Integrated D4Z4 structural, epigenetic and exome-based evaluation of facioscapulohumeral muscular dystrophy in a tertiary referral cohort from Türkiye

Background Diagnosing facioscapulohumeral muscular dystrophy (FSHD) requires integrated evaluation of D4Z4 repeat size, permissive haplotype status, epigenetic context and alternative molecular aetiologies, particularly in borderline, non-contracted or structurally complex cases. Methods We evaluated 135 unrelated referrals with suspected FSHD at a tertiary referral centre in Türkiye between 2019 and 2026. First-line testing included single-molecule D4Z4 repeat sizing, haplotyping and structural analysis using molecular combing or optical genome mapping. DR1 methylation profiling and whole-exome sequencing (WES) were used as second-line tests in unresolved, borderline, non-contracted or clinically atypical cases. Results FSHD was confirmed in 121/135 referrals (89.6%): FSHD1 in 109/121 (90.1%), FSHD1+2 in 4/121 (3.3%) and FSHD2 in 8/121 (6.6%). Of the remaining referrals, two had confirmed alternative molecular diagnoses, one had a candidate DES -related myopathy, six were FSHD excluded and five remained unresolved. Single-molecule analysis identified mosaicism, allelic imbalance, homozygous contracted genotypes and complex 4q configurations. Second-line DR1 methylation refined borderline and non-contracted cases, while WES identified six SMCHD1 variants, including four identified in this study, and candidate alternative or dual diagnoses. Exploratory analyses showed that age-corrected severity captured repeat length and age-at-onset-related gradients better than raw severity scores. Conclusion These findings support the real-world diagnostic value of combining established structural, epigenetic and sequencing-based methods for suspected FSHD, particularly in diagnostically challenging referrals. The study further provides cohort-level data from Türkiye, an under-represented population in the FSHD literature, and may inform future diagnostic interpretation of borderline, non-contracted and complex locus configurations.