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

Study objectives: We aim to analyse the microbiota composition in RLS patients and its relationship with the different RLS phenotypes. Methods: We recruited idiopathic RLS (RLS) and insomnia (INS) patients and healthy subjects (CTRL). Validated questionnaires (PSQI, IRLS, ISI, BDI-II) were administered in the RLS and INS. Fecal microbiota was analysed 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. Results: 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 IRLS and PSQI scores. Conclusions: Although only a few previous studies have reported the presence of small intestinal bacterial overgrowth (SIBO) 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.