Pathology in Practice

Cristian de la Fuente Department of Animal Medicine and Surgery, Veterinary School, Autonomous University of Barcelona, 08193 Bellaterra (Barcelona), Spain.

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 DVM, MSc
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G. Diane Shelton Department of Pathology, School of Medicine, University of California-San Diego, La Jolla, CA 92093.

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Jessica Molín Department of Animal Medicine and Surgery, Veterinary School, Autonomous University of Barcelona, 08193 Bellaterra (Barcelona), Spain.

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Martí Pumarola Department of Animal Medicine and Surgery, Veterinary School, Autonomous University of Barcelona, 08193 Bellaterra (Barcelona), Spain.

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Ling T. Guo Department of Pathology, School of Medicine, University of California-San Diego, La Jolla, CA 92093.

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Sònia Añor Department of Animal Medicine and Surgery, Veterinary School, Autonomous University of Barcelona, 08193 Bellaterra (Barcelona), Spain.

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 DVM, PhD, DACVIM

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History

A 5-month-old sexually intact male French Bulldog was evaluated because of a 3-month history of dysphagia, regurgitation, and intermittent episodes of bilateral mucous nasal discharge and cough. Mild exercise intolerance and delayed growth were also reported by the owners. Regurgitation had been noticeable since the dog was obtained from the breeder, and clinical signs were slowly progressive.

Clinical and Gross Findings

Initial physical examination of the dog revealed poor body condition; small body size, compared with that expected for a healthy 5-month-old French Bulldog puppy; and presence of a soft tissue bulge in the submandibular region (Figure 1). Partial trismus was also present. Careful inspection of the oral cavity revealed hypertrophy of the pharyngeal muscles and macroglossia. Hypertrophy of the dorsal cervical musculature and generalized atrophy of the muscles of both pelvic limbs were also observed. No signs of pain were elicited via skeletal muscle palpation. Results of a neurologic examination were unremarkable except for signs of mild exercise intolerance.

Figure 1—
Figure 1—

Photograph of a 5-month-old male French Bulldog with a 3-month history of dysphagia, regurgitation, and intermittent episodes of mucous nasal discharge and cough. Notice the hypertrophy of the dorsal cervical musculature and generalized atrophy of pelvic limb muscles (A) and soft tissue bulging in the submandibular region caused by hypertrophied lingual muscles (asterisk; B).

Citation: Journal of the American Veterinary Medical Association 240, 12; 10.2460/javma.240.12.1423

Results of a CBC and serum biochemical and bile acids analyses were unremarkable except for moderately high alanine aminotransferase (630 U/L; reference range, 21 to 102 U/L) and aspartate aminotransferase (579 U/L; reference range, 23 to 66 U/L) activities and markedly high creatine kinase activity (36,277 U/L; reference range, 10 to 150 U/L). Thoracic radiography revealed diaphragm asymmetry, with a flattened and cranially displaced left crus as well as a soft tissue mass dorsal to the caudal cervical and first thoracic vertebrae. Positive-contrast esophagography revealed the presence of a dynamic hiatal hernia. Abdominal ultrasonography did not reveal any remarkable findings, except for small thickened areas in the diaphragm that were evident on parasagittal views.

During an electromyographic evaluation, complex repetitive discharges were detected in all muscles tested in the pelvic limbs, thoracic limbs, and tongue. Motor and sensory nerve conduction velocities of the sciatic and ulnar nerves were within reference limits. Magnetic resonance imaging of the head revealed severe enlargement of the extrinsic tongue muscles and the intrinsic lingual muscle.

The dog was anesthetized, and biopsy specimens from the quadriceps femoris, cranial tibial, and triceps brachii muscles were collected and fixed in 4% paraformaldehyde for routine histologic examination. Muscle biopsy specimens that remained unfixed were used for immunohistochemical analysis. Six weeks after diagnosis, because of progressive clinical signs and poor prognosis, the owners requested euthanasia of the dog and allowed necropsy.

Gross findings on postmortem examination included generalized atrophy of the pelvic limb muscles with marked hypertrophy of sublingual and dorsal cervical muscles. A hiatal hernia with a small part of the gastric fundus located inside the thoracic cavity and diaphragm hypertrophy were also observed.

Formulate differential diagnosis from the history, clinical findings, and Figure 1—then turn page

Histopathologic Findings

Histopathologic abnormalities in muscle biopsy samples were dystrophic in nature (Figure 2), and the large proximal limb muscles were more markedly affected than the distal limb muscles. The lingual and diaphragm muscles were the most affected. There was moderate to marked myofiber size variability, multifocal areas of myonecrosis, and large groups of small regenerating fibers. Numerous calcium deposits were present in the necrotic areas but could also be seen scattered throughout the muscle biopsy sections.

Figure 2—
Figure 2—

Photomicrographs of cryosections of the quadriceps femoris muscle of the dog in Figure 1. A typical dystrophic histologic phenotype is evident in the muscle sections, including excessive variability in myofiber size and calcific deposits (A); areas of degeneration (myonecrosis; A) and groups of regenerating fibers (B) are also visible. H&E stain; bar = 50 μm (applies to both A and B).

Citation: Journal of the American Veterinary Medical Association 240, 12; 10.2460/javma.240.12.1423

Immunohistochemical analysis for dystrophin and dystrophy-associated proteins was performed again on frozen sections of muscle specimens collected at necropsy. Although this had been already performed on antemortem biopsy samples, results had not been conclusive owing to sample autolysis. Sections of diaphragm, tongue, and triceps brachii and quadriceps femoris muscles were collected and sent refrigerated to a neuromuscular laboratory for analysis. Cryosections of the quadriceps femoris muscle were incubated with several commercially available monoclonal and polyclonal antibodies against the rod domain (DYS1) and carboxy terminal (DYS2) of dystrophin, developmental myosin heavy chain, utrophin, spectrin, laminin α2, α-sarcoglycan, caveolin 3, and dysferlin. Compared with archived canine control quadriceps femoris muscle, staining with antibodies against DYS1 and DYS2 of dystrophin was not observed in the sections of quadriceps femoris muscle collected from the puppy (Figure 3). Results of staining with the antibody against developmental myosin heavy chain were indicative of robust regeneration, and there was increased sarcolemmal staining with the antibody against utrophin, compared with control muscle. Immunoblotting of a muscle protein extract from the submitted postmortem biopsy specimens and monoclonal antibodies against DYS1 and DYS2 also revealed undetectable dystrophin.

Figure 3—
Figure 3—

Results of immunohistochemical analysis of cryosections and immunoblotting of a protein extract from a quadriceps femoris muscle biopsy specimen obtained from the dog in Figure 1. A—Immunofluorescence staining of cryosections of the quadriceps femoris muscle biopsy specimen (obtained after euthanasia) from the dystrophic French Bulldog and archived canine control quadriceps femoris muscle. Sections were incubated with antibodies against the rod domain and carboxy terminal (C-terminus) of dystrophin, developmental myosin heavy chain (for detection of regenerating fibers [dMHC]), utrophin, laminin α2, α-sarcoglycan, caveolin 3, and dysferlin. An antibody against spectrin was used to confirm sarcolemmal membrane integrity. As a control for background staining, only the secondary antibody was used without a primary antibody (2nd only). Compared with control muscle findings, staining for the rod and carboxy terminal of dystrophin was undetectable and staining for utrophin and regenerating fibers was more pronounced in sections of muscle obtained from the puppy. Bar = 50 μm (applies to all images). B—Immunoblots of a muscle protein extract from a control dog (lane c) and a muscle protein extract from the quadriceps femoris muscle of the dystrophic French Bulldog (lane d). Following electrophoresis and western blotting, membranes were reacted with monoclonal antibodies against the rod domain (DYS1) and carboxy terminal (DYS2) of dystrophin. Notice the absence of dystrophin in the extract from the affected dog. By use of an antibody against β-actin, equal protein loading in all lanes was confirmed.

Citation: Journal of the American Veterinary Medical Association 240, 12; 10.2460/javma.240.12.1423

Morphologic Diagnosis

Dystrophin-deficient muscular dystrophy.

Comments

Muscular dystrophies are a heterogeneous group of inherited, degenerative, mostly noninflammatory disorders characterized by progressive muscle weakness and wasting.1 The most common form of muscular dystrophy in dogs, cats, and humans is caused by dystrophin deficiency.2 Dystrophin is a large protein (400 kDa) that connects the muscle fiber cytoskeleton to the extracellular matrix through the cell membrane to stabilize the sarcolemma during contraction, and it is encoded by a large gene located in the X chromosome.3 Owing to the large size of the gene and to its location, mutations are common and males are predominantly affected.4 Although dystrophin-deficient muscular dystrophy is best characterized in Golden Retrievers, dystrophinopathies in several other breeds have been reported2,5–7 but not French Bulldogs, to our knowledge. Dystrophin deficiency in dogs is the genetic homologue of human Duchenne and Becker muscular dystrophy and is investigated as a model of these human diseases.8

Clinical signs of dystrophin-deficient muscular dystrophy usually become noticeable at 6 to 9 weeks of age and are slowly progressive, although a case of late-onset disease in a 2-year-old Weimaraner has been reported.5 Clinical signs include gait abnormalities and exercise intolerance as well as atrophy of limb muscles, trismus, and tongue hypertrophy.2 In affected animals, the lack or impairment of dystrophin function leads to sarcolemmal membrane instability and facilitates creatine kinase leakage outside myofibers; as a result, markedly high serum creatine kinase activity is a consistent finding among animals with dystrophinopathies.1

The radiographic abnormalities detected in Golden Retrievers with muscular dystrophy have been well described.9 Thoracic abnormalities include diaphragm asymmetry, hiatal hernia, and thickened diaphragm. Similar abnormal features were identified in the dog of this report. Electrodiagnostic evaluation in dystrophic dogs typically reveals spontaneous muscle activity, consisting primarily of complex repetitive discharges and, less frequently, myotonic discharges (detected via electromyography), with normal nerve conduction velocities.1,2,10

Histologically, dystrophic features include myofiber degeneration and regeneration, excessive variability in myofiber size, fibrosis, myonecrosis, and calcific deposits.2 The pathological changes, although dystrophic in nature, are not specific for dystrophinopathies; thus, additional diagnostic procedures are always necessary to identify a missing or mutated protein and obtain an accurate diagnosis for future targeted molecular assessments. Immunohistochemical staining of fresh frozen muscle biopsy specimens with dystrophy-associated monoclonal or polyclonal antibodies and immunoblotting of muscle extracts from affected dogs can specifically detect the lack of dystrophin or other proteins, and those findings are essential to reach a definitive diagnosis.11 For the dog of the present report, results of immunohistochemical analysis of cryosections and immunoblotting of muscle extracts from the affected and control muscle tissues confirmed a diagnosis of dystrophin-deficient muscular dystrophy.

There is no definitive treatment for muscular dystrophy, and the prognosis for dystrophic dogs is poor owing to the progressive worsening of clinical signs. Treatment with prednisone may be attempted,12 but it is not usually successful.

For the dog of this report, further information regarding clinical signs of the disease in its parents or any siblings was unavailable. In the absence of a pedigree analysis, it was not possible to elucidate whether the puppy had an inherited form of dystrophin deficiency or a spontaneous mutation of the dystrophin-encoding gene.

References

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