559. A Pseudo-Exon Derived from an Intronic Insertion Is Responsible for Duchenne-Like Muscular Dystrophy in the Welsh Corgi Dog

Duchenne Muscular Dystrophy (DMD) is the most common X- linked inherited disease, with an incidence of 1 in 3500 male births. This disease is relentlessly progressive and usually fatal by early adulthood. A wide range of mutations in the dystrophin gene have been characterized for DMD, as well as the less severe form, Becker's Muscular Dystrophy. DMD presents gene therapy challenges due to the size of the gene and resulting cDNA and the wide variety and complexity of the mutations involved. Animal models for DMD have been described in the mouse, cat and dog. The disease in mice and cats is significantly less severe than that seen in humans, while the canine disease, characterized by muscle hypertrophy, followedby muscle loss, fibrosis and death, closely mimics disease progression in affected humans. As is expected from the frequency of mutation in the human population, DMD has been recognized in several breeds of dogs including the Golden Retriever, German Short-Haired Pointer, Rottweiler, Labrador Retriever, West Highland White and Welsh Corgi. Where the mutation has been determined, each breed has demonstrated a new mutation. Here we report the identification of the mutation responsible for DMD in the Welsh Corgi. Affected dogs can be recognized at, or shortly after, birth by increased serum creatine kinase levels. These dogs show progressive muscle atrophy and fibrosis, stunted growth, contractures and consequent debilitation. However, several of the affected males have lived to sexual maturity. Muscle biopsies from affected Corgis show that most fibers remain unstained for dystrophin, while rare fibers show dystrophin staining using a variety of antibodies. Sequence analysis of the cDNA indicates a 166 base insertion between exons 13 and 14, based on the human exon structure. The inserted sequence shows a high degree of sequence similarity to a canine LINE-1 element. Sequence analysis of intron 13, which is approximately 25kb, indicates that there is an insertion in the intron. The insertion is immediately downstream of an AG dinucleotide pair and contains the 166 bases seen in the cDNA followed by a GT dinucleotide pair and additional inserted sequence. Thus the insertion utilizes a putative intronic 3’ splice acceptor site and a 5’ splice donor to form a novel exon, which is spliced into the mature mRNA. An in-frame stop codon in this novel exon results in a truncated dystrophin protein, which is non-functional. Staining of occasional fibers with antibodies to epitopes located downstream of this insertion indicates that this exon may be skipped or that alternative splicing and/or promoter use may produce a product. This large animal model, with its defined mutation, will be useful in a variety of ways, including the use of gene repair approaches to skip the inserted exon, studies of spicing mechanisms in dystrophin, and studies of other gene therapy approaches.

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