Gemma Walmsley

A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient Cavalier King Charles Spaniels is amenable to exon 51 skipping

Background 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 dystrophin 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 5′ 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.

Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte.

The molecular identity of platelet Ca2+ entry pathways is controversial. Furthermore, the extent to which Ca2+‐permeable ion channels are functional in these tiny, anucleate cells is difficult to assess by direct electrophysiological measurements. Recent work has highlighted how the primary megakaryocyte represents a bona fide surrogate for studies of platelet signalling, including patch clamp recordings of ionic conductances. We have now screened for all known members of the transient receptor potential (TRP) family of non‐selective cation channels in murine megakaryocytes following individual selection of these rare marrow cells using glass micropipettes. RT‐PCR detected messages for TRPC6 and TRPC1, which have been reported in platelets and megakaryocytic cell lines, and TRPM1, TRPM2 and TRPM7, which to date have not been demonstrated in cells of megakaryocytic/platelet lineage. Electrophysiological recordings demonstrated the presence of functional TRPM7, a constitutively active cation channel sensitive to intracellular Mg2+, and TRPM2, an ADP‐ribose‐dependent cation channel activated by oxidative stress. In addition, the electrophysiological and pharmacological properties of the non‐selective cation channels stimulated by the physiological agonist ADP are consistent with a major role for TRPC6 in this G‐protein‐coupled receptor‐dependent Ca2+ influx pathway. This study defines for the first time the principal TRP channels within the primary megakaryocyte, which represent candidates for Ca2+ influx pathways activated by a diverse range of stimuli in the platelet and megakaryocyte.

The association between Chiari-like malformation, ventriculomegaly and seizures in cavalier King Charles spaniels.

Cavalier King Charles spaniels (CKCSs) with Chiari-like malformation (CM) and associated seizures are frequently diagnosed with idiopathic epilepsy. There could be an association between ventriculomegaly (V) or caudal fossa overcrowding (CCFP) and seizures. A retrospective case-control study was performed using MRI to investigate the possible association between these morphological abnormalities and seizures. Seizure semiology and, where possible, electroencephalographic (EEG) abnormalities were documented. Eighty-five CKCS with CM were included, 27 with seizures. There was no association between V or CCFP and seizures (P=0.10 and 0.71, respectively). Seizures were classified as having partial onset in 61% of individuals in the study population (95% CI 42.41-76.43%). Another cause of recurrent seizures in CKCS (such as familial epilepsy) is suspected, as previously reported.

Facial myokymia in a puppy

MYOKYMIA is a muscular disorder that causes a vermicular, writhing motion visible through the skin overlying the affected muscles, which is diagnosed by its characteristic clinical features and distinctive appearance on electromyography (Shapiro and others 2002). Generalised and focal forms are recognised in human beings; focal myokymia most commonly affects the face and is a rare sign associated with a variety of conditions, including intra-axial pontine lesions and demyelinating conditions such as multiple sclerosis and Guillain-Barre syndrome (Gutmann and Gutmann 2004). To the authors’ knowledge facial myokymia has not previously been described in dogs. This short communication describes a case of a puppy with facial myokymia. A six-month-old male cocker spaniel puppy was referred to the Queen’s Veterinary School Hospital, Cambridge University, with a two-month history of facial muscle twitching. This movement was continuous and persisted when the dog was sleeping; it had initially been restricted to the left side of the muzzle but by the time of presentation had progressed to involve the periorbital muscles and right side of the face. The owners did not report any other problems, and the puppy was able to eat normally. A possible diagnosis of otitis media had been investigated by the referring veterinary surgeon, but the condition had failed to respond to a course of antibiotics. During the clinical examination, there was a persistent, worm-like, writhing motion over the dog’s upper lip and eyelids, which was more pronounced on the left side. A similar pattern of muscle activity could also occasionally be seen over the left shoulder region. A neurological examination detected cranial nerve deficits: there was a bilaterally absent menace response, weak palpebral reflexes, abnormal vestibular eye movements and apparently poor hearing. The puppy had a tendency to tilt its head to the left and seemed generally slightly incoordinated; for example, it could not easily catch objects in its mouth. There were no deficits in postural reactions or segmental spinal reflexes. Electromyography (EMG) recorded characteristic neuromyokymic discharges (with an intraburst frequency of approximately 300 Hz) from the levator nasolabialis, orbicularis oris and orbicularis oculi muscles on the left and right sides (Fig 1a) (Anon 2006). A brainstem auditory evoked response could not be recorded at 100 dB from either ear. The electrodiagnostic tests were repeated two months later when clinical reassessment found that the dog’s condition was unchanged. The EMG recording (Fig 1b) demonstrated a distinct change in the pattern of activity: in addition to the previously observed myokymic activity there were positive sharp waves and fibrillation potentials, suggesting axonal disease. Routine haematology and serum biochemistry (including measurement of the activity of creatine kinase) were unremarkable. Serological tests were negative for immunoglobulin G and M antibodies to Toxoplasma species, but showed an elevated titre of antibodies to canine distemper virus (the puppy had been routinely vaccinated). Cerebrospinal fluid (CSF) contained an increased amount of protein (total protein 0·38 g/l, reference range 0·00 to 0·25 g/l) and increased numbers of nucleated cells (10/μl, reference range 0 to 8/μl); lymphocytes and monocytes were the predominant cell types. Magnetic resonance imaging (MRI) detected mild herniation of the caudal cerebellar tonsils through the foramen magnum and scattered equivocal areas of hypointensity were observed throughout the brain on T1-weighted and fluid-attenuated inversion recovery scans. Because the CSF analysis suggested inflammation, a trial treatment with corticosteroids was initiated, but did not eliminate the clinical signs; the owners declined further attempts at treatment. The clinical appearance and EMG findings in the puppy were diagnostic for focal myokymia. This focal condition has not previously been reported in dogs, but there have been previous reports of episodic involvement of the entire musculature, predominantly in adult Jack Russell terriers (Reading and McKerrell 1993, Van Ham and others 2004). In the series reported by Van Ham and others (2004), the dogs presented with generalised signs including ataxia and episodic collapse, during which there was continuous muscle activity. This generalised condition appears to be much more severe than the present case of focal myokymia, since four of the six cases described either died or were euthanased because of their ataxia or episodes of collapsing. There is considerable evidence to implicate abnormalities of the axonal voltage-gated potassium channels as the underlying cause of myokymia. These abnormalities can arise as a result of, for example, genetic abnormalities (Browne and others 1994), toxins (Lewis and Gutmann 2004) or autoimmune disease (Hart and others 2002). Continuous myokymia restricted to the face is an uncommon presentation in human medicine, and has been associated with structural lesions of the pons, demyelinating conditions, toxins, and genetic and autoimmune channelopathies; a benign, postfatigue form, affecting the periorbital musculature, is also recognised (Gutmann and Gutmann 2004). The duration of clinical signs is very variable, depending on the aetiology: in polyradiculoneuropathy it may last for several weeks or months, but in pontine glioma it may persist for the lifespan of the patient (Gutmann and Gutmann 2004). The diagnosis of hearing deficits and evidence of vestibular disturbances in combination with myokymia in the present case might suggest that a structural lesion of the caudal brainstem could underlie the clinical signs. However, although MRI did reveal a Chiari type I malformation, there were no other well defined anomalies. Nevertheless, many defects in neural development, such as neuronal migration disorders (Sarnat and Flores-Sarnat 2002), are not visible on MRI scans, and so a microscopic structural disorder cannot be ruled out. Indeed, defects in the formation of cranial nerve nuclei have been assoShort Communications

Multi-focal cerebral oligoastrocytoma in a puppy

A nine-month-old puppy was presented for investigation of seizures. Neurological deficits were found localising to the prosencephalon, mesencephalon and myelencephalon. Magnetic resonance imaging identified multiple, large lesions involving both cerebral hemispheres. Management with antiepileptic and immunomodulatory drugs was instituted; however, the seizures became progressively refractory and the puppy was euthanased. Histopathology following post-mortem examination found a mixed glial cell tumour with discrete areas where neoplastic cells appeared oligodendroglial or astrocytic. Primary intracranial neoplasia is a rare cause of neurological disease in young dogs. Moreover, this case is unusual in terms of both the mixed glial nature of the neoplasm and also its multi-focal distribution.

HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth

The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.

IMAGING DIAGNOSIS—SPINAL CORD HEMANGIOMA IN TWO DOGS

Intramedullary masses are a dilemma due to the limited access for a nonsurgical biopsy, thus, accurate imaging characterization is crucial. Magnetic resonance imaging findings of two confirmed canine thoracic intramedullary hemangiomas are described. A capillary hemangioma was of mixed intensity but predominantly T2-hyperintense and mildly T1-hyperintense to spinal cord with strong contrast enhancement. A cavernous hemangioma had a target-like appearance in both T1-weighted (T1w) and T2-weighted (T2w) images. In T2w images there was a small isointense center surrounded by a relatively large hyperintense area. In T1w images, there was a large isointense centre with a relatively small hyperintense periphery. Such characteristics should prioritize hemangioma as a consideration in a progressive myelopathy due to an intramedullary mass.

Neurological, imaging and pathological features of a meningeal inflammatory pseudotumour in a Maltese terrier

A five-year eight-month-old Maltese terrier was presented with a 3-week history of progressive paraparesis and pelvic limb ataxia. Neurological examination was consistent with a lesion involving the T3-L3 spinal cord segments. Myelogram and magnetic resonance imaging revealed a spherical, intradural-extramedullary mass lesion at T13/L1. A dorsal laminectomy, durotomy and debulking of the mass were performed. Histopathologic examination revealed a highly cellular tissue, most likely of mesenchymal origin, infiltrated by many lymphocytes, macrophages and neutrophils. The pathological diagnosis of an inflammatory pseudotumour was made. Postsurgical analgesia was achieved with opioids and 2 mg/kg carprofen twice daily for 5 days. When the histopathological diagnosis was made, a tapering course of 1 mg/kg prednisolone twice daily was prescribed, with dose reduction by approximately 50% every 4 to 6 weeks over a 4-month period. Magnetic resonance imaging was repeated at 22, 32 and 85 weeks postsurgery; no signs of regrowth could be detected and the patient recovered with residual mild paraparesis. Inflammatory pseudotumour has not been documented previously at this site in dogs and, although rare, should be considered in the differential diagnosis of a focal mass lesion affecting the spinal cord. Surgical debulking and immunomodulatory therapy can be curative.

Management of tail pull injuries in cats

Feline tail pull injury is a challenging condition that is commonly encountered in first-opinion practice. This article discusses the key factors in the diagnosis, prognosis and management of tail pull injuries, including the use of radiography to confirm sacrocaudal luxation, and the identification and appropriate treatment of associated urinary and faecal dysfunction. It also highlights the importance of good owner communication, as currently the best prognostic indicators at the time of presentation for the return of continence (the conscious perception of noxious stimuli applied to the perineum or tail base) are not infallible and recovery can take weeks.

Vacuolar myopathy in an adult Warmblood horse

Histopathological interpretation of semimembranosus muscle samples from an adult Warmblood mare with clinical signs suggestive of exertional rhabdomyolysis and intermittent mild elevations in muscle enzyme activities revealed abundant sarcoplasmic vacuoles in all fibre-types containing fine, apparently proteinaceous debris. Vacuolar contents stained lightly with PAS, but did not appear to contain amylopectate, lipid or acid phosphatase and their periphery was unstained with dystrophin immunohistochemistry. Electron microscopy revealed that vacuoles were not membrane bound. No vacuoles were detected in muscle samples evaluated at post mortem following 4 months of rest. To our knowledge, this is the first report of a presumed primary vacuolar myopathy in a horse.