Fmr1 mutation reshapes gut microbiome structure, diversity, intestinal barrier integrity, and function in a sex and genotype-dependent manner

Fragile X Syndrome (FXS) is the leading monogenic cause of autism spectrum disorder (ASD), caused by mutation in the Fmr1 gene. In addition to cognitive and behavioral challenges, FXS patients often experience altered gut microbiome-induced gastrointestinal (GI) problems. Evidence suggests that an altered gut microbiome can disrupt mucosal barrier and promote inflammation, leading to impaired intestinal barrier integrity and function. However, the mechanisms by which the gut microbiome alters the barrier integrity and contributes to GI pathology in FXS remain poorly understood. To address this gap, we tested the hypothesis that Fmr1 mutation reshapes the gut microbiome composition, altering transcriptional markers of intestinal barrier regulation and gut barrier physiology in mice. To test our hypothesis, we used an Fmr1 knockout (KO) mouse model with wild-type (WT) littermates as controls. First, we performed 16S ribosomal RNA sequencing to characterize gut microbial community structure and diversity across genotypes and sexes. Among the alpha-diversity metrics, only Chao1 showed significant differences across female genotypes in both fecal and cecal contents. Additionally, qRT-PCR analysis of ileal samples revealed reduced barrier and mucosal defense gene expression in female KO and Het mice compared with WT females. Next, we utilized a Ussing chamber assay to test gut epithelial permeability and function. Our physiological data showed that female Het mice had increased transepithelial resistance compared to WT females, indicating a tighter epithelial barrier. Overall, FXS is associated with modest genotype and sex-specific microbiome variation, impaired gut barrier integrity, and altered epithelial barrier function in female mice.