Fmr1 mutation is associated with gut microbiome structure and diversity as well as intestinal barrier integrity and function in a sex- and genotype-dependent manner

ABSTRACT 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 is associated with 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 used an 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. IMPORTANCE Fragile X syndrome (FXS), the most common inherited cause of autism, affects not only brain function but also physical health, including chronic gastrointestinal (GI) problems that are often overlooked. This study reveals that a single genetic mutation linked to FXS is associated with the gut microbiome and the intestinal barrier—the body’s frontline defense against inflammation and disease—in a sex-specific manner. Using a mouse model, we show that females carrying the Fmr1 mutation exhibit changes in gut microbes, reduced expression of genes that protect the intestinal lining, and altered gut barrier function. These findings highlight a critical gut-genetic connection in FXS and point to the intestine as an important, yet underappreciated, site of disease impact. By linking genetic risk for neurodevelopmental disorders to gut health, this work opens new avenues for understanding—and potentially treating—the gastrointestinal symptoms experienced by individuals with FXS.