Background and ObjectivesLittle is known of the functional potential of the gut microbiome in pediatric-onset multiple sclerosis (MS). We performed metagenomic analyses using stool samples from individuals with pediatric-onset MS and unaffected controls.MethodsPersons ≤21 years old enrolled in the Canadian Pediatric Demyelinating Disease Network providing a stool sample were eligible. Twenty patients with MS (McDonald criteria) with symptom onset <18 years were matched to 20 controls by sex, age (±3 years), stool consistency, and race. Microbial taxonomy and functional potentials were estimated from stool sample–derived metagenomic reads and compared by disease status (MS vs controls) and disease-modifying drug (DMD) exposure using alpha diversity, relative abundance, and prevalence using Wilcoxon rank sum, ALDEx2, and Fisher exact tests, respectively.ResultsIndividuals with MS were aged 13.6 years (mean) at symptom onset and 8 were DMD-naive. Mean ages at stool sample were 16.1 and 15.4 years for MS and control participants, respectively; 80% were girls. Alpha diversity of enzymes and proteins did not differ by disease or DMD status (p > 0.20), but metabolic pathways, gene annotations, and microbial taxonomy did. Individuals with MS (vs controls) exhibited higher methanogenesis prevalence (odds ratio 10, p = 0.044) and Methanobrevibacter abundance (log₂ fold change [LFC] 1.7, p = 0.0014), but lower homolactic fermentation abundance (LFC −0.48, p = 0.039). Differences by DMD status included lower phosphate butyryl transferase for DMD-naive vs exposed patients with MS (LFC −1.0, p = 0.033).DiscussionThe gut microbiome's functional potential and taxonomy differed between individuals with pediatric-onset MS vs controls, including higher prevalence of a methane-producing pathway from Archaea and depletion of the lactate fermentation pathway. DMD exposure was associated with butyrate-producing enzyme enrichment. Together these findings indicate that the gut microbiome of individuals with MS may have a disturbed functional potential.