“Introduction: Facioscapolohumeral muscular dystrophy (FSHD) is the third most common dystrophy. A wide range of phenotypes are observed, that could show a different disease progression and/or imply distinct genetic mechanisms.
To date, the diagnostic criteria for FSHD are based on the detection of the genetic signature of the disease. However, the molecular diagnosis and the interpretation of the genetic test cannot ignore a careful correlation with the phenotype.

Methods: We present data of a cohort of 43 patients from 24 families selected by phenotypic features, characterized by incomplete penetrance/atypical phenotypes according to the Comprehensive Clinical Evaluation Form (CCEF), in which a short D4Z4 allele segregated.
Assessment of 4q subtype, DNA methylation levels,WES and segregation analysis.

Results: Methylation levels displayed high variability in relation to the disease phenotype. In more than half of the atypical phenotypes, despite the detection of the FSHD genetic signature, WES analysis identified VUS or likely pathogenic/pathogenic variants in other genes associated with neuromuscular disorders, compatible with the observed phenotype, or known FSHD-modifying genes. A definitive alternative diagnosis was obtained in 5 families.

Conclusion: Our results further support the need to perform a detailed phenotypic characterization of patients with a suspect of FSHD, and, in cases of atypical phenotypes, to combine the D4Z4 sizing with other procedures such as WES. In this regard, methylation analysis represents a valuable tool to provide a preliminary evidence for FSHD to be confirmed by further testing.
Nevertheless, a correct diagnosis based on a comprehensive evaluation of patients is needed for familial counseling, prognosis, management and clinical trials readiness.”