A L2HGDH initiator methionine codon mutation in a Yorkshire terrier with L-2-hydroxyglutaric aciduria.

BACKGROUND: L-2-hydroxyglutaric aciduria is a metabolic repair deficiency characterized by elevated levels of L-2-hydroxyglutaric acid in urine, blood and cerebrospinal fluid. Neurological signs associated with the disease in humans and dogs include seizures, ataxia and dementia. CASE PRESENTATION: Here we describe an 8 month old Yorkshire terrier that presented with episodes of hyperactivity and aggressive behavior. Between episodes, the dog's behavior and neurologic examinations were normal. A T2 weighted MRI of the brain showed diffuse grey matter hyperintensity and a urine metabolite screen showed elevated 2-hydroxyglutaric acid. We sequenced all 10 exons and intron-exon borders of L2HGDH from the affected dog and identified a homozygous A to G transition in the initiator methionine codon. The first inframe methionine is at p.M183 which is past the mitochondrial targeting domain of the protein. Initiation of translation at p.M183 would encode an N-terminal truncated protein unlikely to be functional. CONCLUSIONS: We have identified a mutation in the initiation codon of L2HGDH that is likely to result in a non-functional gene. The Yorkshire terrier could serve as an animal model to understand the pathogenesis of L-2-hydroxyglutaric aciduria and to evaluate potential therapies.

Clinical characteristics of non-infectious inflammatory myopathy in the boxer dog.

OBJECTIVES: To evaluate the clinical characteristics, treatment, outcome and potential association between non-infectious inflammatory myopathy and malignancy in boxer dogs. MATERIALS AND METHODS: Boxer dogs histologically diagnosed with non-infectious inflammatory myopathy at the Comparative Neuromuscular Laboratory, University of California San Diego from 2010 to 2018 and with complete medical records were included in this retrospective study. Signalment, history, clinical signs, clinicopathologic findings, treatment and outcome were documented. RESULTS: Twenty-eight boxer dogs with non-infectious inflammatory myopathy, aged 1 to 11 years, were included. Eighteen were male (16 neutered; two entire) and 10 were female (seven spayed; three entire). Clinical signs included generalised weakness (n=17), dysphagia (n=11) and weight loss (n=10). Serum creatine kinase activity was elevated in all 20 cases tested (range 908 to 138,000 IU/L). One dog had undifferentiated round cell neoplastic infiltration within the muscle at the time of inflammatory myopathy diagnosis. Five dogs historically had mast cell tumours and 21 dogs were not diagnosed with neoplasia prior, at the time of or after inflammatory myopathy diagnosis. Treatment included glucocorticoid monotherapy (n=12), cyclosporine monotherapy (n=1) or multiple immune-suppressive medications (n=14). Six dogs neurologically improved, 11 improved but relapsed while on treatment, seven did not improve. Eight dogs were euthanased, one died, four were lost to follow-up. CLINICAL SIGNIFICANCE: Boxer dogs with non-infectious inflammatory myopathy can present for generalised weakness and dysphagia; long-term successful outcome is uncommon. The relationship between neoplasia and non-infectious inflammatory myopathy in boxer dogs remains unclear; future prospective studies evaluating a larger cohort are warranted.

Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy.

Cypher is a member of a recently emerging family of proteins containing a PDZ domain at their NH(2) terminus and one or three LIM domains at their COOH terminus. Cypher knockout mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles. Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function. In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components. These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

Encephalomyelopathy and polyneuropathy associated with neuronal vacuolation in two Boxer littermates.

Neuronal vacuolation and spinocerebellar degeneration is a rare, presumably inherited condition that is reported only in Rottweilers and in crossbred dogs with known or potential Rottweiler heritage. Gross and histopathologic findings include laryngeal muscle atrophy, neuronal vacuolation, and a combined central and peripheral axonopathy. Two 6-month-old Boxer puppies from the same litter were referred for evaluation of progressive pelvic limb paresis and ataxia, upper airway stridor, and visual deficits. Examination of each dog suggested a combined myelopathy and peripheral neuropathy, as well as congenital ocular disease. Gross lesions were limited to atrophy of the intrinsic laryngeal muscles. Histopathologically, there was diffuse loss of axons and myelin in the dorsolateral and ventral funiculi throughout the spinal cord and extending into the caudal aspect of the brain stem. Vacuolation of scattered neuronal cell bodies was present in the spinal cord and selected brain stem nuclei. Multifocal axonal degeneration and demyelination was observed in the recurrent laryngeal nerve, sciatic nerve, and brachial plexus and was most severe in the recurrent laryngeal nerve. Ocular abnormalities included microphthalmia, cataracts, and retinal dysplasia. The findings in these Boxer dogs, unrelated to the Rottweiler breed, are analogous to the syndrome of neuronal vacuolation and spinocerebellar degeneration reported in Rottweilers.

Late-onset Becker-type muscular dystrophy in a Border terrier dog.

A 9-year-old Border terrier was presented to a referral hospital after a 1-year history of progressive stiffness and exercise intolerance. Neurological examination was consistent with a neuromuscular disorder. Serum creatine kinase activity was mildly elevated. A myopathy was suspected based on MRI findings and electrophysiological examination. Muscle histopathology was consistent with a severe non-inflammatory myopathy of a dystrophic type. Immunofluorescence and western blotting confirmed a dystrophinopathy with an 80-kDa truncated dystrophin fragment similar to Becker muscular dystrophy in people. To our knowledge, this is the first description of a late-onset Becker-type muscular dystrophy in a dog, and the first description of a dystrophinopathy in a Border terrier. Muscular dystrophy in dogs should not be ruled out based on late onset clinical signs and only mildly elevated creatine kinase.

Dilatation of the right atrium in a dog with polymyositis and myocarditis.

A nine-year-old, female, neutered golden retriever was referred for investigation of weakness. Immune-mediated polymyositis with mixed mononuclear cell inflammation and fibrosis was diagnosed in muscle biopsy specimens from several sites. A soft tissue opacity was noted radiographically cranial to the cardiac silhouette. The dog failed to respond to treatment with prednisolone and was euthanased. At post-mortem examination, the structure observed radiographically was showed to be an enlarged right atrium. Histopathological examination of samples collected at post-mortem confirmed polymyositis. Additionally, mononuclear cell infiltrations and fibrosis were observed in areas of endocardium and/or myocardium in the walls of all four cardiac chambers. Myocarditis has not been reported previously in canine polymyositis but is well recognised in people. Enlargement of the right atrium has also been reported in association with polymyositis and myocarditis in people.

Adult onset acquired myasthenia gravis in three Great Dane littermates.

Acquired canine myasthenia gravis is an autoimmune disease in which autoantibodies are directed against muscle postsynaptic nicotinic acetylcholine receptors. Three adult great dane littermates were evaluated over a four month time period for an acute onset of generalised neuromuscular signs. All three dogs had elevated serum acetylcholine receptor antibody titres, which were considered diagnostic for acquired myasthenia gravis. Identification of three littermates with acquired myasthenia gravis in a breed with a low relative risk of developing the disease suggests a familial and possibly a genetic predisposition to myasthenia gravis in this family of dogs.

Autoimmune myasthenia gravis and dysautonomia in a dog.

A two-year-old male entire border collie dog was evaluated for a short history of mixed bowel diarrhoea, coughing, vomiting and stranguria. Physical examination revealed dyspnoea with increased ventral lung sounds and a flaccidly distended bladder. Neurological examination revealed poor pupillary light reflexes, an absent gag reflex and a poor anal tone. Thoracic radiography was consistent with megaoesophagus and aspiration pneumonia. Clinicopathological testing revealed an elevated muscular nicotinic acetylcholine receptor antibody titre. The dog was euthanased because of clinical deterioration. Cerebrospinal fluid (CSF) collected immediately post-mortem revealed macrophagic pleocytosis. Post-mortem histopathological examination was consistent with dysautonomia. This is the first report of coexisting autoimmune myasthenia gravis and dysautonomia in a non-human species. The concomitant diseases may suggest a common immunopathological aetiology.

Dysphagia secondary to focal inflammatory myopathy and consequent dorsiflexion of the tongue in a dog.

A 14-month-old female pitbull terrier mix was presented for evaluation of dysphagia of 8 months' duration secondary to intermittent dorsiflexion of the tongue apex. Physical and neurological examinations were unremarkable with the exception of the dorsiflexed tongue. Serum creatine kinase activity was increased (703 IU/L, reference interval: 55 to 257 IU/L), and electromyography of the tongue demonstrated areas of fibrillation potentials. Histopathology of the tongue showed myopathic changes with excessive variability in myofibre size and endomysial fibrosis. Cytochemical stains verified mixed mononuclear cells throughout the endomysium and perimysium consistent with a chronic inflammatory myopathy. No improvement was reported following prednisone administration; although the dog was able to prehend kibble, it needed assistance when drinking water. This is the first report documenting a focal lingual myopathy in a non-corgi breed and highlights the utility of determining creatine kinase activity and obtaining tongue biopsies when warranted in dysphagic animals.

Myositis, Ganglioneuritis, and Myocarditis with Distinct Perifascicular Muscle Atrophy in a 2-Year-Old Male Boxer.

A 2-year-old male, intact Boxer was referred for chronic diarrhea, hyporexia, labored breathing, weakness and elevated creatine kinase, and alanine aminotransferase activities. Initial examination and diagnostics revealed a peripheral nervous system neurolocalization, atrial premature complexes, and generalized megaesophagus. Progressive worsening of the dog's condition was noted after 36 h; the dog developed aspiration pneumonia, was febrile and oxygen dependent. The owners elected humane euthanasia. Immediately postmortem biopsies of the left cranial tibial and triceps muscles and the left peroneal nerve were obtained. Postmortem histology revealed concurrent myositis, myocarditis, endocarditis, and ganglioneuritis. Mixed mononuclear cell infiltrations and a distinct perifascicular pattern of muscle fiber atrophy was present in both muscles. This is a novel case of diffuse inflammatory myopathy with a distinct perifascicular pattern of atrophy in addition to endocarditis, myocarditis, and epicarditis.

Canine NAPEPLD-associated models of human myelin disorders.

Canine leukoencephalomyelopathy (LEMP) is a juvenile-onset neurodegenerative disorder of the CNS white matter currently described in Rottweiler and Leonberger dogs. Genome-wide association study (GWAS) allowed us to map LEMP in a Leonberger cohort to dog chromosome 18. Subsequent whole genome re-sequencing of a Leonberger case enabled the identification of a single private homozygous non-synonymous missense variant located in the highly conserved metallo-beta-lactamase domain of the N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD) gene, encoding an enzyme of the endocannabinoid system. We then sequenced this gene in LEMP-affected Rottweilers and identified a different frameshift variant, which is predicted to replace the C-terminal metallo-beta-lactamase domain of the wild type protein. Haplotype analysis of SNP array genotypes revealed that the frameshift variant was present in diverse haplotypes in Rottweilers, and also in Great Danes, indicating an old origin of this second NAPEPLD variant. The identification of different NAPEPLD variants in dog breeds affected by leukoencephalopathies with heterogeneous pathological features, implicates the NAPEPLD enzyme as important in myelin homeostasis, and suggests a novel candidate gene for myelination disorders in people.

Dystrophin-deficient muscular dystrophy in a Weimaraner.

A 2-year-old, male Weimaraner with muscular dystrophy was presented with generalized muscle atrophy of the limbs; hypertrophy of the neck, infraspinatus, and lingual muscles; dysphagia; and regurgitation. Unilateral cryptorchidism, unilateral renal agenesis, and hiatal hernia were also detected. Spontaneous muscle activity was identified on myography. Serum creatine kinase was markedly elevated. Immunohistochemical staining for dystrophin was restricted to suspected revertant (characteristics of immaturity) fibers. Histologically, skeletal myofiber degeneration, endomysial fibrosis, and mineralization were present. Following euthanasia, necropsy revealed hypertrophy of the diaphragm and cardiac muscle fibrosis. This case of muscular dystrophy represents a slowly progressive form with organ agenesis.

What's new in muscle and peripheral nerve diseases?

It is likely that most neuromuscular diseases that are described in humans will have a counterpart in our companion animals. With the advent of molecular genetics and the completion of the canine and feline genomes, an ever expanding number of DNA-based tests should become available for the diagnosis of muscle and peripheral nerve diseases. Molecular testing procedures should enable us to continue to unravel the molecular basis of neuromuscular diseases for which the cause is still unknown. It is important that accurate clinical evaluations and diagnostic testing, including muscle and peripheral nerve biopsies, are performed in order to reach these goals. This review focuses on recently identified inherited neuromuscular diseases in companion animals.

Clinical Phenotype of Musladin-Lueke Syndrome in 2 Beagles.

Musladin-Lueke syndrome (MLS), previously termed Chinese Beagle syndrome, is an autosomal-recessive connective tissue disorder characterized by extensive fibrosis of the skin and joints that was first identified in Beagles in the 1970s. Recent research identified a founder mutation (c.660C>T; p.R221C) in the ADAMTSL2 gene in Beagles with MLS. Here, we report the detailed clinical phenotype and laboratory findings in 2 Beagles affected with MLS. We discuss these findings in relation to the human disorder geleophysic dysplasia (GD), which also arises from recessive ADAMTSL2 mutations, and recent findings in Adamtsl2-deficient mice.

Myopathy in horses with pituitary pars intermedia dysfunction (Cushing's disease).

Fifteen horses with pituitary pars intermedia dysfunction were studied. The horses were of various breeds and between 15 and 28 years of age. Control horses matched for breed and age were studied for comparison. Evaluations included complete blood cell count and serum biochemical analysis, electromyography, and gluteus medius muscle biopsies for histochemical, morphometric, and ultrastructural analysis. No differences were found between groups of horses on routine laboratory analysis or electromyography. We demonstrated that muscle wasting in diseased horses was the result of atrophy of types 2A and 2B muscle fibers and loss of type 2B myofibers. Mild non-specific non-inflammatory myopathic alterations such as myofiber size variation, internal nuclei, perimysial, endomysial and sarcoplasmic fat accumulation were observed. At the ultrastructural level, subsarcolemmal mitochondrial accumulation and increased lipid droplets were evident. Similar to other species, this study confirmed atrophy of type 2 fibers as the cause of muscle mass loss in horses with Cushing's disease.

Inherited myopathy of great Danes.

A hereditary, non-inflammatory myopathy occurring in young great Danes with distinctive histological features in muscle biopsy specimens is reviewed. Onset of clinical signs is usually before one year of age and both sexes are affected. Clinical signs are characterised by exercise intolerance, muscle wasting, and an exercise-induced tremor. Although most affected dogs have a severe form of the disease, occasional dogs may have a less pronounced form and survive into adulthood with an acceptable quality of life. Litters containing affected puppies are born to clinically unaffected parents, and an autosomal recessive pattern of inheritance is likely. All recorded cases have had fawn or brindle coat coloration. Elevated serum creatinine kinase concentrations and spontaneous electrical activity in skeletal muscles are frequently found. While originally reported (Targett and others 1994) as a central core myopathy in this breed, the histochemical characteristics of the distinct cytoarchitectural structures differ from those of the well-characterised central core myopathy in human beings. In fact, these structures differ from any known myopathy in human beings and likely represents a unique non-inflammatory myopathy affecting dogs. Until this myopathy is characterised further, the name inherited myopathy in great Danes is suggested.

A Homozygous RAB3GAP1:c.743delC Mutation in Rottweilers with Neuronal Vacuolation and Spinocerebellar Degeneration.

BACKGROUND: A variety of presumed hereditary, neurologic diseases have been reported in young Rottweilers. Overlapping ages of onset and clinical signs have made antemortem diagnosis difficult. One of these diseases, neuronal vacuolation and spinocerebellar degeneration (NVSD) shares clinical and histological features with polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV), a recently described hereditary disease in Black Russian Terriers (BRTs). Dogs with POANV harbor mutations in RAB3GAP1 which codes for a protein involved in membrane trafficking. HYPOTHESIS: Rottweilers with NVSD will be homozygous for the RAB3GAP1:c.743delC allele associated with POANV in BRTs. ANIMALS: Eight Rottweilers with NVSD confirmed at necropsy, 128 Rottweilers without early onset neurologic signs, and 468 randomly selected dogs from 169 other breeds. METHODS: Retrospective case-control study. Dogs were genotyped for the RAB3GAP1:c.743delC allele with an allelic discrimination assay. RESULTS: All 8 NVSD-affected dogs were homozygous for the RAB3GAP1:c.743delC allele while the 128 NVSD-free Rottweilers were either homozygous for the reference allele (n = 105) or heterozygous (n = 23) and the 468 genotyped dogs from other breeds were all homozygous for the reference allele. CONCLUSIONS AND CLINICAL IMPORTANCE: The RAB3GAP1:c.743delC mutation is associated with a similar phenotype in Rottweilers and BRTs. Identification of the mutation permits a DNA test that can aid in the diagnosis of NVSD and identify carriers of the trait so that breeders can avoid producing affected dogs. Disruption of membrane trafficking could explain the neuronal vacuolation seen in NVSD and other spongiform encephalopathies.

Major Histocompatibility Complex I and II Expression and Lymphocytic Subtypes in Muscle of Horses with Immune-Mediated Myositis.

BACKGROUND: Major histocompatibility complex (MHC) I and II expression is not normally detected on sarcolemma, but is detected with lymphocytic infiltrates in immune-mediated myositis (IMM) of humans and dogs and in dysferlin-deficient muscular dystrophy. HYPOTHESIS/OBJECTIVES: To determine if sarcolemmal MHC is expressed in active IMM in horses, if MHC expression is associated with lymphocytic subtype, and if dysferlin is expressed in IMM. ANIMALS: Twenty-one IMM horses of Quarter Horse-related breeds, 3 healthy and 6 disease controls (3 pasture myopathy, 3 amylase-resistant polysaccharide storage myopathy [PSSM]). METHODS: Immunohistochemical staining for MHC I, II, and CD4+, CD8+, CD20+ lymphocytes was performed on archived muscle of IMM and control horses. Scores were given for MHC I, II, and lymphocytic subtypes. Immunofluorescent staining for dysferlin, dystrophin, and a-sarcoglycan was performed. RESULTS: Sarcolemmal MHC I and II expression was detected in 17/21 and 15/21 of IMM horses, respectively, and in specific fibers of PSSM horses, but not healthy or pasture myopathy controls. The CD4+, CD8+, and CD20+ cells were present in 20/21 IMM muscles with CD4+ predominance in 10/21 and CD8+ predominance in 6/21 of IMM horses. Dysferlin, dystrophin, and a-sarcoglycan staining were similar in IMM and control muscles. CONCLUSIONS AND CLINICAL IMPORTANCE: Deficiencies of dysferlin, dystrophin, and a-sarcoglycan are not associated with IMM. Sarcolemmal MHC I and II expression in a proportion of myofibers of IMM horses in conjunction with lymphocytic infiltration supports an immune-mediated etiology for IMM. The MHC expression also occured in specific myofibers in PSSM horses in the absence of lymphocytic infiltrates.

Sarcoplasmic masses in equine skeletal muscle.

Sarcoplasmic masses in humans have been associated with various myopathies, although the significance remains elusive. Similar structures have also been observed in equine muscle. The aim of this study was to determine the frequency of such structures in normal and abnormal equine muscle, and to characterize these structures using histological, histochemical, immunohistochemical, ultrastructural, and morphometric analyses. The histological and histochemical appearance was similar to that of human sarcoplasmic masses with a central or subsarcolemmal distribution. Of interest was a predilection for the gluteus medius muscle in younger horses and type 2B fibers. Ultrastructurally they contained disorganized myofibrils and other cellular components that were not membrane bound and were present in both normal and abnormal equine muscle without a specific disease association, suggesting they are a non-pathological degenerative structure. The relatively frequent occurrence of sarcoplasmic masses in horses may make this species a good model for studying the pathogenesis of these structures.