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

Two novel missense mutations in the myostatin gene identified in Japanese patients with Duchenne muscular dystrophy

BACKGROUND: Myostatin is a negative regulator of skeletal muscle growth. Truncating mutations in the myostatin gene have been reported to result in gross muscle hypertrophy. Duchenne muscular dystrophy (DMD), the most common lethal muscle wasting disease, is a result of an absence of muscle dystrophin. Although this disorder causes a rather uniform pattern of muscle wasting, afflicted patients display phenotypic variability. We hypothesized that genetic variation in myostatin is a modifier of the DMD phenotype. METHODS: We analyzed 102 Japanese DMD patients for mutations in the myostatin gene. RESULTS: Two polymorphisms that are commonly observed in Western countries, p.55A>T and p.153K>R, were not observed in these Japanese patients. An uncommon polymorphism of p.164E>K was uncovered in four cases; each patient was found to be heterozygous for this polymorphism, which had the highest frequency of the polymorphism observed in the Japanese patients. Remarkably, two patients were found to be heterozygous for one of two novel missense mutations (p.95D>H and p.156L>I). One DMD patient carrying a novel missense mutation of p.95D>H was not phenotypically different from the non-carriers. The other DMD patient was found to carry both a novel mutation (p.156L>I) and a known polymorphism (p.164E>K) in one allele, although his phenotype was not significantly modified. Any nucleotide change creating a target site for micro RNAs was not disclosed in the 3' untranslated region. CONCLUSION: Our results indicate that heterozygous missense mutations including two novel mutations did not produce an apparent increase in muscle strength in Japanese DMD cases, even in a patient carrying two missense mutations

Targeted Skipping of Human Dystrophin Exons in Transgenic Mouse Model Systemically for Antisense Drug Development

Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD/mdx mouse, a transgenic model carrying the full-length human dystrophin gene with mdx background, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD/mdx mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD/mdx mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement