Dystrophin-Deficient Muscular Dystrophy in Two Male Juvenile Brittanys
ABSTRACT
A 6 mo old and a 7 mo old male intact Brittany were presented for progressive exercise intolerance, failure to grow, and dysphagia. Creatine kinase activity was markedly and persistently elevated in both dogs. Based on the neurological examination, clinical signs localized to the neuromuscular system. Electromyography revealed complex repetitive discharges in multiple muscle groups. Immunofluorescence of biopsies confirmed dystrophin-deficient muscular dystrophy. This is the first report describing dystrophin-deficient muscular dystrophy in the Brittany breed. Currently, no specific therapies are available for this form of myopathy. The presence of dystrophin deficiency in the two dogs suggests an inherited myopathy rather than a spontaneous mutation. The location of the dogs in the United States and Japan suggests a wide distribution of this dystrophy and should alert clinicians to the existence of this myopathy in the Brittany breed. A mutation in the DMD gene has not yet been identified.
Introduction
Dystrophin-deficient muscular dystrophy (DMD) is an X-linked inherited myopathy that generally affects young purebred males, although females and mixed-breed dogs can be rarely affected.1,2 Clinical signs of DMD are first noted between 8 and 10 wk of age and include weakness, stiff gait, difficult prehension, and inability to fully open the jaw. Dystrophinopathies have been well characterized in golden retrievers, although several other breeds have been affected or suspected to be affected.3–14 Dystrophin-deficient muscular dystrophy was first described in the golden retriever breed and the mutation in the DMD gene identified.3 Many breeds have since been identified with DMD and additional mutations in the DMD gene identified including but not limited to those in the corgi, German short-haired pointer, Labrador retriever, Japanese spitz, and miniature poodle.10,13,15–17 This report is the first confirmation of DMD in male Brittany puppies, one from the United States and another from Japan. A previous case report from Belgium in 1995 described a congenital myopathy in a Brittany puppy with a decreased range of jaw mobility, a hypertrophied tongue, and a markedly elevated creatine kinase (CK) activity.18 The dog was ultimately euthanized without confirmation of dystrophin deficiency. In this report we confirm the diagnosis of DMD and suggest a wide distribution in this breed.
Case Reports
Case 1
A 6 mo old, 8.5 kg, intact male Brittany from the United States was presented for progressive exercise intolerance and failure to grow. He had been the smallest puppy in his litter, and clinical signs were noted by his owner at adoption at 8 wk of age. A chemistry panel performed by the referring veterinarian showed markedly elevated aspartate aminotransferase (AST 501 U/L; reference range 23–65 U/L) and CK (30,136 U/L; reference range 46–467 U/L) activities. The dog was referred to the University of Pennsylvania School of Veterinary Medicine for evaluation of a possible neuromuscular disease.
At referral, the dog was severely muscle wasted, most notably over the masticatory muscles, pelvic limbs, and thorax. There was intermittent focal muscle pain and an inability to completely open the jaw. The tongue showed decreased movement, and the base of the tongue appeared hypertrophied. Neurologic examination showed a short-strided gait with severe diffuse muscle atrophy and decreased withdrawal and myotactic reflexes in all four limbs. Cranial nerves and postural reactions were normal. The neuroanatomic localization was to the neuromuscular system.
Thoracic radiographs revealed a subjectively flattened, undulant diaphragm and normal cardiac size. Abdominal ultrasound showed a mottled spleen and several large intra-abdominal lymph nodes (likely age-related). Repeat chemistry panel showed persistently elevated AST (3066 U/L) and CK (350,190 U/L) activities, as well as an elevated phosphorus and calcium (likely due to the patient’s young age). Neospora caninum and Toxoplasma gondii antibody titers were negative, as was a comprehensive tick panel (polymerase chain reaction [PCR] and serology). Ophthalmology consultation showed mild diffuse altered tapetal reflectivity, which was considered a normal variation in this breed. Cardiac auscultation revealed a sinus tachycardia.
Electromyography was performed under general inhalational anesthesia and showed complex repetitive discharges in the supraspinatus, extensor carpi, biceps femoris, vastus lateralis, and tongue. Unfixed chilled and formalin fixed biopsies were collected from the left vastus lateralis muscle and shipped by an overnight express service under refrigeration to the Comparative Neuromuscular Laboratory, University of California, San Diego, California, where they were snap frozen in isopentane precooled in liquid nitrogen and stored at −80°C until further processed. Cryosections were evaluated with a standard panel of histochemical stains and reactions.19 A degenerative and regenerative myopathy with mild mixed mononuclear cell infiltrations was observed consistent with a dystrophic phenotype (Figure 1). Additional cryosections were cut and stained for indirect immunofluorescence as previously described using monoclonal antibodies against the rod (1:100, NCL-DYS1a) and carboxy-terminus (1:100, NCL-DYS2a) of dystrophin, utrophin (1:20, NCL-DRP2a), and developmental myosin heavy chain for regenerating fibers (1:20, NCL-MHCda); a monoclonal antibody against caveolin 3b (1:100), and polyclonal antibodies against laminin α2 (1:200), α-sarcoglycan (1:200), and collagen VI (direct apply, monoclonal antibody 3G7), all gifts from Professor Eva Engvall.20 Compared with archived control muscle, staining was absent for the rod- and carboxy-terminus of dystrophin with mild upregulation of utrophin consistent with a diagnosis of a dystrophin-deficient muscular dystrophy (Figure 2).
Citation: Journal of the American Animal Hospital Association 58, 6; 10.5326/JAAHA-MS-7255
Citation: Journal of the American Animal Hospital Association 58, 6; 10.5326/JAAHA-MS-7255
The dog continued to decline over the next month and the owners elected humane euthanasia. Necropsy showed multifocal chronic myofiber degeneration, regeneration, interstitial fibrosis, and calcific deposits, consistent with a form of muscular dystrophy in the diaphragm, tongue, and skeletal muscles. The heart was histologically normal. DNA was purified from skeletal muscle and tested by PCR for the splice site point mutation in exon 6 that results in skipping of exon 7 in dystrophin-deficient golden retrievers.3 The variant was not identified, and the genotype was confirmed as wild type. This suggests that the mutation for DMD in Brittanys is different than that in golden retrievers. The dog was not tested for dystrophin variants confirmed in other breeds.
Case 2
A 7 mo old male intact Brittany from Japan was referred for decreased level of activity, slow and unsteady gait, low neck carriage, reluctance with head elevation, and dysphagia. Clinical signs appeared at 3 mo of age, then gradually progressed over time. Before the referral, complete blood count and serum chemistry profile revealed marked elevations in CK >20,000 U/L (reference range 49–166 U/L), alanine aminotransferase 633 U/L (reference range 17–78 U/L), and AST 1120 U/L (reference range 17–44 U/L) activities. Serum antibody titers for N caninum and T gondii were negative. Thoracic radiographs were unremarkable.
At referral, the dog weighed 10.0 kg and physical examination revealed poor body condition score (2/5), attributed to a generalized myopathy and oropharyngeal dysphagia. A short-strided gait was evident in all four limbs with low head carriage. Neurologic examination revealed loss of gag reflex and dysphagia with normal mentation, proprioception, and spinal reflexes in all four limbs (Supplementary Video I).
An electrodiagnostic evaluationc including electromyography and measurement of motor nerve conduction velocities, followed by skeletal muscle biopsies, was performed under general inhalational anesthesia. Laryngeal function was subjectively assessed upon induction of anesthesia and considered normal.
Electromyography was performed on multiple muscles from the right side of the body. Increased insertional activity and widespread spontaneous activity consisting predominantly of complex repetitive discharges was detected (Figure 1B). Motor nerve conduction velocity evaluated from the right peroneal nerve with a surface recording electrode on the extensor digitorum brevis muscle resulted in a normal motor nerve conduction velocity (52 m/sec in the distal segment and 67 m/sec in the proximal segment) with compound muscle action potential amplitudes (onset-to-peak amplitudes of 3.8 mV, 4.1 mV, and 6.0 mV with stimulation at hock, stifle, and coxofemoral joints, respectively; Figure 1C).
Unfixed chilled and fixed biopsies were collected from the left triceps brachii, infraspinatus, and biceps femoris muscles immediately following the electrodiagnostic evaluation. Biopsies were shipped under refrigeration by an express service to the Comparative Neuromuscular Laboratory, University of California, San Diego, California, where they were processed as for Case 1. Like Case 1, a dystrophic phenotype was observed in all muscles evaluated and dystrophin-deficient muscular dystrophy was identified by immunofluorescent studies on muscle cryosections. At the time of writing, this dog was still alive at 8 mo of age with slowly declining neurologic function under supportive care consisting of small meals of soaked kibble three times a day, daily weight check, and light exercise.
Informed consent was obtained from both case owners before manuscript preparation. Both cases were clinically managed according to contemporary standards of care.
Discussion
The clinical signs, markedly elevated CK activity, and electromyographic changes in the Brittanys of this report were consistent with a neuromuscular disorder. A form of muscular dystrophy was confirmed through pathological changes in muscle biopsies and further immunofluorescent staining documenting dystrophin deficiency (Figure 2). Muscle biopsies were essential for identification of a form of muscular dystrophy causing the markedly and persistently elevated CK activities as elevated CK activity can also be associated with inflammatory and necrotizing myopathies that are nondystrophic in nature. Once a pathological diagnosis of a muscular dystrophy is confirmed, a further search can be performed by immunofluorescent staining of muscle cryosections to identify the missing or altered protein. Such studies can then focus the search for a specific gene mutation and limit the scope of further genetic testing. In this case, the dystrophin gene would be the candidate for sequencing.
Dystrophin mutations in dogs are varied and include exonic substitutions, frameshift insertions, and deletions and splice site mutations.13 Testing of Case 1 for the golden retriever mutation by PCR showed a wild-type sequence, indicating that the Brittany mutation differs from that of the golden retriever. Additional testing was not performed to evaluate dystrophin mutations identified in other breeds in either Case 1 or Case 2. Further genetic studies are required.
Presenting clinical signs in both cases were consistent with a chronic and progressive myopathy. A thickened tongue with calcific deposits and a thickened diaphragm are relatively common findings in dogs with DMD. Radiographically, the thorax can have a striking appearance.21 Thoracic changes that can be seen on radiography include a flattened diaphragm, pulmonary hyperinflation, hiatal hernia, pectus excavatum, bronchopneumonia, and megaesophagus.22 Case 1 had a flattened diaphragm, though no other specific changes were noted. Case 2 presented with oropharyngeal dysphagia, which has been described in muscle diseases.
The prognosis for DMD in dogs is generally grave, although mild forms with minimal clinical signs have been described in Labrador retrievers.10 At this time there are no successful treatments, though there are some reports that L-carnitine and coenzyme Q10 can increase muscle strength and prolong survival.12 Corticosteroid (prednisone) therapy is documented to improve various functional outcome measures in both human and golden retriever muscular dystrophy. In boys with muscular dystrophy, prednisone or deflazacort therapy may prolong ambulation and delay other complications including respiratory compromise and myocardial disease.23,24 Golden retrievers with DMD show dose-dependent improvement in muscle strength and a reduction of muscular calcific deposits, but it is unclear whether this leads to improved overall survival time.23,24
Conclusion
Dystrophinopathies are well described in golden retrievers and several other breeds.3–14 This is the first confirmation of DMD in the Brittany breed. The early report of a suspected muscular dystrophy in a Brittany from Belgium, and our confirmed cases from the United States and Japan, suggests that this form of dystrophy may have a widespread distribution in the Brittany breed and should alert clinicians to the possibility of DMD in young puppies with a myopathic presentation and markedly and persistently elevated CK activities.18
(A) Representative cryosection (8 mm) from the vastus lateralis muscle of a 6 mo old Brittany (Case 1) with persistently and markedly elevated creatine kinase activity, poor growth, and a thickened tongue. Myofiber degeneration and regeneration are prominent with calcific deposits consistent with a dystrophic phenotype (hematoxylin and eosin stain). (B) Electromyogram tracing from Case 2 showing a run of complex repetitive discharges. (C) Measurement of motor nerve conduction velocity from the peroneal nerve of Case 2.
Immunofluorescent staining of the vastus lateralis muscle of the 6 mo old Brittany (Case 1) and archived control muscle using monoclonal or polyclonal antibodies against the rod and carboxy terminus of dystrophin, utrophin, developmental myosin heavy, laminin α2, α-sarcoglycan, collagen VI, and caveolin. Compared with archived control muscle, staining for the rod and carboxy terminus of dystrophin was absent, confirming a diagnosis of dystrophin-deficient muscular dystrophy.
Contributor Notes
The online version of this article (available at jaaha.org) contains supplementary data in the form of one video.
