MUTATIONAL ANALYSIS OF DYSTROPHIN DEFICIENT MUSCULAR DYSTROPHY IN CAVALIER KING CHARLES SPANIELS. G. L. Walmsley1, V.Arechavala-Gomeza2, M. Fernandez-Fuente1,2, N. Nagel3, R. Stanley1, K. Chandler1, F. Muntoni2, G. D. Shelton4 and R.J. Piercy1,2 1. Department of Veterinary Clinical Sciences, Royal Veterinary College, London, UK; 2. Dubowitz Neuromuscular Centre, UCL Institute of Child Health London, UK; 3. Northdale Veterinary Practice, West Sussex, UK; 4.Department of Pathology, School of Medicine, University of California San Diego, La Jolla, CA.
Canine dystrophin-deficient muscular dystrophy, analogous to Duchenne muscular dystrophy of humans, is a severe inherited degenerative disorder of striated muscle. This debilitating and ultimately fatal condition results in a progressive destruction of skeletal and cardiac muscle due to mutations in the gene encoding dystrophin, a structural protein that links the contractile apparatus to the sarcolemma. The disorder is seen in several canine breeds but the genetic cause has only been reported in the Golden Retriever, German Short-haired Pointer and Rottweiler. Here we present the findings of clinical, histopathological and molecularcharacterisation of this condition in Cavalier King Charles Spaniels.A 10 month old male neutered client-owned Cavalier KingCharles Spaniel from the United Kingdom was presented witha chronic progressive history of lethargy, exercise intolerance and dysphagia. The dog was tetraparetic with poor skeletal muscle mass (body condition score = 2/9), reduced spinal reflexes, macroglossia and restricted jaw movement. Investigations documented a marked elevation in creatine kinase activity (33,695U/l; 61 – 394U/l) and electromyography revealed spontaneous activity indicative of a primary muscle disorder (complex repetitive discharges and pseudomyotonia). Dystrophin-deficient muscular dystrophy was diagnosed on the basis of skeletal muscle histopathology, immunohistochemistry and immunoblotting using monoclonal antibodies to the dystrophin rod and carboxy termini. Oligonucleotide primer pairs designed for RT-PCR to amplify overlapping regions of dystrophin cDNA identified, following sequencing, an exon deletion and a frame-shift not present in control cDNA, that is predicted to result in premature termination of the protein product. Sequencing the associated genomic DNA confirmed the causative (and novel) mutation. The ability of antisense oligonucleotide induced exon skipping to restore the reading frame was demonstrated in vitro in cultured myoblasts from the affected dog. Sequencing of amplified DNA from an additional Cavalier King Charles Spaniel with dystrophin-deficient muscular dystrophy from North America identified the same mutation. In conclusion, dystrophin deficient muscular dystrophy in the Cavalier King Charles Spaniel may provide an excellent model for Duchenne muscular dystrophy due to the potential application for trials of antisense oligonucleotide-mediated exon skipping – one of the more promising research directions for genetic therapy in this fatal disorder.