Fig. 1: Study design and analysis workflow.

This study investigates the associations between epigenome-wide DNA methylation (794,125 high-quality CpG sites), measured using the Illumina Infinium MethylationEPIC array, in blood samples from infants with severe bronchiolitis, and the subsequent risk of recurrent wheezing (before 3 years of age, n = 448, 170 cases) and asthma (at 6 years of age, n = 506, 112 cases) during childhood. The analytical data were collected from participants of the 35th Multicenter Airway Research Collaboration (MARC-35) study. Thirty-one differentially methylated regions (DMRs) were associated with recurrent wheezing, and thirty-three DMRs were associated with asthma, taking into account potential interactions with rhinovirus infection during infancy. In silico blood cell-type deconvolution and chromatin state enrichment analyses were conducted to identify specific blood cell types likely driving the associations at these DMRs. The chromatin state annotation was obtained from the Roadmap Epigenomics Consortium. We then explored the relationship between these DMRs and the levels of 347 proteins measured in the same bronchiolitis infant blood samples using the Olink multiplex platform. For proteins associated with the DMRs, we constructed a protein-protein interaction (PPI) network leveraging data from the STRING database, performed two-sample mendelian randomization analysis using protein quantitative trait loci (pQTLs, UK Biobank and deCODE) and GWAS summary statistics from published studies, and conducted pathway enrichment analysis using data from the Gene Ontology database. These analyses aim to provide additional biological insights into the early development of childhood respiratory diseases in infants with severe bronchiolitis underlying the DNA methylation differences.