Multiple sclerosis (MS) is the most common inflammatory neurodegenerative disease of the central nervous system (CNS) in young adults and results in progressive neurological defects. The relapsing-remitting phenotype (RRMS) is the most common disease course in MS, which ultimately progresses to secondary progressive MS (SPMS), while primary progressive MS (PPMS) is a type of MS that worsens gradually over time without remissions. There is a gap in knowledge regarding whether the relapsing form can be distinguished from the progressive course, or healthy subjects (HS) based on an altered serum metabolite profile. In this study, we performed global untargeted metabolomics with the 2D GC-GC-MS platform to identify altered metabolites between RRMS, PPMS, and HS. We profiled 235 metabolites in the serum of patients with RRMS (n = 41), PPMS (n = 31), and HS (n = 91). A comparison of RRMS and HS patients revealed 22 significantly altered metabolites at p < 0.05 (false-discovery rate [FDR] = 0.3). The PPMS and HS comparisons revealed 28 altered metabolites at p < 0.05 (FDR = 0.2). Pathway analysis using MetaboAnalyst revealed enrichment of four metabolic pathways in both RRMS and PPMS (hypergeometric test p < 0.05): (1) galactose metabolism; (2) amino sugar and nucleotide sugar metabolism; (3) phenylalanine, tyrosine, and tryptophan biosynthesis; and (4) aminoacyl-tRNA biosynthesis. The Qiagen IPA enrichment test identified the sulfatase 2 (SULF2) (p = 0.0033) and integrin subunit beta 1 binding protein 1 (ITGB1BP1) (p = 0.0067) genes as upstream regulators of altered metabolites in the RRMS vs. HS groups. However, in the PPMS vs. HS comparison, valine was enriched in the neurodegeneration of brain cells (p = 0.05), and heptadecanoic acid, alpha-ketoisocaproic acid, and glycerol participated in inflammation in the CNS (p = 0.03). Overall, our study suggests that RRMS and PPMS may contribute metabolic fingerprints in the form of unique altered metabolites for discriminating MS disease from HS, with the potential for constructing a metabolite panel for progressive autoimmune diseases such as MS.

Disease-modifying therapies (DMT)s) for relapsing-remitting multiple sclerosis (RRMS) act through distinct immunological mechanisms, yet the within-patient molecular response programs associated with these therapies remain incompletely defined. Here, we reanalyzed publicly available peripheral blood mononuclear cell (PBMC) miRNA microarray data (GSE230064) using a longitudinal, robustness-focused framework to compare therapy-associated miRNA response patterns following cladribine versus ocrelizumab treatment. Baseline (t0) and 6-month post-treatment (t1) samples were paired within individuals and technical replicates consolidated prior to analysis, yielding a final paired cohort of four cladribine-treated and six ocrelizumab-treated patients. Within each treatment arm, we quantified per-patient Δ-miRNA (t1 − t0) values and prioritized therapy-associated response features using a multi-evidence framework integrating effect direction, magnitude, directional consistency across individuals, and leave-one-out sensitivity. Cladribine treatment was associated with a highly coordinated, directionally concordant upregulation of five miRNAs including hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-503-5p, hsa-miR-148a-3p, and hsa-miR-26a-5p, all exhibiting 100% directional stability across patients and mean Δ-expression values ranging from +0.77 to +1.38. These miRNAs target pathways relevant to MS pathophysiology, including Th17/Treg balance, Wnt-β-catenin signaling, macrophage polarization, and epigenetic immune regulation. In contrast, ocrelizumab elicited a more selective response pattern, with five miRNAs including hsa-miR-100-5p, hsa-miR-410-3p, hsa-miR-432-5p, hsa-miR-296-5p, and hsa-miR-485-3p showing moderate directional stability (83%) and greater inter-individual heterogeneity, consistent with the more targeted mechanism of CD20+ B-cell depletion. Notably, the two treatment-associated signatures were non-overlapping, with hsa-miR-27b-3p representing the only miRNA shared with prior cross-sectional analyses of this dataset. The identified ocrelizumab-associated miRNAs implicate pathways including mTOR/IGF1R signaling, NF-κB regulation, RNA editing, and mitochondrial biogenesis, several of which are dysregulated in progressive MS. Together, these findings demonstrate that cladribine and ocrelizumab induce distinct, treatment-specific miRNA response architectures that reflect their divergent immunological mechanisms. This work establishes a stability-aware analytic template for extracting reproducible longitudinal miRNA signals from small paired RRMS cohorts and provides a ranked set of biologically plausible candidate miRNAs for prospective validation and mechanistic investigation.