A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping

Abstract

BackgroundDuchenne 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 FindingsHere 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/SignificanceGiven 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.

Duchenne muscular dystrophy gene therapy in the canine model

“…19,91,92 Local injection also resulted in exon skipping in GRMD and beagle-background GRMD dogs. 92,93 While a single AON seems sufficient for exon skipping in myoblasts, 19,92 interestingly, a cocktail of AONs is required for efficient exon skipping in canine muscle in vivo. 92 To test whole-body exon skipping, Yokota et al delivered the PMO cocktail to beaglebackground GRMD dogs by intravenous injection.…”

“…15 Since then, dystrophin-deficient dogs have been described in many other breeds. 15,[17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] The majority of these reports are descriptive case studies. Dystrophin mutations have been determined in some breeds.…”

Relevant aspects of golden retriever muscular dystrophy for the study of Duchenne muscular dystrophy in humans

“…Spontaneous mutations in the dystrophin-encoding gene, resulting in muscular dystrophy linked to the X chromosome, have been reported in several dog breeds (COLLINS & MORGAN, 2003;SHELTON & ENGVALL, 2005;NAKAMURA & TAKEDA, 2011). Genetic mutations in the Golden Retriever, Rottweiler, German Shorthaired Pointer, Cavalier King Charles Spaniels, Beagle and Weimaraner breeds were described (COOPER et al, 1988;SHARP et al, 1992;SCHATZBERG et al, 1999;SHIMATSU et al, 2003;BALTZER et al, 2007;WALMSLEY et al, 2010). All canine models of DMD, type of mutation, and site of each dystrophin gene mutation are described in table 1.…”

Detection of Mutation-Hotspots and Exon-Deletions using Digital Signal Processing in Duchenne Muscular Dystrophy (DMD) Gene

“…The exons representing deletion hotspots in mutated DMD were found out from various references through literature search in Tan [15]. Of these 22 hotspot exons (1, 3-5, 8, 13, 19, 40-45, 47, 48, 50-55, 60) were studied using antinotch filter.…”