The Role of Whole‐Exome Sequencing and Methylation Analysis in Untangling Complex Facioscapulo‐Humeral Muscular Dystrophy Cases

ABSTRACT Facioscapulo‐humeral muscular dystrophy is characterized by a distinctive phenotype, although a wide range of clinical expressions is observed, possibly reflecting different disease progression rates or complex genetic mechanisms. To date, the diagnostic criteria for FSHD rely on identifying the genetic signature of the disease (reduced D4Z4 allele, permissive 4q allele, hypomethylation, and in some cases variants in modifier genes). However, interpreting genetic data requires careful correlation with the phenotype, especially in atypical cases. The study included a cohort of 42 patients with a D4Z4 contraction or belonging to a pedigree in which DRAs segregated but who were selected due to presenting atypical clinical features or an unexpected disease severity according to the Comprehensive Clinical Evaluation Form (CCEF). The 42 underwent 4q subtype analysis, DNA methylation assessment, whole‐exome sequencing (WES) and segregation analysis. In 24 cases, WES identified likely pathogenic or pathogenic variants in genes associated with different neuromuscular disorders, in some cases possibly compatible with the observed phenotype. Methylation analysis proved useful in distinguishing asymptomatic and atypical cases, prompting differential diagnosis. Our results emphasize the importance of a detailed phenotypic characterization of patients with a suspicion of FSHD and, in the case of atypical phenotypes, the combination of D4Z4 sizing with other procedures such as WES.