A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides

Abstract

ObjectiveMutations in dysferlin (DYSF), a Ca2+-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.MethodsWe 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.ResultsWe 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.InterpretationThis 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.

Progress and challenges in diagnosis of dysferlinopathy

“…A similar pathogenic effect has been proven to occur from the first deep intronic mutation (c.4886+1249G>T, intron 44) described in this gene . This latter mutation was found to recur in compound heterozygosity in patients with only 1 mutation detected (in 2 of 8 cases in 1 study and in 1 of 33 cases in another). Large genomic rearrangements have been reported to be pathogenic, the most frequent of which is the c.2512‐?_3174+?del, and others are quoted in public mutation databases.…”

“…Several studies have used mRNA analysis to demonstrate that many intronic mutations actually disrupt correct mRNA splicing . A similar pathogenic effect has been proven to occur from the first deep intronic mutation (c.4886+1249G>T, intron 44) described in this gene . This latter mutation was found to recur in compound heterozygosity in patients with only 1 mutation detected (in 2 of 8 cases in 1 study and in 1 of 33 cases in another).…”

Genetic landscape and novel disease mechanisms from a large LGMD cohort of 4656 patients

“…This splicing causes an in‐frame insertion of 59 additional amino acids in the dysferlin protein 39. This intronic variant segregated in a compound heterozygous state with the other pathogenic variant in two siblings (Fig.…”

Exon 32 Skipping of Dysferlin Rescues Membrane Repair in Patients’ Cells

“…Another group has also demonstrated that a mutant dysferlin pseudoexon could be skipped [34]. Such an approach has already been successfully applied in the case of DMD.…”

“…Exon skipping could also be used to remove pseudoexons. Recently, a mutation creating a pseudoexon between exons 44 and 45 of DYSF has been identified [34, 46]. Exon skipping using an AON targeting this pseudoexon restored a normal mRNA and increased dysferlin expression, opening the way for such an approach in dysferlinopathies [34].…”