Whole genome sequencing reveals a 7 base-pair deletion in DMD exon 42 in a dog with muscular dystrophy

Dystrophin is a key cytoskeletal protein coded by the Duchenne muscular dystrophy (DMD) gene located on the X-chromosome. Truncating mutations in the DMD gene cause loss of dystrophin and the classical DMD clinical syndrome. Spontaneous DMD gene mutations and associated phenotypes occur in several other species. The mdx mouse model and the golden retriever muscular dystrophy (GRMD) canine model have been used extensively to study DMD disease pathogenesis and show efficacy and side effects of putative treatments. Certain DMD gene mutations in high-risk, the so-called hot spot areas can be particularly helpful in modeling molecular therapies. Identification of specific mutations has been greatly enhanced by new genomic methods. Whole genome, next generation sequencing (WGS) has been recently used to define DMD patient mutations, but has not been used in dystrophic dogs. A dystrophin-deficient Cavalier King Charles Spaniel (CKCS) dog was evaluated at the functional, histopathological, biochemical, and molecular level. The affected dog’s phenotype was compared to the previously reported canine dystrophinopathies. WGS was then used to detect a 7 base pair deletion in DMD exon 42 (c.6051-6057delTCTCAAT mRNA), predicting a frameshift in gene transcription and truncation of dystrophin protein translation. The deletion was confirmed with conventional PCR and Sanger sequencing. This mutation is in a secondary DMD gene hotspot area distinct from the one identified earlier at the 5′ donor splice site of intron 50 in the CKCS breed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00335-016-9675-2) contains supplementary material, which is available to authorized users

Clinical and genetic characterisation of dystrophin-deficient muscular dystrophy in a family of Miniature Poodle dogs

Four full-sibling intact male Miniature Poodles were evaluated at 4–19 months of age. One was clinically normal and three were affected. All affected dogs were reluctant to exercise and had generalised muscle atrophy, a stiff gait and a markedly elevated serum creatine kinase activity. Two affected dogs also showed poor development, learning difficulties and episodes of abnormal behaviour. In these two dogs, investigations into forebrain structural and metabolic diseases were unremarkable; electromyography demonstrated fibrillation potentials and complex repetitive discharges in the infraspinatus, supraspinatus and epaxial muscles. Histopathological, immunohistochemical and immunoblotting analyses of muscle biopsies were consistent with dystrophin-deficient muscular dystrophy. DNA samples were obtained from all four full-sibling male Poodles, a healthy female littermate and the dam, which was clinically normal. Whole genome sequencing of one affected dog revealed a >5 Mb deletion on the X chromosome, encompassing the entire DMD gene. The exact deletion breakpoints could not be experimentally ascertained, but we confirmed that this region was deleted in all affected males, but not in the unaffected dogs. Quantitative polymerase chain reaction confirmed all three affected males were hemizygous for the mutant X chromosome, while the wildtype chromosome was observed in the unaffected male littermate. The female littermate and the dam were both heterozygous for the mutant chromosome. Forty-four Miniature Poodles from the general population were screened for the mutation and were homozygous for the wildtype chromosome. The finding represents a naturally-occurring mutation causing dystrophin-deficient muscular dystrophy in the dog

Long-Term Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Golden Retriever Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal, X-linked recessive disease affecting 1 in 3,500 newborn boys for which there is no effective treatment or cure. One novel strategy that has therapeutic potential for DMD is inhibition of myostatin, a negative regulator of skeletal muscle mass that may also promote fibrosis. Therefore, our goal in this study was to evaluate systemic myostatin inhibition in the golden retriever model of DMD (GRMD). GRMD canines underwent liver-directed gene transfer of a self-complementary adeno-associated virus type 8 vector designed to express a secreted dominant-negative myostatin peptide (n =4) and were compared with age-matched, untreated GRMD controls (n =3). Dogs were followed with serial magnetic resonance imaging (MRI) for 13 months to assess cross-sectional area and volume of skeletal muscle, then euthanized so that tissue could be harvested for morphological and histological analysis. We found that systemic myostatin inhibition resulted in increased muscle mass in GRMD dogs as assessed by MRI and confirmed at tissue harvest. We also found that hypertrophy of type IIA fibers was largely responsible for the increased muscle mass and that reductions in serum creatine kinase and muscle fibrosis were associated with long-term myostatin inhibition in GRMD. This is the first report describing the effects of long-term, systemic myostatin inhibition in a large-animal model of DMD, and we believe that the simple and effective nature of our liver-directed gene-transfer strategy makes it an ideal candidate for evaluation as a novel therapeutic approach for DMD patients

Are mice good models for human neuromuscular disease? Comparing muscle excursions in walking between mice and humans

The mouse is one of the most widely used animal models to study neuromuscular diseases and test new therapeutic strategies. However, findings from successful pre-clinical studies using mouse models frequently fail to translate to humans due to various factors. Differences in muscle function between the two species could be crucial but often have been overlooked. The purpose of this study was to evaluate and compare muscle excursions in walking between mice and humans

Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV-microdystrophin gene therapy in DMD patients

International Veterinary Epilepsy Task Force recommendations for systematic sampling and processing of brains from epileptic dogs and cats

Traditionally, histological investigations of the epileptic brain are required to identify epileptogenic brain lesions, to evaluate the impact of seizure activity, to search for mechanisms of drug-resistance and to look for comorbidities. For many instances, however, neuropathological studies fail to add substantial data on patients with complete clinical work-up. This may be due to sparse training in epilepsy pathology and or due to lack of neuropathological guidelines for companion animals. The protocols introduced herein shall facilitate systematic sampling and processing of epileptic brains and therefore increase the efficacy, reliability and reproducibility of morphological studies in animals suffering from seizures. Brain dissection protocols of two neuropathological centres with research focus in epilepsy have been optimised with regards to their diagnostic yield and accuracy, their practicability and their feasibility concerning clinical research requirements. The recommended guidelines allow for easy, standardised and ubiquitous collection of brain regions, relevant for seizure generation. Tissues harvested the prescribed way will increase the diagnostic efficacy and provide reliable material for scientific investigations

Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia

Background Preclinical testing of potential therapies for Duchenne muscular dystrophy (DMD) is conducted predominantly of the mdx mouse. But lack of a detailed quantitative description of the pathology of this animal limits our ability to evaluate the effectiveness of putative therapies or their relevance to DMD. Methods Accordingly, we have measured the main cellular components of muscle growth and regeneration over the period of postnatal growth and early pathology in mdx and wild-type (WT) mice; phalloidin binding is used as a measure of fibre size, myonuclear counts and BrdU labelling as records of myogenic activity. Results We confirm a two-phase postnatal growth pattern in WT muscle: first, increase in myonuclear number over weeks 1 to 3, then expansion of myonuclear domain. Mdx muscle growth lags behind that of WT prior to overt signs of pathology. Fibres are smaller, with fewer myonuclei and smaller myonuclear domains. Moreover, satellite cells are more readily detached from mdx than WT muscle fibres. At 3 weeks, mdx muscles enter a phase of florid myonecrosis, accompanied by concurrent regeneration of an intensity that results in complete replacement of pre-existing muscle over the succeeding 3 to 4 weeks. Both WT and mdx muscles attain maximum size by 12 to 14 weeks, mdx muscle fibres being up to 50% larger than those of WT as they become increasingly branched. Mdx muscle fibres also become hypernucleated, containing twice as many myonuclei per sarcoplasmic volume, as those of WT, the excess corresponding to the number of centrally placed myonuclei. Conclusions The best-known consequence of lack of dystrophin that is common to DMD and the mdx mouse is the conspicuous necrosis and regeneration of muscle fibres. We present protocols for measuring this in terms both of loss of muscle nuclei previously labelled with BrdU and of the intensity of myonuclear labelling with BrdU administered during the regeneration period. Both measurements can be used to assess the efficacy of putative antinecrotic agents. We also show that lack of dystrophin is associated with a number of previously unsuspected abnormalities of muscle fibre structure and function that do not appear to be directly associated with myonecrosis

Care for patients with severe mental illness: the general practitioner's role perspective

BACKGROUND: Patients with severe mental illness (SMI) experience distress and disabilities in several aspects of life, and they have a higher risk of somatic co-morbidity. Both patients and their family members need the support of an easily accessible primary care system. The willingness of general practitioners and the impeding factors for them to participate in providing care for patients with severe mental illness in the acute and the chronic or residual phase were explored. METHODS: A questionnaire survey of a sample of Dutch general practitioners spread over the Netherlands was carried out. This comprised 20 questions on the GP's 'Opinion and Task Perspective', 19 questions on 'Treatment and Experiences', and 27 questions on 'Characteristics of the General Practitioner and the Practice Organisation'. RESULTS: 186 general practitioners distributed over urban areas (49%), urbanised rural areas (38%) and rural areas (15%) of the Netherlands participated. The findings were as follows: GPs currently considered themselves as the first contact in the acute psychotic phase. In the chronic or residual phase GPs saw their core task as to diagnose and treat somatic co-morbidity. A majority would be willing to monitor the general health of these patients as well. It appeared that GP trainers and GPs with a smaller practice setting made follow-up appointments and were willing to monitor the self-care of patients with SMI more often than GPs with larger practices.GPs also saw their role as giving support and information to the patient's family.However, they felt a need for recognition of their competencies when working with mental health care specialists. CONCLUSION: GPs were willing to participate in providing care for patients with SMI. They considered themselves responsible for psychotic emergency cases, for monitoring physical health in the chronic phase, and for supporting the relatives of psychotic patients