Limits...
Evaluation of exon-skipping strategies for Duchenne muscular dystrophy utilizing dystrophin-deficient zebrafish.

Berger J, Berger S, Jacoby AS, Wilton SD, Currie PD - J. Cell. Mol. Med. (2011)

Bottom Line: By utilizing antisense oligonucleotides, splicing of the dystrophin transcript can be altered so that exons harbouring a mutation are excluded from the mature mRNA.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.Novel dmd mutations were identified to enable the design of phenotype rescue studies via morpholino administration.

View Article: PubMed Central - PubMed

Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.

Show MeSH

Related in: MedlinePlus

Novel dmd mutants identified in a non-complementation screen. (A) Birefringence of the muscle of a 3 dpf WT larva, evoked by polarized light, indicates an ordered array of myofilaments. (B–D) All novel dmd mutants display reduced muscle birefringence. (A’) Immunofluorescence revealed that dystrophin (green) is present at the somite borders (arrows) in siblings, (B’–D’) but absent in the homozygous dmd mutants. (B”–D”) Genomic sequencing of the dmd mutants revealed premature stop codons within exon 21 of dmdpc1/pc1, exon 32 of dmdpc2/pc2 and exon 53 of dmdtm90c/tm90c. (E) Arrows in the schematic diagram of zebrafish dystrophin point to the location of the premature stop codon encoded in each indicated mutant. Amino acid positions are displayed in the scale bar below; calponin-homology domains are in red, hinge domains in green and spectrin-like repeats in blue.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4373433&req=5

fig01: Novel dmd mutants identified in a non-complementation screen. (A) Birefringence of the muscle of a 3 dpf WT larva, evoked by polarized light, indicates an ordered array of myofilaments. (B–D) All novel dmd mutants display reduced muscle birefringence. (A’) Immunofluorescence revealed that dystrophin (green) is present at the somite borders (arrows) in siblings, (B’–D’) but absent in the homozygous dmd mutants. (B”–D”) Genomic sequencing of the dmd mutants revealed premature stop codons within exon 21 of dmdpc1/pc1, exon 32 of dmdpc2/pc2 and exon 53 of dmdtm90c/tm90c. (E) Arrows in the schematic diagram of zebrafish dystrophin point to the location of the premature stop codon encoded in each indicated mutant. Amino acid positions are displayed in the scale bar below; calponin-homology domains are in red, hinge domains in green and spectrin-like repeats in blue.

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 [12], 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 [2]. Thus, the mutations evident in the novel dystrophin alleles dmdpc1, dmdpc2 and dmdtm90c, lie within regions suitable for exon-skipping protocols (Fig. 1E).


Evaluation of exon-skipping strategies for Duchenne muscular dystrophy utilizing dystrophin-deficient zebrafish.

Berger J, Berger S, Jacoby AS, Wilton SD, Currie PD - J. Cell. Mol. Med. (2011)

Novel dmd mutants identified in a non-complementation screen. (A) Birefringence of the muscle of a 3 dpf WT larva, evoked by polarized light, indicates an ordered array of myofilaments. (B–D) All novel dmd mutants display reduced muscle birefringence. (A’) Immunofluorescence revealed that dystrophin (green) is present at the somite borders (arrows) in siblings, (B’–D’) but absent in the homozygous dmd mutants. (B”–D”) Genomic sequencing of the dmd mutants revealed premature stop codons within exon 21 of dmdpc1/pc1, exon 32 of dmdpc2/pc2 and exon 53 of dmdtm90c/tm90c. (E) Arrows in the schematic diagram of zebrafish dystrophin point to the location of the premature stop codon encoded in each indicated mutant. Amino acid positions are displayed in the scale bar below; calponin-homology domains are in red, hinge domains in green and spectrin-like repeats in blue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4373433&req=5

fig01: Novel dmd mutants identified in a non-complementation screen. (A) Birefringence of the muscle of a 3 dpf WT larva, evoked by polarized light, indicates an ordered array of myofilaments. (B–D) All novel dmd mutants display reduced muscle birefringence. (A’) Immunofluorescence revealed that dystrophin (green) is present at the somite borders (arrows) in siblings, (B’–D’) but absent in the homozygous dmd mutants. (B”–D”) Genomic sequencing of the dmd mutants revealed premature stop codons within exon 21 of dmdpc1/pc1, exon 32 of dmdpc2/pc2 and exon 53 of dmdtm90c/tm90c. (E) Arrows in the schematic diagram of zebrafish dystrophin point to the location of the premature stop codon encoded in each indicated mutant. Amino acid positions are displayed in the scale bar below; calponin-homology domains are in red, hinge domains in green and spectrin-like repeats in blue.
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 [12], 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 [2]. Thus, the mutations evident in the novel dystrophin alleles dmdpc1, dmdpc2 and dmdtm90c, lie within regions suitable for exon-skipping protocols (Fig. 1E).

Bottom Line: By utilizing antisense oligonucleotides, splicing of the dystrophin transcript can be altered so that exons harbouring a mutation are excluded from the mature mRNA.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.Novel dmd mutations were identified to enable the design of phenotype rescue studies via morpholino administration.

View Article: PubMed Central - PubMed

Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.

Show MeSH
Related in: MedlinePlus