NATURAL DISEASE Spontaneous Mucolipidosis in a Cat: An Animal Model of Human I-Cell Disease
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Vet Pathol33:l-13 (1996)
NATURAL DISEASE
Spontaneous Mucolipidosis in a Cat: An Animal Model
of Human I-Cell Disease
N. U. BOSSHARD,M. HUBLER,S. ARNOLD,J. BRINER,M. A. SPYCHER,
H.-J. SOMMERLADE, AND R. GITZELMANN
K. VON FIGURA,
Departments of Pediatrics (NUB, RG) and Pathology (JB, MAS), Faculty of Medicine,
and Department of Reproduction, Faculty of Veterinary Medicine (MH, SA), University of Zurich, Zurich,
Switzerland; Department of Biochemistry 11, Georg-August-University, Gottingen, Germany (HJS, KVF)
Abstract. A 7-month-old female cat was seen for abnormal facial features and abnormality of gait. Facial
dysmorphism, large paws in relation to body size, dysostosis multiplex, and poor growth were noted, and
mucopolysaccharidosiswas suspected. A negative urine test for sulfated glycosaminoglycansand extreme stiffness
of skin indicated a mucolipidosis hitherto unknown in animals. Deficiency of UDP-N-acetylglucosamine:
lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17) ac-
tivity was demonstrated in leukocytes and cultured fibroblasts, which had the appearance of inclusion cells (I-
cells). Activities of a set of lysosomal hydrolases were abnormally low in fibroblasts and excessive in blood
plasma. Postmortem morphology revealed lysosomal inclusions predominantly in fibroblasts but also in en-
dothelial cells and chondrocytes, i.e., in cells of mesenchymal origin. Storage lysosomes contained oligosac-
charides, mucopolysaccharides,and lipids. Tissues most affectedwere bones, cartilage, skin, and other connective
tissues such as those in heart valves, aortic wall, and vocal cords. Parenchymal cells of liver and kidney were
unaffected, as was skeletal muscle. Only a few of the cerebral cortical neurons had lipid inclusions; in sciatic
nerve some axons were affected, but other peripheral nerves were normal. There were striking clinical, bio-
chemical, and morphologic similarities between the disorder in this cat and the human I-cell disease.
Key words: Animal model; cats; I-cell disease; lysosomal storage disease; mucolipidosis.
I-cell disease (mucolipidosis I1 [ML 113) is an auto- facial features, hardened skin, severe skeletal dysplasia,
soma1 recessive genetic disorder in humans resulting stiffening of the joints, growth failure, and psycho-
from the abnormal transport of lysosomal enzymes, motor retardation. 16,23 Early onset and death usually
most apparent in cells of mesenchymal rigi in.'^,^^ The in the first decade distinguishes this disorder from the
disorder is caused by the activity deficit of the Golgi biochemically related mucolipidosis I11 (ML 111, pseu-
enzyme UDP-N-acetylglucosamine : lysosomal en- do-Hurler polydystrophy), which although caused by
zyme N-acetylglucosamine- 1-phosphotransferase the same enzyme deficiency is milder and presents
(GlcNAc-phosphotransferase).This enzyme is respon- later, with survival possible into adulthood. Here, we
sible for the attachment of phosphate groups to man- present a cat affected with a mucolipidosis resembling
nose residues of soluble lysosomal enzymes. The re- human I-cell disease for which hitherto no animal
sulting mannose-6-phosphate (M-6-P) recognition site model has been described.
(together with the M-6-P receptors) directs most sol-
Case Report
uble lysosomal enzymes to the lysosomes. If the M-6-P
recognition site is missing, those lysosomal enzymes, A 7-month-old female domestic short-haired cat was re-
which depend on such targeting, are not properly traf- ferred to the Small Animal Clinic, University of Zurich, be-
ficked to the lysosomes and are released into extracel- cause of abnormal physical features and progressive abnor-
lular space instead. Hence, affected cells are deficient mality ofgait. The mother and three littermates were healthy.
The affected cat was small for its age, weighed 1.75 kg, and
in these enzymes and lack the facility to disassemble preferred to sit with its hind limbs to one side or to lie on
and dispose Of macromolecules~which then remain its side. It walked with its forelegs, but was unable to use its
visible in the form of lysosomal inclusions (I-cells). In hind legs, which it draggedbehind. The range ofmotion was
contrast, Serum and other extracellular fluids have el- limited in its hind legs, but deep pain sensation was present
evated activity levels of these enzymes. in all four legs. There was generalized muscle atrophy, which
I-cell disease in humans is characterized by coarse was most severe in the hind legs. Paws were large in relation
1
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Fig. 1. Face; cat, 8 months of age. Left. Note the broad face with hypertelorism, thickened eye lids, and small ears.
Right. Note frontal bossing.
to body size. There was reduced mobility of the spine. Ab- hours at 4 C. The membrane pellets were resuspended in 20
normal facial features included a flat, broad face with hy- pl H,O and stored at -20 C until assayed.
pertelorism, frontal bossing, a depressed nasal bridge, short For the study of the intracellular distribution of /3-glucu-
ears, and marked thickening of the eyelids (Fig. l), features ronidase, fibroblasts were fractionated into lysosomal and
suggestive of mucopolysaccharidosis. However, the consis- microsomalGolgi fractions by osmotic shock treatment.26
tency of the skin suggested mucolipidosis. The skin felt ex- Cross-contamination between the microsomalGolgi and
tremely hard, stiff, and somewhat thickened, and it fitted lysosomal fractions, as monitored by the activities of the
tightly, especially at the nape. It was impossible to lift the marker enzymes a-mannosidase (lyso~omes),~ NADH-cy-
cat by its nape. tochrome c reductase (microsomes)," and galactosyltrans-
The cat was generally inactive but responsive to stimuli. ferase ( G ~ l g i )was
, ~ ~below 10%.
Vision seemed impaired; the menace reflex was negative bi- In plasma and fibroblast homogenates, lysosomal enzymes
laterally. Ophthalmologic examination revealed a diffuse were assayed as previously described.6
generalized retinal degeneration. Movements became pro- GlcNAc-phosphotransferase (EC 2.7.8.17) activity was
gressively difficult, and the cat had to be hand fed. Eating assayed36using arylsulfatase A (ASA) as acceptor. Unless
provoked severe singultus. The cat was euthanatized at age otherwise stated, the assay contained 0-3 pg of ASA, 22 pM
11 months, and a necropsy was done. /3-[32P]-UDP-GlcNAc(3-6 pCi/mmol), 1Yo (v/v) Triton
X-100, 2 mM ADP, 10 mM CDP-choline, 10 mM MgCl,,
5 mM dimercaptopropanol, 0.125 mM leupeptin, 35 mM
Materials and Methods sodium phosphate (pH 6.7), and up to 10 pl of microsomal
Leukocytes were prepared from 5 ml of heparinized blood membranes in a final volume of 20 pl. After incubation for
with dextran (Macrodex; Pharmacia LKB, Uppsala, Swe- 2 hours at 37 C, the reaction was stopped by addition of 20
den),39skin fibroblasts were cultured by standard methods, p1 of 250 mM Tris/HCl (pH 6.8), 2% (v/v) sodium dodecyl
and proteins were measured according to Lowry. sulphate (SDS), 20 mM dithiotreitol, and 20% glycerol and
For the determination of GlcNAc-phosphotransferase and heating for 5 minutes at 95 C. The radioactively labeled
galactosyltransferase,microsomal membranes were prepared proteins were separated by SDS-polyacrylamide gel electro-
from leukocytes and fibroblasts as follows. Fibroblasts were phoresis in the presence of dithiothreitol and visualized by
harvested by trypsin at confluency, and frozen leukocytes fluorography (exposure up to 10 days). The amount of 32P
were thawed. Cells were suspended in 1 ml of 50 mM Tns- transferred to ASA was quantified by densitometry with a
HC1 (pH 7.4) and 250 mM sucrose and homogenized in a laserscan densitometer (LKB, Bromma, Sweden) and ex-
1-ml glass homogenizer (Wheaton) using a tightly fitting pes- pressed as absorption units x millimeters. The transfer of
tle (pestle A) with 100 strokes on ice. The homogenate was 32Pwas linear for up to 3 hours of incubation and up to 100
transferred to 1s - m l reaction vials and spun for 5 min at pg of microsomal membranes. /3-[32P]-UDP-N-acetylglucos-
4,000 rpm at 4 C in a Eppendorff centrifuge. Supernatants amine (/3-[32P]-UDP-Gl~NA~), the substrate, was synthe-
were transferred to new tubes, and the centrifugation was sized.28The specific activity of the [32P]-UDP-GlcNacwas
repeated. Supernatants were centrifuged at lo5 x g for 1.5 3,000 Ci/mmol, adjusted for the in vitro phosphorylation
Downloaded from vet.sagepub.com by guest on May 14, 2015Vet Pathol 33:1, 1996 Feline Mucolipidosis 3
Fig. 3. Cervical spine, lateral view; cat. Note fusion of
Fig. 2. Fibroblasts in culture; cat. Top. Control cat. Bot- vertebrae. Bar = 5 mm. (Courtesy of James Hayden, R.B.P.,
tom. Affected cat. The cytoplasm is filled with an increased School of Veterinary Medicine, University of Pennsylvania,
number of highly contrasting, spherical lysosomal inclusions. Philadelphia, PA).
Bar = 10 pm. (Differential interference phase contrast, cour-
tesy of Prof. T. Bachi, Institute of Medical Microbiology,
University of Zurich, Zurich, Switzerland.) tervertebral articulations, and dorsal fusion of the cer-
vical spine (Fig. 3). There was marked hip dysplasia
reaction to 3-6 Ci/mmol by the addition of unlabeled UDP- with bilateral luxation of the femoral heads, which
GlcNAc. ASA, the acceptor, was purified from baby hamster were flattened. Both elbow and knee joints were ab-
kidney (BHK2 1) cells overexpressing ASA using an immu- normally shaped and mildly subluxated. Facial bones
noaffinity chromatography method based on the monoclonal were shortened and broadened.
ASA-antibody 20B 1 covalently coupled to Affigel 10 (Biorad)
as previously described.30 Biochemical findings
A urine sample gave a negative toluidine blue test
Galactosyltransferase (EC 2.4.1.38), the reference enzyme,
was measured by a modification of a previously described
for sulfated glycosaminoglycans. In blood plasma, ac-
method.29 tivities of lysosomal acid hydrolases were elevated 7-
Portions of the various tissues removed at necropsy were
97-fold, with the exception of acid lipase and phos-
fixed in buffered neutral 4% formalin, embedded in paraffin,
phatase (Table 1).
and stained with hematoxylin and eosin (HE), elastin-van
Leukocyte GlcNAc-phosphotransferase activity was
Gieson, alcian blue, and periodic acid-Schiff (PAS). For elec-
only 8% of a control, amounting to 11% when related
tron microscopy, specimens were fixed in 2.5% phosphate-
to the reference enzyme galactosyltransferase (Table 2).
buffered glutaraldehyde followed by phosphate-buffered os-
In cultured fibroblasts, GlcNAc-phosphotransferase
mium tetroxide. After dehydration in graded ethanol, tissues
activity was not detectable, whereas the activity of the
were embedded in Epon 8 12, cut on a Reichert Ultrotome,
reference enzyme galactosyltransferase was normal
stained with uranyl acetate and lead citrate, and viewed in
a Philips CM- 10 electron microscope. (Table 2). A large group of lysosomal acid hydrolases
Control cats were domestic short-haired cats of both sexes
had low activity, i.e., 8-5 1% of controls (Table 1); this
7-9 months of age seen at the Small Animal Clinic for ster-
group corresponded with the group of enzymes with
ilization or castration. excessive activity in plasma. A minor group of en-
Results zymes appeared normal.
In contrast to findings in human I-cell disease, fi-
Skin biopsy broblast P-glucuronidase activity was not subnormal
A skin biopsy was taken from the right lateral ab- but was even higher than that of control cells, although
dominal wall. Light and electron microscopy revealed in plasma it was excessive. The kinetic study in fibro-
vacuoles in a great number of cells. Cultured skin fi- blast homogenates of the proposita and a control cat
broblasts showed cytoplasmic inclusions as described showed no difference in K, (0.096 mM versus 0.105
in human I-cells (Fig. 2). mM), whereas v,, was higher in the proposita (21.7
versus 7.5 nmol/minute/mg protein). As revealed by
Radiology osmotic shock cell fractionation, the excessive activity
Radiographs of the entire axial skeleton showed of fibroblast p-glucuronidase was only in part in the
heavily deformed cervical, thoracic, and lumbar ver- microsomal Golgi fraction (1 9% versus 7% in the con-
tebrae with widened vertebral canal, ankylosis of in- trol) and thus not exclusively microsomal.
Downloaded from vet.sagepub.com by guest on May 14, 20154 Bosshard, Hubler, Arnold, Briner, Spycher, Sommerlade, von Figura, and Gitzelmann Vet Pathol 33:1, 1996
Table 1. Lysosomal enzyme activities in blood plasma and cultured fibroblasts of affected and control cats.
Plasma* Fibroblasts?
~
Enzymes Controls Controls
Affected Affected
...~-
Range n Ranae n
~~ ~
Affected
(3-N-acetylgalactosaminidase 17.3 0.60-2.32 3 3.52 24.0-65.6 3
a-N-acetylglucosaminidase 99.6 0.33-3.14 3 0.05 0.34-0.53 3
Arylsulfatase A 64.8 0.20-0.9 1 3 0.55 5.51-6.63 3
Arylsulfatase B 12.1 0.64-1.28 2 0.77 1.28-3.5 1 3
a-fucosidase 233 2.47-3.24 3 0.19 4.46-8.22 3
a-galactosidase 9.25 1.1 2-1.60 3 0.51 0.99-3.79 3
(3-galactosidase 19.2 0.21-0.3 1 3 0.68 11.1-18.3 3
(3-hexosaminidaseA B + 70.2 1.67-8.64 3 19.4 80.7-3 14 3
Iduronate 2-sulfatase* 18.5 1.68-3.87 3 32.9 299 1
a-iduronidase 3.87 0.04-0.09 3 0.30 2.90-3.50 3
a-mannosidase 793 63.5- 121 3 0.96 2.13-5.07 3
(3-mannosidase 64.2 0.66-1.33 3 0.09 1.03 1
Apparently unaffected
a-N-acetylgalactosaminidase 1.97 3.80 1
a-glucosidase (pH 4.0) 0.39 0.29-0.41 3
(3-glucosidase(+ taurocholate) 9.19 2.53-5.85 3
(- taurocholate) 1.88 0.67-1.66 3
Acid lipase 374 169-20 1 2 5.80 4.60-15.7 3
Acid phosphatase 4.33 1.45-2.83 2 108 35.3-6 9.4 3
Sphingomyelinase 1.58 2.624.15 3
Other
p-glucuronidase 189 2.18 1 23.5 8.25- 14.9 2
* Measurement units are nmol/minute/ml plasma.
~
t Measurement units are nmol/minute/mg protein.
f Measurement units are U/mg protein.
Necropsy findings predominantly affecting the cartilage of bones (Fig. 4),
larynx (Fig. 5), and the tracheobronchial tree, but also
A lateral photographic view of the cervical spine is the large blood vessels (Fig. 6), the cardiac valves, and
shown in Figure 3. Histologic examination revealed the fibrous tissue mainly of the dermis. Chondrocytes
widespread storage material staining with alcian blue, were ballooned, and the cytoplasm was filled with nu-
Table 2. Activities of GlcNAc-phosphotransferase and the reference enzyme galactosyltransferase in leukocytes and
cultured fibroblasts of affected and control cats.
Enzymes Affected Control
Leukocytes
GlcNAc-phosphotransferase (AU.mm/pg protein/hour)* 1.8 x 23.0 x
Galactosyltransferase (fmol 'H Gallpg protein/hour)t 0.38 0.54
Specific activity (AU.mm/fmol 3H Gal)$ 4.74 x 10-2 42.6 x
Fibroblasts
GlcNAc-phosphotransferase (AU .mm/pg protein/hour)§ not detectable 9.6 x 10-3
Galactosyltransferase (fmol 3H Gal/pg protein/hour)t 28.8 28.4
Specific activity (AU.mm/fmol 3H Gal)* not detectable 1.35 x 1 0 - 4
* 20 pg microsomes and 3 pg arylsulfatase A as acceptor were incubated under standard conditions with [P3*P]UDP-GlcNAcas donor.
AU.mm = absorption units x mm.
t 20 pg microsomes were incubated with 2.2, lo5 dpm 'H UDP-GlcNAc under standard conditions.
$ Activity quotient GlcNAc-phosphotransferase/galactosyltransferase.
5 25 pg microsomes and 2.6 pg arylsulfatase A as acceptor were incubated under standard conditions with p32P]UDP-GlcNAcas donor.
Downloaded from vet.sagepub.com by guest on May 14, 2015Vet Pathol 33:1, 1996 Feline Mucolipidosis 5
Fig. 4. Rib; cat. Enchondral ossification is preserved. The Fig. 5. Larynx; cat. The chondrocytes ofthe ary-epiglottic
chondrocytes are enlarged but columnation is regular. The cartilage and the perichondral fibroblasts (arrow) contain a
perichondrium is thickened (arrowheads) because of a large large amount of storage material. E = squamous epithelium.
number of storage cells (arrows; see also Fig. 11). A wide HE. Bar = 60 pm.
peripheral zone of cartilage is poorly stained (asterisk). HE.
Bar = 70 wm.
7). Liver, brain, medulla oblongata, peripheral nerves,
esophagus, thymus, spleen, kidneys, uterus, ovaries,
merous vacuoles, the contents of which stained with myocardium, and skeletal muscles were unremarkable
alcian blue and to a minor degree with PAS; nucleoli macroscopically and by light microscopy.
were small and eccentric. Storage was not confined to Electron microscopic examination revealed lysoso-
chondrocytes but was also present in perichondral fi- ma1 storage, which was widespread although not pan-
broblasts. Enchondral ossification appeared to be reg- cytic (Figs. 8-14). With few exceptions, the storage
ular; however, chondrocytes were enlarged and peri- material was membrane bound. Inclusion bodies had
chondral and periostal fibroblasts contained inclu- contents of great variety: floccular material, at times
sions. The walls of the pulmonary artery and aorta associated with globular and electron-opaque material,
were thickened and contained a large number of storage membranous zebra bodies (Fig. lo), and membranous
cells, predominantly in the media but also in the in- cytoplasmic (Fig. lo), curvilinear (Fig. 15), and fin-
tima; cells were arranged in groups, had vacuolated gerprint (Fig. 15) inclusions. Throughout the body, fi-
cytoplasm (Fig. 6 ) , and contained alcian blue-positive broblasts were the most affected cell type. The epi-
material. Aortic and pulmonary valves were myxoid, dermis was normal. Dermal, perineural, and perivas-
thickened, and studded with enlarged fibroblasts con- cular fibroblasts were filled with electron-lucent inclu-
taining storage material. Skin architecture appeared sions also containing more dense material (Fig. 8).
normal; however, collagen fibers were abundant, and Epithelial cells of apocrine glands also contained abun-
fibroblasts were occasionally enlarged and filled with dant inclusions resembling those in fibroblasts. Blood
vacuoles. Cells of the apocrine glands had expanded vessels had inclusions in endothelial cells and pericytes
cytoplasmic area due to numerous small vacuoles (Fig. (Fig. 9). Inclusions in vascular smooth muscle cells
Downloaded from vet.sagepub.com by guest on May 14, 20156 Bosshard, Hubler, Arnold, Bnner, Spycher, Sommerlade, von Figura, and Gitzelmann Vet Path0133 I , 1996
.-)’;-
,.--c .
J
i
Fig. 6. Aorta; cat. Intima and media of the aorta harbor Fig. 7. Skin; cat. The epithelial cells of apocrine glands
a large number of ballooned cells, smooth muscle cells, and (arrow) contain numerous small vacuoles. Ill-defined en-
macrophages, the cytoplasm of which contains vacuoles of larged fibroblasts (arrowheads) lie between collagen fibers of
various sizes. HE. Bar = 150 um. the dermis. Vacuoles of both cell types contain PAS-positive
material. PAS. Bar = 25 urn.
were exclusively in the form of zebra bodies and mem-
branous cytoplasmic bodies (Figs. 10, 13). Skeletal
vacuolation only sporadically. A minority of tubular
muscle cells were unaffected; myocardial myocytes oc-
epithelial cells were loaded with storage lysosomes; the
casionally contained large electron-lucent vacuoles.
contents were of the clear type in the distal epithelia,
Chondrocytes of hyaline cartilage were filled with cy-
toplasmic inclusions (Fig. 1 1). Heterogeneous inclu- whereas in the proximal ones, curvilinear and finger-
print-like structures predominated (Fig. 15). Vascular
sions were seen in cerebral cortical neurons. In the
endothelia differed in the content of storage material,
sciatic nerve, most axons looked normal and were well
independent of organ and tissue; most cells were free
myelinated. Others were filled with polymorphous in-
of inclusions, and excessive storage was not observed.
clusions and had meager myelin sheeths (Fig. 12). Such
In short, lesions were strikingly similar to those seen
changes were not observed in the peripheral nerves of
in children with I-cell disease.
skin, muscle, and liver. Hepatocytes, endothelial cells,
and Kupffer cells were essentially normal; Kupffer cells
rarely contained storage lysosomes. In the portal fields, Discussion
fibroblasts were filled with inclusions contrasting with Several lysosomal disorders have been described in
epithelial cells of bile ductules, which were free of stor- cats;9 they include the counterparts of human muco-
age vacuoles (Fig. 13). Collagen was abundant. Rare polysaccharidoses I, VI, and VII,6 of the sphingolipi-
perivascular lymphocytes lacked inclusions, whereas doses GM, -gangliosidosis, GM,-gangliosidosis, Nie-
plasma cells had some that were always electron dense mann-Pick and Krabbe diseases, and of a-mannosi-
(Fig. 14). In kidney, glomerular endothelia and po- dosis. In all these disorders, neurologic symptoms have
docytes were normal; parietal epithelium cells showed been de~cribed.~ The existence of neurologic involve-
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Fig. 8. Electron micrograph. Skin; cat. Dermal fibroblast stuffed with mostly electron-lucent inclusions also containing
electron-dense membranous and amorphous material. C = collagen. Bar = 0.25 pm.
ment was not excluded in the cat in this report; how- Biochemical, histopathologic, ultrastructural, and
ever, facial dysmorphism and dysostosis multiplex were radiologic findings closely matched those of the human
the leading symptoms and signs. Because these features I-cell disease patients. The deficiency of the GlcNAc-
are not typical of the sphingolipidoses, mucopolysac- phosphotransferase was readily demonstrated in leu-
charidosis was considered but rejected in view of the kocytes and fibroblasts (Table 2). Residual activity in
extreme stiffness of the skin and a negative urine test leukocytes amounted to 10% of that in controls as
for sulfated glycosaminoglycans. A disorder corre- compared with 1-10% in human mucolipidosis I1 and
sponding to human mucolipidosis was suspected in- I11 patient^.^"^,^^ In fibroblasts, no residual GlcNAc-
stead and proven biochemically by excessive extra- phosphotransferase activity was detected. In human
cellular activity of a number of lysosomal hydrolases, patients, residual activity ranges from 0% to 30% of
reduced cellular activity of the same enzymes, and de- in some cases even up to 63°/0,21,22and
ficiency of GlcNAc-phosphotransferase, which was the varies with the acceptor used in the assay, either en-
primary defect. dogenous or exogenous.
Fig. 9. Electron micrograph. Skin; cat. Dermal blood capillary-Endothelial cell (E) with numerous lysosomes with
amorphous, globular electron-dense material. Pericyte with zebra and membranous cytoplasmic bodies (arrows). * =
erythrocyte. Bar = 1 pm.
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Downloaded from vet.sagepub.com by guest on May 14, 2015Vet Pathol 33:1, 1996 Feline Mucolipidosis 9
Fig. 12. Electron micrograph. Sciaticnerve cross section; cat. One axon is poorly myelinated and filled with polymorphous
inclusions. Bar = 0.7 pm.
In human I-cell disease, those lysosomal hydrolases d e n t ~ ,was
~ . ~not
~ substantiated by cell fractionation
that depend on posttranslational modification by the experiments, and a kinetic anomaly was not shown.
action of GlcNAc-phosphotransferase do not find their Nevertheless, the primary enzyme defect and the sec-
'intracellular targets because they are not equipped with ondary anomalies of lysosomal enzyme distribution in
their mannose-6-phosphate marker. Consequently, they this cat matched closely those in I-cell disease patients.
are lost to the extracellular space, as first described by The lysosomal enzymes investigated here have the same
Wiesmann et al.38and confirmed by ~ t h e r ~ . ~ , ~ ,requirements
~ ~ , ~ ~ , ~for~ correct
, ~ ~ ,intracellular
~~ trafficking in hu-
Exempt from this trafficking are acid phosphatase and mans and in cats.
a- and p-glucosidases because they are directed to ly- In I-cell disease patients, the GlcNAc-phosphotrans-
sosomes by mannose-6-phosphate-independent mech- ferase deficiency has been demonstrated in liver, kid-
anisms. 1,27,32 Compared with human I-cell disease pa- ney, spleen, and brain, whereas activities of lysosomal
tients, this GlcNAc-phosphotransferase-deficientcat hydrolases, with the exception of P-galacto~idase,~~J~
had the same two sets of lysosomal enzymes, one of are close to n ~ r m a l . *Thus,
~ , ~ ~it appears that targeting
which appeared to depend on GlcNAc-phosphotrans- of many lysosomal enzymes in parenchymal cells of
ferase modification and had excessive activity in blood these organs does not depend on the mannose-6-phos-
plasma and low activity in fibroblasts and the second phate marker; perhaps, enzymes are taken up by en-
of which was exempt, with the possible addition of two docytosis from the p l a ~ r n a . Although
~ ~ . ~ ~ enzyme ac-
or three more hydrolases (Table 1). tivities in the respective organs of this cat were not
p-glucuronidase seemed to hold an intermediate po- assayed, it was obvious from the ultrastructural study
sition; its activity was excessive in plasma, but it was that parenchymal cells were generally free of storage,
not deficient in fibroblasts. The suspicion that in the in contrast to cells of mesenchymal origin, most of
fibroblasts of this cat we had observed a microsomal which were heavily affected. The sparing of ludney
rather than lysosomal 0-glucuronidase, as seen in ro- parenchyma from storage may explain the absence of
c
Fig. 10. Electron micrograph. Skin; cat. Smooth muscle cells of arterial wall containing numerous lysosomes with
inclusions in the form of zebra bodies (arrowhead) and membranous cytoplasmic bodies (arrow). Bar = 0.5 pm.
Fig. 11. Electron micrograph. Rib, resting cartilage; cat. Two neighboring chondrocytesare filled with storage lysosomes.
Bar = 10 wm.
Downloaded from vet.sagepub.com by guest on May 14, 201510 Bosshard, Hubler, Arnold, Briner, Spycher, Sommerlade, von Figura, and Gitzelmann Vet Pathol 33:1, 1996
Fig. 13. Electron micrograph. Liver, portal field; cat. Clear Fig. 14. Electron micrograph. Liver, portal field; cat. He-
lysosomal vacuoles in fibroblasts (arrowheads) with poly- patocytes (H) lack storage lysosomes. Plasma cells with poly-
morphous contents. Smooth muscle cells of arterial wall con- morphous inclusion bodies (arrows). F = fibroblast, C =
tain storage lysosomes (arrows) with lamellar material. Ep- collagen. Bar = 1 pm.
ithelial cells of bile ductulus (D) are unaffected. Note abun-
dance of collagen (C). M = macrophage. Bar = 2 pm.
was the cartilage of the larynx and the tracheobronchial
tree. Heart valves, perineurium, vocal cords, hepatic
excess mucopolysacchariduria (evidenced by the neg- portal fields, and perivascular sheeths, i.e., structures
ative toluidine test for sulfated glycosaminoglycans) in rich in fibroblasts, were also markedly involved. Skel-
our cat, as it is in patients with I-cell disease.23 etal muscle was essentially unaffected. Muscle cells of
With rare exceptions, the cell morphology in our cat small blood vessels and of the myocardium had few
was that observed in I-cell disease patients. 14,23 Fibro- inclusion bodies. Similarly, little storage was seen in
blasts were predominantly affected, but endothelial cells parietal epithelial cells of glomerula and in a minority
and chondrocytes were also affected. They had nu- of renal tubular cells, and mesangial and endothelial
merous membrane-bound vacuoles filled with PAS- cells were free of it. Glomerular podocytes were inclu-
and alcian blue-positive inclusions consisting of elec- sion-free, as was reported in one case of human I-cell
tron-lucent and-dense material. These dense inclusions disease.20The central nervous system was normal on
had membranous cytoplasmic, curvilinear, or finger- light microscopic examination, and electron micros-
print aspects or appeared as zebra bodies. Thus, it is copy revealed only few neurons of the cerebral cortex
likely that storage lysosomes held oligosaccharides, with zebra bodies and membranous cytoplasmic bod-
glycosaminoglycans, and lipids. ies. Alterations in the peripheral nerves were restricted
As in I-cell patients who have dysostosis multiplex, to the sciatic nerve, where some axons were affected,
the skeletal system of the cat was severely affected, as but peripheral nerves of skin, muscle, and liver were
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Fig. 15. Electron micrograph. Kidney cortex; cat. Proximal tubular epithelial cell containing storage material with
curvilinear (arrowhead) and fingerprint (arrow) patterns, apparently not membrane bound. Bar = 0.3 pm.
not affected. On the whole, pathology findings in this 2 Ben-Yoseph Y, Baylerian MS, Nadler H L Radiometric
cat were remarkably similar to those in humans with assay of N-acetylglucosaminylphosphotransferase and
I-cell disease.23 a-N-acetylglucosaminyl phosphodiesterase with sub-
As far as symptoms and clinical signs can be com- strates labeled in the glucosamine moiety. Anal Biochem
142~297-304, 1984
pared, the homology between this cat and humans with
3 Ben-Yoseph Y, Mitchell DA, Yager RM, Wei JT, Chen
I-cell disease14was striking: facial dysmorphism, stiff- TH, Shih LY: Mucolipidoses I1 and I11 variants with
ness of skin, broadening of paws, reduced mobility of normal N-acetylglucosamine 1-phosphotransferase ac-
the spine, developmental delay, congenital hip dislo- tivity toward a-methylmannoside are due to nonallelic
cation, poor growth, retinal changes, and a rapid course. mutations. Am J Hum Genet 50:137-144, 1992
We believe that this cat represents a true animal model 4 Fishman WH, Goldman SS, DeLellis R: Dual localiza-
of human I-cell disease. (The mother cat was unavail- tion of P-glucuronidase in endoplasmic reticulum and in
able for enzyme studies; further offspring are being lysosomes. Nature 213:457-460, 1967
bred at the Laboratory of Pathology [Dr. M. Haskins], 5 Galjaard H: Genetic Metabolic Diseases. Early Diag-
School of Veterinary Medicine, University of Penn- nosis and Prenatal Analysis. ElseviedNorth-Holland
sylvania, Philadelphia.) Biomedical Press, Amsterdam, The Netherlands, 1980
6 Gitzelmann R, Bosshard NU, Superti-Furga A, Spycher
MA, Briner J, Wiesmann U, Lutz H, Litschi B: Feline
Acknowledgments mucopolysaccharidosis VII due to P-glucuronidase de-
ficiency. Vet Pathol31:435-443, 1994
We thank Dr. M. Haskins, University of Pennsylvania, 7 Glaser JH, McAlister WH, Sly WS: Genetic heteroge-
Philadelphia, for reviewing the manuscript and acknowledge neity in multiple lysosomal hydrolase deficiency. J Pe-
the cooperation of the owners of the cat. A report on this diatr 85192-198, 1974
study was presented in part as a poster at the Ninth Workshop 8 Gusina NB, Tsukerman G L AKTHBHOCTH JIH~OCOMH~IX
of the European Study Group on Lysosomal Diseases, Del- rHApOJIa3 llJIa3MbI ti Jle%KOqHTOBy TOMO- H reTepO3HrOT
phi, Greece, 1993. C pa3JIIIYHbIMH BapUDITaMki I-KJIeTOYHOk 6 o n e 3 ~ [AC-
~.
tivity of lysosomal hydrolases in plasma and leukocytes
in homo- and heterozygotes with different variants of
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Request reprints from Prof. R. Gitzelmann, Department of Pediatrics, University of Zurich, Kinderspital, Steinwiesstrasse
75, 8032 Zurich (Switzerland).
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