Objective: Comprehensively characterize the epigenetic profile of isolated REM Sleep Behaviour Disorder (iRBD) using genome-wide and gene-targeted approaches, linking it to clinical measures and  phenoconversion.

Background: iRBD is a prodromal state of an underlying α-synucleinopathy, preceding neurodegenerative disorders by years. Epigenetic alterations, particularly DNA methylation (DNAm), have emerged as potential biomarkers for neurodegenerative diseases, yet with limited clinical validation. Investigating DNAm changes in iRBD may provide insights into disease mechanisms and early phenoconversion detection.

Method: We prospectively recruited a cohort of 43 iRBD patients (mean age 68.9±6.4 years) and 155 age- and sex-matched controls (mean age 66.2±9.2 years) (Figure,Table 1). Blood samples were collected at baseline and annualy for iRBDs. An epigenome-wide association study (EWAS) was performed, followed by targeted bisulphite sequencing of seven genomic regions (Figure 2). iRBDs underwent cognitive, motor, and autonomic evaluations until phenoconversion. ANOVA and mixed-effects models compared DNAm levels between groups, assess associations with clinical features, evaluate longitudinal changes, and determine risk for phenoconversion.

Results: EWAS identified differentially methylated regions in EPHA10, DTD1, EXOC3L2, SLC52A3, MAPK8, and SLC7A11. Targeted sequencing also analyzed intron 1 of SNCA and the genomic region surrounding cg26033520. Compared to controls, iRBD patients exhibited significant hypomethylation in specific CpG sites within SLC52A3, EXOC3L2, and EPHA10 (Figure 3). Notably, hypomethylation of SLC52A3 correlated with cognitive impairment in iRBD and progressed over time (Figure 4). However, baseline methylation levels were not significantly different between converted (9-20.9%) and non-converted patients.

Conclusion: Our findings suggest distinct epigenetic signatures in iRBD, characterized by hypomethylation of genes involved in riboflavin transport (SLC52A3) and Alzheimer’s susceptibility (EXOC3L2). The observed association between SLC52A3 hypomethylation and cognitive impairment, along with its progressive hypomethylation over time, underscores the potential of DNAm as a biomarker for disease progression. Further studies in larger cohorts are warranted to validate these findings and refine the role of epigenetic markers in tracking phenoconversion trajectories.

Cohort’s structure.

General clinical features of iRBD patients.

Biomolecular and clinical workflow.

Significant amplicons of targeted sequencing.

Correlation with cognition and conversion.

References: 1) Miglis MG, Adler CH, Antelmi E, Arnaldi D, Baldelli L, Boeve BF, Cesari M, Dall’Antonia I, Diederich NJ, Doppler K, Dušek P, Ferri R, Gagnon JF, Gan-Or Z, Hermann W, Högl B, Hu MT, Iranzo A, Janzen A, Kuzkina A, Lee JY, Leenders KL, Lewis SJG, Liguori C, Liu J, Lo C, Ehgoetz Martens KA, Nepozitek J, Plazzi G, Provini F, Puligheddu M, Rolinski M, Rusz J, Stefani A, Summers RLS, Yoo D, Zitser J, Oertel WH. Biomarkers of conversion to α-synucleinopathy in isolated rapid-eye-movement sleep behaviour disorder. Lancet Neurol. 2021 Aug;20(8):671-684. doi: 10.1016/S1474-4422(21)00176-9. PMID: 34302789; PMCID: PMC8600613.

2) Baldelli L, Pirazzini C, Sambati L, Ravaioli F, Gentilini D, Calandra-Buonaura G, Guaraldi P, Franceschi C, Cortelli P, Garagnani P, Bacalini MG, Provini F. Epigenetic clocks suggest accelerated aging in patients with isolated REM Sleep Behavior Disorder. NPJ Parkinsons Dis. 2023 Mar 30;9(1):48. doi: 10.1038/s41531-023-00492-2. PMID: 36997543; PMCID: PMC10063653.

3) Stefani A, Antelmi E, Arnaldi D, Arnulf I, During E, Högl B, Hu MMT, Iranzo A, Luke R, Peever J, Postuma RB, Videnovic A, Gan-Or Z. From mechanisms to future therapy: a synopsis of isolated REM sleep behavior disorder as early synuclein-related disease. Mol Neurodegener. 2025 Feb 11;20(1):19. doi: 10.1186/s13024-025-00809-0. PMID: 39934903; PMCID: PMC11817540.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/epigenome-wide-and-gene-targeted-epigenetic-profile-of-isolated-rem-sleep-behaviour-disorder-a-clinico-molecular-study/