Epigenetic Association Analyses and Risk Prediction of RLS

Background: As opposed to other neurobehavioral disorders, epigenetic analyses and biomarkers are largely missing in the case of idiopathic restless legs syndrome (RLS). Objectives: Our aims were to develop a biomarker for RLS based on DNA methylation in blood and to examine DNA methylation in brain tissues for dissecting RLS pathophysiology. Methods: Methylation of blood DNA from three independent cohorts (n = 2283) and post-mortem brain DNA from two cohorts (n = 61) was assessed by Infinium EPIC 850 K BeadChip. Epigenome-wide association study (EWAS) results of individual cohorts were combined by random-effect meta-analysis. A three-stage selection procedure (discovery, n = 884; testing, n = 520; validation, n = 879) established an epigenetic risk score including 30 CpG sites. Epigenetic age was assessed by Horvath's multi-tissue clock and Shireby's cortical clock. Results: EWAS meta-analysis revealed 149 CpG sites linked to 136 genes (P < 0.05 after Bonferroni correction) in blood and 23 CpG linked to 18 genes in brain (false discovery rate [FDR] < 5%). Gene-set analyses of blood EWAS results suggested enrichments in brain tissue types and in subunits of the kainate-selective glutamate receptor complex. Individual candidate genes of the brain EWAS could be assigned to neurodevelopmental or metabolic traits. The blood epigenetic risk score achieved an area under the curve (AUC) of 0.70 (0.67–0.73) in the validation set, comparable to analogous scores in other neurobehavioral disorders. A significant difference in biological age in blood or brain of RLS patients was not detectable. Conclusions: DNA methylation supports the notion of altered neurodevelopment in RLS. Epigenetic risk scores are reliably associated with RLS but require even higher accuracy to be useful as biomarkers.