Multi-Omic Dissection of Autism Reveals Dominant Effects of Family, Sex, and Host-Microbe Metabolic Interactions

Autism spectrum disorder (ASD) has been associated with gut microbiome and metabolic alterations, but reported biomarkers are inconsistent and often inadequately account for family and shared environment. We analysed 620 children, including 334 with ASD and 286 neurotypical siblings, with a median age of 6.0 years (IQR 4.0-8.0). Faecal and urine samples were collected monthly up to eight times. After quality control, 869 microbiome, 754 faecal metabolome and 787 urine metabolome samples were analysed using 16S rRNA sequencing, NMR and LC-MS metabolomics. Mixed models accounted for family, repeated sampling and biological sex. Family membership explained 46.1% of variation across microbiome and metabolome profiles, compared with 8.0% attributable to ASD. After family adjustment, ASD explained only 0.1% to 0.3% of microbiome beta diversity. No stable taxonomic biomarkers were identified, and microbial classification was poor (AUROC <0.75). Urinary metabolomics identified elevated 5-hydroxy-L-tryptophan and altered phenylalanine metabolism. Structural equation modelling found no direct associations between selected taxa and metabolites. These findings argue against a universal ASD microbiome and indicate that family context contributes more strongly than diagnosis to microbial and metabolic variation.