Evaluation of exon-skipping strategies for Duchenne muscular dystrophy utilizing dystrophin-deficient zebrafish.
Bottom Line: Although this approach has been shown to be effective to restore partially functional dystrophin protein, the level of dystrophin protein that is necessary to rescue a severe muscle pathology has not been addressed.As zebrafish dystrophin mutants (dmd) resemble the severe muscle pathology of human patients, we have utilized this model to evaluate exon skipping.Novel dmd mutations were identified to enable the design of phenotype rescue studies via morpholino administration.
Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.Show MeSH
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Mentions: To identify novel dystrophin alleles, a non-complementation screen based on the dmdta222a fish was performed. Adult male zebrafish were treated with ENU and subsequently crossed to WT female to generate F1 founders. The F1 founders were subsequently crossed to heterozygous dmdta222a/+ fish. Resulting offspring were screened for their levels of birefringence at 3 dpf. Non-complementation of the dmdta222a allele, indicated by a reduction in birefringence, identified 2 novel dmd mutants. In addition, the zebrafish mutant dmdtm90c (synonym: sapje, saptm90c), previously identified in a large scale screen but yet to be molecularly characterized , was also confirmed not to complement the dmdta222a allele (Fig. 1A–D). Whole mount immunohistochemistry with antibody against dystrophin indicated loss of dystrophin in all three mutants and each of the mutants possessed a similar level of phenotypic severity compared to the original (Fig. 1A’–D’). Subsequent sequencing of the genomic dystrophin coding region in the mutants and comparison with the WT sequence led to the discovery of premature stop codons within the exons 21, 32 and 53 for the alleles named dmdpc1, dmdpc2 and dmdtm90c, respectively (Fig. 1B”–D”). The identified mutations are widely distributed across the repetitive spectrin repeat region of dystrophin, which is often deleted within BMD patients and is also largely dispensable for the functional rescue of the mdx mouse by mini-dystrophin . Thus, the mutations evident in the novel dystrophin alleles dmdpc1, dmdpc2 and dmdtm90c, lie within regions suitable for exon-skipping protocols (Fig. 1E).
Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.