Dystrophin-Deficient Muscular Dystrophy in a Labrador Retriever

Introduction

Muscular dystrophy is a term that is broadly used to refer to any primary skeletal muscle disease that results in progressive degeneration, limited regeneration, and fibrosis of myofibers.¹² With the advent of immuno-histochemical analyses and molecular diagnostics, more specific classifications of the various muscular dystrophies are now possible.³⁴ Canine X-linked muscular dystrophy (CXMD) was first described in the golden retriever.⁵⁶ Variations of this disease have since been documented in multiple breeds including the Samoyed,⁷ Brittany spaniel,⁸ Irish terrier,⁹ Groenendaeler shepherd,¹⁰ miniature schnauzers,¹¹ Pembroke Welsh corgi,¹² German shorthaired pointer,¹³ and a rat terrier.¹⁴ While all result from a deficiency of the membrane-associated cytoskeletal protein dystrophin, not all dystrophin-deficient myopathies are the result of the same mutation.⁶¹³ Dystrophin is a 400-kilodalton protein that functions to stabilize the muscle membrane during contraction.¹⁵ It is encoded by a very large gene located on the X-chromosome.¹⁶ Since the dystrophin gene is so large, it is a relatively frequent target of mutations, and because males have only one X-chromosome, dystrophin deficiency predominantly affects males. Deficiency of this protein is responsible for both Duchenne and Becker’s muscular dystrophies in human boys. A similar type of X-linked muscular dystrophy has been reported in the cat.¹⁷

While not the only differential diagnosis in a young puppy, the most common heritable neuromuscular disease in young Labrador retrievers is a condition called heritable myopathy in Labrador retrievers (HMLR).^18–21 Early investigations described a type II fiber deficiency associated with an autosomal recessive mode of inheritance.¹⁸¹⁹ Due to the variable histopathological changes including dystrophic abnormalities, fiber type deficiency, and neurogenic atrophy, definitive classification of this disorder has proven difficult and the precise molecular defect(s) are not known.

The following case report describes a Labrador retriever puppy with early-onset muscle weakness associated with dystrophin deficiency. Differentiation of this disorder from HMLR is critical, as prognosis and inheritance patterns differ. To the authors’ knowledge, this is the first report of X-chromosome-associated muscular dystrophy in this breed.

Case Report

A 3.5-month-old, 10.3-kg, male intact Labrador retriever was presented to the teaching hospital for evaluation of dysphagia, generalized weakness, and inspiratory stridor. The owners reported that the dog was the runt of the litter and had always had difficulty swallowing when eating. No information was available about littermates. They also thought he had problems moving his tongue, drooled excessively, and retained food in his mouth [Figure 1]. In addition, they noted stertorous breathing and exercise intolerance.

On physical examination, the dog had poor body condition and generalized muscle atrophy. The dog was able to prehend and chew but dropped food during swallowing due to inappropriate tongue movement. Likewise, he was not able to drink normally but had to extend his head and neck to swallow water. A decreased gag reflex, hypertrophied muscles of the throat and cranial neck, and an enlarged nonmobile tongue were found on neurological examination. Segmental spinal reflexes were normal. Based on these findings, a primary myopathy was suspected. Rule-outs included inflammatory (e.g., infectious secondary to Toxoplasma gondii [T. gondii] or Neospora caninum [N. caninum]; immune-mediated) and noninflammatory (e.g., congenital; metabolic) muscle diseases.

Results of complete blood count were within reference ranges. Increases in serum phosphorus (7.2 mg/dL; reference range, 2.6 to 6.0 mg/dL) and calcium (11.8 mg/dL; reference range, 9.5 to 11.6 mg/dL) were attributed to the young age of the dog. Elevations in alanine aminotransferase (437 U/L; reference range, 13 to 88 U/L) and potassium (5.4 mEq/L; reference range, 3.4 to 4.6 mEq/L) were observed and may have been related to leakage secondary to muscle necrosis.²²²³ Serum creatine kinase (CK) was 56,482 U/L (reference range, 69 to 708 U/L). A fecal flotation was negative for parasites. A fluoroscopic esophagram using both liquid and food-mixed barium paste demonstrated that the dog was unable to swallow liquid barium or to form a food and barium bolus. This appeared to result from inability to pass food over the tongue. This abnormality indicated a problem with the oral preparatory phase of deglutition. Both the pharyngeal and esophageal phases of deglutition were normal. An awake needle electromyogram (EMG) showed complex repetitive discharges in all appendicular muscles examined (i.e., gastrocnemius, cranial tibial, quadriceps, epaxial, triceps, biceps, extensor carpi radialis, infraspinatus, and supraspinatus). The EMG findings were consistent with a myopathic disease or denervation. Pending protozoal titer results, the dog was sent home on empirical clindamycin (7.3 mg/kg body weight, per os q 8 hours for 4 days) therapy and frequent elevated feedings of food in a gruel consistency. Serum titers for T. gondii and N. caninum were negative.

The dog’s clinical signs were unchanged during the next 2 weeks, at which time a needle EMG was again performed under general anesthesia. Complex repetitive discharges were found in thoracic and pelvic limb muscles, epaxial musculature, head muscles, and the tongue. These consisted of trains of positive sharp waves that began abruptly and stopped abruptly. The overall motor nerve conduction velocity (MNCV) along the left tibial/sciatic nerve (i.e., stimulation sites, coxofemoral joint, stifle and tarsus; recording site, plantar interosseous muscle) was within reference range for a dog of this age (47 meters per second; reference range, 47 to 59 meters per second). The evoked compound muscle action potentials were normal in both amplitude (hock, 13.8 mV; stifle, 13.0 mV; hip, 11.4 mV) and configuration, based on subjective comparisons with results obtained from normal animals.²⁴²⁵ Normal amplitudes have not been established for dogs of this age in the authors’ laboratory.

A surgical biopsy was taken from the vastus lateralis muscle and shipped overnight on cold packs to the Comparative Neuromuscular Laboratory, University of California, San Diego. The muscle was frozen in isopentane precooled in liquid nitrogen, sectioned in a cryostat, and stained with a standard panel of histochemical stains and enzyme reactions²⁶ and by indirect immunofluorescence technique. The following antibodies (dilution within parentheses) were used: monoclonal antibodies against the rod domain (1:25, NCL-DYS1) and carboxy terminus (1:50, NCL-DYS2) of dystrophin; against spectrin (1:50), α-sarcoglycan (1:50, NCL-a-SARC), β-sarcoglycan (1:50, NCL-b-SARC), and γ-sarcoglycan (1:50, NCL-g-SARC);^a and antibody against α-dystroglycan (1:100).^b Additional monoclonal antibodies were used against laminin α2 (1B4 and 4F11) and laminin γ1 as previously characterized.²⁷ Rabbit polyclonal antiserum was used against β-dystroglycan and α- and β-sarcoglycans as described,²⁸ and against δ-sarcoglycan (1:600).^c All dilutions of primary antibodies were made in 3% bovine serum albumin in phosphate-buffered saline, and incubations were for 1 hour at 37°C. Secondary antibodies used were goat anti-rabbit IgG-FITC (1:200)^d and goat anti-mouse IgG-FITC (1:200),^e and incubation was for 1 hour at room temperature. In addition to biopsy specimens from the dog of this report, sections of vastus lateralis muscle from a normal dog were similarly processed.

Muscle histopathological changes included variability in myofiber size, endomysial fibrosis, necrosis, phagocytosis, and basophilic regenerating fibers. Numerous calcific deposits were scattered throughout the biopsy sections [Figure 2]. The calcific nature of the deposits were confirmed using the alizarin red stain (not shown). Immunohistochemical staining for dystrophin and related proteins [Figure 3] showed absence of staining for dystrophin [Figure 3B] and dystroglycans [Figure 3D], with a few small fibers positively stained for sarcoglycans [Figure 3F]. Membrane integrity was confirmed by normal staining with the antibody against spectrin (not shown), laminin α2 [Figure 3H], and laminin γ1 (not shown). Staining was present in the normal dog muscle with all antibodies [Figures 3A, 3C, 3E, 3G]. The histopathological changes and immunohistochemical staining were consistent with a myopathy associated with dystrophin deficiency and secondary loss of the dystrophin-related proteins, dystroglycan and sarcoglycan.

The dog was discharged with instructions for supportive care. At 7 months of age, the dog was reevaluated due to progression of clinical signs and respiratory difficulties. The dog weighed 9.5 kg and had severe generalized muscle wasting. The dog had severe bloody diarrhea the night before presentation, and a fecal parvovirus test performed at an emergency clinic was negative. A neurological examination found slightly depressed spinal reflexes, a decreased gag reflex, and hypertrophy of the tongue. No abnormalities were noted by echocardiography. Creatine kinase was elevated at 4,704 U/L. An awake EMG again showed complex repetitive discharges in all appendicular muscles examined.

The dog was euthanized at the owner’s request. Necropsy findings included severe emaciation with a body condition score of 1/5 and slight hydronephrosis. Other than changes in skeletal muscle, there were no other significant abnormalities. Skeletal muscle changes in the vastus lateralis, tongue, and esophagus were similar to those described for the initial muscle biopsy. No changes were found in the cardiac muscle.

Discussion

To the authors’ knowledge, this report is the first description of muscular dystrophy associated with abnormalities of dystrophin and dystrophin-associated proteins in the Labrador retriever. The documentation of dystrophin deficiency is important, because it demonstrates the occurrence of a heritable neuromuscular disease in addition to HMLR in this breed. This finding is of further importance because earlier pathological studies of HMLR describe widely variable histopathological findings, including dystrophic abnormalities.²⁰ It is possible that some of the dogs included in earlier studies may have indeed had CXMD and not HMLR.

Histopathological evaluation of fresh-frozen and fixed muscle biopsy specimens is critical to the diagnosis of muscle diseases in young puppies. Myopathies affecting young puppies can be noninflammatory and include dystrophies, metabolic and mitochondrial disorders, and nemaline rod and central core myopathies; or they can be inflammatory secondary to immune-mediated disease or infection with T. gondii or N. caninum. An accurate diagnosis is critical for prognosis, for determination of appropriate therapy if available, and for elimination of genetic diseases from breeding populations.

The term “muscular dystrophy” covers a diverse group of inherited disorders, and the marked phenotypic heterogeneity in human patients with muscular dystrophy has made exact classification difficult. The classification of the muscular dystrophies has greatly benefited from the identification of several genes shown to cause different forms of muscular dystrophy,²⁹ and from the development of several monoclonal and polyclonal antibodies against the protein products of these genes useful in clinical diagnostic assays.³⁰ For clinical evaluation, the use of protein expression by immunohistochemistry is a relatively simple and highly informative adjunct to a histopathological diagnosis and can be performed directly on the fresh-frozen biopsy specimens used for the histopathological diagnosis.³⁰ Immunohistochemistry for the localization of proteins such as dystrophin, the sarcoglycans, and laminin α2 along with proper controls is a cost-effective and sensitive method. Using such methodology, deficiency of laminin α2 has recently been identified in two cats³¹ and a female dog.³² Such classifications would be important to breeders and geneticists to guide mutation analysis, particularly in disorders such as the limb-girdle muscular dystrophies that are a group of diseases very difficult to differentiate on clinical grounds alone. Limb-girdle muscular dystrophy is a disease classification used to describe several distinct pathologies that result in the same phenotype.³³³⁴ For owners, this is also important information, because disease progression and prognosis will differ for the different forms of muscular dystrophy.

The clinical and histopathological characteristics of the puppy in this report are consistent with a diagnosis of CXMD. Since HMLR would be a primary differential, it is important for clinicians to discriminate between these disorders. There are multiple differences in the clinical features of the two disorders [see Table]. Similar to the puppy of this report, dogs with CXMD show clinical signs at birth or early in life, consisting of stiff pelvic limbs, excessive salivation, enlarged tongue, and dysphagia. Weakness is progressive. Initially, spinal reflexes are intact but gradually become depressed. Also, there may be hypertrophy of proximal limb muscles.⁵ In HMLR, there is a variable onset of clinical signs from 6 weeks up to 6 to 7 months of age, although signs are usually noted by 3 to 4 months.¹⁸ Dogs with HMLR display muscle weakness, abnormalities of gait and posture, ventroflexion of the neck, an arched back, a short and stilted bunny-hopping gait, and abnormal joint posture. There may be generalized atrophy of muscles, especially of the proximal limb muscles. Spinal reflexes are initially reduced.¹⁸

As a result of myonecrosis and the absence of membrane dystrophin and related proteins that are consistent findings in CXMD, the serum CK concentration is usually dramatically elevated with levels ≥20,000 U/L (reference range, <708 U/L).⁵ Since myonecrosis is not a consistent finding in HMLR and dystrophin and associated proteins are not absent,²¹³⁵ the serum CK concentration may be normal or mildly elevated or increase with exercise.¹⁸

Electrophysiological abnormalities have been reported in both diseases and do not allow the differentiation of the two. Various EMG changes reported with CXMD include complex repetitive discharges (previously referred to as pseudomyotonic potentials), positive sharp waves, and fibrillation potentials.⁵³⁶³⁷ These EMG changes tend to increase until 4 months of age. The EMG changes associated with HMLR are similar.³⁸ These abnormalities may be less severe in mildly affected animals. The proximal limb, head, and thoracolumbar paraspinal muscles may show the most changes. In both diseases, motor nerve conduction velocities are within reference ranges.³⁷³⁸

Histopathological changes in the dog of this report were dystrophic in nature, with necrosis, phagocytosis, variability in myofiber size, and calcific deposits within muscle fibers. The absence of immunohistochemical staining for dystrophin and dystrophin-associated proteins confirmed the diagnosis of a dystrophin-associated muscular dystrophy.

Conclusion

The puppy in this case report had clinical, biochemical, histochemical, immunohistochemical, and electrophysiological abnormalities consistent with CXMD. Deficiency of dystrophin and related proteins was confirmed immunohistochemically. This case is significant for several reasons. Canine X-linked muscular dystrophy has not been reported in the Labrador retriever, and this should now be considered as a potential cause of an inherited myopathy in this breed. Although dystrophinopathies are X-linked recessive diseases, there is a high mutation rate due to the large size of the dystrophin gene that leads to many isolated cases where the mother is not a carrier.²⁹ The authors cannot say, based on the occurrence in one dog, whether this is an inherited dystrophy or a spontaneous mutation, since neither a mutational analysis nor breeding studies were performed. However, this finding should alert clinicians, pathologists, and researchers to the possibility of an X-linked disorder in this breed.

Acknowledgments

This work was partially supported by grants provided to Dr. Eva Engvall from the National Institution of Health and the Muscular Dystrophy Association. The authors thank Norma Prades for expert technical assistance.