Stepwise Diagnostic Strategy Integrating Long-Read Sequencing for the Interpretation of Phenotype-Genotype Discordance in Dystrophinopathy.

Introduction: Pathogenic variants in the DMD gene maintaining the open reading frame typically cause Becker muscular dystrophy. Here, we report a 7.7-year-old boy exhibiting a severe Duchenne muscular dystrophy phenotype, despite an in-frame deletion of DMD exons 50-51 identified by initial genetic testing, representing a notable exception to the conventional reading-frame rule. Methods: To elucidate his phenotype-genotype discordance, muscle biopsy and subsequent dystrophin protein and mRNA analyses were conducted, followed by long-read sequencing of DMD gene and splicing analysis. Results: Muscle biopsy revealed a dystrophic pattern and negative expression of dystrophin-N and dystrophin-C. The dystrophin mRNA analysis identified two out-of-frame DMD transcripts, which were different from the in-frame deletion of DMD exons 50-51 and can explain his severe phenotype. Long-read sequencing uncovered a novel deletion variant (~97kb) in DMD gene, which produced the two out-of-frame transcripts through aberrant splicing. Conclusion: This case underscores the necessity of a stepwise molecular analysis strategy for the interpretation of phenotype-genotype discordance in dystrophinopathy. This stepwise diagnostic approach is essential for accurately characterizing DMD variants, guiding patient management, and genetic counseling.