Predictive value of D4Z4 methylation levels for phenotypic heterogeneity and disease progression in Facioscapulohumeral Muscular Dystrophy with borderline D4Z4 repeat units: a retrospective cohort study

Background Facioscapulohumeral muscular dystrophy (FSHD) patients carrying borderline D4Z4 repeat units (DRUs) (8–10) represent a molecularly ambiguous group overlapping FSHD1 and FSHD2, characterized by pronounced phenotypic heterogeneity. This study aimed to determine the association between methylation levels and disease severity, progression in this undercharacterized cohort. Methods This single-center and retrospective cohort study (ClinicalTrials.gov: NCT04369209), was conducted at the Fujian Neuromedical Centre (FNMC), China. Methylation levels were quantified using bisulfite sequencing in all participants. Whole-exome sequencing (WES) was performed for all probands. Phenotypic classification followed the Comprehensive Clinical Evaluation Form (CCEF). Motor function was assessed using the FSHD clinical score, clinical severity scale (CSS), and age-corrected CSS. Key functional endpoints were defined as follows: (1) lower extremity involvement, CSS ≥ 3.0; and (2) independent ambulation loss, CSS 4.5–5. Results The patients carrying borderline DRUs exhibited milder phenotypes, broader phenotypic variability and lower methylation levels compared to those carrying 4–7 DRUs. The mean methylation levels of the 10 CpG sites and CpG6 methylation levels showed significant negative correlations with FSHD clinical score, CSS, and age-corrected CSS. Those methylation thresholds effectively discriminated symptomatic from asymptomatic patients with borderline DRUs. Furthermore, patients with lower methylation levels exhibited higher disease penetrance and an increased risk of progressing to lower extremity involvement. In a multigenerational pedigree, cooccurrence of a pathogenic SMCHD1 variant exacerbated hypomethylation and clinical severity. Conclusions Hypomethylation of the distal D4Z4 array serves as a robust biomarker for phenotypic penetrance and disease progression in borderline-allele FSHD. The co-presence of mutations in epigenetic modulators ( e.g. , SMCHD1 ) and D4Z4 hypomethylation is correlated with more severe clinical phenotypes, underscoring a compound epigenetic-genetic disease mechanism.