The diagnostic role of next-generation sequencing on facioscapulohumeral muscular dystrophy(FSHD) and related phenotypes

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common genetic neuromuscular disorder exhibited by progressive muscle weakness, primarily affecting the face, scapular, and especially upper arm muscles. In approximately 95% of cases, classified as type 1 (FSHD1), the disease results from contractions of the D4Z4 macrosatellite repeats on chromosome 4q35. The remaining cases, classified as type 2 (FSHD2), are primarily caused by pathogenic variants in SMCHD1, with a smaller subset involving DNMT3B and LRIF1 genes. These variants not only lead to D4Z4 hypomethylation and aberrant DUX4 activation but may also influence the disease severity in FSHD1 patients with borderline D4Z4 repeat contractions. The diagnostic algorithm of FSHD involves the examination of D4Z4 Repeat Units (RU) and the analysis of the FSHD-associated genes to confirm or exclude diagnosis. The Molecular Combing (MC) technique has been a crucial tool for the precise and effective analysis of the D4Z4 region, while NGS provides a broader genetic landscape, specific to FSHD2 cases. This approach enables the identification of variants in SMCHD1, LRIF1, and DNMT3B, as well as the detection of variants in other neuromuscular disease-related genes. It enhances differential diagnosis, prevents misclassification, and improves the interpretation of genotype-phenotype correlations. A total of 96 unrelated patients' D4Z4 RUs were determined using MC. In this group, 82 patients had contracted D4Z4 RUs (67/82 with pathogenic 1–7 RUs and 15/82 with borderline 8–10 RUs). Of the remaining 14 patients, 12 presented with ≥11 RUs while in the remaining 2 cases, only the 4qB allele was detected. Twelve patients from this uncontracted group were selected for Whole Exome Sequencing (WES) analysis based on their disease severity and complex MC results. One unaffected and two affected relatives of one patient were included in this study group for segregation and confirmation purposes. Pathogenic variants in SMCHD1 were detected in three patients confirming the clinical diagnosis of FSHD. Pathogenic and/or likely pathogenic CAPN3 variants were identified in two patients, leading to a revised diagnosis of Limb-Girdle Muscular Dystrophy 2A (LGMD2A). In one patient, variant of unknown significance in FHL1 gene were identified. The results of this study largely align with the literature regarding the prevalence of contracted D4Z4 alleles in patients with a clinical diagnosis of FSHD and underscore the significance of NGS for ascertaining a definitive diagnosis in the small subset without contracted D4Z4 alleles. It is crucial to explore the impact of NGS to reveal overlapping genetic factors that may contribute to the clinical heterogeneity of FSHD and its differential diagnoses.