Analysis of gut microbiota in Restless Legs Syndrome: searching for a metagenomic signature

<h4>Study objectives</h4>We aim to analyze the microbiota composition in Restless Legs Syndrome (RLS) patients and its relationship with the different RLS phenotypes.<h4>Methods</h4>We recruited idiopathic RLS (RLS) and insomnia (INS) patients and healthy subjects (CTRL). Validated questionnaires (Pittsburg Sleep Quality Index, International Restless Legs Syndrome Study Group Rating Scale, Insomnia Severity Index, Beck Depression Inventory-II) were administered in the RLS and INS. Fecal microbiota was analyzed by 16S rRNA gene sequencing according to Illumina metagenomics standard procedure on MiSeq Platform. Dada2 pipeline was used to process sequencing data, while DESeq2 and Aldex2 tools were used to calculate differential abundance taxa, correcting for age, sex, body mass index, sequencing run, and presence of mood disorders.<h4>Results</h4>The sample included 37 RLS (28 females, mean age 64.78 years), 31 INS (22 females, mean age 60.64 years), and 33 CTRL (24 females, mean age 62.54 years). Differential abundance analysis revealed a statistically significant decrease in the abundance of Lachnoclostridium and Flavonifractor genera in RLS compared to CTRL and INS, but not in the INS compared to CTRL. Lachnoclostridium abundance tended to decrease with long disease duration and a predominant motor phenotype. In the RLS group, several genera were identified as significantly associated with International Restless Legs Syndrome Study Group Rating Scale and Pittsburg Sleep Quality Index scores.<h4>Conclusions</h4>Although only a few previous studies have reported the presence of small intestinal bacterial overgrowth in RLS, to the best of our knowledge this is the first study to highlight significant differences in the gut microbiota composition of RLS compared to both CTRL and INS, identifying a specific RLS metagenomic signature. Statement of Significance This is the first study to comprehensively characterize the gut microbiota in patients with Restless Legs Syndrome (RLS), identifying a distinct microbial signature compared to insomnia patients and healthy controls. We observed alterations alpha and beta diversity and specific changes in bacterial families and genera, some of which significantly correlated with RLS clinical features. In particular, Lachnoclostridium genus was significantly reduced in RLS and tended to be less abundant in patients with longer disease duration and without sensory symptoms. This genus is known to modulate systemic inflammation through the production of short-chain fatty acids, suggesting a potential link between gut dysbiosis, inflammation, and dopaminergic dysfunction. These findings support a role for gut microbiota alterations in RLS pathogenesis and open new avenues for microbiota-based diagnostic and therapeutic strategies.