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©Barbara J. Skelly and Robin J.M. Franklin, Recognition and Diagnosis of Lysosomal Storage Diseases in the Cat and Dog J Vet Intern Med 2002;16:133–141

Lysosomal storage diseases result from a deficiency of enzyme(s) within the lysosomal catabolic pathway. Most lysosomal storage diseases are inherited in an autosomal recessive pattern and involve single enzyme deficiencies. Other, more complex diseases arise from cofactor deficiencies (galactosialidosis and AB variant of GM2 gangliosidosis) or from deficiencies of proteins required for the correct synthesis and targeting of many separate enzyme activities (I-cell disease). Lysosomes serve as subcellular compartments within which the macromolecular constituents of the cell are degraded. They provide a suitable environment for enzyme-catalysed hydrolysis while the other cellular structures are protected from the harmful effects of unconfined degradative enzymes. The enzymes involved in lysosomal catabolism are acid hydrolases, especially exoglycosidases, which act sequentially to catalyse the breakdown of the oligosaccharide side chains of complex macromolecules. Proteases and nucleases are also active within lysosomes, but no clinical syndromes have been described that are associated with their deficiency because of (1) the overlapping substrate specificities of the proteases, and (2) the likelihood that nuclease insufficiency is incompatible with life. Ceroid lipofuscinosis is usually classified as a lysosomal storage disease, and this disease is a proteinosis.

Although some forms of ceroid lipofuscinosis in humans have been shown to be due to deficiencies in lysosomal enzymes, this may not be true for all forms, and no specific deficiencies have been characterized in cats. The inactivity of 1 or more enzymes within the metabolic cascade induces a catabolic blockade and results in storage of substrates of the deficient enzyme. The lysosomal storage disease group comprises more than 30 different diseases organized into subgroup classifications on the basis of the metabolic pathways affected and the type of storage found. The main subgroups are the glycoproteinoses, the oligosaccharidoses, the sphingolipidoses, the mucopolysaccharidoses (MPS), and the proteinoses. Because enzyme specificity is toward the type of glycosidic linkage rather than a specific substrate molecule, there is some overlap among diseases assigned to these separate groups, enabling GM1 gangliosidosis, for example, to be classified as both a glycoproteinosis and a lipidosis. Lysosomal storage diseases have been extensively studied in human patients, and most have been subject to a full biochemical and histopathological investigation. In addition, the availability of more rapid cloning and mutation detection techniques has allowed the molecular basis of disease phenotypes to be elucidated. This level of understanding has helped the process of defining similar if not identical diseases in animals. There are over 50 naturally occurring examples of lysosomal storage diseases in nonhuman species, many of which are described in the cat. The study of this group of diseases is desirable because, although the fundamental biochemical defect may appear simple, the pathophysiological mechanisms leading to the disease phenotype are far from being well understood.

In cat breeding populations, a genetic defect, once recognized, may be bred out of affected families, and therapy is usually not attempted. This is not possible in human families, and extensive research has been directed toward designing therapies in animals that may be adapted to treat human patients. Therapies directed at single enzyme deficiencies include enzyme replacement, bone marrow transplantation, and gene replacement protocols. Bone marrow transplantation was carried out in a human fucosidosis patient35 on the basis of the favourable response to the same procedure in dogs. Similarly, cats with alpha-mannosidosis have received bone marrow transplants that markedly increased concentrations of alpha-mannosidase in the brain and dramatically slowed the disease progression. At present, enzyme replacement therapy is being evaluated for MPS VI in cats before it is attempted in human patients.

Lysosomal storage diseases are all autosomal recessive traits, with the exceptions of Hunter and Fabry diseases. These diseases have not been described in dogs or cats apart from 1 isolated report of Hunter disease in a dog from the United States. The inbred populations of pedigree dog breeds, in particular, provide an ideal environment for the amplification of deleterious alleles, especially when a mutation co-segregates with a desirable breed-specific trait. As many lysosomal storage diseases have been described in domestic short-hair cats, these diseases should not be ruled out in non-pedigree animals. When several animals from subsequent generations have been affected, examination of pedigree information can be informative, and a founder effect can be established.

Lysosomal storage diseases usually do not have a gender predilection, and they affect males and females with equal frequency. The age of onset of the clinical signs can be variable and reflects the severity and consequences of the underlying mutation. Classically, the human diseases were described as type I (early onset with severe phenotype and rapid deterioration) or type II (later onset, milder phenotype, and slow disease progression). Although this phenotypic separation is no longer used, a spectrum of disease severity exists both for individuals carrying different mutations within the same gene and, more surprisingly, family members with the same molecular defect. Thus, phenotypic heterogeneity is a common observation, and delayed or adult onset diseases are frequently encountered in humans. When a storage disease is suspected, the breed of the dog or cat can give clues as to the classification of the disease present. Storage diseases can, however, occur in any breed, and new mutations can be identified at any time, so the appearance of clinical signs in the ‘‘wrong’’ breed should not preclude a diagnosis.

Although the onset of clinical signs can be insidious, deterioration is progressive once established—albeit variably so—both among diseases and within the same disease. Unlike inflammatory or infectious diseases of the central nervous system, however, cats and dogs with neuronal storage do not show rapid deterioration, and many animals survive months to years after the onset of clinical signs. Most storage diseases in cats and dogs are recognized in the immature animal, although some, particularly canine fucosidosis and the adult onset forms of ceroid lipofuscinosis, affect mature animals. It is probable that later onset diseases are recognized less frequently, as inherited disorders are usually suspected in the young. Recent descriptions of storage diseases in a 5-year-old Labrador Retriever (Hunter disease) and a 10-year-old Tibetan Terrier (type unknown) suggest that delayed onset diseases do occur and that their frequency is underestimated. In the latter case, the dog had mild persistent neurological problems over a period spanning several years before diagnosis.

The clinical signs of individual lysosomal storage diseases reflect the abundance of the enzyme substrate within a particular tissue of the body, since substrate accumulation leads, directly or indirectly, to cellular dysfunction. The link between storage and the development of clinical signs is poorly understood, particularly in the nervous system, where storage can lead to diverse and distant consequences. That the accumulation of storage material triggers the neurological dysfunction in some of the lysosomal storage diseases is reinforced by the ability of enzyme replacement through bone marrow transplantation to prevent clinical deterioration in cats deficient in alpha-mannosidase. In other neuronal storage diseases, intraneuronal storage may prove the most benign manifestation in an array of abnormalities, including meganeurite formation, axonal spheroid development, and neuronal death. Axonal spheroids (axonal enlargements containing the same material regardless of the nature of the storage disease) are thought to be responsible for many of the clinical neurological manifestations found in the animal models of storage diseases, although their relationship to the primary enzyme defect remains obscure. One neurological disease for which a reasonably coherent pathophysiological mechanism exists is globoid cell leukodystrophy, where high psychosine concentrations, secondary to the catabolic blockade, lead to oligodendrocyte death. In tissues other than the central nervous system, bone, and cartilage, pathophysiological changes may be caused directly by an increase in the number and size of lysosomes. An example of the way in which cellular distortion due to the accumulation of storage material can lead to clinical pathology is provided by the development of atrioventricular and aortic valvular incompetence in dogs with MPS I and VII. The connective tissue of the heart valves becomes swollen and misshapen in dogs with these storage diseases because of the accumulation of mucopolysaccharide, and cardiac murmurs and the development of congestive heart failure can follow. Again, these clinical signs are alleviated when the accumulation of mucopolysaccharide is prevented by bone marrow transplantation. Most lysosomal enzyme activities are expressed ubiquitously and have a ‘‘house-keeping’’ function; consequently, in the absence of an enzyme, storage products accumulate universally and can be detected in most tissues. The following paragraphs summarize the clinical signs specific to individual body systems.

The diversity of neurological impairment can be daunting to the diagnostician and may give the appearance of several separate disease processes. Animals may show behavioral changes, loss of learned behavior, vacancy, stereotypical behavior, ataxia, proprioceptive deficits, apparent blindness, deafness, and seizure activity. Frequently, however, many of the neuronal storage diseases begin with cerebellar or cerebellovestibular signs such as tremor, ataxia, dysmetria, and nystagmus with progression to paresis and paralysis. These signs are prevalent early in the clinical course for gangliosidoses in dogs and cats, Niemann-Pick disease types A and C and globoid cell leukodystrophy, canine Gaucher disease, and feline alpha-mannosidosis. Later, behavioral abnormalities and seizures may be seen, although these changes may also be found earlier in diseases such as globoid cell leukodystrophy in the Poodle, fucosidosis, and ceroid lipofuscinosis. Neurovisceral phenotypes, so common in humans, are encountered in cats (examples include Niemann-Pick type C and some cases of alpha-mannosidosis) and dogs (the canine GM1 gangliosidoses). Phenotypic heterogeneity is such, however, that a milder form of alpha-mannosidosis has been reported in domestic long-hair cats that lacks the skeletal pathology, ocular abnormalities, and hepatomegaly found in other feline models of the disease.

Diseases such as Niemann-Pick type A and, to some extent, globoid cell leukodystrophy may present with features of a peripheral neuropathy. Both of these diseases feature abnormalities of myelination in the peripheral and central nervous systems. Three cats with Niemann-Pick type A were reported with a demyelinating polyneuropathy, although other cats with Niemann-Pick type A and cats with Niemann-Pick type C showed predominantly cerebellar signs or signs suggestive of more generalized central nervous system pathology. The ceroid lipofuscinoses are a complex group of disorders that tend to be classified according to the age of onset of the (primarily neurological) clinical signs and also according to whether or not subunit c of mitochondrial adenosine triphosphate synthase predominates as a storage product. Three clinical subclasses are suggested to be a separate prepubertal-protracted disease from early adult onset, rapidly progressive, and adult onset, slowly progressive diseases. Clinical signs include visual impairment, ataxia, hypermetria, tremors, seizures, and behavioral abnormalities. Retinal degeneration has been reported in Miniature Schnauzers, Tibetan Terriers, and Cocker Spaniels with ceroid lipofuscinosis, although in younger dogs, blindness appears to be due to a central lesion rather than to a retinal disease. Ceroid lipofuscinosis differs most markedly from many of the other neuronal storage diseases in that signs of diffuse forebrain disease and central blindness can be among the earliest clinical signs recognized. Dogs and cats with glycogenoses have an array of clinical signs peculiar to them, which reflect the distribution of glycogen within skeletal and cardiac muscle as well as in nervous tissue. Glycogenosis type II or Pompe’s disease has been reported in Lapland dogs and is characterized by progressive muscle weakness, megaesophagus, and cardiac abnormalities.

A thorough ophthalmologic examination is recommended when a lysosomal storage disease is suspected, because many diseases manifest with ocular pathology. Progressive corneal opacification or cataract formation has been reported as storage products accumulate, and animals may become quite visually impaired. As with all clinical signs, clinical heterogeneity is common, and there are no pathognomic ocular changes for individual diseases.

Skeletal or connective tissue pathology is recognized in several of the different classes of lysosomal storage disease including, most notably, MPS. Skeletal pathology is also prominent, however, in some types of gangliosidosis (eg, GM1 gangliosidosis in the English Springer Spaniel) and in some glycoproteinoses (eg, alpha-mannosidosis in domestic short-hair, but not Persian cats). Dogs with fucosidosis appear skeletally normal, in contrast to their human analogs. Phenotypic heterogeneity within storage disease groups is emphasized in a comparison between GM1 gangliosidosis in the English Springer Spaniel and the Portuguese water dog in which, although both dogs had skeletal dysplasia, dwarfism and coarse facial features were seen only in the English Springer Spaniel. Human patients with MPS are recognized for their ‘‘gargoyle’’ appearance, which involves the coarsening of the facial features. Although the facial changes in cats are more subtle, cats with MPS VI can be identified as looking dysmorphic in comparison to their littermates. Other skeletal abnormalities include deformation of the long bones, development of dysostoses multiplex, and collapse of the intervertebral disc spaces. Dogs and cats suffering from a deficiency of alpha-L-iduronidase (MPS I) and cats with arylsulfatase B (MPS VI) deficiency display a range of skeletal abnormalities. The spectrum of clinical signs and the presence or absence of skeletal or connective tissue abnormalities can thus help narrow the differential diagnosis list but cannot conclusively categorize an unknown storage disease into a subgroup classification.

In the live animal, a thorough neurological examination will be revealing, and when a multifocal neurological disease has been recognized, lysosomal storage diseases should be among the differential diagnoses.

Haematological and routine biochemical assessment is usually unremarkable, and no consistent parameter abnormalities have been recorded. Examination of blood smears can be informative and can reveal the presence of storage vacuoles within leukocytes. Although storage material may be present in all cells with organelles, not all cells will be vacuolated, since storage reflects the distribution and abundance of substrate material, and the examination of cells from several different tissues may be required. It is not true that storage diseases with a predominantly neurological phenotype require the examination of nervous tissue to allow the identification of vacuolated cells. If a storage disease is suspected, and the peripheral blood smear has proved unremarkable, lymphoid tissue, including the spleen, can show evidence of vacuolation, and lymph node aspirates or biopsies can be diagnostically helpful. When there is gross hepatomegaly, liver aspirates or needle biopsies can help characterize whether a storage disease is likely and can help identify storage material. Even if there is no palpable or radiographic evidence of hepatic pathology, hepatocytes may be extensively vacuolated. If vacuolation is present, then it serves as a reliable indicator of abnormal storage.

Fig 1. Siamese cats with mucopolysaccharidosis (MPS) VI showing facial deformity and frontal bossing of the skull. (b) Neutrophils from the cats in ‘‘a’’ showing multiple, dark-staining, cytoplasmic inclusion bodies (toluidine blue staining, 1,000). (c) Radiograph showing the apparent fusion of lateral cervical and cranial thoracic spine from a cat with MPS VI. (These pictures were provided by Prof Mark Haskins, University of Pennsylvania.)

Radiographic examination can help show bony malformations in animals that appear dysmorphic or have evidence of skeletal pathology (pain, gait abnormalities, and history of fractures). Bony and connective tissue abnormalities frequently characterize MPS (see above Figure), although abnormalities of this nature can also be seen in other classes of storage disease, eg, in some forms of gangliosidosis or glycoproteinosis. The involvement of bones and soft tissues in different classes of lysosomal storage disease has been shown to be mutation-dependent in human studies in which genotype-phenotype correlations have been possible.

Many of the lysosomal storage diseases that present with a neurological phenotype will include a cerebrospinal fluid analysis as part of their investigation. In some, including globoid cell leukodystrophy and fucosidosis, the sample can identify vacuolated macrophages or lymphocytes that are full of storage material.

Peripheral nerve biopsy has been advocated as a diagnostic test for the identification of the characteristic pathology associated with storage diseases. Globoid cell leukodystrophy is the most frequently diagnosed disease by this method, and the technique is well documented. Animals with several other lysosomal storage diseases including Niemann-Pick type A, alpha-mannosidosis, and fucosidosis have demonstrable peripheral nerve pathology, and, in dogs with fucosidosis, the ulnar nerve may be palpably enlarged.

This is perhaps most useful in the diagnosis of a glycogen storage disease, as members of this group manifest, at least initially, muscle pathology and show spontaneous electrical activity during electromyographic examination. Muscle biopsies taken from animals with glycogenoses demonstrate characteristic pathological changes and may be stained for glycogen accumulation within membrane-bound vesicles. Not all glycogen storage diseases are members of the lysosomal storage disease group, however, and in nonlysosomal glycogen storage diseases, glycogen accumulates in the cytosol and not within lysosomal compartments.

Fig 2. Accumulation of dark-staining storage material (arrows) within the cell bodies of neurons in the brain stem of a X-bred dog with GM1 gangliosidosis (toluidine blue–stained resin section, scale bar 20 m). (b) High power electronmicrograph of membranous cytoplasmic bodies (open arrow) and zebra bodies (closed arrow) within a brain stem neuronal cell body from the same dog as illustrated in ‘‘a.’’ The ultrastructural appearance of these inclusions is characteristic of gangliosidoses but is not specific for a particular disease (scale bar 1.5 m).

Although in human medicine, the magnetic resonance imaging (MRI) characteristics of many of the reported lysosomal storage diseases have been described, few veterinary patients have been investigated in this way, and there is a dearth of information about MRI changes found in specific storage diseases in animals. Cairn and West Highland White Terriers with globoid cell leukodystrophy have, however, been studied in more detail, and the MRI changes are reported. Changes consist of diffuse, symmetrical increases in the signal intensity of white matter shown on T2-weighted images. These signal intensity abnormalities correlate with regions of demyelination.

Another, rather more specific diagnostic route attempts to identify evidence of abnormal storage through the analysis of urinary excretory products. Thin-layer chromatography is used to separate abnormal oligosaccharides and glycopeptides in the urine, again by means of normal samples as controls. Characteristic excretory profiles are described for individual storage diseases, particularly in human patients with MPS. Although less commonly measured, dogs with fucosidosis have been found to excrete fucoglycoconjugates,60 whereas cats with mannosidosis have urine containing mannose-rich oligosaccharides. Both enzyme analysis and urinary excretory product analysis require the assistance of a laboratory skilled in the techniques involved. However, the MPS spot test, which involves staining urine on filter paper with toluidine blue, can give a crude indication that the urine contains increased concentrations of glycosaminoglycans.

If a lysosomal storage disease is strongly suspected, then 1 of 2 main routes can be taken to pursue a specific diagnosis in the live animal. The first relies on the identification of the deficient enzyme by assaying for the activities of a selection of lysosomal enzymes, including those known to have been reported as deficient in the breed of animal under investigation. This method provides a definitive diagnosis for the majority of lysosomal storage diseases. Ideally, an age-matched control should be assayed in parallel to provide a normal set of control values. For autosomal recessive diseases, an affected homozygote would be expected to have severely depleted enzyme activity (typically 0–5% of normal), whereas heterozygotes should have approximately 50% of the normal activity. Thus, heterozygotes may be diagnosed through enzyme assay, particularly when pedigree information can assist in the interpretation of results. Even if the precise enzyme is not identified in a preliminary screen, the secondary increase observed in the activities of other lysosomal enzymes when one is deficient is enough to merit further investigation. Suitable substrates for antemortem enzyme analysis include whole-blood leukocytes, liver and kidney biopsy samples, and cultured skin fibroblasts. It is rarely necessary to set up fibroblast cultures from affected animals for the purpose of enzyme analysis, although if complex diseases such as Niemann-Pick type C are to be investigated, cholesterol transport assays usually require cultured fibroblasts.61 As the level of conservation of lysosomal enzyme structure between species is high, enzyme activities in dogs and cats are investigated by the same techniques and artificial substrates as those used in their human counterparts.

The increasing accessibility of molecular genetic technology over the past decade has led to the investigation of the molecular basis of many storage diseases. The 2nd specific diagnostic method thus relies on the identification of the genetic defect. Lysosomal storage diseases are rewarding subjects to study because they are single gene defects and are inherited in an autosomal recessive pattern, making heterozygote detection by methods other than enzyme assay very desirable. To date, molecular genetic tests are available for canine fucosidosis,62 globoid cell leukodystrophy in the Cairn and West Highland White Terriers, MPS I in the dog, MPS VII in the dog, and MPS VI in the cat. New forms of the same disease may be recognized, however, and the experience in human medicine is that each affected family has a novel mutation; therefore, excluding the reported mutation in an unrelated case with suspicious clinical signs cannot categorically rule out the presence of a specific disease. Detailed pedigree information about the animal under investigation can make the interpretation of molecular genetic tests easier.

As the clinical consequences of lysosomal storage diseases are extremely debilitating and the deterioration remorseless, affected animals are usually euthanized, and the disease is examined postmortem. Again, the feature of widespread vacuolation, representing lysosomal storage, is prominent. The nature of the storage, its reaction with specific stains, and its ultrastructural features can go some way toward classifying the disease into one of the subgroups listed in Table 1. Ultrastructurally, the accumulation of soluble substrates gives the appearance of ‘‘empty’’ vacuoles, whereas amphipathic lipid-containing substrates often assume complex conformations known as membranous cytoplasmic bodies or zebra bodies. Differential staining techniques and the use of lectins have enabled storage products to be characterized for each different disease. The ceroid lipofuscinoses are novel among the lysosomal storage disease group in that the nature of the major type of storage material does not readily allow identification of the biochemical defect. At gross postmortem examination of dogs with ceroid lipofuscinosis, there is brain atrophy, and yellow discoloration may be observed. Yellow-brown discoloration of the bowel is also a feature of the disease in Cocker Spaniels.50 Microscopically, there is an accumulation of a fluorescent lipopigment in neurons and other tissues of the body. Subunit c of mitochondrial adenosine triphosphate synthase is the protein component of the stored material in many but not all forms of ceroid lipofuscinosis. Examination of the ultrastructural features of affected tissues reveals electron-dense granular or membranous inclusions that vary in appearance among clinical syndromes.

Fig. 1. Skeletal muscle, thigh; Abyssinian cat. Transverse section of muscle with many PAS-positive, diastase-resistant intrasarcoplasmic inclusions (dark granules) in several muscle fibers. Note the marked variation in fiber diameter and the presence of internalized nuclei. PAS with diastase. Bar = 50 µm.
Fig. 2. Skeletal muscle, thigh; Abyssinian cat. Higher magnification of the polysaccharide inclusions (arrowheads) in the sarcoplasm of two myofibers. Methylene blue-azure II. Plastic embedded semithin section. Bar = 25 µm.
Fig. 3. Spinal cord; Abyssinian cat. Numerous spherical polysaccharide inclusions (arrowheads) are scattered throughout the gray matter. PAS with diastase. Bar = 75 µm.
Fig. 4. Electron micrograph. Myofiber from the skeletal muscle of the thigh; Abyssinian cat. Several variably sized inclusions (arrowheads) are shown in the sarcoplasm of a myofiber. Degeneration of the affected fiber is indicated by Z-band streaming (arrows) and myofilament disarray. Uranyl acetate–lead citrate stain. Bar = 1.0 µm.
Fig. 5. Electron micrograph. Myofiber from the skeletal muscle of the thigh; Abyssinian cat. Nonmembrane-bound, electron-dense, finely granular, and filamentous storage material within the sarcoplasm. Uranyl acetate–lead citrate stain. Bar = 0.16 µm.
Fig. 6. Electron micrograph. Spinal cord; Abyssinian cat. A single round, nonmembrane-bound polysaccaride inclusion (asterisk) nearly completely replaces the cytoplasm in an unidentified cell process. The inclusion is composed of tangled, electron-dense filaments similar to those seen in the skeletal muscle. Uranyl acetate–lead citrate stain. Bar = 0.5 µm.

Glycogen storage disease in cats