Circulating cell-free DNA methylation profiles as noninvasive multiple sclerosis biomarkers: A proof-of-concept study

In multiple sclerosis (MS), there is a critical need for non-invasive biomarkers to concurrently classify disease subtypes, evaluate disability severity, and predict long-term progression. In this proof-of-concept study, we performed low-coverage whole-genome bisulfite sequencing (WGBS) on 75 plasma cell-free DNA (cfDNA) samples and assessed the clinical utility of cfDNA methylation as a single assay for distinguishing MS patients from non-MS controls, identifying MS subtypes, estimating disability severity, and predicting disease trajectories. We identified thousands of differentially methylated CpGs and hundreds of differentially methylated regions (DMRs) that significantly distinguished MS from controls, separated MS subtypes, and stratified disability severity levels. These DMRs were highly enriched in immunologically and neurologically relevant regulatory elements ( e.g., active promoters and enhancers) and contained motifs associated with neuronal function and T-cell differentiation. To distinguish MS subtypes and severity groups, we achieved area-under-the-curve (AUC) values ranging from 0.67 to 0.81 using DMRs and 0.70 to 0.82 using inferred tissue-of-origin patterns from cfDNA methylation, significantly outperforming benchmark neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in the same cohort. Finally, a linear mixed-effects model identified “prognostic regions” where baseline cfDNA methylation levels were associated with disease progression and predicted future disability severity (AUC=0.81) within a 4-year evaluation window. As we plan to generate higher-depth WGBS data and validation in independent cohorts, the present findings suggest the potential clinical utility of circulating cfDNA methylation profiles as promising noninvasive biomarkers in MS diagnosis and prognosis.