C101-10 Epigenetic Signatures of Sputum Eosinophils in COPD

Abstract Rationale Eosinophilic airway inflammation in COPD is associated with more frequent exacerbations,lower lung function,and corticosteroid responsiveness.DNA methylation captures information about genetic factors and environmental exposures, but is under-studied in eosinophilic COPD.We hypothesized that sputum cell methylation patterns reflect genomic regulation of inflammatory endotypes in COPD by DNA sequence variation. Methods We analyzed DNA previously extracted from induced sputum cells for a prior study(PMID38261629) for which sufficient quantity DNA remained for methylation arrays.For this analysis,we focused on n = 132 SPIROMICS participants with ≥20 pack-years smoking history.Samples were stratified into tertiles based on sputum eosinophil proportions,previously determined from sputum cell differential analysis by the centralized sputum analysis core for SPIROMICS(Univ of North Carolina).We assessed proportion of DNA methylation (β values) spanning 936,866 CpG sites using the Illumina Infinium Methylation EPICv2.0 BeadChip Array,which we transformed into M values(meffil R package). To identify differentially methylated regions (DMRs)(FDR0.05 in minimum of 2 CpG sites per gene region),we contrasted CpG β values between the high-eosinophil and low-eosinophil tertiles(N = 44 each),using the DMRcate package.We tested for enrichment of genes overlapping with significant DMRs using Enrichr (GWAS Catalog 2025 gene set library).To identify genetic variants associated with DNA methylation levels,we performed Methylation Quantitative Trait Loci (meQTL) analysis using TensorQTL.For each CpG site,we tested associations with SNPs within a ± 500 kb window (cis-meQTL).The linear regression model included age,sex,BMI,smoking status,smoking pack-years,study site,inhaled corticosteroid use, the first two genetic principal components, cell-type proportions (macrophages/monocytes, neutrophils, squamous epithelial cells, lymphocytes), and eosinophil tertile . Significant meQTLs were identified at an FDR0.05 using Benjamini-Hochberg correction. Results A total of 532 eosinophil-associated DMRs (FDR 0.05) were identified between the high- and low-eosinophil groups,encompassing 446 unique genes.The top enriched pathways related to counts of eosinophils (adjusted p = 0. 00002464), lymphocytes (adjusted p = 0.0001860), and the sum of eosinophils-plus-basophils (adjusted p = 0.0001860).We identified 18,432 significant SNP-CpG/meQTL pairs (FDR 0.05),suggesting extensive genetic influence on airway DNA methylation patterns.Among the n = 521 eosinophil-associated CpGs that overlapped with meQTLs,rs55687040 (TENT2,a gene involved in mRNA and miRNA processing) is a GWAS locus for FEV1/FVC ratio (PMID: 26634245), suggesting possible genetic control of airway eosinophilic methylation signatures. Conclusions Our findings disclose distinct DNA methylation signatures associated with sputum eosinophil proportion in COPD.A subset of these CpGs are regulated by genetic variants,suggesting an integrated genetic-epigenetic mechanism underlying airway eosinophilic inflammation with the potential to influence COPD risk and severity. This abstract is funded by: SPIROMICS was supported by contracts from the NIH/NHLBI (HHSN268200900013C, HHSN268200900014C, HHSN268200900015C, HHSN268200900016C, HHSN268200900017C, HHSN268200900018C, HHSN268200900019C, HHSN268200900020C), grants from the NIH/NHLBI (U01 HL137880, U24 HL141762, R01 HL182622, and R01 HL144718), and supplemented by contributions made through the Foundation for the NIH and the COPD Foundation from Amgen; AstraZeneca/MedImmune; Bayer; Bellerophon Therapeutics; Boehringer-Ingelheim Pharmaceuticals, Inc.; Chiesi Farmaceutici S.p.A.; Forest Research Institute, Inc.; Genentech; GlaxoSmithKline; Grifols Therapeutics, Inc.; Ikaria, Inc.; MGC Diagnostics; Novartis Pharmaceuticals Corporation; Nycomed GmbH; Polarean; ProterixBio; Regeneron Pharmaceuticals, Inc.; Sanofi; Sunovion; Takeda Pharmaceutical Company; and Theravance Biopharma and Mylan/Viatris.