Marys Medicine


Degenerative myelopathy in a bernese mountain dog with a novel sod1 missense mutation

Case ReportJ Vet Intern Med 2011;25:1166–1170 F.A. Wininger, R. Zeng, G.S. Johnson, M.L. Katz, G.C. Johnson, W.W. Bush, J.M. Jarboe, An 8-year-old female spayed Bernese Mountain Dog sociation with a protein concentration of 30mg/dL and (BMD) presented to the Bush Veterinary Neurology no nucleated cells. Magnetic resonance imaging of the Service (BVNS) for paraparesis. The dog had been seen spinal cord was performed 21 months after the initial at the BVNS 13 months earlier for evaluation of presentation with a 1.5 T unit,a T2-weighted (T2W), T1- progressive pelvic limb weakness. At that time, the dog weighted (T1W), and half Fourier acquisition single-shot had a short-strided gait in the pelvic limbs, mild postural turbo spin echo (HASTE) sagittal sequences were reaction deficits in the right pelvic limb, normal seg- acquired from the foramen magnum to the sacrum.
mental reflexes, and laxity of the right coxofemoral joint.
Transverse T2W, T1W, and T1W postcontrastb weighted The dog was treated with meloxicam (0.15 mg/kg PO images were acquired from T12 to L4. Spondylosis and a q24h) for 3 days, amantidine (2 mg/kg PO q24h) for mildly increased signal within the dorsal funiculus of the 1 month, and doxycycline (6 mg/kg PO q12h) for 2 weeks spinal cord were noted at the T12–13 disc space on T2W with no improvement noted. The dog had a previous images. The T12–13 disc was well hydrated and similar in history of hypothyroidism that had been treated with size to adjacent discs.
0.3 mg levothyroxine PO q12h.
The dog was euthanized 21 months after the initial Asymmetric general proprioceptive ataxia and spastic presentation because the disease had progressed to paraparesis were present on neurologic examination.
include fecal and urinary incontinence. Histopathologic Postural reactions were delayed in both pelvic limbs and evaluation of formalin-fixed spinal cord was performed segmental reflexes were normal. Initial diagnostic tests at the University of Missouri Veterinary Medical Diag- included a CBC, serum biochemistry, and thoracic and nostic Laboratory. Spinal cord cross sections were spinal radiographs. The results of all tests were within stained by standard methods with luxol fast blue (LFB), normal limits. A DNA test for the SOD1:c.118G 4 A and periodic acid Schiff (PAS) as a counter stain.
mutation commonly associated with canine degenerative Immunostaining was performed to detect phosphoryla- myelopathy (DM) was normal (homozygous for the G ted neurofilament and glial fibrillary acid protein allele).1 Physiotherapy was initiated.
(GFAP) as a measure of axonal loss and gliosis, respec- Sixteen months after the initial presentation, signs had tively. Deparaffinized sections were treated with rabbit progressed to paraplegia with preservation of pain per- anti-GFAP antibodyc without pretreatment, and binding ception. Decreased patellar and withdrawal reflexes were was detected with horseradish-peroxidase-bound second- present in both pelvic limbs and suggested neurodegen- eration that had progressed to include the lower motor Phosphorylated neurofilament in axons was detected neurons associated with the femoral and sciatic nerves.
with a mouse monoclonal antibody,e an analogous Analysis of cerebrospinal fluid obtained by atlanto- mouse detection system and similar chromogen. Sections occipital puncture revealed mild albuminocytologic dis- were lightly counterstained with hematoxylin; other sec-tions were stained identically excluding the primaryantibody.
From the Department of Veterinary Medicine and Surgery (Win- In affected tissue, bilateral areas of sclerosis nearly inger, Coates), the Department of Veterinary Pathobiology (Zeng, devoid of myelin and with decreased axonal density were G.S. Johnson, G.C. Johnson), University of Missouri, Columbia, MO noted in the dorsal portion of the lateral funiculus, adja- Department of Veterinary Pathobiology, Mason Eye Institute, Uni-versity of Missouri, Columbia, MO (Katz); Bush Veterinary cent to the entry of the dorsal roots (Fig 1). Swollen axon Neurology Service, Leesburg, VA (Bush, Jarboe). Clinical assess- sheaths with occasional digestion chambers were present ment and magnetic resonance imaging were performed at the Bush in all white matter tracts. The GFAP staining was more Veterinary Neurology Service. All genetic and histopathologic studies homogenous and intense in these regions of myelin and were performed at the University of Missouri. Some of these data were axon loss, without an obvious increase in astrocytic presented in poster format at the Advances in Canine and Feline nuclei. The associated spinal nerves appeared normal.
Genomics and Inherited Diseases 5th International Conference inBaltimore, MD, September 23, 2010.
Although the distribution of axonal dropout and gliosis Corresponding author: Fred A. Wininger, Department of Veteri- was more diffuse and less concentrated in the dorsal fun- nary Medicine and Surgery, College of Veterinary Medicine, iculi than is typical of DM in SOD1:c.118A homozygous University of Missouri, 900 E. Campus Drive, VMTH-Clydesdale dogs, the overall pattern of histologic lesions, including Hall, Columbia, MO 65211; e-mail: the distribution of axon loss and demyelination in the Submitted March 3, 2011; Revised March 23, 2011; Accepted spinal cord strongly supported a diagnosis of DM.
June 3, 2011.
Resequencing all 5 SOD1 exons in DNA from the Copyright r 2011 by the American College of Veterinary Internal affected BMD revealed a homozygous c.52A 4 T trans- version, which predicts a p.T18S substitution. Nucleotide Novel Mutation of SOD1-Associated Degenerative Myelopathy Microscopic appearance of thoracic spinal cord cross-sections. (A) Low magnification of the spinal cord highlights astrocyte processes by immunohistochemistry to detect glial fibrillary acidic protein reagent and Romulin Red chromogen. Poorly defined foci of fibrillary as-trogliosis are situated lateral to the dorsal root entry zones, and localized staining of the fasciculus gracilis in the dorsal-most aspect of thesection. Bar 5 300 mm. (B) A similar section stained with Luxol fast blue demonstrates myelin loss in the areas of gliosis, which is also mostsevere in the dorsolateral funiculus. (C) Higher magnification on one of the dorsolateral lesions demonstrates GFAP positive astrocyte pro-cesses between remaining axons, next to more normal tissue in the upper left. Staining similar to (A), bar 5 100 mm. (D) Demonstrates thesame areas stained by immunohistochemistry to detect phosphorylated neurofilament M, found in axons. There is reduction in the density ofaxons in the center of the affected tissue, with occasional dilated axons and empty axons sheaths. Bar 5 100 mm, All immunohistochemistriesin this figure ultilize Romulin Red chromogen and hematoxylin counter stain.
sequence electrophorograms from the affected dog and cord sections from the dog, along with corresponding from a normal dog are shown in Figure 2. Spinal cord sections from 7 age-matched control dogs known to be motor neurons from dogs of various breeds that were confirmed to have suffered from DM associated with the immunostained simultaneously under identical condi- SOD1:c.118G 4 A mutation exhibited accumulations of tions. Two of the negative controls had been presented cytoplasmic aggregates that were immuno-positive for for chronic thoracolumbar myelopathies, whereas the SOD1.1 To determine whether the c.52A 4 T mutation other 5 were euthanized for unrelated, nonneurologic was the cause of the neurodegeneration, sections of disease. Each specimen was processed with and without thoracic spinal cord were evaluated by immunohisto- primary antibody to verify the specificity of the staining.
chemistry for the presence of similar SOD1-containing Motor neurons in the thoracic spinal cord of the ventral cytoplasmic aggregates as described previously.1 Spinal gray matter of the propositus contained cytoplasmicaggregates that stained darkly with the anti-SOD1 anti-body (Fig 3). These aggregates appeared to be similar tothose previously described in dogs with DM that werehomozygous for the SOD1:c.118A allele.1,2 No aggre-gates were detected in the 7 control dogs. Patches ofSOD1 immunostaining more intense than the generalbackground were observed in the neuropil of spinal cordsections from some but not all affected and control dogs(Fig 3B, C, E, F). Because this pattern of staining waspresent in both control and affected dogs and was seeninconsistently, it was most likely a postmortem artifact.
Canine DM is characterized by a slowly progressive, often asymmetric, general proprioceptive ataxia and up-per motor neuron spastic paresis of the pelvic limbsbeginning in late adulthood. Ultimately, paraplegia and Nucleotide sequence electrophorograms from a normal dog (top) and the BMD with DM (bottom) showing the c.A52 T trans- thoracic limb involvement develop, necessitating eutha- nasia.2,3 The overall prevalence of the disease among all SOD1 immunohistochemistry of thoracic spinal cord motor neurons from a BMD homozygous for the novel missence mutation in SOD1 showing the cytoplasmic aggregates that stained darkly with an anti-SOD1 antibody (A and B). These aggregates are present in thethoracic spinal cord motor neurons from 2 dogs homozygous for the SOD1:c.118A mutation with clinical degenerative myelopathy (C and D).
The aggregates are absent in 2 clinically normal dogs homozygous for the wild-type SOD1:c.118G allele (E and F).
dogs is estimated at 0.19%, but prevalence varies widely likely that the previously described SOD1:c.118G 4 A among breeds.4 Earlier reports indicate that DM occurs mutation is not the only canine SOD1 mutation that can in the BMD.2,5 The pathogenesis appears to be complex, and immunologic, metabolic, nutritional, oxidative, The diagnosis of DM is based on the clinical charac- teristics of the disease and exclusion of other etiologies by normal spinal MRI and CSF. A definitive diagnosis re- Based on histopathology, DM can be best described as quires histopathologic confirmation.2 In this BMD, the a multisystem central and peripheral axonopathy.2 In age at onset and progression of clinical signs were similar general, the spinal cord pathology of DM is consistent to those in previously reported clinical descriptions of with noninflammatory axonal degeneration. Dogs with DM, although the neurologic signs progressed more DM have characteristic patterns of axon cylinder slowly (21 months) than expected.3,6,9 Different ge- vacuolization and loss. Regional axonal loss is severe in notypic causes could underlie phenotypic variation as many DM affected dogs with complete loss of axonal noted in humans with ALS.17 A subtle intraparenchymal and myelin profiles and replacement by large areas of change was noted on MRI of the caudal thoracic seg- astrogliosis. Lesion distribution in the mid to caudal tho- ments. Intervertebral disc herniation was considered, but racic region varies among cases, but typically there are no specific focal pathologic changes were found at this regions of increased lesion severity in the dorsal portion site. Because of its small size and lack of histologic cor- of the lateral funiculi.3,11 The dog described here had a roboration, the MRI finding was considered incidental.
more diffuse distribution of lesions. A similar distribu- The SOD1:c.52A 4 T missense mutation predicts the tion has been noted in many other cases of DM,6 and the substitution of a serine for threonine at position 18 in the axonal dropout and gliosis in this BMD were consistent amino acid sequence of SOD1. Threonine occurs at with a diagnosis of DM.
position 18 in several other mammalian species, whereas Recently, we reported that dogs homozygous for a isoleucine occurs in humans, the orangutan, the common SOD1:c.118G 4 A missense mutation are at high risk of gibbon, the tufted capuchin, and the guinea pig, and developing DM as they age.1 Numerous mutations in valine occurs in the rat and horse (Fig 4). Although this human SOD1 have been shown to underlie amyotrophic conservative amino acid substitution at a site harboring lateral sclerosis (ALS).12 The clinical and genetic similar- different amino acids across other mammalian species ities between DM and ALS suggest that these diseases are could be a neutral sequence variant unrelated to DM, it analogous. Despite the similarities between DM and appears more likely to be the cause of DM in this BMD ALS, substantial pathological and clinical differences because motor neurons contained cytoplasmic aggre- are observed. DM involves proprioceptive pathways as well as the upper motor neuron tracts in contrast to the aggregates resemble those found in DM cases caused by predominantly motor disease of humans.6,9 The neuro- homozygosity of the SOD1:c.118A allele, in human nal cell body degeneration and loss in the ventral horn of fALS cases associated with many different SOD1 muta- the spinal cord of ALS patients is not a prominent histo- tions, and in transgenic murine ALS models expressing pathlogic finding in DM, although lower motor neuron mutant human SOD1.18–20 The aggregates are thought to signs develop late in the disease.6,9 form because amino acid substitutions force SOD1 to Familial ALS (fALS) accounts for 5–10% of all ALS assume an unstable conformation. It is unclear if the cases.13,14 At least 9 genes and 6 additional mapped loci aggregates cause or contribute to the neurodegeneration have been associated with fALS.15 Since the initial dis- or are a byproduct of other neurodegenerative processes.
covery that mutations in SOD1 can cause ALS,16 more Aggregates binding anti-SOD1 antibodies have been than 145 SOD1 mutations have been identified in ALS consistently absent from spinal cords of healthy dogs patients Thus, it seems lacking known SOD1 mutations. On the other hand, Novel Mutation of SOD1-Associated Degenerative Myelopathy The N-terminal amino acid sequences for superoxide dismutase 1 from 20 mammalian species.
SOD1 antigen-containing aggregates have been detected in spinal cords from some clinically normal dogs hetero- The authors acknowledge Liz Hansen and Corrine zygous for SOD1:c.118, which may be indicative of Mann for their technical assistance.
preclinical or subclinical neurodegenerative processes.1 The study was supported by ACORN grants from the Furthermore, antibodies that bind SOD1 with an aber- American Kennel Club Canine Health Foundation rant conformation have been used to detect similar (1,212, 1,213) and by an unrestricted grant from Re- aggregates in patients that have sporadic ALS without search to Prevent Blindness Inc.
SOD1 mutations.21 Thus, the occurrence of SOD1-containing aggregates in the BMD suggests but does notprove that the disease was caused by homozygosity for the SOD1:c.52 T allele.
1. Awano T, Johnson GS, Wade CM, et al. Genome-wide as- In summary, we have described DM in a BMD that is sociation analysis reveals a SOD1 mutation in canine degenerative typical of previously described DM except for a slower myelopathy that resembles amyotrophic lateral sclerosis. Proc Natl than average rate of disease progression and the DNA Acad Sci USA 2009;106:2794–2799.
test result indicating homozygosity for the wild-type G 2. Coates JR, Wininger FA. Canine degenerative myelopathy.
allele at SOD1:c.118. This finding serves as a reminder Vet Clin North Am Small Anim Pract 40:929–950.
that direct DNA tests indicate the presence or absence of 3. Averill DR Jr. Degenerative myelopathy in the aging Ger- disease-causing alleles but cannot be used to rule out a man Shepherd dog: Clinical and pathologic findings. J Am Vet Med diagnosis because other sequence variants in the same gene or in a different gene might produce a similar dis- 4. Coates JR, March PA, Oglesbee M, et al. Clinical charac- terization of a familial degenerative myelopathy in Pembroke Welsh ease phenotype. A definitive conclusion as to whether or Corgi dogs. J Vet Intern Med 2007;21:1323–1331.
not homozygosity of the SOD1:c.52 T allele was the 5. Kathmann I, Cizinauskas S, Doherr MG, et al. Daily con- cause of the DM in the BMD will require clinical and trolled physiotherapy increases survival time in dogs with suspected pathological evaluations of additional canine SOD1:c.52 degenerative myelopathy. J Vet Intern Med 2006;20:927–932.
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