Sarcoglycan-Deficient Muscular Dystrophy in a Boston Terrier

Abstract

A 4-month-old, 3.8 kg, intact male Boston Terrier was presented to the Iowa State University Veterinary Teaching Hospital (ISU VTH) for a 2-month history of progressive gait abnormalities and regurgitation. A mild right thoracic limb lameness initially was observed 48 hours after vaccinationa and progressed to bilateral thoracic limb stiffness over 5 days. At this time, appetite was normal without vomiting or regurgitation. Treatment with prednisolone was initiated at 2.5 mg (1 mg/kg) PO q24h for a presumed vaccine reaction. There was no improvement in clinical signs over the next 4 days, and the dog again was examined by the referring veterinarian. In addition to the stiff thoracic limb gait, decreased appetite, vomiting or regurgitation, and weight loss were now reported. Physical examination revealed stertor and mildly enlarged submandibular lymph nodes. Thoracic auscultation was normal. A CBC (reference ranges are for adult dogs) showed a mildly regenerative anemia (29.3%; reference range, 37–55%; corrected reticulocyte count, 1.4%), decreased hemoglobin (9.6 g/dL; reference range, 12–18 g/dL), and thrombocytosis (695 × 109/L; reference range, 175–500 × 109/L). Serum chemistry abnormalities (all reference ranges are for adult dogs) included increased alanine aminotransferase activity (ALT, 381 U/L; reference range, 8–75 U/L) and hypochloremia (103 mEq/L; reference range, 105–119 mEq/L). Serum creatine kinase (CK) activity was not measured. Thoracic radiographs (lateral and ventrodorsal views) showed mild air distension of the thoracic esophagus resulting in mild ventral deviation of the cranial thoracic trachea. Serum chemistry was evaluated 3 weeks later and showed persistently increased ALT activity (255 U/L; reference range, 4–125 U/L), hyperglycemia (144 mg/dL; reference range, 67–139 mg/dL), hyperphosphatemia (9.3 mg/dL; reference range, 2.7–8.0 mg/dL), and hypercalcemia (12.8 mg/dL; reference range, 7.7–11.0 mg/dL). Serum creatinine concentration was decreased (<0.20 mg/dL; reference range, 0.3–1.2 mg/dL). Serum CK and aspartate aminotransferase (AST) activities were both increased above the detectable range of the analyzer. Over the next month, the puppy was examined for continued frequent vomiting or regurgitation of both food and water. The dog was referred to the ISU VTH. On presentation to the internal medicine service, the dog had a stiff, stilted gait consisting of bilateral thoracic limb abduction without ataxia. Nonpainful, nonfirm enlargement of the parotid and submandibular salivary glands was also noted. Increased referred upper airway noise and generalized symmetrical muscle hypertrophy, more pronounced in the muscles of the thoracic limbs (pectorals, supraspinatus, biceps, and triceps), were noted. The remainder of the physical examination was within normal limits. With the exception of the stilted gait and muscle hypertrophy, the neurologic assessment (including level of consciousness, cranial nerves, conscious proprioception, and spinal reflexes) was normal. Spinal hyperpathia was not noted. Based on the clinical findings of stilted gait, muscle hypertrophy, and regurgitation with possible esophageal dilatation (based on referral radiographs), and the markedly increased serum CK and AST activities, a diffuse neuromuscular disorder, most likely of myopathic origin, was suspected. Differential diagnoses included infectious, immune-mediated, and congenital muscle diseases. Blood samples were collected for CBC, serum biochemistry, and repeated determination of serum CK activity. Urine was collected by cystocentesis. Right lateral and dorsoventral thoracic radiographs were obtained. The CBC showed thrombocytosis (552,000/μL; reference range, 200,000–500,000/μL), lymphocytosis (6.508 × 103/μL; reference range, 1.0–4.8 × 103/μL), and eosinophilia (0.813 × 103/μL; reference range, 0.0–0.75 × 103/μL). Serum biochemistry analysis indicated increased ALT activity (475 U/L; reference range, 24–105 U/L), hypercholesterolemia (314 mg/dL; reference range, 140–270 mg/dL), hypercalcemia (12.3 mg/dL; reference range, 9.2–11.2 mg/dL), hyperphosphatemia (8.0 mg/dL; reference range, 3.2–6.2 mg/dL), and hypochloremia (101 mEq/L; reference range, 107–124 mEq/L). CK activity was markedly increased (9,179 U/L; reference range, 60–270 U/L). Urinalysis revealed 3+ blood and trace proteinuria. Thoracic radiographs showed a normal position of the trachea, with no esophageal dilatation. A focal, poorly marginated soft tissue opacity was seen in the area of the caudal esophagus. The thoracic trachea was decreased in diameter and considered possibly hypoplastic, with a tracheal lumen diameter to thoracic inlet distance ratio of 0.125 (reference ratio for brachycephalic dogs other than Bulldogs is 0.160).1 Differential diagnoses for the poorly marginated soft-tissue opacity were gastroesophageal intussusception or herniation. There were no other radiographic abnormalities. Esophageal function was evaluated with a 3-phase (liquid, paste, and meal) barium esophagram. The esophagram showed a persistent region of narrowing in the caudal thoracic esophagus, approximately midway between the base of the heart and the diaphragm. Although this area consistently allowed passage of solid food, it persistently reverted to a narrowed dimension and a consistent curved shape. There was extension of the cardia region of the stomach cranial to the diaphragm predominantly with administration of the paste and food consistency barium. This finding coincided with the soft tissue opacity identified on the lateral survey films. These observations were considered consistent with a hiatal hernia (Fig 1). A stricture immediately cranial to the hiatal hernia was suspected. The dog was discharged with instructions that it should be fed small amounts of canned food several times daily. The owners also were instructed to maintain the dog in an upright position while eating and drinking, and for several minutes thereafter. Metoclopramide (0.7 mg PO q6h) and famotidine (1.9 mg PO q12h) were prescribed. Esophagoscopy, electromyography (EMG),b and muscle biopsy were recommended. Lateral view of a thoracic radiograph taken during the paste-consistency phase of the barium esophagram. Note extension of the cardia region of the stomach cranial to the diaphragm, consistent with a hiatal hernia. Megaesophagus was not present. The dog was readmitted and esophagoscopy was performed. A mildly dilated esophagus filled with foam without strictures or erosions was found. The gastric and duodenal mucosa appeared grossly normal. There was no evidence of pyloric outflow obstruction. Serum CK activity again was markedly increased (8,919 U/L). The EMG showed abnormal spontaneous activity, including trains of positive sharp waves, fibrillation potentials of variable frequency and amplitude, and complex repetitive discharges in muscles of the thoracic (supraspinatus, biceps, and triceps) and pelvic (quadriceps, semimembranosus, semitendinosus, and cranial tibial) limbs. Based on these findings and the persistently increased serum CK activity, muscle biopsies were performed. Unfixed and fixed biopsies from the left infraspinatus, triceps, and cranial tibial muscles were submitted under refrigeration to the Comparative Neuromuscular Laboratory, University of California, San Diego. Chilled, unfixed muscle samples were flash frozen in isopentane precooled in liquid nitrogen and evaluated in frozen sections using a standard panel of histochemical stains and reactions.2 Fixed muscle specimens were processed in paraffin for routine histopathology. Pathologic changes were similar in severity in all 3 muscles, with abnormal variability in myofiber size, multifocal areas of myonecrosis, and scattered calcific deposits (Fig 2A). Muscular dystrophy (MD) or, less likely, an infectious, predominantly necrotizing myopathy, was suspected based on histopathology. Recovery from anesthesia was uneventful, and the dog was discharged with continuation of elevated feedings, small frequent meals, and metoclopramide and famotidine. Histopathology and immunohistochemical analysis of dystrophin and sarcoglycans (SGs) in the triceps muscle from the Boston Terrier with muscular dystrophy. A typical dystrophic phenotype was present within muscle biopsy specimens with groups of degenerating myofibers and calcific deposits (H&E, A). Sarcolemmal staining was normal using monoclonal antibodies against the rod (B) and carboxy terminus (C) of dystrophin, but absent using monoclonal antibodies against β- (D) and γ- (E) SGs. Staining with a monoclonal antibody against spectrin (F) was normal, which confirmed that the sarcolemma was intact. Scale bar = 100 μm for all figures. On examination 5 weeks later, the dog was eating canned food and body weight had increased to 4.8 kg. Thoracic limb stiffness, abnormal gait, and swollen salivary glands were still evident. Serum biochemistry and CK activity were reevaluated. Serum ALT activity was still increased (317 U/L), and serum CK activity was increased from previous measurements (17,982 U/L). Toxoplasma gondii IgG and IgM titers were negative by immunofluorescent assay at the 1 : 40 dilution, and Neospora caninum serology IFA was negative at the 1 : 50 dilution. Because a form of MD was suspected, immunohistochemical staining (Fig 2B–2F) and immunoblotting (Fig 3) for dystrophin and associated proteins were performed on serial frozen biopsy sections and extracts from the triceps muscle. Sections and extracts were incubated with monoclonal antibodiesc against the rod (1 : 20, NCL-DYS1) and carboxy terminus (1 : 20, NCL-DYS2) of dystrophin, β-sarcoglycan (SG) (1 : 50, NCL-b-SARC), γ-SG (1 : 50, NCL-g-SARC), and spectrin (1 : 100, NCL-SPEC2) as a control for membrane integrity, or with a polyclonal antibody against α-SG3 (1 : 1,000). Staining of the muscle sarcolemma was not detected with any of the SG antibodies (Fig 2D, 2E), whereas staining for dystrophin (Fig 2B, 2C) and spectrin (Fig 2F) was considered normal. Absence of α-, β-, and γ-SGs with normal amounts of dystrophin was also found on immunoblotting (Fig 3). These findings confirmed SG-deficient MD. Immunoblot analysis of muscle from the Boston Terrier with muscular dystrophy and a control dog. Similar to the results of immunohistochemical staining, staining for the rod (DYS1) and carboxy terminus (DYS2) of dystrophin was present, whereas staining for α-sarcoglycan (α-SG), β-SG, and γ-SG was absent. A monoclonal antibody against β-actin was used as a loading control. The MDs comprise a varied group of genetic, degenerative diseases characterized by progressive muscle wasting.4, 5 More than 30 different types of MD have been described in humans, with the most common form associated with dystrophin deficiency. The dystrophin-glycoprotein complex (DGC) spans the muscle plasma membrane, linking the intracellular actin cytoskeleton with the extracellular matrix.5 A mutation in any of the associated proteins may result in MD and weakness.5 Only a few forms of MD have been reported to date in companion animals, including dystrophin deficiency in many breeds of dogs and in cats,6-12 laminin α2 deficiency,13-15 and SG deficiency.d,4, 5 Similar to humans, dystrophin deficiency is the most common type of MD in dogs and cats.4, 5 In humans, SG-deficient MD is part of the complex of limb-girdle muscular dystrophies (LGMDs).16 The SG complex consists of 4 transmembrane glycoproteins: α-, β-, γ-, and δ-SG, which form a subcomplex within the DGC. Loss of each of the SG glycoproteins is associated with a specific form of autosomal recessive LGMDs (LGMD2C-F).16 Humans with dystrophin deficiency (Duchenne's MD) or SG deficiency can also have concurrent loss of other components of the DGC.16-18 However, in the case reported here, a deficiency in the other components was not found. MD associated with SG deficiency previously has been reported in an abstractd and reviews4, 5 in 3 young dogs, including a Chihuahua, Cocker Spaniel, and a 7-month-old castrated male Boston Terrier. Clinical signs in all 4 cases of SG deficiency included failure to thrive, lethargy, exercise intolerance, and diffuse neuromuscular signs. The hiatal hernia found in our case may be related to skeletal muscle weakness involving the diaphragm, as has been reported in dogs with Duchenne's MD.19 Impaired respiratory muscle strength has been demonstrated in humans with LGMDs,20, 21 and α-SG-null mice have histopathologic changes in the diaphragm consistent with the dystrophic phenotype.22 Regurgitation and dysphagia can also occur as a result of hypertrophy of the pharyngeal, esophageal, or lingual musculature in animals with various forms of MD.23 In addition, the dog of this report had decreased tracheal luminal diameter, which could increase intrathoracic pressure and contribute to development of the hiatal hernia. It is also possible that the hernia is unrelated, and could simply be a congenital hiatal hernia found incidentally. Similar to the dog in this report, all previously described dogs diagnosed with sarcoglycanopathy had increased ALT, CK, and AST activities (when measured).d,4, 5 EMG was abnormal in our Boston Terrier and in the previously described Cocker Spaniel with spontaneous activity including fibrillation potentials and complex repetitive discharges in several muscle groups.d,4, 5 Complex repetitive discharges on EMG have also been reported in dogs with dystrophin-deficient MD.24 In this Boston Terrier, immunohistochemical staining and immunoblotting confirmed an absence of α-, β-, and γ-SGs with normal amounts of dystrophin (2, 3), a pattern similar to that found in the previously described Boston Terrier.d,4, 5 Commercial antibodies that recognize canine δ-SG currently are not available. It is difficult to predict from immunohistochemical staining or immunoblotting alone which SG protein is defective, because a deficiency in any one of the SGs typically leads to a secondary deficiency of the entire SG complex. Screening of the SG genes for mutations is needed to determine the specific form of LGMDs and for the development of DNA-based testing.4, 5, 17 For clinical purposes, immunohistochemical staining and immunoblotting confirm the diagnosis of a sarcoglycanopathy. Although clinical manifestations are variable, muscle weakness and wasting are typical of MD,4, 5 as in the dogs with the sarcoglycanopathies.d,4, 5 Hypertrophy of various muscle groups has also been described in dogs and cats with MD6, 9, 14, 15 as was seen in the dog reported here. Hypertrophy of the calves and deltoids has been described in humans with LGMDs.16, 25 Dramatically increased serum CK activity is common and helpful in establishing a differential diagnosis in young animals with muscle disease.4 A typical dystrophic phenotype is found on histopathologic examination of muscle biopsy specimens, including clusters of degenerating and regenerating fibers, variable fibrosis, and tissue calcification.4 As performed in our case, immunohistochemical testing of muscle biopsy sections from clinically affected animals for the presence or absence of dystrophy-related proteins is available and can readily confirm a diagnosis of specific forms of MD. Based on the results of immunohistochemical testing, specific mutational analyses can be performed and genetic tests developed. In summary, SG-deficient MD occurs in dogs, and it is of interest that 2 of the 4 reported cases were Boston Terriers. Recently, one of the authors (G.D.S.) confirmed 2 additional, young, related female Boston Terrier dogs with SG deficiency with a dystrophic phenotype in muscle biopsy sections and absence of staining for SG proteins by immunohistochemistry (Shelton, unpublished observations). The 2 related dogs were presented primarily for dysphagia, an enlarged tongue, and muscle wasting. Serum CK activities were dramatically and persistently increased. It is possible that the Boston Terrier breed is at risk for an inherited form of SG-deficient MD. Additional molecular studies must be performed, including mutational analysis, to determine which subunit or subunits is involved in canine SG deficiency and to establish genetic-based testing to advise breeding programs. In conclusion, MD should be a differential diagnosis for any young dog presenting with muscle atrophy or hypertrophy, gait abnormalities, dysphagia, and dramatically increased CK, AST, and ALT activities. aVanguard Plus 5/CV-L, Pfizer Inc, New York, NY bNeuropack MEB-9200/9300, Nihon Kohden Corp, Tokyo, Japan cNovocastra, Newcastle-upon-Tyne, UK dSchatzberg S, Whittemore J, Morgan E, et al. Sarcoglycanopathy in 3 dogs. 21st Annual Internal Medicine Forum, 2003:88 (abstract)