma and DNAm levels at this CpG in nasal samples from African American patients [7]. Moreover, DNAm levels at FKBP5 have been associated with bronchiolitis severity in blood samples, which constitutes a risk factor for childhood asthma [8]. We acknowledge that patients in our study come from diverse geographic, demographic, and ethnic backgrounds, which may entail differential exposure to environmental risk factors for asthma exacerbations (e.g., air pollution, viral infections, and allergens). In addition, age- and sex-related biological differences may also influence susceptibility to asthma exacerbations and interact with genetic and epigenetic mechanisms. Although we adjusted our EWAS models for genetic ancestry, age, and sex, residual confounding due to unmeasured environmental exposures, gene–environment interactions, and ancestry variability may still influence our findings. Future studies with larger and diverse populations, harmonized environmental data across cohorts, and using integrative approaches will be essential to further disentangle these complex relationships. In conclusion, our study reported novel epigenetic associations with AEs in minoritized admixed and multi-ancestry populations and validated previous findings in Europeans. We conducted the largest multi-ethnic meta-EWAS of AEs by employing robust and standardized statistical methods to control confounders, multiple testing, and biases in DNAm. However, cross-sectional associations do not provide information about causality. Further research will be needed to assess epigenetic biomarkers' clinical applicability in asthma management. M.P.-Y. was involved in the conceptualization and design of the study; J.W., S.H., C.E., D.H., R.G.-P., J.M.H.-P., P.P.-G., O.S., I.S.-.M, E.M.-L., P.C.-E., J.V., J.R.-S., E.Z., and E.G.-B. in data acquisition; E.M.-G., J.P.-G., M.M.-A., F.L.-D., and M.P.-Y. in data curation, formal analysis, and/or interpretation of the data; M.P.-Y. in project supervision. E.M.-G., J.P.-G., and M.P.-Y. participated in the preparation and writing of the original draft. All authors were involved in revising the manuscript and approving the final version. All authors have read and agreed to the published version of the manuscript. We acknowledge the patients, families, recruiters, healthcare providers, and community clinics for their participation. We thank Sandra Salazar for her support as GALA II study coordinator. We thank the investigators and teams at the New York Genome Center and the Northwest Genomics Center for whole-genome sequencing sample preparation, quality control, data generation, and data processing. We gratefully acknowledge the studies and participants who provided biological samples and data for TOPMed. We also gratefully acknowledge the contributions of the investigators of the NHLBI TOPMed Consortium (www.topmed.nhlbi.nih.gov/topmed-banner-authorship). The generation of molecular data for the TOPMed program was supported by the National Heart, Lung, and Blood Institute (NHLBI). RNA-seq for the NHLBI TOPMed Genes-Environments and Admixture in Latino Asthmatics Study (GALA II; phs000920) and Study of African Americans, Asthma, Genes, and Environments (SAGE; phs000921) was performed at the Broad Institute Genomics Platform (HHSN268201600034I). The authors thank the Centro Nacional de Genotipado-Plataforma de Recursos Biomoleculares-Instituto de Salud Carlos III (CeGen-PRB3-ISCIII; www.cegen.org) for providing genotyping and DNAm assays services for the GEMAS studies. E.M.-G. reports a fellowship from the Canarian Agency for Research, Innovation, and the Information Society of the Counseling of Universities, Science and Innovation and Culture. J.P.-G. reports a fellowship from the MICIU. M.M.-A. reports a fellowship from the Spanish Ministry of Science, Innovation and Universities. M.P.-Y. reports grants from the Instituto de Salud Carlos III, Madrid, Spain. M.P.-Y. reports grants from the Spanish Ministry of Science, Innovation, and Universities (MICIU/AEI/10.13039/501100011033) and also received grant support from CSL Behring for a project outside of this work. The rest of the authors declare no conflicts of interest or other interests that might be perceived to influence the interpretation of the article. No supporting institution may gain or lose financially through this publication. The data that support the findings of this study are openly available in Zenodo Repository at https://zenodo.org, reference number https://doi.org/10.5281/zenodo.14851968. Figure S1: Quantile–quantile plots for the EWAS of the association between asthma exacerbations and DNA methylation in (A) Latinos, (B) African Americans, and (C) Meta-analysis of Latinos and African Americans. The observed p-value (− log10 p-value) is shown on the y-axis along with the expected p-value (− log10 p-value) represented on the x-axis. Figure S2: Manhattan plot of the meta-EWAS of asthma exacerbations in blood samples from the (A) Minoritized admixed populations meta-analysis (Latinos and African Americans) and (B) multi-ancestry population meta-analysis (Latinos, African Americans, and Europeans). The blue and red lines represent the false discovery rate < 5% and the genome-wide (p-value < 9 × 10−8) significance thresholds, respectively. Gene annotation is represented for the top-hit CpGs. Table S1: Characteristics of the study participants included in the meta-EWAS of asthma exacerbations in minoritized admixed populations. Table S2: Summary of the quality control of methylation data from Latinos and African Americans. Table S3: Summary results of the eQTM analysis of CpGs in Latinos, African Americans, and meta-analysis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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