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GM2 Gangliosidosis in British Jacob Sheep
J. Comp. Path. 2014, Vol. 150, 253e257
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ScienceDirect www.elsevier.com/locate/jcpa
SPONTANEOUSLY ARISING DISEASE
GM2 Gangliosidosis in British Jacob Sheep M. E. Wessels*, J. P. Holmes†, M. Jeffrey‡, M. Jacksonx, A. Mackintoshk, E. H. Kolodny{, B. J. Zeng{, C. B. Wang{ and S. F. E. Scholes‡ * AHVLA Preston, Barton Hall, Garstang Road, Barton, Preston PR3 5HE, † AHVLA Shrewsbury, Kendal Road, Harlescott, Shrewsbury SY1 4HD, ‡ AHVLA Lasswade, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, x GSTS Pathology, Genetics Centre, Guy’s Hospital, London SE1 9RT, k AHVLA Carmarthen, Job’s Well Road, Johnstown, Carmarthen SA31 3EZ, UK and { Division of Neurogenetics, Department of Neurology, New York University School of Medicine, 400 East 34th Street, Room 311, New York, NY 10016, USA
Summary GM2 gangliosidosis (TayeSachs disease) was diagnosed in 6- to 8-month-old pedigree Jacob lambs from two unrelated flocks presenting clinically with progressive neurological dysfunction of 10 day’s to 8 week’s duration. Clinical signs included hindlimb ataxia and weakness, recumbency and proprioceptive defects. Histopathological examination of the nervous system identified extensive neuronal cytoplasmic accumulation of material that stained with periodic acideSchiff and Luxol fast blue. Electron microscopy identified membranous cytoplasmic bodies within the nervous system. Serum biochemistry detected a marked decrease in hexosaminidase A activity in the one lamb tested, when compared with the concentration in age matched controls and genetic analysis identified a mutation in the sheep hexa allele G444R consistent with TayeSachs disease in Jacob sheep in North America. The identification of TayeSachs disease in British Jacob sheep supports previous evidence that the mutation in North American Jacob sheep originated from imported UK stock. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. Keywords: GM2 gangliosidosis; Jacob sheep; TayeSachs disease
Lysosomal storage diseases are a group of genetic and, less frequently, induced disorders characterized by the accumulation of intralysosomal material (Maxie and Yousef, 2007). This can be the result of an absence or defect in a specific lysosomal enzyme, aberrant trafficking of enzymes or a lack of cofactors or transport proteins (Walkley, 1998). The subsequent accumulation of partly hydrolysed molecules leads to the formation of abnormal intracellular inclusions or storage bodies. No clear explanation is available as to how these defects lead to cellular dysfunction, but a number of theories have been suggested including direct toxic effects of accumulated substrate, reduction of precursor molecules leading to dysfunction of metabolic pathways, formation of meganeurites and ectopic dendrites altering electrical
Correspondence to: M. E. Wessels (e-mail: [email protected]. uk). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2013.10.003
properties of neurons, neuronal apoptosis and inflammation (Walkley, 1998; Jeyakumar et al., 2003). GM2 gangliosidosis is the prototypical lysosomal storage disease that was first described clinically in man by Tay (1881) and clinicopathologically by Sachs (1887). Defects in the b-hexosaminidase (Hex) enzyme and cofactor system lead to the accumulation of GM2 ganglioside in lysosomes. This system consists of two major isoenzymes, composed of two subunits e a and b (Hex A [ab] and Hex B [bb]), a minor isoenzyme S and the GM2 activator protein (Suzuki and Suzuki, 2008). Defects in Hex A lead to TayeSachs disease, those involving Hex B cause Sandhoff’s disease and GM2 activator protein deficiency results in the AB variant. These diseases are characterized by progressive neurological dysfunction with variable onset and severity related to the degree of enzyme deficiency. In animals, naturally occurring GM2 gangliosidosis has been described in cats (Cork et al., 1978; Neuwelt Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
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et al., 1985), dogs (Karbe, 1973; Cummings et al., 1985), pigs (Kosanke et al., 1978), Muntjak deer (Fox et al., 1999), American flamingo (Zeng et al., 2008), Jacob sheep in the USA (Porter et al., 2011) and a rabbit (Rickmeyer et al., 2013). We describe the clinical, pathological, molecular and clinical biochemical presentation of GM2 gangliosidosis in British Jacob sheep. Pedigree Jacob sheep from two flocks were investigated for neurological disease. In October and November 2008, two 8-month-old male lambs (A and B) were submitted for post-mortem examination. Lamb A was reported to have been born with mild contraction of the forelimb flexor tendons. At 7 months of age intermittent periods of recumbency, ataxia, abduction of the forelimbs and poor weight gain were noted. Lamb B was submitted alive and presented with ataxia, a mild swaying gait and hindlimb weakness with partial flexure of the hock joints. The lamb had marked difficulty in rising from sternal recumbency and proprioceptive deficits in the forelimbs and had deteriorated over a period of 8 weeks. In November 2011, two 6-month-old lambs (one female and one castrated male; lambs C and D) were submitted from a second unrelated flock, presenting with progressive hindlimb weakness over a 10-day period with ataxia, involuntary twitching, star gazing and decrease in body condition. Lambs A, C and D were killed by intravenous injection of pentobarbitone before submission. Lamb B was submitted alive, allowing the collection of heparinised, EDTA and clotted blood samples prior to death. Plasma and serum were harvested from these samples and stored at 70 C. Air-dried blood spots on filter paper were taken from the spleen of lamb B. On post-mortem examination, the lambs were in fair to poor body condition, weighing 20 kg (A), 26 kg (B) 19 kg (C) and 21 kg (D). Lamb A was found to be cryptorchid with a small intra-abdominal left testis. No other significant findings were present on gross examination of the lambs. For lambs B, C and D, total brain weights were 86.0 g, 100.5 g and 100.9 g, respectively, and cerebellar weights (expressed as % of whole brain weight) were 9.9 g (11.3%), 9.9 g (9.9%) and 11.2 g (11.1%), respectively. Diffuse weak autofluorescence of the cerebral cortex was seen when the brains of lambs C and D were examined under ultraviolet (UV) light. Samples of brain, spinal cord, pituitary gland, trigeminal ganglion and nerve, eye, liver, pancreas, thyroid gland, trachea, lung, kidney, skeletal muscle (semitendinosus and triceps) and heart were collected and fixed in 10% neutral buffered formalin and processed routinely. Sections were stained with haematoxylin and eosin (HE). Additional sections of brain
were stained using periodic acideSchiff (PAS), Sudan black (SB) and Luxol fast blue (LFB). Frozen sections were obtained from brain cryoprotected in 30% sucrose and stained with PAS, SB, LFB and oil red O (ORO). Cryoprotected unstained sections were examined with UV light (330e385 nm) using a DM400 dichroic mirror. Neurofilament immunohistochemistry (IHC) was performed on selected central nervous system (CNS) sections using clone N52 recognizing NF200 independent of phosphorylation status (catalogue number N0142; Sigma, St Louis, Missouri, USA) following antigen retrieval in citrate buffer (pH6.0 at 97 C) for 10 min. Sites of primary antibody binding were ‘visualized’ using a polymerbased immunoperoxidase method (EnVision+; Dako, Glostrup, Denmark). Microscopically, within the brain and spinal cord there was diffuse marked neuronal perikaryonal swelling with pale amphophilic granular material or microvacuolar change associated with eccentric displacement of nuclei and Nissl substance (Fig. 1). Spinal, trigeminal and retinal ganglion cells were similarly affected. There was occasional clear vacuolation of neuron cytoplasm and rare neuronal necrosis. Meganeurite formation was occasionally detected in the substantia nigra. Multifocal swelling of nerve processes was highlighted by neurofilament IHC (Fig. 2). Changes were most striking in the cerebellum, involving Purkinje cells with axonal spheroid formation in the internal granular layer and in folial and deep white matter (Fig. 3). Rare scattered axonal spheroids were present within the white matter of the spinal cord. Many astrocytes had increased cytoplasmic eosinophilia and
Fig. 1. Many neurons are distended by faintly stained amphophilic cytoplasmic storage material that displaces the Nissl substance. There is meganeurite formation (arrowheads) and astrocytic hypertrophy (arrows). Substantia nigra. HE. Bar, 25 mm.
GM2 Gangliosidosis in Jacob Sheep
Fig. 2. Segmental swellings within neuronal process with evidence of accumulation of storage material (arrowheads). Substantia nigra. IHC. Bar, 50 mm.
Fig. 3. Purkinje neurons and their dendrites are distended by storage material and multiple spheroids are present in the internal granule cell layer and folial white matter. Inset shows intense staining of storage material in neurons including Purkinje cells and their dendrites in frozen section. Cerebellum. PAS. Bar, 100 mm.
prominent processes (Fig. 1). Small numbers of lymphocytes and macrophages were seen multifocally in neuroparenchymal perivascular sites and in cerebellar meninges. Small foci of mineralization of the neuroparenchyma and similar moderate change in the leptomeninges were noted. LFB staining of cryoprotected brain sections showed intense staining of neuronal intracytoplasmic material and neuronal processes. No storage material was evident within axonal spheroids in LFB-stained sections. PAS staining of routinely processed brain showed only weak staining. However, cryoprotected frozen sections showed intense PAS and equivocal SB intraneuronal staining (Fig. 3). ORO failed to stain material in
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frozen sections. Examination of cryoprotected unstained sections with UV light found autofluorescence of stored material in neurons. For electron microscopical examination, a cerebellar hemisphere was collected from lamb B and fixed in 4% paraformaldehyde solution. Tissue blocks (1 mm3) were post-fixed in osmium tetroxide, dehydrated and embedded in araldite. Tissue sections were studied using a Jeol 1200EX electron microscope. Ultrastructurally, neuronal perikarya and dendrites contained numerous membranous cytoplasmic bodies. The cytoplasmic bodies each consisted of parallel or concentric lamellae and often entirely filled the perikaryon (Fig. 4). Similar, but less frequent inclusions were also present in astroglia. Total hexosaminidase was assayed in both serum and plasma from lamb B using 4-methylumbelliferyl-b-D-N-acetylglucosamine (4MUG) as the synthetic substrate and Hex A activity was measured in plasma using the specific sulphated substrate (4MUGS) for this enzyme (Landels et al., 1991). Samples from four clinically normal sheep (North Country Cheviot breed) were tested simultaneously as controls. Hex A activity was only 2.6% in lamb B compared with the control sheep, indicating a marked deficiency. Total Hex activity in both plasma and serum of lamb B was 16.9% and 37.3% that of the controls, respectively (Table 1). Genetic analysis as described by Torres et al. (2010) undertaken on air-dried blood spots on filter paper from lamb B found this animal to have a homozygous recessive missense mutation at nucleotide position 1330, consistent with the TayeSachs Jacob sheep hexa mutant allele G444R. The clinical, pathological, biochemical and molecular genetic findings associated with GM2 gangliosidosis have been reported in Jacob sheep in North America (Torres et al., 2010; Porter et al., 2011). Although only lamb B was confirmed to have GM2 gangliosidosis, the clinical and pathological presentation of the other three lambs suggests a similar presumptive diagnosis. Our findings are consistent with those seen in American Jacob sheep, with only minor differences noted in the pathology. Cryoprotected sections of CNS were not examined in the cases from North America, but intense PAS and equivocal SB staining was evident in the CNS tissue from the UK cases using this technique. Routine processing to paraffin wax appears to remove much of the stored material as demonstrated both in our own case series and those reported from North America in which PAS staining was weak. Where cryoprotected sections were examined in other species with GM2 gangliosidosis there was strong PAS and SB
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trophic change was more evident in the UK sheep. This is unlikely to be a reflection of differences in the duration of the disease process as animals were of comparable age. No explanation can be given for the foci of mineralization in the leptomeninges and neuroparenchyma, but necrosis with subsequent dystrophic mineralization secondary to the degenerative changes within the neuroparenchyma are a possible cause. A novel observation in Jacob sheep was the detection of autofluorescence grossly and microscopically. This is a feature of some of the sphingolipidoses (Jolly and Walkley, 1997). A definitive diagnosis of GM2 gangliosidosis relies on the measurement of tissue or blood b-hexosaminidase and isoenzyme activity plus analysis of brain ganglioside content. The ready availability of serum and plasma enzyme analysis in the UK led to these tests being undertaken in preference to tissue analysis. The blood enzyme values obtained for the one lamb tested are typical of those seen in human patients and results are most consistent with the diagnosis of GM2 gangliosidosis (TayeSachs variant), as although there was a reduction in total Hex activity this was not as low as would be expected with the Sandhoff variant. Although based on analysis of only one animal, these findings do raise the possibility of using blood as an ante-mortem test as opposed to relying on post-mortem tissues, which have been analysed most frequently in previous reports involving animals. At the time of the initial investigation, a molecular genetic test was not available in the UK. With this test becoming more widely available, biochemical testing has been superseded by detection of the gene mutation, which means that the diagnosis of this recessively inherited disorder can be made in the live animal and a breeding programme introduced to reduce/eliminate the mutant gene from the flock/population. Torres et al. (2010) describe in detail the biochemistry and molecular genetics in North American Jacob sheep. A missense mutation of the hexa cDNA results in diminished Hex A activity accounting for the
Fig. 4. TEM of granule cell neuron (cerebellum) showing numerous membranous cytoplasmic bodies. Bar, 1 mm.
positivity (Karbe, 1973; Cork et al., 1978; Fox et al., 1999; Suzuki and Suzuki, 2008). In contrast to the American cases, we did not find unequivocal evidence of decreased intensity of myelin staining, while our investigations found evidence of accumulation of storage material in nerve processes, which is reported in man and may play a part in the pathophysiology of TayeSachs disease (Walkley, 1998; Suzuki and Suzuki, 2008). The lack of storage material within axonal spheroids, as seen in LFB-stained sections, is a feature of many lysosomal storage diseases and suggests a common pathogenic mechanism. These consist of accumulated organelles normally transported along axons (Walkley, 2004). A similar pattern of astrocytosis was seen, but hyper-
Table 1 Biochemical analysis of total Hex and Hex A activity in plasma and serum for lamb B and control sheep Blood constituent
Enzyme
Enzyme activity* Jacob sheep B
Serum Plasma Plasma *
Total b-hexosaminidase† Total b-hexosaminidase† b-hexosaminidase A‡
269 92 1
Control sheep
Jacob sheep (B) as % of mean control
1
2
3
4
Control mean
637 452 37
705 511 35
959 715 49
586 500 31
722 544 38
Absolute values as nmol substrate degraded per hour and per ml of serum and plasma respectively (nmol/h/ml). Determined with 4MUG substrate. ‡ Determined with 4MUGS substrate. †
37.3 16.9 2.6
GM2 Gangliosidosis in Jacob Sheep
biochemical and subsequent pathological changes seen in affected Jacob sheep. This was confirmed using restriction site analysis in lamb B for the UK case. Pedigree analysis of North American Jacob sheep suggests that this mutation was introduced following the introduction of an imported ram into the USA from the UK (F. Horak, unpublished information). It is likely therefore that this condition has been present in the UK for some time, but has not been reported previously.
Acknowledgements We wish to acknowledge the help of technical staff at AHVLA, GSTS Pathology and the Division of Neurogenetics of the Department of Neurology, New York University School of Medicine. The help of F. Horak is acknowledged. This work was carried out as part of scanning surveillance activities funded by Defra’s food and farming group.
References Cork LC, Munnell JF, Lorenz MD (1978) The pathology of feline GM2 gangliosidosis. American Journal of Pathology, 90, 723e734. Cummings JF, Wood PA, Walkley SU, de Lahunta A, DeForest ME (1985) GM2 gangliosidosis in a Japanese spaniel. Acta Neuropathologica, 67, 247e253. Fox J, Li Y-T, Dawson G, Allerman A, Johnsrude J et al. (1999) Naturally occurring GM2 gangliosidosis in two Muntjak deer with pathological and biochemical features of human classical TayeSachs disease (type B GM2 gangliosidosis). Acta Neuropathologica, 97, 57e62. Jeyakumar M, Thomas R, Elliot-Smith E, Smith DA, van der Spoel AC et al. (2003) Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis. Brain, 126, 974e987. Jolly RD, Walkley SU (1997) Lysosomal storage diseases of animals: an essay in comparative pathology. Veterinary Pathology, 34, 527e548. Karbe E (1973) GM2 gangliosidosis (amaurotic idiocies) types I, II and III. Animal model: canine GM2 gangliosidosis. American Journal of Pathology, 71, 151e154. Kosanke SD, Pierce KR, Bay WW (1978) Clinical and biochemical abnormalities in porcine GM2 gangliosidosis. Veterinary Pathology, 15, 685e699.
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Landels EC, Ellis IH, Bobrow M, Fensom AH (1991) TayeSachs disease heterozygote detection: use of a centrifugal analyser for automation of hexosaminidase assays with two different artificial substrates. Journal of Medical Genetics, 28, 101e109. Maxie MG, Yousef S (2007) Nervous system. In: Pathology of Domestic Animals, 5th Edit., Vol. 1, MG Maxie, Ed., Elsevier Saunders, Philadelphia, pp. 281e457. Neuwelt EA, Johnson WG, Blank NK, Pagel MA, MasienMcClure C et al. (1985) Characterization of a new model of GM2 gangliosidosis (Sandhoff’s disease) in Korat cats. Journal of Clinical Investigation, 76, 482e490. Porter BF, Lewis BC, Edwards JF, Alroy J, Zeng BJ et al. (2011) Pathology of GM2 gangliosidosis in Jacob sheep. Veterinary Pathology, 48, 807e813. Rickmeyer T, Schoniger S, Petermann A, Harzer K, Kustermann-Kuhn B et al. (2013) GM2 gangliosidosis in an adult pet rabbit. Journal of Comparative Pathology, 148, 243e247. Sachs B (1887) On arrested cerebral development with special reference to its cortical pathology. Journal of Nervous and Mental Diseases, 14, 541e553. Suzuki K, Suzuki K (2008) Lysosomal diseases. In: Greenfield’s Neuropathology, 8th Edit., S Love, DN Louis, DW Ellison, Eds., Hodder Education, London, pp. 515e599. Tay W (1881) Symmetrical changes in the region of the yellow spot in each eye of an infant. Transactions of the Ophthalmological Society of the United Kingdom, 1, 53e64. Torres PA, Zeng BJ, Porter BF, Alroy A, Horak F et al. (2010) TayeSachs disease in Jacob sheep. Molecular Genetics and Metabolism, 101, 357e363. Walkley SU (1998) Cellular pathology of lysosomal storage disorders. Brain Pathology, 8, 175e193. Walkley SU (2004) Secondary accumulation of gangliosides in lysosomal storage disorders. Seminars in Cell and Developmental Biology, 15, 433e444. Zeng BJ, Torres PA, Viner TC, Wang ZH, Raghavan SS et al. (2008) Spontaneous appearance of TayeSachs disease in an animal model. Molecular Genetics and Metabolism, 95, 59e65.
July 8th, 2013 ½ Received, Accepted, October 14th, 2013
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
SPONTANEOUSLY ARISING DISEASE
GM2 Gangliosidosis in British Jacob Sheep M. E. Wessels*, J. P. Holmes†, M. Jeffrey‡, M. Jacksonx, A. Mackintoshk, E. H. Kolodny{, B. J. Zeng{, C. B. Wang{ and S. F. E. Scholes‡ * AHVLA Preston, Barton Hall, Garstang Road, Barton, Preston PR3 5HE, † AHVLA Shrewsbury, Kendal Road, Harlescott, Shrewsbury SY1 4HD, ‡ AHVLA Lasswade, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, x GSTS Pathology, Genetics Centre, Guy’s Hospital, London SE1 9RT, k AHVLA Carmarthen, Job’s Well Road, Johnstown, Carmarthen SA31 3EZ, UK and { Division of Neurogenetics, Department of Neurology, New York University School of Medicine, 400 East 34th Street, Room 311, New York, NY 10016, USA
Summary GM2 gangliosidosis (TayeSachs disease) was diagnosed in 6- to 8-month-old pedigree Jacob lambs from two unrelated flocks presenting clinically with progressive neurological dysfunction of 10 day’s to 8 week’s duration. Clinical signs included hindlimb ataxia and weakness, recumbency and proprioceptive defects. Histopathological examination of the nervous system identified extensive neuronal cytoplasmic accumulation of material that stained with periodic acideSchiff and Luxol fast blue. Electron microscopy identified membranous cytoplasmic bodies within the nervous system. Serum biochemistry detected a marked decrease in hexosaminidase A activity in the one lamb tested, when compared with the concentration in age matched controls and genetic analysis identified a mutation in the sheep hexa allele G444R consistent with TayeSachs disease in Jacob sheep in North America. The identification of TayeSachs disease in British Jacob sheep supports previous evidence that the mutation in North American Jacob sheep originated from imported UK stock. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. Keywords: GM2 gangliosidosis; Jacob sheep; TayeSachs disease
Lysosomal storage diseases are a group of genetic and, less frequently, induced disorders characterized by the accumulation of intralysosomal material (Maxie and Yousef, 2007). This can be the result of an absence or defect in a specific lysosomal enzyme, aberrant trafficking of enzymes or a lack of cofactors or transport proteins (Walkley, 1998). The subsequent accumulation of partly hydrolysed molecules leads to the formation of abnormal intracellular inclusions or storage bodies. No clear explanation is available as to how these defects lead to cellular dysfunction, but a number of theories have been suggested including direct toxic effects of accumulated substrate, reduction of precursor molecules leading to dysfunction of metabolic pathways, formation of meganeurites and ectopic dendrites altering electrical
Correspondence to: M. E. Wessels (e-mail: [email protected]. uk). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2013.10.003
properties of neurons, neuronal apoptosis and inflammation (Walkley, 1998; Jeyakumar et al., 2003). GM2 gangliosidosis is the prototypical lysosomal storage disease that was first described clinically in man by Tay (1881) and clinicopathologically by Sachs (1887). Defects in the b-hexosaminidase (Hex) enzyme and cofactor system lead to the accumulation of GM2 ganglioside in lysosomes. This system consists of two major isoenzymes, composed of two subunits e a and b (Hex A [ab] and Hex B [bb]), a minor isoenzyme S and the GM2 activator protein (Suzuki and Suzuki, 2008). Defects in Hex A lead to TayeSachs disease, those involving Hex B cause Sandhoff’s disease and GM2 activator protein deficiency results in the AB variant. These diseases are characterized by progressive neurological dysfunction with variable onset and severity related to the degree of enzyme deficiency. In animals, naturally occurring GM2 gangliosidosis has been described in cats (Cork et al., 1978; Neuwelt Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
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et al., 1985), dogs (Karbe, 1973; Cummings et al., 1985), pigs (Kosanke et al., 1978), Muntjak deer (Fox et al., 1999), American flamingo (Zeng et al., 2008), Jacob sheep in the USA (Porter et al., 2011) and a rabbit (Rickmeyer et al., 2013). We describe the clinical, pathological, molecular and clinical biochemical presentation of GM2 gangliosidosis in British Jacob sheep. Pedigree Jacob sheep from two flocks were investigated for neurological disease. In October and November 2008, two 8-month-old male lambs (A and B) were submitted for post-mortem examination. Lamb A was reported to have been born with mild contraction of the forelimb flexor tendons. At 7 months of age intermittent periods of recumbency, ataxia, abduction of the forelimbs and poor weight gain were noted. Lamb B was submitted alive and presented with ataxia, a mild swaying gait and hindlimb weakness with partial flexure of the hock joints. The lamb had marked difficulty in rising from sternal recumbency and proprioceptive deficits in the forelimbs and had deteriorated over a period of 8 weeks. In November 2011, two 6-month-old lambs (one female and one castrated male; lambs C and D) were submitted from a second unrelated flock, presenting with progressive hindlimb weakness over a 10-day period with ataxia, involuntary twitching, star gazing and decrease in body condition. Lambs A, C and D were killed by intravenous injection of pentobarbitone before submission. Lamb B was submitted alive, allowing the collection of heparinised, EDTA and clotted blood samples prior to death. Plasma and serum were harvested from these samples and stored at 70 C. Air-dried blood spots on filter paper were taken from the spleen of lamb B. On post-mortem examination, the lambs were in fair to poor body condition, weighing 20 kg (A), 26 kg (B) 19 kg (C) and 21 kg (D). Lamb A was found to be cryptorchid with a small intra-abdominal left testis. No other significant findings were present on gross examination of the lambs. For lambs B, C and D, total brain weights were 86.0 g, 100.5 g and 100.9 g, respectively, and cerebellar weights (expressed as % of whole brain weight) were 9.9 g (11.3%), 9.9 g (9.9%) and 11.2 g (11.1%), respectively. Diffuse weak autofluorescence of the cerebral cortex was seen when the brains of lambs C and D were examined under ultraviolet (UV) light. Samples of brain, spinal cord, pituitary gland, trigeminal ganglion and nerve, eye, liver, pancreas, thyroid gland, trachea, lung, kidney, skeletal muscle (semitendinosus and triceps) and heart were collected and fixed in 10% neutral buffered formalin and processed routinely. Sections were stained with haematoxylin and eosin (HE). Additional sections of brain
were stained using periodic acideSchiff (PAS), Sudan black (SB) and Luxol fast blue (LFB). Frozen sections were obtained from brain cryoprotected in 30% sucrose and stained with PAS, SB, LFB and oil red O (ORO). Cryoprotected unstained sections were examined with UV light (330e385 nm) using a DM400 dichroic mirror. Neurofilament immunohistochemistry (IHC) was performed on selected central nervous system (CNS) sections using clone N52 recognizing NF200 independent of phosphorylation status (catalogue number N0142; Sigma, St Louis, Missouri, USA) following antigen retrieval in citrate buffer (pH6.0 at 97 C) for 10 min. Sites of primary antibody binding were ‘visualized’ using a polymerbased immunoperoxidase method (EnVision+; Dako, Glostrup, Denmark). Microscopically, within the brain and spinal cord there was diffuse marked neuronal perikaryonal swelling with pale amphophilic granular material or microvacuolar change associated with eccentric displacement of nuclei and Nissl substance (Fig. 1). Spinal, trigeminal and retinal ganglion cells were similarly affected. There was occasional clear vacuolation of neuron cytoplasm and rare neuronal necrosis. Meganeurite formation was occasionally detected in the substantia nigra. Multifocal swelling of nerve processes was highlighted by neurofilament IHC (Fig. 2). Changes were most striking in the cerebellum, involving Purkinje cells with axonal spheroid formation in the internal granular layer and in folial and deep white matter (Fig. 3). Rare scattered axonal spheroids were present within the white matter of the spinal cord. Many astrocytes had increased cytoplasmic eosinophilia and
Fig. 1. Many neurons are distended by faintly stained amphophilic cytoplasmic storage material that displaces the Nissl substance. There is meganeurite formation (arrowheads) and astrocytic hypertrophy (arrows). Substantia nigra. HE. Bar, 25 mm.
GM2 Gangliosidosis in Jacob Sheep
Fig. 2. Segmental swellings within neuronal process with evidence of accumulation of storage material (arrowheads). Substantia nigra. IHC. Bar, 50 mm.
Fig. 3. Purkinje neurons and their dendrites are distended by storage material and multiple spheroids are present in the internal granule cell layer and folial white matter. Inset shows intense staining of storage material in neurons including Purkinje cells and their dendrites in frozen section. Cerebellum. PAS. Bar, 100 mm.
prominent processes (Fig. 1). Small numbers of lymphocytes and macrophages were seen multifocally in neuroparenchymal perivascular sites and in cerebellar meninges. Small foci of mineralization of the neuroparenchyma and similar moderate change in the leptomeninges were noted. LFB staining of cryoprotected brain sections showed intense staining of neuronal intracytoplasmic material and neuronal processes. No storage material was evident within axonal spheroids in LFB-stained sections. PAS staining of routinely processed brain showed only weak staining. However, cryoprotected frozen sections showed intense PAS and equivocal SB intraneuronal staining (Fig. 3). ORO failed to stain material in
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frozen sections. Examination of cryoprotected unstained sections with UV light found autofluorescence of stored material in neurons. For electron microscopical examination, a cerebellar hemisphere was collected from lamb B and fixed in 4% paraformaldehyde solution. Tissue blocks (1 mm3) were post-fixed in osmium tetroxide, dehydrated and embedded in araldite. Tissue sections were studied using a Jeol 1200EX electron microscope. Ultrastructurally, neuronal perikarya and dendrites contained numerous membranous cytoplasmic bodies. The cytoplasmic bodies each consisted of parallel or concentric lamellae and often entirely filled the perikaryon (Fig. 4). Similar, but less frequent inclusions were also present in astroglia. Total hexosaminidase was assayed in both serum and plasma from lamb B using 4-methylumbelliferyl-b-D-N-acetylglucosamine (4MUG) as the synthetic substrate and Hex A activity was measured in plasma using the specific sulphated substrate (4MUGS) for this enzyme (Landels et al., 1991). Samples from four clinically normal sheep (North Country Cheviot breed) were tested simultaneously as controls. Hex A activity was only 2.6% in lamb B compared with the control sheep, indicating a marked deficiency. Total Hex activity in both plasma and serum of lamb B was 16.9% and 37.3% that of the controls, respectively (Table 1). Genetic analysis as described by Torres et al. (2010) undertaken on air-dried blood spots on filter paper from lamb B found this animal to have a homozygous recessive missense mutation at nucleotide position 1330, consistent with the TayeSachs Jacob sheep hexa mutant allele G444R. The clinical, pathological, biochemical and molecular genetic findings associated with GM2 gangliosidosis have been reported in Jacob sheep in North America (Torres et al., 2010; Porter et al., 2011). Although only lamb B was confirmed to have GM2 gangliosidosis, the clinical and pathological presentation of the other three lambs suggests a similar presumptive diagnosis. Our findings are consistent with those seen in American Jacob sheep, with only minor differences noted in the pathology. Cryoprotected sections of CNS were not examined in the cases from North America, but intense PAS and equivocal SB staining was evident in the CNS tissue from the UK cases using this technique. Routine processing to paraffin wax appears to remove much of the stored material as demonstrated both in our own case series and those reported from North America in which PAS staining was weak. Where cryoprotected sections were examined in other species with GM2 gangliosidosis there was strong PAS and SB
M.E. Wessels et al.
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trophic change was more evident in the UK sheep. This is unlikely to be a reflection of differences in the duration of the disease process as animals were of comparable age. No explanation can be given for the foci of mineralization in the leptomeninges and neuroparenchyma, but necrosis with subsequent dystrophic mineralization secondary to the degenerative changes within the neuroparenchyma are a possible cause. A novel observation in Jacob sheep was the detection of autofluorescence grossly and microscopically. This is a feature of some of the sphingolipidoses (Jolly and Walkley, 1997). A definitive diagnosis of GM2 gangliosidosis relies on the measurement of tissue or blood b-hexosaminidase and isoenzyme activity plus analysis of brain ganglioside content. The ready availability of serum and plasma enzyme analysis in the UK led to these tests being undertaken in preference to tissue analysis. The blood enzyme values obtained for the one lamb tested are typical of those seen in human patients and results are most consistent with the diagnosis of GM2 gangliosidosis (TayeSachs variant), as although there was a reduction in total Hex activity this was not as low as would be expected with the Sandhoff variant. Although based on analysis of only one animal, these findings do raise the possibility of using blood as an ante-mortem test as opposed to relying on post-mortem tissues, which have been analysed most frequently in previous reports involving animals. At the time of the initial investigation, a molecular genetic test was not available in the UK. With this test becoming more widely available, biochemical testing has been superseded by detection of the gene mutation, which means that the diagnosis of this recessively inherited disorder can be made in the live animal and a breeding programme introduced to reduce/eliminate the mutant gene from the flock/population. Torres et al. (2010) describe in detail the biochemistry and molecular genetics in North American Jacob sheep. A missense mutation of the hexa cDNA results in diminished Hex A activity accounting for the
Fig. 4. TEM of granule cell neuron (cerebellum) showing numerous membranous cytoplasmic bodies. Bar, 1 mm.
positivity (Karbe, 1973; Cork et al., 1978; Fox et al., 1999; Suzuki and Suzuki, 2008). In contrast to the American cases, we did not find unequivocal evidence of decreased intensity of myelin staining, while our investigations found evidence of accumulation of storage material in nerve processes, which is reported in man and may play a part in the pathophysiology of TayeSachs disease (Walkley, 1998; Suzuki and Suzuki, 2008). The lack of storage material within axonal spheroids, as seen in LFB-stained sections, is a feature of many lysosomal storage diseases and suggests a common pathogenic mechanism. These consist of accumulated organelles normally transported along axons (Walkley, 2004). A similar pattern of astrocytosis was seen, but hyper-
Table 1 Biochemical analysis of total Hex and Hex A activity in plasma and serum for lamb B and control sheep Blood constituent
Enzyme
Enzyme activity* Jacob sheep B
Serum Plasma Plasma *
Total b-hexosaminidase† Total b-hexosaminidase† b-hexosaminidase A‡
269 92 1
Control sheep
Jacob sheep (B) as % of mean control
1
2
3
4
Control mean
637 452 37
705 511 35
959 715 49
586 500 31
722 544 38
Absolute values as nmol substrate degraded per hour and per ml of serum and plasma respectively (nmol/h/ml). Determined with 4MUG substrate. ‡ Determined with 4MUGS substrate. †
37.3 16.9 2.6
GM2 Gangliosidosis in Jacob Sheep
biochemical and subsequent pathological changes seen in affected Jacob sheep. This was confirmed using restriction site analysis in lamb B for the UK case. Pedigree analysis of North American Jacob sheep suggests that this mutation was introduced following the introduction of an imported ram into the USA from the UK (F. Horak, unpublished information). It is likely therefore that this condition has been present in the UK for some time, but has not been reported previously.
Acknowledgements We wish to acknowledge the help of technical staff at AHVLA, GSTS Pathology and the Division of Neurogenetics of the Department of Neurology, New York University School of Medicine. The help of F. Horak is acknowledged. This work was carried out as part of scanning surveillance activities funded by Defra’s food and farming group.
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July 8th, 2013 ½ Received, Accepted, October 14th, 2013