Abstract
X-linked myotubular myopathy (XLMTM) is a severe congenital myopathy due to mutations in MTM1 encoding a 3-phosphoinositides phosphatase myotubularin. XLMTM patients display severe generalized hypotonia at birth accompanied by respiratory insufficiency and pathologically show small-size fibers with peripheral halo, centrally located nuclei, disorganized perinuclear organelles in the muscle. Among 78 patients with pathologically diagnosed as XLMTM in our cohort, causative mutations were not identified in 20 cases by targeted re-sequencing panel for congenital myopathy, MTM1 Sanger sequencing nor Whole-exome sequencing. In order to validate deep intronic mutations and aberrant splicing events, we performed RNA-seq analysis on 14 cases who had unreported variants or no-mutations in exonic regions of MTM1. Sashimi plot revealed retentions of intronic sequence in patient1 (between exon 5 and 6) and patient 2 (between exon 11 and 12), respectively. In patient 3, exon 4 skipping was observed due to a synonymous mutation in exon 4. These aberrant splicing were predicted to cause frameshift and subsequently create premature stop codon in these patients. The in vitro splicing experiments with artificial hybrid minigene constructs confirmed those mutations exclusively produced abnormal splicing products. Lastly, Myoblasts from patient 2 showed perinuclear distribution of mitochondria in addition to the abnormal splicing, and treatment with antisense morpholino oligonucleotides rescued those phenotypes. In conclusion, RNA-seq analysis can uncover deep intronic mutations and synonymous mutations which lead abnormal splicing events as the cause of muscle diseases such as XLMTM. We propose that antisense therapy is potentially practical for diseases arising from this type of mutation.
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