Muscular Dystrophy and
the Cavalier King Charles Spaniel
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Some cavalier King Charles spaniels suffer from a genetic form of muscular dsytrophy (MD) caused by a mutation of the dystrophin gene. Dystrophin is a protein that acts to stabilize skeletal and cardiac muscles and provides mechanical stability during the muscles’ contractions. The dystrophin gene encodes that protein. The mutations in the dystrophin gene causes an absence of dystrophin in the muscles, which results in progressive degeneration of muscle fibres.
Muscular dystrophy is hereditary in the cavalier but is carried only by the X chromosome. Since males have only one X chromosome, they are more susceptible to inheriting MD because they need only one parent to pass the gene to them. For females to inherit the gene, both parents must carry it. Females are more often only carriers of the gene.
The genetic cause of muscular dystrophy in the CKCS was identified in a 2009 report by a research team at the UK Royal Veterinary College, led by Dr. Richard. J. Piercy and Gemma L. Walmsley. This study is on-going, with the cooperation of Dr. G. Diane Shelton at the Comparative Neuromuscular Laboratory.
In a 2010 study, the same group of researchers have opined that all affected cavaliers may be related to a single founder. The extent of this disorder in the breed is not known. In a study examining 96 female CKCSs in the UK, there were no carriers identified. So, MD is suspected to be present at only a low level within the breed.
The type of MD in cavaliers is very similar to Duchenne muscular dystrophy (DMD) in humans. It reportedly afflicts one in 3,500 boys, and therefore it is one of the most common genetic disorders of children.
Since muscular dystrophy results in a lack of stability of the sketetal muscles, classic signs of MD are exercise intolerance, a stiff gait, and a lack of muscle mass. These symptoms usually are detectable at an early age, in some dogs, as young as six weeks to three months.
Other signs include: overall weakness, difficulty swallowing, an enlarged tongue, gagging when eating, breathing difficulties, muscle spasms, leg deformities, and tremors. The swallowing difficulty and gagging may result in aspiration pneumonia.
Symptoms in females usually are milder than in males.
Some mouth symptoms are similar to masticatory muscle myositis, another neuromuscular disease affecting the muscles used by the dog to chew – the jaw and temporal muscles.
Based upon the dog’s symptoms, usually some sort of a neuromuscular disorder is suspected. The veterinarian should test the dog’s reflexes, tongue, and jaw movement.
A blood test would reveal that serum creatine kinase (CK) is highly elevated. A muscle biopsy, under general anesthesia, should be taken for evaluation. Immunohistochemistry analysis of the biopsy is an essential test to determine the absence of dystrophin in the muscles.
Research laboratories are able to determine whether cavaliers carry the mutation of the gene. UK's Animal Health Trust, with the Royal Veterinary College have identified the genetic mutation. VetNostic Laboratories has developed a test based on the mutation, and it is in the process of validating that test. Click here for more information about the test.
There is no cure for muscular dystrophy in the cavalier King Charles spaniel. Nor is there any medical therapy for maintenance of the disorder or to slow its progression. An exception to this could be anabolic steroid supplements or growth hormone treatments, which may temporarily slow the progression, but these drugs have potentially damaging side effects and may not be used long term.
Researchers are examining the efficacy of transplanting stem cells into dogs with muscular dystrophy. One such research project has reported increases in the dogs’ level of dystrophin and regained muscle strength and mobility.
Other researchers are studying gene therapy to inactivate the offending gene (in this case the "exon 51") and restoring the expression of dystrophin. This is called antisense therapy. In a 2010 study, the researchers reported that "Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected [cavalier] dog restored the reading frame and protein expression". Translation: that is good, promising news.
For now, however, the prognosis is that affected cavaliers will not survive very long after severe symptoms appear. The lack of dystrophin eventually will begin to affect the contractibility of the dog’s heart.
This form of muscular dystrophy is an hereditary disease passed by at least parent to its litters. Carriers can be identified.
August 2011: Cavaliers' DNA needed for muscular dystrophy test validation. VetNostic Laboratories needs 50 cavalier King Charles spaniels to contribute DNA samples to validate a test for dystrophin-deficient muscular dystrophy in the breed (CKCS-MD). Collection is performed using a cheek swab kit which consists of three swabs per dog. A self addressed, postage paid, return envelope will be provided. Once the lab receives the swabs, it will take about a week to obtain the results. Testing is free and result certificates will be provided. To obtain a collection kit along with instructions, please email Rob Mason email@example.com or call 877-255-9208. Website www.vetnostic.com
Muscular dystrophy in Cavalier King Charles spaniels. Piercy, Richard. J. and Walmsley, Gemma. Vet Rec. 2009 165 (2), p. 62. Quote: "We have recently identified the genetic cause of a form of muscular dystrophy in CKCS. The causative mutation is in the dystrophin gene and the X-linked disease is associated with weakness, muscle atrophy and exercise intolerance, detectable from a few months of age. Prominent signs in affected dogs are dysphagia [the symptom of difficulty in swallowing] and macroglossia (enlarged tongue)[tongue enlargement that leads to functional and cosmetic problems]. Serum creatine kinase is usually markedly elevated. Male dogs with the mutation [are] clinically affected and female dogs with the mutation are silent carriers. We are also keen to hear from veterinary surgeons who believe they may have seen an affected dog in their practice, in order to estimate the prevalence of this disease and limit its spread by genetic testing." Contact Dr. Piercy at the Royal Veterinary College's Comparative Neuromuscular Diseases Laboratory at firstname.lastname@example.org
Mutational Analysis of Dystrophin-deficient Muscular Dystrophy in Cavalier King Charles Spaniels. G. L. Walmsley, V. Arechavala-Gomeza, M. Fernandez-Fuente, N. Nagel, R. Stanley, K. Chandler, F. Muntoni, G. D. Shelton, and R.J. Piercy. J Vet Intern Med. 2009; 23(3):741 (ACVIM 27th Ann. Vet. Med. Forum Abstract Program: Abstract 189). Quote: "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 molecular characterisation of this condition in Cavalier King Charles Spaniels. A 10 month old male neutered client-owned Cavalier King Charles Spaniel from the United Kingdom was presented with a chronic progressive history of lethargy, exercise intolerance and dysphagia. The dog was tetraparetic with poor skeletal muscle mass (body condition score 52/9), 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."
A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping. Gemma L. Walmsley, Virginia Arechavala-Gomeza, Marta Fernandez-Fuente, Margaret M. Burke, Nicole Nagel, Angela Holder, Rachael Stanley, Kate Chandler, Stanley L. Marks, Francesco Muntoni, G. Diane Shelton, Richard J. Piercy. J. Plos One Jan2010; 5(1):e8647. Quote: "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 dys trophin 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 59 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."
Mammalian Models of Duchenne Muscular Dystrophy: Pathological Characteristics and Therapeutic Applications. Akinori Nakamura and Shin'ichi Takeda. J.Biomed.Biotechnol.; Jan 2011; 2011:184393. Quote: "5.3. Cavalier King Charles Spaniels with Muscular Dystrophy (CKCS-MD) -- 5.3.1. Pathological Characteristics -- Very recently, it was reported that another dystrophic dog, the Cavalier King Charles Spaniel with dystrophin-deficient muscular dystrophy (CKCS-MD) has a severe phenotype. This canine model has a missense mutation in the 5′ donor splice site of exon 50 resulting in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. 5.3.2. Therapeutic Applications -- The therapeutic strategy of exon skipping in the GRMD or CXMDJ models provides a significant advantage in clinical trials, but also has the unfavorable characteristic that the disease-causing mutation does not include the region of the DMD gene that is most commonly mutated in human DMD patients. About 60% of DMD patients harbor deletions in exons 45–55 of the DMD gene [55, 56]. As described previously, the exon 51 skipping technique, which is being employed in clinical trials [30, 31], is applicable to DMD patients which have deletions of exon 50, exon 52, exons 48–50, or exons 49-50, among others, and is feasible for more patients than other exon skipping strategies [27, 57, 58]. AO-mediated skipping of exon 51 in cultured myoblasts of the CKCS-MD dog restored the reading frame and protein expression . This observation suggests that the use of this canine model would be valuable in the preclinical trials of therapy based upon exon 51 skipping."
Genetic Connection: A Guide to Health Problems in Purebred Dogs, Second Edition. Lowell Ackerman. July 2011; AAHA Press; pg 127. Quote: "Cavalier King Charles spaniels ...are susceptible to an X-linked myopathy similar to Duchenne-type muscular dystrophy in humans. ... The defect has been characterized in the Cavalier King Charles spaniel ... and so it is likely that other genetic tests will eventually become available."
Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies. Joe N. Kornegay, Janet R. Bogan, Daniel J. Bogan, Martin K. Childers, Juan Li, Peter Nghiem, David A. Detwiler, C. Aaron Larsen, Robert W. Grange, Ratna K. Bhavaraju-Sanka, Sandra Tou, Bruce P. Keene, James F. Howard Jr., Jiahui Wang, Zheng Fan, Scott J. Schatzberg, Martin A. Styner, Kevin M. Flanigan, Xiao Xiao, Eric P. Hoffman. Mamm. Genome. Jan. 2012;23(1):85-108. Quote: "The progression of clinical disease and serum creatine kinase (CK) levels in canine X-linked muscular dystrophy (CXMD) was studied in 7 dogs from birth to 12-14 months and in 18 dogs at varying intervals from birth to 8 weeks. One affected male was studied from age 3.5 to 6 years, and all pups were descendants of this dog. A lethal neonatal form was recognized in some pups. In the more typical form, clinical signs of stunting, weakness and gait abnormalities were evident by 6-9 weeks and were progressive, leading to marked muscle atrophy, fibrosis and contractures by 6 months. Serum CK levels were markedly elevated, such that affected pups could be identified by 1 week. CK values increased until 6-8 weeks, then plateaued at approx. 100 times normal. Affected females and beagle-cross dogs were less severely affected than large breed-cross dogs. In the 2 adult dogs with cardiac insufficiency CK levels had decreased to 5-15 times normal. These studies show that CXMD and Duchenne muscular dystrophy have striking phenotypic as well as genotypic similarities. In addition, these studies of CXMD suggest that in females and in smaller dogs the same genetic defect results in a less severe clinical disease."
Inhibition of CD26/DPP-IV enhances donor muscle cell engraftment and stimulates sustained donor cell proliferation. Maura H Parker, Carol Loretz, Ashlee E Tyler, Lauren Snider, Rainer Storb, Stephen J Tapscott. Skelet Muscle. 2012; 2: 4. Published online 2012 February 16. doi: 10.1186/2044-5040-2-4 Quote: "Background: Transplantation of myogenic stem cells possesses great potential for long-term repair of dystrophic muscle. In murine-to-murine transplantation experiments, CXCR4 expression marks a population of adult murine satellite cells with robust engraftment potential in mdx mice, and CXCR4-positive murine muscle-derived SP cells home more effectively to dystrophic muscle after intra-arterial delivery in mdx5cv mice. Together, these data suggest that CXCR4 plays an important role in donor cell engraftment. Therefore, we sought to translate these results to a clinically relevant canine-to-canine allogeneic transplant model for Duchenne muscular dystrophy (DMD) and determine if CXCR4 is important for donor cell engraftment. Methods: In this study, we used a canine-to-murine xenotransplantation model to quantitatively compare canine muscle cell engraftment, and test the most effective cell population and modulating factor in a canine model of DMD using allogeneic transplantation experiments. Results: We show that CXCR4 expressing cells are important for donor muscle cell engraftment, yet FACS sorted CXCR4-positive cells display decreased engraftment efficiency. However, diprotin A, a positive modulator of CXCR4-SDF-1 binding, significantly enhanced engraftment and stimulated sustained proliferation of donor cells in vivo. Furthermore, the canine-to-murine xenotransplantation model accurately predicted results in canine-to-canine muscle cell transplantation. Conclusions: Therefore, these results establish the efficacy of diprotin A in stimulating muscle cell engraftment, and highlight the pre-clinical utility of a xenotransplantation model in assessing the relative efficacy of muscle stem cell populations."
Cardiac structure and function in female carriers of a canine model of Duchenne muscular dystrophy. A.M. Kane, T.C. DeFrancesco, M.C. Boyle, D.E. Malarkey, J.W. Ritchey, C.E. Atkins, J.M. Cullen, J.N. Kornegay, B.W. Keene. Research in Vet. Sci. Dec. 2012. Quote: "This investigation tested the hypothesis that carriers of golden retriever muscular dystrophy (GRMD), a genetically homologous condition of Duchenne muscular dystrophy (DMD), have quantifiable abnormalities in myocardial function, structure, or cardiac rhythm. Eleven GRMD carriers and four matched controls had cardiac evaluations and postmortem examinations. 24-h ECG Holter monitoring disclosed ventricular ectopy in 10 of 11 carriers and 2 of 4 controls. Conventional echocardiography failed to demonstrate significant differences between carriers and controls in systolic function. All carriers had multifocal, minimal to marked myofiber necrosis, fibrosis, mineralization, inflammation, and/or fatty change in their hearts. Immunohistochemistry revealed a mosaic dystrophin deficiency in scattered cardiac myofibers in all carriers. No controls had cardiac histologic lesions; all had uniform dystrophin staining. Despite cardiac mosaic dystrophin expression and degenerative cardiac lesions, GRMD carriers at up to 3 years of age could not be distinguished statistically from normal controls by echocardiography or 24-h Holter monitoring."