Nicholas J.H. Sharp

Molecular analysis of a spontaneous dystrophin `knockout' dog

We have determined the molecular basis for skeletal myopathy and dilated cardiomyopathy in two male German short-haired pointer (GSHP) littermates. Analysis of skeletal muscle demonstrated a complete absence of dystrophin on Western blot analysis. PCR analysis of genomic DNA revealed a deletion encompassing the entire dystrophin gene. Molecular cytogenetic analysis of lymphocytes from the dam and both dystrophic pups confirmed a visible deletion in the p21 region of the affected canine X chromosome. Utrophin is up-regulated in the skeletal muscle, but does not appear to ameliorate the dystrophic canine phenotype. This new canine model should further our understanding of the physiological and biochemical processes in Duchenne muscular dystrophy.

Alternative dystrophin gene transcripts in golden retriever muscular dystrophy

Golden retriever muscular dystrophy (GRMD), the canine model of Duchenne muscular dystrophy (DMD), is caused by a splice site mutation in the dystrophin gene. This mutation predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. Western blot analysis of skeletal muscle from GRMD dogs reveals a slightly truncated 390‐kD protein that is approximately 91% the size of normal dystrophin. This 390‐kD dystrophin suggests that GRMD dogs, like some DMD patients, employ a mechanism to overcome their predicted frameshift. Reverse‐transcriptase polymerase chain reaction on GRMD muscle has revealed two in‐frame dystrophin transcripts which lack either exons 3–9 or exons 5–12. Both transcripts could be translated into a dystrophin protein of approximately 390 kD. An understanding of how truncated dystrophin is produced in GRMD may allow this mechanism to be manipulated toward a potential therapy for DMD.

Magnetic resonance imaging characteristics of necrotizing meningoencephalitis in Pug dogs.

BACKGROUND The magnetic resonance imaging (MRI) characteristics of necrotizing meningoencephalitis (NME) are not well documented. OBJECTIVES To describe common MRI features of NME, to compare the MRI features to histopathologic findings, and to determine whether or not MRI lesions are predictive of survival time. ANIMALS Eighteen Pugs with NME. METHODS Retrospective MRI case study of Pugs identified by a search of medical records at 6 veterinary institutions. Eighteen dogs met inclusion criteria of histopathologically confirmed NME and antemortem MRI exam. MRI lesions were characterized and compared with histopathology with the kappa statistic. Survival times were compared with MRI findings by use of Mann-Whitney U-tests and Spearmans rho. RESULTS Twelve of 18 lesions were indistinctly marginated with mild parenchymal contrast enhancement. Prosencephalic (17/18) lesion distribution included the parietal (16/18), temporal (16/18), and occipital (16/18) lobes. There were cerebellar (4/18) and brainstem (3/18) lesions. Asymmetric lesions were present in both gray and white matter in all dogs. Falx cerebri shift was common (11/18), and 6 dogs had brain herniation. Leptomeningeal enhancement was present in 9/18 dogs. A moderate positive association was found between parenchymal contrast enhancement and both necrosis (kappa= 0.45; P= .045) and monocytic inflammation (kappa= 0.48; P= .025). Higher MRI lesion burden was correlated with longer time from disease onset to MRI (P= .045). MRI lesion burden did not correlate to survival time. CONCLUSIONS AND CLINICAL IMPORTANCE Asymmetric prosencephalic grey and white matter lesions with variable contrast enhancement were consistent MRI changes in Pugs with confirmed NME. While not pathognomonic for NME, these MRI characteristics should increase confidence in a presumptive diagnosis of NME in young Pugs with acute signs of neurologic disease.

Notexin-induced muscle injury in the dog

Notexin, a myotoxic phospholipase, was used to induce focal necrosis in the sartorius muscles of normal mixed-breed adult dogs and in 12-week-old beagles. Notexin injury caused pathologic changes similar to those of Duchenne muscular dystrophy (DMD) and its canine homologue, golden retriever muscular dystrophy (GRMD). All three conditions are characterized by increased serum creatine kinase (CK) levels, sarcolemmal defects, delta lesions, hyaline degeneration of myofibers, calcium-positive myofibers, and minimal effects on neurovascular structures. Four and 24 h after exposure to notexin, serum CK levels were elevated, and many myofibers were necrotic. In addition, by 24 h the necrotic areas were heavily invaded by mononuclear cells, and calcium-positive myofibers were prominent. Capillaries appeared intact even in areas of marked myonecrosis. Massive cellular infiltrate and myotube formation was evident at 3 days post injury. By 7 days, most affected fascicles were occupied by small immature myofibers. Regeneration was largely complete at 21 days. Our results suggest that notexin-induced muscle injury in dogs will be useful in the evaluation of potential therapies for DMD such as myoblast transplantation.

Contraction tension and kinetics of the peroneus longus muscle in golden retriever muscular dystrophy

Contraction tension and kinetics of the peroneus longus muscle were studied in dogs with the Duchenne homologue, golden retriever muscular dystrophy (GRMD), in advance of evaluating localized therapies such as myoblast transplantation. Absolute and both muscle- and body-weight-corrected twitch tension in GRMD dogs were low compared to normal litter mates at 3 months of age (p < 0.0005 for all). Tetanic tension was affected similarly. However, whereas absolute values were still reduced at 6 months (p < 0.0005 for twitch and 0.005 for tetany), twitch and tetanic tension corrected for either muscle or body weight was not statistically different, suggesting that the peroneus longus may be relatively spared in GRMD. Post-tetanic potentiation was more pronounced in GRMD versus normal dogs at both 3 (p < 0.0001) and 6 (p < 0.01) months. The degree of positive staircase at 3 months of age did not differ. Twitch contraction and relaxation times were dramatically prolonged, and there was concomitant sustained electrical activity, at, or before, 6 months of age in some severely affected dogs. Relatively few carriers were evaluated at these ages, but their values were similar to those of normal dogs. Apparent sparing of the peroneus longus muscle may limit application of this technique to evaluation of therapies administered early in life or in combination with toxins. Treatment to alter changes in contraction kinetics could also be assessed.

Golden Retriever Muscular Dystrophy: Monitoring for Success

There are many phenotypic features that can be monitored to, in the end, assess the success of any form of treatment for muscular dystrophy (Table 1). Of course, in this setting, we are speaking principally of myoblast transplantation.

α‐Dystroglycan deficiency correlates with elevated serum creatine kinase and decreased muscle contraction tension in golden retriever muscular dystrophy

The dystrophin—glycoprotein complex was examined in dystrophin‐deficient dogs with golden retriever muscular dystrophy (GRMD) using immunoblot and immunofluorescence analysis. The dystrophin‐associated proteins were substantially reduced in muscle from dogs with GRMD. Interestingly, regression analysis revealed a strong correlation between the amount of α‐dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.

Treatment of canine nasal aspergillosis

Nicholas Sharp Nick Sharp graduated from the RVC in 1979, where hespenttwo years ashouse surgeon. He did a masters degree in neurology at Glasgow before spending three years at Liverpool. He moved to the USA in 1985 and worked both at the University of Pennsylvania and North Carolina veterinary colleges. He became a Diplomate of the American College of Veterinary Surgery in 1988. He isnow completing his PhD at Duke University, North Carolina, working on a canine model for Duchenne mus-

Molecular Markers for Myoblast Transplantation in GRMD

Golden retriever muscular dystrophy (GRMD) has been proposed to be an animal model for Duchenne muscular dystrophy (DMD) (Kornegay et al., 1989). We have been studying GRMD to determine the underlying defect in this model using methodology and cDNA probes developed for the study of DMD (Koenig et al., 1987). Observed molecular differences between normal and affected animals may be useful for monitoring myoblast transfer in this model similar to what Ron Worton proposed as a means of defining tissue-specific markers for myoblast transplantation in DMD families (Worton reference this book). To illustrate the potential use of these markers of transplantation, an example of a human pedigree with a restriction fragment polymorphism which differs between individuals is illustrated in Figure 1. In this particular pedigree, there are two patients with deletions. Two potential markers would be useful for testing persistence of donor myoblasts after transplantation. The molecular markers in this case would be the presence of the deleted portion of the gene found in normal donor myoblasts, and if the donor was a non-carrier female, a gene dosage analysis which discriminates between the relative dosage of the X and Y-specific sequences would identify the donor cells.

Dystrophin mRNA Processing in the Canine Homologue of Duchenne Muscular Dystrophy: An Authentic Model for Gene Therapy

Duchenne muscular dystrophy (DMD) is a fatal, X-linked, recessive disease of humans that afflicts 1 in 3500 live-born males (Koenig et al. 1988). Approximately two thirds of DMD patients carry detectable deletions in the gene encoding dystrophin (Walker et al. 1989; Koenig et al. 1987), which is a muscle cytoskeletal protein (Koenig et al. 1988). Dystrophin protein (Hoffmann et al. 1988) and transcript (Oronzi Scott et al. 1988) are either absent or severely deficient in DMD patients (Koenig et al. 1988).