A possible new inherited myopathy in a young Labrador retriever

Abstract

A 5-month-old, male, Labrador retriever was evaluated for progressive weakness and muscle atrophy. Histologic evaluation of fresh frozen muscle revealed distinct cytoarchitectural changes and central mitochondrial accumulations indistinguishable from those found in the inherited myopathy described in Great Danes. Multiple male littermates and half-siblings were similarly affected.

A 5-month-old, 12.7 kg, intact male, chocolate Labrador retriever was referred to the Western College of Veterinary Medicine (WCVM) for a 3-week history of progressive weakness. Two weeks earlier, the puppy had been evaluated by a veterinarian for a 1-week history of weakness, lethargy, inappetance, and muscle atrophy. Cephalexin (Novocephalexin; Nu-Pharm, Richmond Hill, Ontario), 22 mg/kg bodyweight (BW), PO, q8h, and meloxicam (Metacam; Boehringer-Ingelheim, Burlington, Ontario), 0.1 mg/kg BW, PO, q24h, had been prescribed for chin folliculitis noted during the examination. Five days later, when there was no improvement in the weakness or the skin condition, a skin biopsy was obtained revealing chronic, neutrophilic, folliculitis with intralesional Staphylococcus spp. bacteria. Meloxicam administration was discontinued and treatment was initiated with prednisone (Apo-prednisone; Apotex, Toronto, Ontario), 2 mg/kg BW, PO, q24h. During 10 d of corticosteroid therapy, the muscle atrophy and weakness increased substantially, until the puppy could stand for only brief periods and exhibited constant ventroflexion of the neck. The puppy was referred to the WCVM for evaluation.

The owner reported that prior to this 3-week progressive course of weakness, muscle atrophy, and lethargy, the puppy had been normally active and had not exhibited any abnormalities of gait or any systemic illness. Routine vaccinations were current. In consultation with the breeder of this puppy and the breeder’s veterinarian, additional information was obtained regarding this litter and previous breedings of the dam. This puppy was born in a litter of 7 males and 4 females. Four littermates, all males, had developed similar clinical signs between 12 and 16 wk of age and had been euthanized without further evaluation. Three and 4 y earlier, 2 litters from the same bitch bred to a different male had resulted in multiple male pups with similar clinical findings. Female puppies were all clinically normal. One affected male pup from the 2nd litter had been examined at the WCVM, where progressive muscle atrophy, weakness, and absent patellar reflexes led to a presumptive diagnosis of centronuclear myopathy (CNM) (1,2); the puppy had been euthanized without further evaluation.

Case description

Physical examination at the WCVM revealed a small puppy with severe generalized muscle atrophy (Figure 1) and a body condition score of 3/9. The puppy stood with a ventroflexed neck and an arched spine, keeping both hind limbs tucked under the abdomen (Figure 2). The puppy walked with a short, choppy stride and collapsed after a few steps. Results of an orthopedic examination were normal and no focal region of spinal, bone, or muscular pain could be identified. A mild healing pustular chin dermatitis was noted. Results from the remainder of the physical examination and fundic examinations were normal.

Figure 1.

Marked temporal and masseter muscle atrophy in a 5-month-old Labrador retriever with an inherited myopathy.

Figure 2.

A 5-month-old Labrador retriever with an inherited myopathy stands with a ventroflexed neck and an arched spine, keeping both hind limbs tucked under the abdomen.

On neurological examination, mentation, cranial nerves, and conscious proprioception were normal, as assessed by response to knuckling and hopping. The palpebral reflex was not fatigable. Muscle tone was normal to slightly decreased. Spinal reflexes were all subjectively diminished and both patellar reflexes were consistently absent. Skin sensation was present. Tail function, anal tone, urinary function, and perineal reflex were normal.

A complete blood (cell) count (CBC) revealed a mild leukopenia (4.5 × 10⁹/L; reference range 4.8 to 13.9 × 10⁹/L), characterized by neutropenia (2.295 × 10⁹/L; reference range 3.0 to 10 × 10⁹/L). The neutropenia could represent a normal value for this puppy; or could have been caused by a focal site of ongoing inflammation. A serum chemical profile revealed mild elevation in alanine aminotransferase (170 U/L; reference range 19 to 59 U/L), indicating hepatocellular damage. Serum alkaline phosphatase was mildly elevated (103 U/L; reference range 9 to 90 U/L), consistent with an age related increase of bone isoenzyme or corticosteroid induction. Increases in total protein (65 g/L; reference range 47 to 59 g/L) and albumin (43 g/L; reference range 28 to 38 g/L) were attributed to mild dehydration. Creatinine was slightly decreased (41 μmol/L; reference range 61 to 97 μmol/L), likely reflecting decreased muscle mass. Creatine kinase (CK) was slightly increased (972 U/L; reference range 51 to 418 U/L), indicating muscle injury. Results of a urinalysis were normal, except for minimal concentration (specific gravity 1.018) in a sample collected after initiating oral fluid therapy.

Thoracic radiographs showed a very mild diffuse bronchointerstitial pattern that was not investigated further pre-mortem. Since there were no respiratory signs, and the puppy was being treated with antibiotics, a transtracheal wash for cytological examination and culture was not performed. Megaesophagus was not evident on plain thoracic radiographs.

Administration of edrophonium chloride (Enlon; Baxter, Toronto, Ontario), 0.2 mg/kg BW, IV, did not improve the puppy’s strength, and circulating serum antibodies against acetylcholine receptors were normal (0.21 nmol/L; reference range < 0.6 nmol/L), making a diagnosis of myasthenia gravis unlikely. Results from serological testing for antibodies to Toxoplasma gondii and Neospora caninum were negative. Results from cytological examination and protein content of cerebrospinal fluid were normal. Electromyography (EMG) was not available. Cheek swabs submitted for DNA testing for CNM (CNM Project; Alfort School of Veterinary Medicine, Alfort, France) were negative (not affected or carrier). These results were not confirmed through a 2nd laboratory.

Based on the clinical and laboratory findings, a congenital myopathy or neuropathy was strongly suspected. Cephalexin and prednisone therapy were discontinued at the time of admission. Treatment with clindamycin (Ratiopharm, Mississauga, Ontario), 20 mg/kg BW, PO, q12h, was initiated for possible protozoal myositis, while waiting for the serologic results. The puppy could eat and drink when held in sternal position. Nursing and supportive care were provided, including periodic turning and passive physiotherapy.

The puppy’s condition progressively deteriorated over the next 7 d. He became unable to stand or maintain himself in a sternal position, and he could not lift his head to eat or drink. His bark became hoarse, and during an unsedated laryngeal examination, no arytenoid cartilage abduction was observed during maximal inspiration. Due to his rapidly worsening condition and the poor prognosis for recovery, the owner elected to have the puppy euthanized.

A necropsy was performed. Unfixed nerve and muscle biopsies, under refrigeration, and formalin-fixed peripheral nerves were submitted to the Comparative Neuromuscular Laboratory (University of California-San Diego, La Jolla, California, USA) for histological, histochemical, and immunohistochemical analyses.

Results from the gross postmortem examination were normal, except for severe muscle atrophy and atelectatic pulmonary tissues. Histological examination of lungs, brain, and spinal cord tissues were normal. Peripheral nerves (peroneal, laryngeal, phrenic, femoral, and sciatic), evaluated in 1-μm thick, resin embedded sections, were judged to be normal with subjectively appropriate density of myelinated fibers, and no evidence of axonal degeneration, demyelination, or abnormalities of supporting structures. All of the muscles examined (lateral vastus, cranial tibial, triceps, temporal, dorsal cricoarytenoid) were abnormal, with the most significant lesions seen in the lateral vastus and cranial tibial muscles (Figure 3). There was marked variability in myofiber size, with atrophic fibers having a round to polygonal shape. Numerous atrophic Type 1 and Type II fibers contained central dense and subsarcolemmal ringed areas that stained basophilically with hematoxylin and eosin (H&E), pale red with the modified Gomori trichrome stain, dark blue with the succinic dehydrogenase reaction, and dark brown with the cytochrome C oxidase (COX) reaction. These central dense areas occupied 10% to 50% of the cross sectional diameter of affected myofibers. Scattered necrotic fibers undergoing phagocytosis were present within the cranial tibial, temporal, and laryngeal muscles. Immunohistochemial staining for dystrophin localization was normal. Electron microscopic examination confirmed that the central dense areas were composed of accumulations of normal appearing and large mitochondria, accumulations of glycogen granules, and disrupted myofibrillar elements.

Figure 3.

Serial fresh frozen biopsy sections from the triceps muscle were evaluated using several histochemical stains and enzyme reactions. Variability in myofiber size and multifocal areas of myonecrosis and phagocytosis were identified histologically (A, hematoxylin and eosin stain). In addition to the areas of myonecrosis, several myofibers showed lightly stained blue to purple subsarcolemmal ringed and central dense areas (B, modified Gomori trichrome stain). The subsarcolemmal ringed and central dense areas were darkly positive with the mitochondrial specific reactions succinic dehydrogenase (SDH, C) and cytochrome C oxidase (COX, D). Atrophic fibers were of both fiber types (E, ATPase reaction at pH 9.8 with type 1 fibers lightly stained and type 2 fibers darkly stained). Lysosomal activity within macrophages clearing necrotic debris was highlighted by the esterase reaction (F). Bar = 150 μm for all images.

The dam of this puppy was presented to the WCVM for euthanasia because of behavioral problems a few months after evaluation of the puppy. Physical and neurologic examinations were normal. Results from histologic examination of muscles and peripheral nerves collected immediately postmortem were normal, while DNA tests for the genetic mutation causing CNM (CNM Project; Alfort School of Veterinary Medicine) and the genetic mutation associated with canine X-linked muscular dystrophy (CXMD) in golden retrievers (HealthGene, Toronto, Ontario) were negative (not carrier or affected).

Discussion

The histopathologic findings of central dense staining structures were identical with previously reported cases of inherited myopathy of Great Danes (IMGD), formerly called central core myopathy (3–6). In affected Great Danes, progressive exercise intolerance, generalized muscle wasting, abnormal posture, and body tremors develop between 6 and 19 mo of age (5,6). Male and female dogs with a fawn or brindle coat can be affected, and profound muscle weakness without myalgia has been reported (6).

Results of neurologic examinations are normal in most dogs, but patellar reflexes are occasionally diminished (6). Most affected dogs have mild to severe increases in serum CK (6). Electromyographs are abnormal, with spontaneous activity including fibrillation potentials and positive sharp waves (6). The severity of signs and the speed of progression are variable, even within litters, with a few dogs becoming totally debilitated within 2 mo and most affected dogs being euthanized within 1 y of diagnosis (5,6). A few dogs with less severe exercise intolerance and muscle atrophy stabilize and survive long-term (5,6).

Diagnosis of this disorder is based upon recognition of the characteristic histopathologic, enzymatic, and ultrastructural features in muscle biopsies. Recently, this disorder has been renamed IMGD to avoid confusion with the central core disease (CCD) described in humans (5). Biopsies from dogs with IMGD and the puppy in this report contain mitochondria and glycogen granules in the central core areas within myofibers, while in CCD, the central core areas are unstained with oxidative enzymes and represent regions of sarcomeric disorganization and mitochondrial depletion (7,8). Greater than 90% of human patients with CCD have a mutation of the gene on chromosome 1 that encodes the skeletal muscle calcium release channel (ryanodine receptor 1: RYR1) (8); the DNA from the Labrador retriever puppy in this report was negative for a mutation at the ryanodine receptor site.

There are 2 previously reported inherited myopathies affecting Labrador retrievers: CNM and CXMD (9,10). Clinical features in this puppy showed some similarities to each, making it difficult to arrive at a clinical diagnosis without a muscle biopsy (Table 1).

Table 1.

Clinical comparisons of centronuclear myopathy (CNM), canine X-linked muscular dystrophy (CXMD), and the puppy in this report (patient)

	CNM	CXMD	Patient
Sex	males and females	male	male
Age at onset	typically 3 to 4 mo6 wk to 7 mo	< 6 wk	4 mo
Clinical signs	weakness, arched back ventroflexion of neck stilted gait generalized muscle atrophy (especially proximal limbs)	pelvic limb stiffness excessive salivation tongue hypertrophy oral dysphagia generalized muscle atrophy (occasional hypertrophy proximal limb muscles)	Weakness arched back ventroflexion of neck stilted gait generalized muscle atrophy
Patellar reflexes	reduced or absent	normal initially may diminish (months)	absent
Creatine kinase	normal to mildly increased	dramatic elevations (> 20 000 U/L)	mildly increased (972 U/L)
Progression	slowly progressive (months) rare rapid progression (weeks) signs may stabilize by 12 mo in mildly affected dogs	slowly progressive (months)	rapid progression to recumbency (weeks)

Centronuclear myopathy is a relatively common disorder in the breed, which was originally described as a type II fiber deficiency (11), then later as hereditary Labrador retriever myopathy (HLRM) (2,12) and autosomal recessive muscular dystrophy of Labradors (13). Affected puppies appear normal at birth. Muscular weakness, an awkward gait, exercise intolerance, and muscle atrophy without myalgia typically become apparent by 3 to 5 mo, with a few puppies showing signs at 6 to 8 wk. The age of onset and severity of clinical signs vary dramatically among affected littermates (2,9,14,15). Severely affected dogs exhibit a low head carriage and a short strided, stilted gait (2,9). Results of neurologic examination are normal, except for consistent patellar hyporeflexia or areflexia (2,9,14,15). Clinical signs are worse with stress, exercise, excitement, or cold temperatures (9). Serum CK is normal to mildly elevated and on EMG examination, spontaneous electrical activity and bizarre high frequency discharges are seen (2,9). Muscular weakness and atrophy are typically slowly progressive, but a few affected puppies will be recumbent within 1 to 2 mo (2,15). Clinical signs stabilize after 12 mo of age in mildly affected dogs (2,9,15). Although pathological changes may be extremely variable in CNM, a marked variability in myofiber size with small and large groups of atrophic fibers having round to angular shapes and fibers of both types is typical. In some cases, type 1 fibers predominate (type 2 fiber deficiency) (11). Myonecrosis is infrequent and inflammation is not found. In 1 study, central nuclei were present in 74% of muscle biopsies, with numbers of fibers containing central nuclei ranging from 1.1% to 23.9% (12). Dogs over 1 y of age contained higher percentages of fibers containing central nuclei (12). Most Labrador puppies with CNM are biopsied at less than 1 y of age, when central nuclei may not appear increased. Loss of patellar reflexes, angular atrophy of both fiber types, and type 1 fiber predominance all suggest that CNM could be primarily a neuropathy, but on microscopic examination, peripheral nerves and spinal cord appear normal (9,12,14–16). Centronuclear myopathy has an autosomal recessive inheritance pattern (1). Recently the causative genetic mutation has been identified and a DNA test made commercially available (17,18).

Muscular dystrophies are a heterogenous group of inherited degenerative noninflammatory muscle disorders (9). Most of the muscular dystrophies recognized in dogs are associated with an absence of the cytoskeletal protein dystrophin, caused by genetic mutation of the dystrophin gene (9). The very large dystrophin gene is located on the X-chromosome, so these disorders are inherited as X-linked recessive traits, clinically apparent in male dogs and transmitted by female dogs that are typically asymptomatic (9,19). Canine X-linked muscular dystrophy has been most completely described in golden retrievers (10,19), but it was reported in a single Labrador retriever puppy in 2002 (10). Dogs with CXMD typically show clinical signs at birth or very early in life consisting of stiff pelvic limbs, weakness, bunny hopping gait, excessive salivation, tongue enlargement, and dysphagia (9,19,20). The Labrador puppy with CXMD was presented primarily for stertor, dysphagia, drooling, and difficulty prehending food (10). Atrophy of the truncal and temporal muscles is common in CXMD, but proximal limb muscles, especially in the thigh, are relatively spared and may actually appear to be hypertrophied (9,19,20). Weakness is progressive, but signs may stabilize after 6 to 12 mo. Initially, spinal reflexes are normal, but they may gradually become depressed due to muscle fibrosis (10,19,20). Serum CK is dramatically increased by 1 d of age, with values increasing steadily and then plateauing at 8 wk of age, when they may approach 100 times that of normal (9,19,20). The Labrador puppy with CXMD had a serum CK of 56 482 U/L (reference range 69 to 708 U/L) at 14 wk of age (10). Histologic features of CXMD include necrosis, phagocytosis, variability in myofiber size, and calcific deposits within muscle fibers (9,10,19). Diagnosis is confirmed by demonstrating an absence of membrane dystrophin with immunohistochemical staining for dystrophin and dystrophin-associated proteins (9,10).

The clinical features of the Labrador puppy reported here included many findings characteristic of CNM, including generalized muscle atrophy, weakness, mildly elevated CK, and diminished spinal reflexes, with normal ability to eat and drink. The early onset of signs and the exclusive involvement of male puppies from 3 litters were more suggestive of CXMD. Evaluation of muscle biopsies resulted in the identification of a novel myopathy most closely resembling the inherited myopathy of Great Danes. Clinicians and pathologists should be aware of the clinical and histological features of this new, rapidly progressive, and debilitating, inherited myopathy of Labrador retrievers.

Since only male puppies were clinically affected, and affected male puppies were present in separate litters from the same dam bred to different sires, the putative inheritance pattern is X-linked recessive.