Epstein–Barr Virus, Lower Vitamin D, Low Sun Exposure, and HLA‐DRB1*1501 Risk Variant Share Common Epigenetic Pathways Leading to Multiple Sclerosis Onset

Objectives Multiple sclerosis (MS) onset risk factors include Epstein–Barr virus (EBV) indices (including host response), lower serum 25‐vitamin D (25(OH)D) levels, low sun exposure, and HLA‐DRB1*1501 . The underlying molecular mechanisms are unclear. Here, we examined mediation through differential DNA methylation (DNAm) to better understand possible epigenetic programming. Methods Two case‐control studies (Ausimmune Study, Australia = 206 cases + 348 controls; and Epidemiologic Investigations of MS [EIMS], Sweden = 140 cases + 139 controls). DNAm was measured using Illumina arrays. Dimension‐reduction methods generated MS‐associated DNAm modules. Pathway enrichment analyses were used to describe DNAm modules’ system‐level biological characteristics. Individual and joint associations with MS risk were assessed using logistic regression. DNAm module mediation of risk factor‐outcome associations were assessed using mediation analysis. A range of temporality analyses were used. Results EBV indices (infectious mononucleosis history and anti‐EBNA IgG titer), lower 25(OH)D, low sun exposure, and HLA‐DRB1*1501 risk variant were individually and jointly associated with MS risk. In each study, 2 DNAm modules were found which mediated multiple exposure‐MS associations. Proportions mediated ranged from 21 to 47% in Ausimmune and 25 to 53% in EIMS. Results were robust to sensitivity analyses. Top‐ranked genomewide association study (GWAS) MS risk‐associated genes were over‐represented in both Ausimmune DNAm modules, A1 3.5‐fold ( p = 0.004) and A2 3‐fold ( p = 0.015). Reactome pathways enriched for DNAm had cross‐study overlap – 45% of pathways enriched in Ausimmune DNAm modules were also enriched in EIMS (4.82‐fold, p < 0.001). Interpretation EBV, lower vitamin D, low sun exposure, and HLA‐DRB1*1501 risk variant act in concert and through common epigenetic pathways to impact MS onset risk. ANN NEUROL 2025