Optimising the molecular investigation of the FSHD locus: an integrated workflow using single molecule optical mapping and Southern blot analysis.

PURPOSE: Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disorder caused by contraction or hypomethylation of the D4Z4 repeat array located at chromosome 4q35. For the disease to manifest, a permissive haplotype is required, as it enables the pathogenic expression of the DUX4 gene. FSHD cases often involve complex rearrangements, such as intrachromosomal rearrangements and translocations, which complicate diagnosis using conventional methods. This study focuses on evaluating the diagnostic potential of single molecule optical mapping (SMOM) for FSHD with complex rearrangements, particularly in cases involving 4q-10q translocations, which present challenges for detection by SMOM alone. Furthermore, we propose an integrated diagnostic strategy, combining SMOM with complementary methods, to improve accuracy in these challenging cases.

METHODS: We reviewed the test results of 238 patients with suspected FSHD, and 25 participants with presumed complex rearrangements were included in this study. SMOM was performed on these participants, and the results were manually reviewed and compared with those obtained from Southern blot (SB) analysis.

RESULTS: Nine patients carrying 4q-10q translocation exhibited discrepancies between the two methods. Linear regression analysis revealed a significant discrepancy in chromosomal assignment between SB and SMOM in cases suspected of translocation.

CONCLUSIONS: Given the complex nature of FSHD, none of the current methods can independently provide a definitive diagnosis. As misdiagnosis may occur when relying on a single technique, we propose an integrated diagnostic approach, with SMOM as the first-line test.