G. Diane Shelton

Hereditary neuromuscular disorders in dogs can be difficult to classify since variants in different genes can result in similar clinical signs or variable phenotypes can be associated with the same DNA sequence variant. A disorder known for many years as Dancing Doberman Disease, suspected to be neuropathy or neuromyopathy, is characterized by repeated lifting and shifting of the pelvic limbs while standing and frequent sitting. More recently, Doberman Pinschers have been identified with a different and more severe phenotype characterized by a crouched stance and bunny hopping gait in the pelvic limbs that is termed duck walking. Dogs with both phenotypes show pelvic limb weakness, muscle atrophy, and fatigue, and clinical signs can progress to involve the thoracic limbs. These distinct phenotypes were evaluated clinically, histologically, and by whole-genome sequencing and genotyping a large cohort of affected and unaffected Doberman Pinschers. The same homozygous missense variant in AGRN (Dog 10K Boxer Tasha chr5:56,346,611,G>A; p.R1710H, XP 038377340.1) was associated with both disorders. AGRN encodes Agrin, an essential synaptic protein, that mediates clustering of acetylcholine receptors on the post-synaptic membrane at the neuromuscular junction. Variants in AGRN are associated with a congenital myasthenic syndrome (CMS) in humans. This is the first report of a CMS in dogs associated with an AGRN variant and expands the spectrum of known CMS genetic risk factors in this species. This study also highlights the importance of whole-genome sequencing (WGS) to accurately classify neuromuscular diseases as forms of CMS, which is not possible based on clinical presentation alone.

A 6‐month‐old male Jack Russell Terrier presented with failure to thrive, lethargy, muscle atrophy, hypersalivation, and exercise intolerance. Similar signs were reported for the only other male puppy in the litter that died prematurely, whereas all the female puppies developed normally. An electromyography (EMG) test revealed abnormal electrical activity, such as complex repetitive discharges and fibrillation potentials. Cryosections of skeletal muscles revealed degenerative and regenerative myopathy with calcific deposits, consistent with a form of muscular dystrophy. Immunohistochemical testing demonstrated a lack of dystrophin, alongside upregulation of utrophin, and a subsequent reduction in dystrophin‐associated proteins, which is consistent with dystrophin‐deficient muscular dystrophy. Whole genome sequencing revealed a complex structural variant including a deletion of approximately 25.5 kb encompassing exons 8 and 9 of the DMD gene, which was absent in more than 1750 unrelated control genomes. This loss‐of‐function variant most likely accounts for the clinical presentation. Subsequent PCR testing revealed the presence of the wild‐type allele in both parents and a normal female littermate. Amplification of the mutant allele could not be achieved. The mutant allele was likely transmitted from the mother to the index case and the presumed affected male littermate. It remains unresolved whether the mutant allele was transmitted over several generations in the maternal line or arose by a de novo mutation event in the germline of the mother. In conclusion, we describe a partial deletion of the DMD gene in a Jack Russell Terrier with a Duchenne‐like muscular dystrophy phenotype due to non‐functional dystrophin.

Radiation-induced peripheral neuropathy (RIPN) is a well-known late adverse effect of radiotherapy (RT) in humans, but to date, is not reported in companion animals. An 11-year-old spayed female beagle was presented for evaluation regarding a progressive left sciatic neuropathy. The dog had received stereotactic RT (20 Gy) following excision of a grade 2 soft tissue sarcoma over the proximo-caudal aspect of the left thigh approximately 19 months prior to the onset of signs. Computed tomography and magnetic resonance imaging of the left sciatic nerve was consistent with RIPN, and histopathology of the sciatic nerve supported severe nerve fiber loss. No improvement was observed with an anti-inflammatory course of corticosteroids, and the affected limb was subsequently amputated.

Here we describe an 8-year-old intact male Jack Russell Terrier with a 9-month history of slowly progressive gait disturbances that advanced over 2 years to generalized stiffness without ataxia and severe bilateral hyperflexion of all limbs. Gait evaluation showed high lifting of both pelvic limbs resulting in a hopping movement in the rear end; the right pelvic limb was kept lifted, making the dog trot on the 3 other limbs. Both thoracic limbs were lifted higher than normal. Complete blood count and serum biochemistry analysis including creatine kinase activity were normal. Electrophysiological examination showed no abnormalities. Muscle biopsy samples collected at a later stage of the disease showed no abnormalities. A homozygous frameshift variant in SETX (senataxin) was identified and is predicted to truncate about 85% of the protein. The SETX variant adds a new gene variant to the list affecting Jack Russell Terriers with gait abnormalities.

X-linked dystrophin-deficient muscular dystrophy (DD-MD) is an uncommon neuromuscular disorder in cats. We described an adult male cat with chronic tongue protrusion, dysphagia, muscle hypertrophy, and a history of rhabdomyolysis associated with anesthesia. Clinical pathology revealed markedly increased CK activity, muscle histopathology demonstrated a dystrophic phenotype, and an absence of dystrophin protein was confirmed by immunofluorescent staining. Whole genome sequencing identified two potential disease-causing variants, including a new missense variant in the DMD gene (c.2207T>C; p.Gln736Arg), which was considered causative of the clinical phenotype. A second variant in the CLIC2 gene was also detected but was considered unlikely to cause myopathic signs. The clinical course remained stable over 1.5 years with supportive management and dietary modification, and no further episodes of rhabdomyolysis occurred. This case expands the known spectrum of feline DMD variants and highlights the value of genetic testing combined with muscle histopathology for diagnosing chronic presentations of MD. Avoidance of inhalant anesthetics may be important in managing affected cats due to the risk of acute muscle injury.

Background: Myopathies caused by genetic abnormalities are increasingly recognized in veterinary medicine. Hypothesis/Objectives: Clinically and genetically characterize a novel creatine deficiency disorder (CDD) myopathy in a family of mixed breed dogs. Animals: Three siblings from the same litter were evaluated and genetically tested, including 2 dogs that were clinically affected and one dog clinically normal. All dogs were client owned. Methods: Case series describing clinical, imaging, electrodiagnostic, histopathologic investigations, and response to treatment. Whole genome sequencing and bioinformatics were performed to identify a causative variant followed by Sanger sequencing to confirm the suspected variant in related dogs. Results: Clinical signs included megaesophagus with generalized muscle atrophy in both affected dogs. One dog showed exercise intolerance. Computed tomography (CT) scan revealed bilateral and symmetrical diffuse hypoattenuating muscle lesions. Electromyography was characterized by nonspecific abnormal spontaneous activity in electrodiagnostically affected muscles. Type 2 fiber atrophy and excessive intramyofiber lipid droplets in type 1 muscle fibers were the predominant findings in histopathology. Both affected dogs were homozygous for a unique GATM p.R414C (NP_001274013.1) missense variant, while the unaffected sibling did not have this variant. All clinical signs improved after 3 days of creatine (800-1500 mg/kg/day) and L-carnitine (80-150 mg/kg) supplementation and remained stable at the time of writing 4 months after diagnosis. Conclusions and clinical importance: This is a report of CDD in dogs characterized by a glycine amidinotransferase (GATM) variant, which showed a good short-term outcome with supplementation with creatine and L-carnitine.

Background: Two Shiba Inu littermates presented for investigation of marked and persistent elevation of creatine kinase activities. Method and Results: Histopathology of muscle biopsy samples revealed a dystrophic phenotype and immunostaining confirmed an absence of dystrophin protein in both cases. Whole genome sequencing of one affected dog revealed a complex deletion in the DMD gene encompassing exon 5. Screening of 27 related dogs confirmed an X-linked inheritance. The variant was identified in three related male dogs. One littermate died from cardiac arrest and the other littermate had no clinical myopathic signs at the time of the manuscript’s preparation. An additional related male dog reportedly died suddenly during grooming. Conclusion: This study adds a new breed to the canine dystrophinopathy spectrum having a ~17 kb deletion that encompasses exon 5 of DMD. This same exon 5 deletion has been identified in human dystrophin-deficient muscular dystrophy patients.

Background: Major genetic risk loci and causative mutations classified as LPN1 (Leonberger polyneuropathy type 1), LPN2 (Leonberger polyneuropathy type 2), and LPPN3 (Laryngeal paralysis polyneuropathy type 3) have been identified in Leonberger and Saint Bernard dogs with laryngeal paralysis and polyneuropathy (LPPN). Other large breed dogs, including the Great Dane, can present clinically with LPPN, and this breed previously was identified as a carrier of the LPPN3 variant. To date, homozygosity for this variant has not been identified in Great Dane dogs. Interventions: Results of neurological examination, electrodiagnostic testing, and muscle and nerve biopsy samples were consistent with lower motor neuron disease associated with axonal degeneration and large nerve fiber loss in two young Great Dane dogs with gait abnormalities and respiratory difficulty. TaqMan genotyping for the three known LPPN variants confirmed a homozygous missense variant in CNTNAP1, the variant associated with LPPN3. Conclusion and Clinical Relevance: A homozygous missense CNTNAP1 variant (p.G937E, XP_548083.3) has been confirmed in young Great Dane dogs that should expand the genetic testing available for LPPN in this breed and aid in the direction of breeding programs. Both dogs were euthanized because of progression of clinical signs approximately 6 months after the original diagnosis.

Earlier, we described a breed-specific inflammatory myopathy in Dutch Kooiker dogs (Het Nederlandse Kooikerhondje), one of the nine Dutch breeds. The disease commonly manifests itself with clinical signs of difficulty walking, muscle weakness, exercise intolerance, and/or dysphagia. In nearly all dogs’ creatine kinase (CK) activity was elevated. Histopathology reveals the infiltration of inflammatory cells within the skeletal muscles. The objective of this study was to further investigate and characterize the histopathological changes in muscle tissue and immunophenotype the inflammatory infiltrates. FFPE fixed-muscle biopsies from 39 purebred Kooiker dogs were included and evaluated histopathologically according to a tailored classification scheme for skeletal muscle inflammation. As in other breed-related inflammatory myopathies, multifocal, mixed, and predominantly mononuclear cell infiltration was present, with an initial invasion of viable muscle fibres and the surrounding stroma leading to inflammation, necrosis, and tissue damage. Immunophenotyping primarily revealed lymphohistiocytic infiltrates, with CD3+ T-cells being the predominant inflammatory cell type, accompanied by CD8+ cytotoxic T-cells. The concurrent expression of MHC-II class molecules on myofibres suggests their involvement in initiating and maintaining inflammation. Additionally, CD20+ B-cells were identified, though in lower numbers compared to T-cells, and IBA-1-positive macrophages were frequently seen. These findings suggest a breed-specific subtype of polymyositis in Kooiker dogs, akin to other breeds. This study sheds light on the immune response activation, combining adaptive and innate mechanisms, contributing to our understanding of polymyositis in this breed.

X‐linked recessive dystrophinopathies are the most common muscular dystrophies (MDs) in humans and dogs. To date, 20 breed‐specific MD‐associated variants are described in the canine dystrophin gene (DMD), including one associated with dystrophin‐deficient MD in the Border Collie mixed breed. Here, we report the diagnosis and follow‐up of mild dystrophin‐deficient MD in a 5‐month‐old male Border Collie, associated with a novel DMD variant. Diagnosis was based on neurological examination and laboratory evaluations including creatine kinase activity, electromyography and muscle biopsies with immunofluorescent staining. Inspection of the Sashimi plots of the RNA‐seq data from the affected muscle biopsy led to the discovery of a 162‐bp L1 pseudoexon in DMD intron 63, introducing a frameshift and a premature stop codon (NM_001003343.1: c.9271_9272insN[162] p.(Ala3091fs*21)). Reduced DMD mRNA levels were detected for both the non‐pseudoexon (50× less) and pseudoexon (3× less) containing transcripts in the affected muscle, compared with the level of the non‐pseudoexon containing transcript in a control muscle, resulting in very low dystrophin protein levels and the upregulation of utrophin. Because the variant was only found in the affected dog, not in the healthy mother and grandmother, or in 108 unrelated Border Collies from the Belgian population (46 males and 62 females), it was considered a de novo variant. Although the prognosis for dystrophinopathy is generally regarded as poor, the dog stabilised at the age of 6 months and is still clinically stable at the age of 2 years.