RNA-seq analysis, targeted long-read sequencing, and in silico prediction to unravel pathogenic intronic events and predict the regulatory mechanisms underlying complex splicing abnormalities in patients with dystrophinopathy

Abstract Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in all cases analyzed. Exonization of intronic sequences was observed in 15 cases and exon skipping in one case; 13 single nucleotide variants and three structural rearrangements were identified as causal genomic variations. DMD transcripts were aberrantly spliced and polyadenylated in two cases in which chromosome rearrangements were detected. In one case, DMD transcripts were terminated due to nucleotide repeat expansion. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.