Integrated Analysis of Gut Microbiota Dysbiosis and microRNA-155/microRNA-20a Expression Across Multiple Sclerosis Disease subtypes: Comparison with Controls

Multiple sclerosis (MS) is a heterogeneous neuroinflammatory disease encompassing four clinical subtypes. Although gut microbiota (GM) dysbiosis and microRNA (miRNA) dysregulation are implicated in MS pathogenesis, their subtype-specific associations remain poorly characterized. This study investigated GM composition and circulating miR-155 and miR-20a across MS subtypes to identify subtype-specific microbiota–miRNA signatures and evaluate their potential to discriminate clinical phenotypes. This case-control study included 240 participants (n = 40 each) with clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), other neurological diseases (OND), or healthy controls (HC). GM phyla/genera and butyryl-CoA gene frequency were quantified by qPCR, alongside serum miR-155 and miR-20a. Diagnostic performance was assessed using ROC analysis and Youden index optimization. SPMS showed the most severe dysbiosis, with reduced Firmicutes and Bacteroidetes, increased Proteobacteria and Akkermansia , and decreased butyryl-CoA (all P < 0.01). miR-155 was increased, whereas miR-20a was decreased across MS subtypes versus HC (P < 0.0001). miR-20a positively correlated with beneficial commensals (Firmicutes: r = 0.384) and negatively with pro-inflammatory taxa, whereas miR-155 showed inverse patterns. Bacteroidetes robustly discriminated SPMS from HC (AUC = 0.993), while miR-20a discriminated RRMS (AUC = 0.893) and PPMS (AUC = 0.935). Combined microbial-miRNA panels achieved AUC = 1.000 for several comparisons, with 85–95% sensitivity and 75–100% specificity. Gut dysbiosis and circulating miR-155/miR-20a profiles are subtype-dependent, with SPMS showing the most pronounced alterations. Integrated microbial-miRNA signatures showed promising cross-sectional discrimination of MS phenotypes, particularly SPMS, but require longitudinal validation before clinical application.