RNA Cargo Profiling of Muscle Extracellular Vesicles Reveals Candidate Biomarkers of Disease Activity and Muscle Degeneration in FSHD

Background Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder characterized by high inter- and intra-individual variability in muscle involvement, disease severity, and rate of progression, even among affected relatives. Remarkably, asymptomatic relatives of FSHD patients, referred to as non-penetrant gene carriers, remain clinically unaffected throughout their lives, despite carrying the genetic background sufficient to cause FSHD. The clinical heterogeneity of FSHD, together with the increasing number of clinical trials involving FSHD patients, underscores the urgent need for reliable biomarkers enabling disease monitoring, stratification of patients and evaluation of treatment efficacy. Extracellular vesicles (EVs) have emerged as promising biomarkers since their cargo reflects physiological state of muscle tissue and remains stable into bloodstream. Methods To explore their potential in FSHD, we isolated EVs from ex-vivo muscle explants obtained from a cross-sectional cohort of 22 FSHD patients, 4 non-penetrant gene carriers and 6 healthy controls. EV-RNA cargo was profiled using small RNA and total RNA sequencing. Results Our study identified distinct EV-RNA signatures, including microRNA, isomiRs and long transcripts, associated with disease activity, assessed by short-tau inversion recovery signal on magnetic resonance imaging. Our analyses also identified EV-RNA profiles linked to muscle degeneration, assessed by T1-weighted signal on magnetic resonance imaging. Additionally, the identified candidate EV-biomarkers discriminated also between FSHD muscles with and without T1 progression at 2-year MRI follow-up. Conclusions In this study, we present a comprehensive characterization of RNA cargo from muscle EVs in FSHD. The identification of EV-RNA signatures linked to disease activity and muscle degeneration paves the way to future use of EV-RNAs as non-invasive biomarkers for disease monitoring and may support clinical trial design. Moreover, these findings provides novel insights into FSHD disease mechanisms.