Dystrophinopathy, including Duchenne and Becker muscular dystrophies (DMD/BMD), is the most common hereditary neuromuscular disease caused by mutations in the DMD gene. It is known that the deletions/duplications spanning one or more exons are the cause of the disease in approximately 70% of patients while the small mutations involving one or at most several nucleotides are responsible for the remaining 30%. Nowadays, a combination of multiplex ligation-dependent probe amplification (MLPA) and targeted resequencing are widely used for the diagnosis of dystrophinopathy. Nevertheless, around 1% of patients still remain undiagnosed and seem to carry deep intronic mutations creating an extra exon or mutations produced by chromosomal rearrangement, leading to abnormal transcript. To decipher such mutations, we performed RNA-seq on 23 undiagnosed DMD/BMD cases in whom no variant was identified albeit dystrophin was deficient on muscle biopsy. We also analyzed 6 DMD/BMD cases who had a missense mutation in DMD gene as we suspected aberrant splicing may be caused by the mutation in these cases. By RNA-seq, abnormal splicing events were seen in 11 of 23 undiagnosed cases, in whom we further confirmed the presence of intronic mutations that led to pseudo exon production. The remaining 12 cases showed low expression of DMD transcript but without obvious abnormal splicing. In addition, in 2 of 6 cases with missense mutation, skipping of the exon harboring the mutation was detected. The variants were located the last or second to last exon in 2 cases. The remaining 4 cases showed normal expression of DMD transcript. Still 12 cases remained unsolved even by RNA-seq. Combination of whole genome sequencing and long-read sequencing will be performed for these cases. Not only RNA-seq analysis complements the routine genetic testing of dystrophinopathy but may help uncover the actual pathomechanisms of "missense" variants.

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