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.
Introduction
Muscular dystrophy has been described in humans, dogs, and cats; it is composed of a heterogeneous group of hereditary, degenerative myopathies.1 The most common form of muscular dystrophy in humans and dogs arises from a mutation in the gene for the dystrophin protein that disrupts the skeletal and cardiac muscles’ dystrophin-glycoprotein complex.1 The human form of dystrophin deficiency is called Duchenne’s muscular dystrophy.2 In dogs, dystrophies have been described in several breeds, including the golden retriever, Labrador retriever, Irish terrier, German shorthaired pointer, rat terrier, Belgian Groenendaeler shepherd, Samoyed, Japanese spitz, Brittany spaniel, and an Old English sheepdog.3–12 A range of clinical phenotypes has been described in humans, including severe forms that progress rapidly and milder forms that have a prolonged course.1 In previously reported dogs with muscular dystrophy, clinical signs usually developed at <6 months of age.5,9,13,14
The purpose of this report is to describe a dystrophin-deficient muscular dystrophy in an adult male Weimaraner. To the authors’ knowledge, this is the first report of a dystrophin-deficiency muscular dystrophy in a Weimaraner.
Case Report
A 2-year-old, male Weimaraner was examined for a 1-year history of regurgitation, exercise intolerance, and dysphagia. The frequency of regurgitation and excessive drooling had increased in the last month, and a hiatal hernia was suspected. Abnormal findings on physical examination included cryptorchidism, poor body condition (body condition score of 1.5/5), and atrophy of most major muscle groups. Muscles of the neck, infraspinatus, and tongue were hypertrophied. Periodically, the dog developed muscle spasms of the neck that lasted 10 to 30 seconds. Gait was short-strided in the thoracic and pelvic limbs, without overt ataxia or neurological deficits. Based on these findings, a primary myopathy was suspected. Differential diagnoses included degenerative disorders (e.g., muscular dystrophy, storage disease), an inflammatory myopathy (e.g., Toxoplasma gondii, Neospora caninum), an immune-mediated disorder (e.g., polymyositis), neoplasia (e.g., lymphoma), and metabolic diseases (e.g., mitochondrial myopathy).
Results of a hemogram indicated a leukocytosis (21,500 cells/μL, reference range 6000 to 17,000 cells/μL) with an absolute neutrophilia (18,060 cells/μL, reference range 3000 to 11,500 cells/μL). Serum creatinine kinase ([CK] 32,672 U/L, reference range 68 to 400 U/L) and alanine aminotransferase ([ALT] 305 U/L, reference range 10 to 130 U/L) were elevated. Serum aspartate aminotransferase (AST) was not analyzed. Serum antibody titers for Toxoplasma gondii were negative for immunoglobulin G (IgG) and did not indicate an active infection on immunoglobulin M (IgM) assay (1:128, active infection >1:1024a). Serum titers for Neospora caninum were negative. Urinalysis revealed myoglobinuria without hemoglobinuria, which was confirmed by microfiltration.b
Radiographs (taken with the dog under general anesthesia with isofluranec in oxygen) of the pharynx identified a soft-tissue swelling near the angle of the mandible, with no bony involvement. Radiographs of the thorax and abdomen revealed the stomach was protruding through the esophageal hiatus into the thorax. A small mass at the ventral margin of the diaphragm was later identified as a thickening of the diaphragm musculature around its central tendon. Echocardiography was performed to assess cardiac muscle function, and no evidence of a cardiomyopathy was seen except for hyperechoic regions within the left ventricular free wall.1 Moderate mitral regurgitation was identified. Systolic function and transaortic and transpulmonic velocities were normal.
Electrophysiology was performed with the dog under general anesthesia (with isofluranec in oxygen). Both the paraspinal and appendicular musculature had grade 4/4 abnormal spontaneous activity consisting of complex repetitive discharges with isolated fibrillation potentials. Peroneal motor-nerve conduction velocity was within normal limits (81 meters per second, reference value >53 meters per second); however, the compound motor-unit action potential amplitude was significantly reduced at the hip, tarsus, and stifle recording sites (14.6 mV, 3.4 mV, and 4.5 mV, respectively).15–17 Electrophysiological results were most consistent with a myopathy.
Muscle biopsies were collected from the infraspinatus, sternohyoideus, and ventral tongue muscles. The biopsies were either fixed in 10% formalin and submitted for general histopathology, or they were refrigerated and shipped overnight for histochemical staining. The fresh biopsy tissue was flash-frozen in isopentane that was precooled in liquid nitrogen, and it was stored at −80°C until further analysis.d,16,18 Specimens from all biopsied muscles were also submitted for aerobic bacterial and fungal cultures. Cultures were negative except for those of the tongue, which grew a coagulase-negative staphylococcus that was likely a contaminant or opportunistic infection.
Histopathological changes typical of a muscular dystrophy were present in all three muscles. Changes included a marked variability in myofiber size with numerous, small, round to angular atrophied fibers; numerous fibers with multiple internal nuclei; extensive endomysial fibrosis; and clusters of degenerating and regenerating fibers with scattered myofiber mineralization [Figure 1A]. The central nuclei in most fibers were indicative of a chronic myopathic change and not regeneration. There were a few basophilic regenerating fibers of very small diameter, but most were large with degenerative changes.
For immunohistochemical analysis of dystrophin, fresh-frozen sections of muscle were incubated with monoclonal antibodies against the rod domain (1:20; NCL-DYS1) and carboxy terminus (1:20; NCL-DYS2) of dystrophine [Figure 1B, 1C]. An antibody against spectrin (1:100; NCL-SPEC2) was used as a control for membrane integrity [Figure 1D]. Compared to the control muscle, dystrophin staining was restricted to the sarcolemma of a few fibers. These fibers may have indicated revertant fibers (those having immature developmental characteristics), which can be seen with abnormalities of dystrophin in various species. Monoclonal antibody staining for dystrophin deoxyribonucleic acid was not performed, however.19,20
Based on the presence of severe muscle weakness and the poor body condition, the dog was euthanized and a necropsy was performed. Necropsy findings included agenesis of the right kidney and ureter and an abdominal right testicle. The musculature of the limbs was grossly atrophied, but the ventral tongue, larynx, and dorsal neck muscles were grossly hypertrophied [Figure 2]. Similar to the biopsy findings, all muscles evaluated (quadriceps, triceps, sternothyroideus, semimembranosus, sternocephalicus, platysma, genioglossus muscles) displayed the same dystrophic pattern of degeneration, regeneration, and fibrosis. The esophagus was grossly normal. The entire stomach was herniated through a diverticulum of the esophageal hiatus of the diaphragm, which expanded to >10 cm. The diaphragmatic musculature was markedly thickened (with prominent striations), which formed a 1.5-cm band around the accentuated central tendon [Figure 3]. Microscopically, the diaphragm contained similar degeneration, regeneration, fibrosis, and mineralization. A trichrome stain of the diaphragm highlighted the marked endomysial fibrosis [Figure 4]. In the heart, the ventricles (especially the left) were moderately dilated, with concentric mural and septal hypertrophy. Microscopically, anastomosing and some-what perivascular myocardial fibrosis with mineralization and myofiber atrophy was seen within the posterobasal left ventricular free-wall myocardium, with sparing of subendocardial and subepicardial regions. No evidence of active myodegeneration or necrosis was seen [Figure 5]. No significant lesions were identified in the central nervous system or in the recurrent laryngeal, sciatic, tibial, peroneal, and radial nerves.
Discussion
Most reported cases of muscular dystrophy in dogs have been associated with dystrophin deficiency rather than expression of an abnormal protein.21 Histologically, typical findings in muscle biopsy specimens have included myofiber degeneration, regeneration, variations in myofiber size, fibrosis, and sometimes calcific deposits.1 All of these changes were identified in the muscles of the Weimaraner in this report.
An unusual aspect of this case was the late presentation (2 years of age) for clinical evaluation. Most dogs with muscular dystrophy are evaluated at <1 year of age because of clinical signs of weakness, exercise intolerance, muscle wasting, and dysphagia that worsen in severity over several months.3,8,9,22 As different mutations in the dystrophin gene can occur, the clinical signs of muscular dystrophy can vary, depending on the size of the deletion or severity of the mutation.10 For example, a truncated form of dystrophin has been described in Japanese spitz dogs that have severe dystrophic changes.10 In these dogs, a 70 to 80 kDa (kilodalton) fragment was recognized by the monoclonal antibody DYS-2.10 Since the smallest functional dystrophin gene has recently been shown to be between 150 and 165 kDa, the 70 to 80 kDa fragment found in the spitz dogs would not have been large enough to protect them from a dystrophic process.23,24 The slower course of the disease in the Weimaraner reported here may have reflected a smaller deletion or a less severe mutation of the dystrophin gene. Further diagnostics beyond the histopathological determination of the dystrophic phenotype and immunohistochemical analysis of muscle biopsy sections for localization of dystrophin and dystrophin-related proteins would have been necessary to determine the precise classification of the dystrophin disorder in this dog.25 Unfortunately, neither Western blotting nor mutational analyses were performed on this case. For routine clinical evaluations, staining of muscle sections for the presence or absence of dystrophin and related proteins has been considered adequate.26
The case reported here displayed many similarities to golden retriever muscular dystrophy, including enlargement of the base of the tongue, diaphragm, and pharyngeal muscles. The dog in this case report had many other similarities to muscular dystrophy of golden retrievers, including clinical, biochemical, histochemical, and immunohistochemical changes.14,27 The elevation in serum ALT may have been related to muscle necrosis.27 Electromyographic findings were also similar, with complex repetitive discharges being the predominant abnormality.28,29 The cardiac changes found on the echocardiogram and necropsy were similar to those identified in affected golden retrievers and in humans with Duchenne’s muscular dystrophy.28,30,31 Histopathological myocardial changes are corroborative of ongoing, gradual myocardial loss and replacement fibrosis. Although sparing of subepicardial regions is more unique to this case, a somewhat perivascular distribution without evidence of mural or luminal changes was similar to previous observations and may indicate vascular factors were involved in the pathogenesis. 28 The hernia identified at necropsy in the esophageal hiatus has also been described in golden retrievers and Labrador retrievers with muscular dystrophy.1,3,32 The progressive nature of the clinical signs reported in the current case was also typical of most reports of dystrophin deficiency in people, dogs, and cats.2,18
In contrast to golden retriever muscular dystrophy and Labrador retriever muscular dystrophy, this case also had right-sided cryptorchidism and agenesis of the right kidney and ureter.1 It was unknown if these problems represented an unrelated mutation or some separate but linked abnormality. The Weimaraner in this report was not stunted in growth, which has been reported in affected golden retrievers. 3,29 The course of the disease and age at death can be variable in the golden retriever, with dogs living several months to several years of age. It was not determined if muscular dystrophy in this case was associated with a spontaneous, new mutation or if it was a typical X-linked, recessive defect. Information was not available on littermates, and mutational analysis was not performed.
Conclusion
Dystrophin-deficient muscular dystrophy was diagnosed in a 2-year-old, male Weimaraner via immunohistochemistry. This dog had a chronic, progressive disease course, and the dystrophy was associated with agenesis of the right kidney. Future studies identifying the dystrophin gene size and exon deletions in clinical cases may be warranted to determine functionality of the dystrophin protein, inheritance pattern in the Weimaraner breed, and the presence of revertant protein.
Infectious Diseases Laboratory, University of Georgia, Athens, GA 30602
Microcon; Millipore Corp., Billerica, MA 01821
Isoflurane; Abbott Laboratories, North Chicago, IL 60064
Comparative Neuromuscular Laboratory; University of California–San Diego, La Jolla, CA 92903
NCL-DYS1, NCL-DYS2, NCL-SPEC2; Novocastra Laboratories—monoclonal antibodies to dystrophin proteins 1 and 2 and spectrin protein 2, respectively; Novocastra Laboratories, Newcastle-upon-Tyne, United Kingdom
Citation: Journal of the American Animal Hospital Association 43, 4; 10.5326/0430227
Citation: Journal of the American Animal Hospital Association 43, 4; 10.5326/0430227
Citation: Journal of the American Animal Hospital Association 43, 4; 10.5326/0430227
Citation: Journal of the American Animal Hospital Association 43, 4; 10.5326/0430227
Citation: Journal of the American Animal Hospital Association 43, 4; 10.5326/0430227
Histopathology and dystrophin immunocytochemistry of the infraspinatus muscle of a 2-year-old, male Weimaraner with muscular dystrophy. (A) Variations in myofiber size, central nuclei, extensive endomysial fibrosis, and myofiber degeneration are consistent with a dystrophic phenotype (Hematoxylin and eosin stain, bar=100 μm). (B) Immunocytochemistry for dystrophin 1 protein (DYS1), showing numerous negatively stained fibers (blue-grey) and small numbers of positively stained fibers (arrow pointing to brown sarcolemmal staining in possible revertant fibers) (bar=100 μm). (C) Sarcolemmal staining was negative (blue-grey) in all muscle fibers following staining for dystrophin protein 2 (DYS2) (bar=100 μm). (D) Positive sarcolemmal staining (brown, arrows) was found following incubation with an antibody (SPEC2) against spectrin (bar=100 μm).
Postmortem photograph of the dog in Figure 1, showing swelling of the neck (A), prominent maxillofacial musculature (B), and thickening of the tongue (C) from muscular hypertrophy. The apparent brachygnathism and glossal retraction are secondary to constriction and fibrosis of the affected musculature. Atrophy of the temporalis (arrow) and masseter muscles can also be seen.
Cross-sectional photograph of unfixed strips of the diaphragm, including the pars costalis and central tendon from a normal, 7-year-old, 20-kg, female, mixed-breed dog (A) and the affected Weimaraner (B) of Figure 2. Both specimens contain comparable areas of the pars costalis and central tendon. The diaphragmatic specimen of the affected Weimaraner is short, thick, and pale primarily from fibrosis (arrow) (marker=1 cm).
This photomicrograph of the diaphragm of the affected Weimaraner with muscular dystrophy demonstrates areas of endomysial fibrosis (blue, A) that separate and surround hypertrophic (B), atrophic (C), and necrotic, fragmented (D) muscle fibers (Masson’s trichrome, bar=85 μm).
This photomicrograph of the myocardium of the affected Weimaraner demonstrates marked myocardial fibrosis (A), which separates and surrounds atrophic cardiac myocytes (B), and areas of mineralization (C) (Hematoxylin and eosin stain, bar=25 μm).
