Facioscapulohumeral muscular dystrophy (FSHD) is caused by pleiotropic contractions of the D4Z4 repeat array on chromosome 4q35 (FSHD1) or by mutations in repressive chromatin regulators of the D4Z4 loci (FSHD2), both resulting in epigenetic dysregulation at the D4Z4 array. DNA methylation of the D4Z4 repeat array has been proposed for diagnosis and prognosis of FSHD disease severity
however, further validation in larger populations is needed. We retrospectively analyzed 247 clinically suspected FSHD with D4Z4 analysis by optical genome mapping (OGM) or molecular combing (MC) and tested the DNA methylation levels for 75 patients and 49 healthy controls. A D4Z4 repeat length-dependent non-linear increase was observed in both distal and global D4Z4 methylation levels. Distal D4Z4 methylation levels identified FSHD1 patients with a sensitivity of 100% and specificity of 97.96% at a cutoff value of 39.66% compared to controls. Distal FSHD1-like hypomethylation was also observed in one subject carrying a special D4Z4 rearrangement resulting in a proximal contracted array. Clinically, distal methylation levels demonstrated a strong correlation with the age-corrected clinical severity score (ACSS) and onset age. Mediation analysis revealed that the influence of distal methylation on ACSS was partially mediated by onset age. This study further confirms the distal 4qA D4Z4 methylation analysis as a valuable complement for differential diagnosis in FSHD suspected patients, including those with complex structural variants.