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

<h4>Introduction</h4>Pathogenic variants in the <i>DMD</i> 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 <i>DMD</i> exons 50-51 identified by initial genetic testing, representing a notable exception to the conventional reading-frame rule.<h4>Methods</h4>To elucidate his phenotype-genotype discordance, muscle biopsy and subsequent dystrophin protein and mRNA analyses were conducted, followed by long-read sequencing of <i>DMD</i> gene and splicing analysis.<h4>Results</h4>Muscle biopsy revealed a dystrophic pattern and negative expression of dystrophin-N and dystrophin-C. The <i>dystrophin</i> mRNA analysis identified two out-of-frame <i>DMD</i> transcripts, which were different from the in-frame deletion of <i>DMD</i> exons 50-51 and can explain his severe phenotype. Long-read sequencing uncovered a novel deletion variant (~97kb) in <i>DMD</i> gene, which produced the two out-of-frame transcripts through aberrant splicing.<h4>Conclusion</h4>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 <i>DMD</i> variants, guiding patient management, and genetic counseling.