title: A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides.
abstract: OBJECTIVE
  Mutations in dysferlin (DYSF), a Ca(2+)-sensitive ferlin family protein important for membrane repair, vesicle trafficking, and T-tubule function, cause Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, and distal myopathy. More than 330 pathogenic DYSF mutations have been identified within exons or near exon-intron junctions. In ~17% of patients who lack normal DYSF, only a single disease-causing mutation has been identified. We studied one family with one known mutant allele to identify both the second underlying genetic defect and potential therapeutic approaches.
  
  
  METHODS
  We sequenced the full DYSF cDNA and investigated antisense oligonucleotides (AONs) as a tool to modify splicing of the mRNA transcripts in order to process out mutant sequences.
  
  
  RESULTS
  We identified a novel pseudoexon between exons 44 and 45, (pseudoexon 44.1, PE44.1), which inserts an additional 177 nucleotides into the mRNA and 59 amino acids within the conserved C2F domain of the DYSF protein. Two unrelated dysferlinopathy patients were also found to carry this mutation. Using AONs targeting PE44.1, we blocked the abnormal splicing event, yielding normal, full-length DYSF mRNA, and increased DYSF protein expression.
  
  
  CONCLUSIONS
  This is the first report of a deep intronic mutation in DYSF that alters mRNA splicing to include a mutant peptide fragment within a key DYSF domain. We report that AON-mediated exon-skipping restores production of normal, full-length DYSF in patients' cells in vitro, offering hope that this approach will be therapeutic in this genetic context, and providing a foundation for AON therapeutics targeting other pathogenic DYSF alleles.
authors.affiliation: Neurology Department, University of Massachusetts Medical School Worcester, Massachusetts, 01605.
authors.forename: Janice A
authors.initials: JA
authors.lastname: Dominov
authors.affiliation: Neurology Department, University of Massachusetts Medical School Worcester, Massachusetts, 01605.
authors.forename: Ozgün
authors.initials: O
authors.lastname: Uyan
authors.affiliation: Neurology Department, University of Massachusetts Medical School Worcester, Massachusetts, 01605.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Sapp
authors.affiliation: Neurology Department, University of Massachusetts Medical School Worcester, Massachusetts, 01605.
authors.forename: Diane
authors.initials: D
authors.lastname: McKenna-Yasek
authors.affiliation: Department of Human Genetics, Emory University School of Medicine Atlanta, Georgia, 30322.
authors.forename: Babi R R
authors.initials: BR
authors.lastname: Nallamilli
authors.affiliation: Department of Human Genetics, Emory University School of Medicine Atlanta, Georgia, 30322.
authors.forename: Madhuri
authors.initials: M
authors.lastname: Hegde
authors.affiliation: Neurology Department, University of Massachusetts Medical School Worcester, Massachusetts, 01605.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Brown