Sphingolipid Remodeling and Extracellular Vesicle Signatures Reflect Disease Severity in Facioscapulohumeral Dystrophy Driven by Mitochondrial Dysfunction and Endoplasmic Reticulum Stress.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a progressive and heterogeneous disorder lacking reliable circulating biomarkers for disease monitoring and therapeutic response. We hypothesized that metabolic dysregulation, lipotoxicity, and endoplasmic reticulum (ER) stress induce sphingolipid remodeling that is reflected in serum and correlates with disease severity. METHODS: We performed RNA sequencing, differential expression, and pathway analyses on biceps brachii muscle biopsies from mild (n = 9) and severe (n = 5) FSHD patients and healthy controls (n = 6). Selected transcripts were validated by OpenArray quantitative PCR. Serum samples from mild (n = 9), severe (n = 13), and control subjects (n = 10) were analyzed by targeted and untargeted LC-MS/MS sphingolipidomics. RESULTS: Severe FSHD exhibited transcriptomic signatures of mitochondrial dysfunction, ER stress, inflammation, and extracellular vesicle biogenesis. RNA-seq revealed activation of the de novo ceramide synthesis pathway, with increased SPTLC1-3, CERS2, CERS5, and DEGS1, reduced SMPD1/4 and SMPDL3A, and dysregulation of cerebroside metabolism. Migrasome/extracellular vesicle markers (TSPAN4, TM4SF1, PIGK, CPQ, ITGA5, and ITGB1) were predominantly upregulated in patients with severe disease. Serum lipidomics showed severity-dependent increases in dihydroceramides, ceramides, sphingomyelins, and dihydrosphingomyelins, while the Cer/HexCer d18:1/18:0 ratio progressively increased with disease severity. CONCLUSIONS: Integrated transcriptomic and lipidomic analyses identify sphingolipid dysregulation and extracellular vesicle biogenesis as hallmarks of severe FSHD and support circulating sphingolipids as candidate biomarkers for disease severity and therapeutic monitoring.