Background and objectives

Little 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.

Methods

Persons ≤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.

Results

Individuals 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).

Discussion

The 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.

Objective

Examine if the gut microbiota composition changes across repeated samples in paediatric-onset multiple sclerosis (MS) or monophasic-acquired demyelinating syndromes (monoADS).

Methods

A total of 36 individuals (18 MS/18 monoADS) with ⩾2 stool samples were included. Stool sample-derived DNA was sequenced. Alpha/beta diversities and genus-level taxa were analysed.

Results

Mean ages at first sample procurement (MS/monoADS) = 18.0/13.8 years. Median time (months) between first/second samples = 11.2 and second/third = 10.3. Alpha/beta diversities did not differ between stool samples (p > 0.09), while one genus - Solobacterium did (p = 0.001).

Conclusions

The gut microbiota composition in paediatric-onset MS and monoADS exhibited stability, suggesting that single stool sample procurement is a reasonable first approach.

OBJECTIVE: The relationship between multiple sclerosis and the gut microbiome has been supported by animal models in which commensal microbes are required for the development of experimental autoimmune encephalomyelitis. However, observational study findings in humans have only occasionally converged when comparing multiple sclerosis cases and controls which may in part reflect confounding by comorbidities and disease duration. The study of microbiome in pediatric-onset multiple sclerosis offers unique opportunities as it is closer to biological disease onset and minimizes confounding by comorbidities and environmental exposures. METHODS: A multicenter case-control study in which 35 pediatric-onset multiple sclerosis cases were 1:1 matched to healthy controls on age, sex, self-reported race, ethnicity, and recruiting site. Linear mixed effects models, weighted correlation network analyses, and PICRUSt2 were used to identify microbial co-occurrence networks and for predicting functional abundances based on marker gene sequences. RESULTS: Two microbial co-occurrence networks (one reaching significance after adjustment for multiple comparisons; q < 0.2) were identified, suggesting interdependent bacterial taxa that exhibited association with disease status. Both networks indicated a potentially protective effect of higher relative abundance of bacteria observed in these clusters. Functional predictions from the significant network suggested a contribution of short-chain fatty acid producers through anaerobic fermentation pathways in healthy controls. Consistent family-level findings from an independent Canadian-US study (19 case/control pairs) included Ruminococaccaeae and Lachnospiraceae (p < 0.05). Macronutrient intake was not significantly different between cases and controls, minimizing the potential for dietary confounding. INTERPRETATION: Our results suggest that short-chain fatty acid producers may be important contributors to multiple sclerosis onset.