Mario Martin-Almeida

Rationale: The role of DNA methylation (DNAm) markers in the upper-airway microbiome related to asthma exacerbations despite inhaled corticosteroid (ICS) treatment is unexplored. Objectives: To identify whole-blood DNAm markers associated with the upper-airway microbiome related to asthma exacerbations despite ICS treatment. Methods: We analyzed salivary, nasal, and pharyngeal microbiome in 242 asthma patients (GEMAS) for discovery. We tested associations between whole-blood DNAm at 703,505 CpGs and 21 exacerbation-related microbiome traits. Replication for the nasal microbiome was evaluated in 272 (Project Viva) and 66 (SysPharmPediA) children with and without asthma. We analyzed differentially methylated regions (DMRs), gene enrichment, associations with blood gene expression (RNA-seq, n=119), and DNAm predictors of plasma proteins. Measurements and Main Results: Whole-blood DNAm at 73 CpGs and 550 DMRs was associated with 17 exacerbation-related microbiome traits (false discovery rate [FDR]ORMDL3 . Most CpGs were associated with Campylobacter (pharyngeal) and Leptotrichia (nasal) abundance. Ten DNAm markers were replicated ( p PTPRR associated with nasal bacterial richness. Enrichment analyses identified genes related to diabetes, obesity, smoking, TGF-β, MAPK, and Wnt signaling and regulated by trichostatin A, NF-κB, and the glucocorticoid receptor (FDRTannerella , was associated with ITM2C whole-blood expression. Nine DNAm-predicted plasma proteins involved in inflammation, tissue remodeling, and TGF-β signaling were associated with seven exacerbation-related microbiome traits, including Streptococcus and Selenomonas . Conclusions: Whole-blood DNAm markers implicated in inflammation, immunity, and asthma comorbidities were associated with the upper-airway microbiome linked to asthma exacerbations.

Background: Polygenic risk scores (PRS) capture inherited susceptibility to asthma, whereas methylation risk scores (MRS) may reflect disease-associated epigenetic variation. We assessed whether these scores differ in patients requiring biologic therapy and whether MRS change after mepolizumab initiation. Methods: Cross-sectional analyses in the GEMAS study included 699 participants with genotype data (428 with asthma, 271 controls) and 668 with blood DNA methylation data (408 with asthma, 260 controls). We evaluated 35 existing asthma PRS and derived six MRS from published epigenomic asthma studies. Longitudinal MRS changes after mepolizumab initiation were assessed in 16 GEMAS patients and were replicated in 20 patients from the MEGA study. Results: Twenty-eight PRS differentiated biologic-treated patients from controls (areas under the curve [AUC], 0.68-0.75), but none distinguished biologic-treated and non-treated patients after multiple-testing correction. MRS was higher in asthma without biologic than in controls (odds ratio [OR], 1.64; 95% CI, 1.34-1.99) and lower in biologic-treated than in asthma without biologic patients (OR, 0.54; 95% CI, 0.35-0.81). MRS did not differ significantly between biologic-treated patients and controls. Following mepolizumab initiation, MRS decreased at 6 and 12 months in GEMAS ( p =4.81×10 ⁻⁵ and p =2.40×10 ⁻⁵ , respectively). The 6-month decrease was also observed in MEGA ( p =3.25×10 ⁻⁹ ). MRS epigenetic markers were predominantly hypermethylated, opposite to their described pattern in asthma risk. Conclusions: Asthma PRS did not differ significantly between biologic-treated and non-biologic-treated patients, but asthma MRS were lower in biologic-treated patients. The MRS decreased after mepolizumab initiation in two independent cohorts, consistent with a reversal of asthma-associated epigenetic patterns.

Background: Extreme-phenotype comparisons allowed the discovery of novel asthma genetic risk loci. However, this approach remains unexplored in epigenome-wide association studies (EWAS). We aimed to identify bulk and cell-specific methylation markers of asthma with severe exacerbations across diverse ancestry groups. Methods: We conducted a meta-EWAS of 739,543 CpGs in whole blood among 1,192 African American and Latino pediatric populations, comparing non-asthmatics and asthma exacerbators. Genome-wide CpGs were followed up for replication in a meta-analysis across 1,516 ethnically diverse participants and in a cross-tissue evaluation of 393 nasal samples. We conducted differentially methylated region (DMRs), cell-type-deconvoluted, and quantitative trait loci analyses (whole-genome sequencing n=1,668; RNA-seq n=1,209). We examined enrichment in traits, pathways, and druggable genes, and analyzed DNAm predictors of plasma proteins and aging. Results: DNAm at 505 CpGs and 119 DMRs in whole blood were associated with asthma exacerbations ( p -8 , λ=1.05). We replicated 25 CpGs in blood cells, cross-validated 7 in nasal samples, and detected 42 cell-specific DNAm markers mainly driven by T cells. DNAm at 134 CpGs was associated with gene expression in whole blood, including 118 associations with T-cell receptor genes, and 446 CpGs were regulated by ≥1 genetic variant. We found enrichment for previous associations with environmental exposures, immune disorders, immune and inflammatory pathways, and druggable genes by developmental drugs. 21 methylation-predicted plasma proteins, involved in host defense, and one lung aging clock were associated with asthma exacerbations. Conclusions: The first meta-EWAS of extreme asthma phenotypes identified hundreds of novel DNAm markers, suggesting novel methylation biomarkers and candidate drugs for asthma and supporting the role of T cells.

Background: Asthma exacerbations (AEs) represent the major contributor to the global asthma burden. Although genetic and environmental factors have been associated with AEs, the role of epigenetics remains uncovered. Objective: This study aimed to identify whole blood DNA methylation (DNAm) markers associated with AEs in Europeans. Methods: DNAm was assessed in 406 blood samples from Spanish individuals using the Infinium MethylationEPIC microarray (Illumina). An epigenome‐wide association study was conducted to test the association of DNAm with AEs at differentially methylated positions, regions, and epigenetic modules. CpGs suggestively associated with AEs (false discovery rate [FDR] < 0.1) were followed up for replication in 222 European individuals, and the genome‐wide significance ( p < 9 × 10 ⁻⁸ ) was declared after meta‐analyzing the discovery and replication samples. Additional assessment was performed using nasal tissue DNAm data from 155 Spanish individuals. The effects of genetic variation on DNAm were assessed through cis‐methylation quantitative trait loci (meQTL) analysis. Enrichment analyses of previous EWAS signals were conducted. Results: Four CpGs were associated with AEs, and two were replicated and reached genomic significance in the meta‐analysis (annotated to ZBTB16 and BAIAP2 ). Of those, CpG cg25345365 ( ZBTB16 ) was cross‐tissue validated in nasal epithelium ( p = 0.003) and associated with five independent meQTLs (FDR < 0.05). Additionally, four differentially methylated regions and one module were significantly associated with AEs. Enrichment analyses revealed an overrepresentation of prior epigenetic associations with prenatal and environmental exposures, immune‐mediated diseases, and mortality. Conclusions: DNAm in whole blood and nasal samples may contribute to AEs in Europeans, capturing genetic and environmental risk factors.