Epigenetic Insights in Pediatric Multiple Sclerosis

Background and objectives: Pediatric-onset multiple sclerosis (PedMS) provides a unique opportunity to investigate MS pathogenesis because of the short interval between environmental exposures and disease onset. This study aimed to identify differentially methylated regions (DMRs) that mediate the interplay between environmental triggers and genomic factors in the early stages of the disease. Methods: In this multicenter study, we analyzed peripheral blood DNA from 175 Italian subjects (122 PedMS patients, 53 healthy controls) using the Infinium Methylation EPIC Array v2. All patients were enrolled within 4 years of onset. The bioinformatic pipeline included batch effect correction, normalization, and differential methylation analysis adjusted for sex and inferred cell-type proportions. DMRs were detected using DMRcate. Network analysis was performed by projecting DMR-overlapping genes onto the STRING interactome using the NDEx tool, followed by pathway enrichment analysis with gProfiler. To ensure robustness, we performed an internal 1/3-2/3 validation and sensitivity analyses on treatment-naïve and low-efficacy treatment (LETA) subgroups. Results: Our analysis revealed 55 DMRs, with 28 affecting promoter regions. Top signals involved genes such as CHI3L2, LPCAT1, CD19, PARP1, CPT1B-CHKB, and IGF2R, including several MS-associated loci. Signals mapping to CHI3L2/DENND2D and LPCAT1 were highly resilient, persisting across all validation and sensitivity subsets. Network analysis identified a subnetwork of 165 nodes where the Epstein-Barr virus (EBV) infection emerged as the top-ranked enriched pathway (p = 3.23 *10-40). High-centrality regulatory hubs included PARP1 and IGF2R, the latter overlapping with established imprinting control regions. Discussion: Our findings identify a distinct epigenetic signature in early-stage PedMS, suggesting that DNA methylation changes are not stochastic but converge on high-centrality regulatory hubs. The stable association with EBV-related pathways and imprinting loci reinforces the hypothesis that epigenetic remodeling serves as a critical interface between early-life environmental exposures and genetic susceptibility in MS onset.