A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient cavalier king charles spaniels is amenable to exon 51 skipping

Abstract

Background: Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot".

Methodology/principal findings: Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression.

Conclusions/significance: Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD.

Figure 1. CKCS-MD phenotype.

(A) Photograph of the index case at 10 months of age. Note the generalised muscle atrophy and the low head carriage, indicative of paresis. (B and C) Representative cryosections of the vastus lateralis muscle from a dystrophic CKCS dog (dog 2) showing excessive variability in myofiber size and typical areas of degeneration (necrosis and phagocytosis, B) and regeneration (small fibers with prominent vesicular central nuclei, C). H&E stain, bar = 100 µm for both B and C. (D) Immunohistochemistry of CKCS-MD skeletal muscle (left column) compared with control dog muscle (right column), labeled with antibodies to the dystrophin rod (Dys1) and carboxy (Dys2) domains, utrophin, developmental myosin heavy chain (dMHC) and β-dystroglycan (bDGC). Note the absence of expression of dystrophin (with the exception of occasional revertant fibers), redistribution of utrophin to the sarcolemma in both regenerating and non-regenerating fibers, expression of developmental myosin heavy chain and reduced/variable expression of β-dystroglycan in the affected dog in comparison with control. Bar = 100 µm for all images. The utrophin and developmental myosin heavy chain labeling is on serial sections. (E and F) Western immunoblot of extracts from skeletal and cardiac muscle from the index case using antibodies to (E) the dystrophin rod (Dys1) and (F) the carboxy (Dys2) domains in comparison with control muscle. Note the total absence of dystrophin in both skeletal and cardiac muscle in the affected dog. The positive bands, detected using an antibody to dysferlin, confirm equal protein loading in each lane.

Figure 2. Genotype of CKCS-MD.

(A and B) RT-PCR of mRNA extracted from skeletal muscle from a control dog (2) and the index case (3) using primer pair 2–10. Lane 1 is a water control. Note the expected amplicon size of 547 bp in the control but a shorter fragment in the affected dog (A) which upon sequencing was determined to be due to deletion of the 109 bp in exon 50 (B). (C) Direct sequencing of genomic DNA from a control (wild type) dog and from the index case (CKCS-MD) revealed a G-T missense mutation (highlighted) in the 5′ consensus splice site (underlined) in intron 50. (D) Restriction fragment length polymorphism (RFLP) assay using restriction enzyme BSTZ17I. The splice site mutation in the 5′ consensus splice site of intron 50 in the index case, dog 2 and dog 3 (lanes 1, 2 and 3 respectively) is identified by the fact that mutant DNA remains undigested (449 bp) whereas wild type DNA is digested into 2 smaller products of 296 and 153 bp. Lane 4 contains restricted PCR products from dog 3's mother, confirming her as a carrier. Lane 5 (dog 3's father) and lane 6 (control dog) contain restricted PCR products that are wild type.

Figure 3. Antisense oligonucleotide mediated skipping of exon 51 restores protein expression.

(A) RT-PCR of mRNA extracted from cultured canine myoblasts derived from the index case in the absence (lane 1) or presence (lane 2) of AVI-4658; lane 3 is a water control. Note the 666 bp product of the unskipped cDNA (upper band) consistent with the deletion of exon 50 (caused by the mutation) and the smaller 433 bp product consistent with skipping of exon 51. Sequences were confirmed in the extracted products (B). (C) Western immunoblot of protein extracts from treated (+) and untreated (−) CKCS-MD myoblasts using antibodies to the dystrophin rod (Dys1) and dysferlin (as a loading control) demonstrating re-expression of dystrophin following exon 51 skipping in treated cells. (D) Sequence alignment of AVI-4658 (above) with part of the canine exon 51 sequence, demonstrating the mismatches (in red) analogous to the differences between the canine and human sequences.