Journal of
J.Neurol. 221, 137--149 (1979)
rqeurao t~) by Springer-Verlag 1979
Original Investigations Adult Type Mucolipidosis with fl-Galactosidase and Sialidase Deficiency Histological and Biochemical Studies* T. Kobayashi ], M. Ohta 2, I. Goto 3, Y. Tanaka 4, and Y. Kuroiwa 3 ~National Akasaka Hospital, Chikugo, Departments of 2Neuropathology and 3Neurology, Kyushu University 60, Fukuoka, and 4Chemistry, Kurume University, Kurume, Japan
Summary. A case of adult type mucolipidosis with fl-galactosidase and sialidase deficiency is described. This patient, a woman aged 20, had mental retardation, macular cherry-red spots, corneal clouding, gargoyle-like face, cerebellar ataxia, myoclonus and convulsions beginning at the age of 14. Bony deformities, vacuoles in the peripheral lymphocyte and foamy cells in the bone marrow were also noted. Biopsy study of the sural nerve and vermiform appendix disclosed many vacuoles in almost every kind of cells, although the accumulated substance in these vacuoles could not be characterized histochemically or ultrastructurally. Deficient leukocyte fl-galactosidase and sialidase were confirmed. There was increased urinary sialoglycopeptide and increased siliac acid and hexosamine in the glycoprotein of lymphocytes. Leukocytes sialidase activities of the parents were 30 to 50% of the control values. These results suggest a genetic defect of sialidase.
Key words: Mucolipidosis - fl-galactosidase deficiency - Sialidase deficiency V a c u o l e - Sialoglycoprotein.
Zusammenfassung. Ein Fall der Mukolipidose im Erwachsenenalter mit flGalaktosidase- und Sialidasemangel wurde bemerkt. Die Patientin, ein zwanzigj/ihriges Friiulein, wies Intelligenzmangel, Kornealtrtibung, Gargoylismus, zerebellare Ataxie, Myoklonus und Konvulsion auf, die im Alter von 14 Jahren auftraten. Die skeletale Deformitgt und die Vakuolenbildung in peripheren Lymphocyten und in f6rmigen Zellen im Knochenmark waren auch bemerkbar. Bei Probeuntersuchungen des N.suralis und Appendix vermiformis wurden viele Vakuolen in fast allen Zellen gefunden, aber das gespeicherte Material in diesen Vakuolen konnte durch enzymhistochemische * This work was supported by a grant from the Japanese Ministry of Health and Welfare Address for offprint requests." Dr. M. Ohta, Department of Neuropathology, Neurological Institute, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan
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T. Kobayashi et al. und ultrastrukturelle Untersuchungen nicht charakterisiert werden. Die Mängel der fl-Galaktosidase und der Sialidase in Leukozyten wurden bemerkt. Es gab vermehrtes Sialylglykopeptid im H a r n und Sialylsäure und Hexosamin im Glykoprotein der Lymphozyten. Leukozytensialidase der Eltern wurde in 3 0 - - 5 0 % der normalen Menge gefunden. Dieses Resultat ergibt, daß unser Fall einen genetischen Defekt der Sialidase aufweist.
Introduction Juvenile or adult cases with macular cherry-red spots, ataxia, myoclonus and gargoyle-like face have recently been reported [ 1 , 3 - - 5 , 7, 10, 11, 15, 20, 21,23]. In these cases fl-galactosidase was deficient in leukocytes or other tissues, but the pathogenesis of this disorder has been uncertain. Wenger et al. [21] reported sialidase as well as fl-galactosidase deficiency in fibroblasts, and suggested that sialidase deficiency was the primary defect. The clinical, morphological and biochemical studies in an adult case with macular cherry-red spot and other characteristic manifestations are described.
Case Report The patient, a woman aged 20, was born of healthy and unrelated Japanese parents. The family history was unremarkable. Her early growth and development were below the average. Generalized convulsive seizures occurred frequently after the age of 14, and 1 year later her extremities moved involuntarily on attempting any purposeful movement such as writing or
Fig. 1. Face of patient with gargoyle-like features Fig. 2. Fundoscopy manifestating cherry-red spot
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Fig. 3. Lymphocyte accompanying several vacuoles in cytoplasm. Giemsa x 1000 Fig. 4. Electron micrograph of lymphocyte with membrane-bound vacuoles containing floccular material. Some also contained membranous, granular and dense materials, x 15,000
reaching for objects. These symptoms were gradually progressive and she could not walk after the age of 17, so, the age of 19, was admitted to the National Akasaka Hospital. She had a coarse face with slight hypertelorism, saddle-shaped nose and thick eyebrows (Fig. 1). The liver and spleen were not palpable. She had numerous angiokeratomas on her soles and palms. On neurological examination, her mentation was retarded (IQ on WAIS was 58). Her vision, 20/50 on the right and 30/50 on the left, was uncorrectable. She had bilateral macular cherry-red spots (Fig. 2). Diffuse granular clouding of cornea was also seen with the slit lamp. Ocular movements were not smooth and horizontal nystagmus was observed on lateral gaze. She had difficulty in speaking, which was explosive and stuttering. There was limb and truncal ataxia with myoclonus and she could neither walk nor stand. Deep reflexes were hyperactive but there were no pathological reflexes and no sensory impairment. Peripheral blood counts and urinalysis were normal. About 30% of peripheral lymphocytes contained cytoplasmic vacuoles (Fig. 3). There were numerous foamy cells with positive PAS staining in the bone marrow. Serum transaminases, alkaline phosphatase, lactic dehydrogenase, total protein and its electrophoretic pattern, cholesterol, phospholipid, triglyceride and urea nitrogen were all normal. There was anterior beaking of L4 and L5 vertebral bodies on X-ray, hut there was no remarkable deformity elsewhere. The electroencephalographic studies showed moderate to marked abnormalities with disorganized background activity and occasional bilateral multiple spikes. The cerebrospinal fluid was normal. Electromyogram and motor and sensory nerve conduction studies were normal. CT scanning revealed no remarkable abnormalities.
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Anticonvulsant medication was effective in controlling myoclonus and generalized convulsions, but visual acuity and mentation deteriorated gradually.
Histological and Electron Microscopic Methods The preparations of peripheral lymphocytes were stained with Giemsa, PAS, toluidine blue, Sudan black B and Sudan III. Acid phosphatase staining was added. For electron microscopic study, 3% cacodylate buffered glutaraldehyde was layed over the buffy coat of heparinized blood and postfixed in 2% osmium tetroxide. A part of the surgically obtained vermiform appendix was immediately frozen in isopentane, cooled by dry ice, for routine histological and histochemical study. Another part was fixed with 3% glutaraldehyde and 1% osmium tetroxide. Each specimen of osmicated venous blood buffy coat, sural nerve and veriform appendix was dehydrated with alcohol and embeded in Epon 812. Thick and thin sections were obtained with Porter-Blum MT2 ultratome. Thin sections were stained with uranyl acetate and lead citrate and examined with an electron microscope, Hitachi HS-9.
Biochemical Methods
1. Analysis of Urinary Glycopeptide The analysis of urinary excretion of acid mucopolysaccharide and glycopeptide was performed according to the previous report with some modifications [17].
2. Biochemical Study of Peripheral Lymphocytes About 100 ml of venous blood with EDTA was obtained from the patient and six age- and sex-matched controls. Lymphocytes were separated by the Ficoll-Conray method. The lymphocytes, dried by lyophilization, were weighed out and delipidized with chloroform methanol (2 : 1 and 1 : 2, v/v), and then digested by in 0.1M tris buffer (pH 8.0) at 45°C for 48h. After deproteinization with 10% trichloroacetic acid, the digested solution, which was not precipitated with 5% cetylpyridinium chloride, was dialyzed against water for 48h. Hexose was estimated by the anthrome method using galactose as a standard. Sialic acid was determined by Warren's method [22] after hydrolysis with 0.1 N sulfuric acid at 80°C for I h. Hexosamine was determined by the indole-hydrochloric acid method after hydrolysis with 3 N hydrochloric acid at 100°C for 8 h in a sealed tube.
3. Leukocyte and Serum Lysosomal Enzymes The enzymes, except for sialidase, were measured according to Troost et al. [19]. Sialidase assay was carried out according to Thomas et al. [18] with some modifications. Substrates for the sialidase assay were fetuin, N-acetyl neuramin lactose and urinary glycopeptide (the first peak of column chromatogram, which was desalted and lyophilized (Fig. 10)). The incubation mixture was composed
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with 150 lal of leukolysate, 40 lal of substrate (fetuin: 25 m g / m l , N-acetyl neuramin lactose: 2.5 m g / m l or urinary glycopeptide: 25 m g / m l ) , 10 lal o f 1 M acetate buffer (pH 4.0). After incubation for 2 to 8 h at 37°C, the released free N-acetyl neuramic acid was determined by W a r r e n ' s m e t h o d [22]. After subtraction of the leukocyte and substrate blanks and correction of the interference o f 2 - - 3 deoxyribose, the enzyme activities were expressed as n m o l / m g protein/h. Protein was determined by the m e t h o d o f L o w r y et al. [8]. All assays were performed in duplicate.
Results
1. Histological and Ultrastructural Studies of Lymphocytes A b o u t 30 to 40% ofperipheral lymphocytes had several vacuoles in their cytoplasm (Fig. 3). Vacuoles were negative on Giemsa staining, PAS reaction, toluidine blue staining and Sudan black B and III, but positive on acid phosphatase staining (Table 1). Ultrastructurally the vacuoles were m e m b r a n e - b o u n d , usually 0.5 la to 1 la, very rarely up to 2 la in diameter and had moderately dense sparse floccular materials, small dense materials up to 0.2la in diameter and very occasionally glycogen granules in electron lucent matrix (Fig. 4). N o lamellated bodies or zebra bodies were encountered. N o abnormality was confirmed in the other cell organelles and in the nucleus.
2. Sural Nerve Examination Biopsied sural nerve contained two fascicles. M a n y vacuoles were seen in the epineurial and endoneurial fibroblasts (Fig. 5). There were no changes suggestive of the nerve fiber degeneration, which was also confirmed by quantitative studies. There were no a b n o r m a l deposits in the endoneurium. The vacuoles were also seen in the Schwarm cell (Fig. 6), and perineurial and endoneurial epithelium. Their ultrastructure was similar to those seen in the circulating lymphocytes. Scanty floccular materials, dense bodies and glycogen granules, especially rich in fibroblasts, were seen (Fig. 6), m a n y of which apposed each other and were frequently confluent. The cells with accumulated vacuoles did not show any other remarkable cytoplasmic changes.
Table 1,, Histochemical findings of various cells
PAS Sudan black B Sudan III Toluidine blue
Lymphocyte vacuoles
Foamy cells in bone marrow
Nerve cells in Auerbach's plexus
-
+ -
+++ + -
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Fig. 5. Transverse section of sural nerve. Vacuole-loaded fibroblasts in endoneurium (arrow). Loss and degenerative changes of myelinated fibers not evident. Toluidine blue x 650 Fig.6. Electron micrograph of sural nerve. Fibroblast and Schwann cell contained similar membrane-bound vacuoles. Many glycogen granules in fibroblast. × 15,000
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Fig. 7. Auerbach's plexus of vermiform appendix. Enlarged nerve cells, some loaded with PAS positive granules. PAS x 140 Fig. 8. Electron micrograph of cytoplasmic inclusions, myelin figure, in nerve cells of Auerbach's plexus, x 15,000
3. Examination of Vermiform Appendix Numerous balooned nerve cells were found in Auerbach's and Meissner's plexuses, which were positive with PAS staining (Fig. 7). Ultrastructurally the abnormally enlarged neurons contained numerous electron dense granules. These were zebra bodies or myelin figures (Fig. 8). Free ribosome, Golgi's complex and mitochondria were also prominent. The satellite cells were also loaded by numerous similar vacuoles and dense bodies. However, these vacuoles depicted somewhat different features from those in lymphocytes and fibroblasts; the content was more dense, more filamentous with frequent dense materials, membrane bodies and lipid droplets. Membranes of many vacuoles were occasionally continuous to the smooth endoplasmic reticulum (Fig. 9). In addition, very occasionally, a lamellar cupshaped wrapping structure was seen around the vacuole, suggesting sequesteration (Fig. 9). Golgi's complex, however, was not so hyperdeveloped in the satellite cells. Many vacuoles, similar to those in the lymphocytes were also present in the smooth muscle cells.
4. Urinary Glycopeptide The amount of sialic acid and hexose in the glycopeptide of the urine was increased 5 to 10 times, compared with those of controls (Fig. 10). However, the
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Fig. 9. Cytoplasmic vacuoles in satellite cells of Auerbach's plexus, membrane-bound. Their content was rauch more filamentous and frequent in membranous structures and lipid droplets. Vacuolar membranes were continuous in some part. Lamellar cup-shaped wrapping structure around vacuoles also seen (arrow). x 26,500
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Polysaccharide in urine
/Jglml
i
40(]
u
20C
os
....
--.%
0
6013
to
trol 0
20
;~ 40 60 tube number
80
~
Fig. 10. DEAE cellulose column chromatogram of urine. After application of 2000 ml urine to Bio-Gel P-6 column and elution with distilled water, eluates positive for cetylpyridinium chloride were applied. First 20 tubes (15 ml/tube) were eluted with 0.1 M tris buffer (pH 7.0) and linear gradient elution was conducted with NaC1 (0 to 1 M), in tris buffer. Hexose by orcinolsulfric acid method; sialic acid by Ehrlich method; uronic acid by carbazole method• First peak (*) was used as substrate for sialidase assay
uronic acid of mucopolysaccharide was not increased. The excretion of uronic acid was 1.56 mg/day, which was within normal range (normal control 0.8--9.3 m g / d a y , n = 15).
5. Biochemical Study of Lymphocytes (Table 2) Sialic acid and hexosamine in chloroform-methanol insoluble residue of lymphocyte were increased 2 to 3 times, which was evident in relation to protein content, dry weight or numbers of lymphocytes.
6. Lysosomal Enzyme Activities (Tables 3 and 4) The leukocyte fl-galactosidase was abnormally low; other lysosomal enzyme activities in leukocyte and serum fl-galactosidase level were normal.
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Table 2. Glycoprotein in lymphocytes Patient
Sialic acid Hexosamine Hexose
Controls
4.4 17.6 218
Mean + S.D.
(Range)
1.4_+ 0.4 5.4_+ 1.2 116 _+48
(1.0- 2.1) (3.5- 7.6) (73 -204)
Values are shown as gg/mg of chloroform-methanol insoluble residue
Table 3. Lysosomal enzyme activities Patient
Leukocyte B-galactosidase a-galactosidase N-acetylB-glucosaminidase B-glucuronidase a-mannosidase a-glucosidase B-glucosidase a-fucosidase Arylsulfatase A Serum B-galactosidase
Father
Mother
Controls (n = 17) Mean _+S.D.
Range
7.9 28.9 916.1
44.4 54.2 1066.7
35.7 41.8 855.9
71.6+ 15.3 36.3+ 11.6 864 _+ 174
(50.0- 92.8) (22.0- 62.2) (606 - 1934)
13.8 58.9 15.8 13.8 34.4 121.0
53.3 77.8 19.6 26.7 26.7 72.3
20.0 85.3 25.3 21.8 26.7 60.0
14.5_+ 6.6 84.8_+ 36.4 15.2_+ 5.0 12.8_+ 4.2 35.7_+ 5.4 68.9_+ 10.6
(1.7- 26.0) (45.5- 151.0) (8.2- 23.3) (5.419.6) (29.1- 43.0) (58.1- 117.0)
5.6
n.d.
n.d.
9.9_+
5.1
(4.0-
18.0)
Values are shown as nmol/mg protein/h in leukocytes and nmol/ml/h in serum. Substrates are p-nitrophenol derivatives except for arylsulfatase A (p-nitrocatechol sulfate) n.d.: not determined
Table 4. Sialidase activities in leukocytes Fetuin
N-acetylneuramin lactose
Urinary glycopeptide
Patient Father Mother
0 0.098 0.074
0.279 n.d. n.d.
0 n.d. n.d.
Controls Number Range
7 0.187--0.574
4 0.163--0.754
3 0.172--0.753
Values are shown as nmol/mg protein/h n.d.: not determined
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The leukocyte fl-galactosidase in the parents was slightly lower than the control ranges. Sialidase in leukocytes was deficient with fetuin and extracted urinary glycopeptide, but normal with N-acetyl neuramin lactose. Leukocyte sialidase activities in the parents were about 30 to 50% of the control values.
Discussion Our case has clinical features of both sphingolipidosis and mucopolysaccharidosis; macular cherry-red spots, angiokeratoma and progressive neurological symptoms as sphingolipidosis, and gargoyle-like face, corneal clouding and bony deformities such as mucopolysaccharidosis. Therefore, this case is compatible with mucolipidosis which Spranger et al. [12] proposed. Fucosidosis, mannosidosis and mucosulfatidosis were denied by enzyme studies. GMl-gangliosidosis and mucolipidosis I, II and III are unlikely because of the age of onset and clinical manifestations. Since Goldberg et al. [3] reported an adult case with macular cherry-red spot, corneal clouding and fl-galactosidase deficiency, several similar cases have been published. Our case had clinical manifestations similar to those previously reported. Most of these cases had vacuoles in lymphocytes, fibroblasts and other tissues. Therefore, we first paid attention to the characteristics of the vacuoles. The previous authors reported that the vacuoles were seen only in lymphocytes, fibroblasts or Kupffer cells in the liver, but in our study, the vacuoles were extensively present in the peripheral lymphocytes, fibroblasts, Schwann cells, perineurial epithelium, smooth muscle cells and endothelial cells, but not in the axons of the peripheral nerves. An essential derangement of the metabolic pathway common to these different kinds of cells might be reasonably considered in this disorder. These vacuoles were not stained with any dyes except for acid phosphatase staining, and electron microscopically, they had no special structures such as lamellated bodies or finger print structures, which are usually seen in ceroid lipofuscinosis or sphingolipidosis [6, 13]. Therefore, the accumulated substance in the vacuoles was obscure, but the vacuoles were positive with acid phosphatase staining. Ultrastructurally they were bound by a unit membrane. They had glycogen granules and dense substance, although very scant. Moreover, they seemed to be autophagiosized, which is compatible to the finding of the sequesteration suggested by Moe et al. [9]. These histochemical and ultrastructural findings suggest that the vacuoles are lysosomes [2]. The inclusions of the nerve cells of the vermiform appendix were stained with PAS and Sudan black B. Ultrastructurally there were many myelin-like figures or lamellated bodies, which had the same ultrastructure of inclusions in various conditions such as sphingolipidosis or mucopolysaccharidosis. These inclusions were found only in the nerve cells and satellite cells. Therefore, the accumulating substance in the neurons may be different from those in the other cells. Enzymatically some authors speculated that fl-galactosidase deficiency was not the primary but the secondary defect inhibited by abnormally accumulated substance, because of normal serum fl-galactosidase activities in the patients and
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n o r m a l leukocyte activities in the p a r e n t s [1, 16, 21]. O n the o t h e r hand, several cases h a d r e d u c e d fl-galactosidase activity in the serum or leukocytes o f the p a r e n t s [10, 23]. In o u r case,/~-galactosidase activity was very low in leukocytes a n d n o r m a l in serum. L e u k o c y t e / ~ - g a l a c t o s i d a s e activity in the p a r e n t s was lower than n o r m a l c o n t r o l values. Therefore, one can n o t conclude at present that flg a l a c t o s i d a s e is s e c o n d a r i l y inhibited by a n o t h e r m e t a b o l i c defect. W e m e a s u r e d sialidase in leukocytes instead o f f i b r o b l a s t s . L e u k o c y t e sialidase activities are lower t h a n those in fibroblasts, b u t they can be easily assayed with the i n c u b a t i o n time o f 2 to 8 h. These sialidase activities d e t e r m i n e d b y W a r r e n ' s m e t h o d are c o m p a t i b l e to those by the i s o t o p e m e t h o d o f Strecker et al. [14]. L e u k o c y t e sialidase o f o u r case was deficient with fetuin a n d extracted u r i n a r y g l y c o p e p t i d e , b u t n o r m a l with N-acetyl n e u r a m i n lactose. A l t h o u g h the exact cause o f this d i s c r e p a n c y is obscure, leukocyte sialidase m a y have enzyme specificity to substrates. T h e r e was an increase o f siliac acid in the g l y c o p r o t e i n o f urine a n d l y m p h o cytes, which suggests the cause to be sialidase deficiency. C o n s i d e r i n g the result o f low sialidase activities o f the p a r e n t s , sialidase deficiency is at least a f u n d a m e n t a l defect in o u r case.
Acknowledgement. The authors wish to thank Drs. M. Kawabuchi and H. Nagara for their cooperation.
References 1. Fukunaga, H., Hirose, K., Beppu, H., Uono, M., Suzuki, Y.: Two siblings with mucolipidosis. Clin. Neurol. (Tokyo) 16, 566--573 (t976) 2. Ghadially, F. N.: Lysosomes. In: Ultrastructural pathology of the cell, pp. 291--368. London, Boston: Butterworths 1975 3. Goldberg, M. F., Cotlier, E., Fichenscher, L. G., Kenyon, K., Enat, R., Borowsky, S. A.: Macular cherry-red spot, corneal clouding, and fl-galactosidase deficiency. Clinical, biochemical, and electron microscopic study of a new autosomal recessive storage disease. Arch. Intern. Med. 128, 387--398 (1971) 4. Goldstein, M., Kolodny, E. H., Gascon, G. G., Gilles, F. H.: Macular cherry-red spot, myoclonic epilepsy, and neurovisceral storage in a 17 year-old girl. Trans. Am. Neurol. Assoc. 99, 110--112 (1975) 5. Kuriyama, M., Umezaki, H., Okada, S., Tanaka, Y., Ishii, N.: Adult mucolipidosis with B-galactosidase deficiency: a clinical report, with studies ofurinary sialic acid-rich substance. Clin. Neurol. (Tokyo) 18, 358--363 (1978) 6. Lazarus, S. S., Vethamany, V. G., Schneck, L., Volk, B. W.: Fine structure and histochemistry of peripheral blood cells in N iemann-Pick disease. Lab. Invest. 17, 155--170 (1967) 7. Loonen, M. C. B., Lugt, L. v. d., Franke, C. L.: Angiokeratoma corporis diffusum and lysosomal enzyme deficiency. Lancet 1974 II, 785 8. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265--275 (1951) 9. Moe, H., Rostgaard, J., Behnke, O.: On the morphology and origin of virgin lysosomes in the intestinal epithelium of the rat. J. Ultrastruct. Res. 12,396--403 (1965) 10. Orii, T., Minami, R., Sukegawa, K., Sato, S., Tsugawa, S., Horino, K., Miura, R., Nakao, T.: A new type of mucolipidosis with fl-galactosidase deficiency and glycopeptiduria. Tohoku J. exp. Med. 107, 303--315 (1972) 11. Shibata, R., Yokota, K., Takashima, S., Mitsudome, A., Kurokawa, T.: A case of mucolipidosis. Brain Develop. (Tokyo) 7, 392--399 (1975)
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12. Spranger, J. W., Wiedemann, H. R.: The genetic mucolipidoses--diagnosis and differential diagnosis. Humangenetik 9, 113--139 (1970) 13. Stekhoven, J. H. S., van Haelst, U. J. G. M., Joosten, E. M. G., Loonen, C. B.: Ultrastructural study of the vacuoles in the peripheral lymphocytes in juvenile amaurotic idiocv. Juvenile form of generalized ceroid lipofuscinosis. Acta Neuropath. (Berl.) 38, 137--142 (1977) 14. Strecker, G., Michalski, J. C., Montreuil, J., Farriaux, J. P.: Deficit in neuraminidase associated with mucolipidosis II (I-cell disease). Biomedicine 25, 238--240 (1976) 15. Suzuki, Y., Nakamura, N.. Shimada, Y., Yotsumoto, H., Endo, H., Nagashima, K.: Macular cherry-red spots and fl-galactosidase deficiency in an adult. An autopsy casewith progressive cerebellar ataxia, myoclonus, thrombocytopathy and accumulation of polysaccharide in liver. Arch. Neurol. 34, 157--161 (1977) 16. Suzuki, Y., Nakamura, N., Fukuoka, K., Shimada, Y., Uono, M.: fl-galactosidase deficiency in juvenile and adult patients. Report of six Japanese cases and review of literature. Hum. Genet. 36,219--229 (1977) 17. Tanaka, Y., Takazono, I., Yasuoka, C., Iwatani, E., Gore, I.: The pattern of urinary chondroitin sulfate and chondroitin excretion with age. Kurume Med. J. 22, 153--157 (1975) 18. Thomas, G. H,, Tiller, G. E., Jr., Reynolds, L. W., Miller, C. S., Bace, J. W.: Increased levels of sialic acid associated with a sialidase deficiency in I-cell disease (mucolipidosis II) fibroblasts. Biochem. Biophys. Res. Commun. 71, 188--195 (1976) 19. Troost, J., van der Heijden, M. C. M., Staal, G. E. J.: Characterization of a-L-fucosidase from two different families with fucosidosis. Clin. Chim. Acta 73, 329--346 (1976) 20. Wenger, D. A., Goodman, S. I., Myers, G. G.: Beta-galactosidase deficiency in young adults. Lancet 1974II, 1319--1320 21. Wenger, D.A., Tarby, T. J., Wharton, C.: Macular cherry-red spots and myoclonus with dementia: Coexistent neuraminidase and fl-galactosidase deficiencies. Biochem. Biophys. Res. Commun. 82, 589--595 (1978) 22. Warren, L.: The thiobarbituric acid assay of siliac acids. J. Biol. Chem, 234, 1971--1975 (1959) 23. Yamamoto, A., Adachi, S., Kawamura, S., Takahashi. M.. Kitani, T., Ohtori, T., Shinji, Y., Nishikawa, M.: Localized fl-galactosidase deficiency. Occurrence in cerebellar ataxia with myoclonus epilepsy and macular cherry-red spot--a new variant of GM~-gangliosidosis.e Arch. Intern. Med. 134, 627--634 (1974) Received December 4, 1978
J.Neurol. 221, 137--149 (1979)
rqeurao t~) by Springer-Verlag 1979
Original Investigations Adult Type Mucolipidosis with fl-Galactosidase and Sialidase Deficiency Histological and Biochemical Studies* T. Kobayashi ], M. Ohta 2, I. Goto 3, Y. Tanaka 4, and Y. Kuroiwa 3 ~National Akasaka Hospital, Chikugo, Departments of 2Neuropathology and 3Neurology, Kyushu University 60, Fukuoka, and 4Chemistry, Kurume University, Kurume, Japan
Summary. A case of adult type mucolipidosis with fl-galactosidase and sialidase deficiency is described. This patient, a woman aged 20, had mental retardation, macular cherry-red spots, corneal clouding, gargoyle-like face, cerebellar ataxia, myoclonus and convulsions beginning at the age of 14. Bony deformities, vacuoles in the peripheral lymphocyte and foamy cells in the bone marrow were also noted. Biopsy study of the sural nerve and vermiform appendix disclosed many vacuoles in almost every kind of cells, although the accumulated substance in these vacuoles could not be characterized histochemically or ultrastructurally. Deficient leukocyte fl-galactosidase and sialidase were confirmed. There was increased urinary sialoglycopeptide and increased siliac acid and hexosamine in the glycoprotein of lymphocytes. Leukocytes sialidase activities of the parents were 30 to 50% of the control values. These results suggest a genetic defect of sialidase.
Key words: Mucolipidosis - fl-galactosidase deficiency - Sialidase deficiency V a c u o l e - Sialoglycoprotein.
Zusammenfassung. Ein Fall der Mukolipidose im Erwachsenenalter mit flGalaktosidase- und Sialidasemangel wurde bemerkt. Die Patientin, ein zwanzigj/ihriges Friiulein, wies Intelligenzmangel, Kornealtrtibung, Gargoylismus, zerebellare Ataxie, Myoklonus und Konvulsion auf, die im Alter von 14 Jahren auftraten. Die skeletale Deformitgt und die Vakuolenbildung in peripheren Lymphocyten und in f6rmigen Zellen im Knochenmark waren auch bemerkbar. Bei Probeuntersuchungen des N.suralis und Appendix vermiformis wurden viele Vakuolen in fast allen Zellen gefunden, aber das gespeicherte Material in diesen Vakuolen konnte durch enzymhistochemische * This work was supported by a grant from the Japanese Ministry of Health and Welfare Address for offprint requests." Dr. M. Ohta, Department of Neuropathology, Neurological Institute, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan
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T. Kobayashi et al. und ultrastrukturelle Untersuchungen nicht charakterisiert werden. Die Mängel der fl-Galaktosidase und der Sialidase in Leukozyten wurden bemerkt. Es gab vermehrtes Sialylglykopeptid im H a r n und Sialylsäure und Hexosamin im Glykoprotein der Lymphozyten. Leukozytensialidase der Eltern wurde in 3 0 - - 5 0 % der normalen Menge gefunden. Dieses Resultat ergibt, daß unser Fall einen genetischen Defekt der Sialidase aufweist.
Introduction Juvenile or adult cases with macular cherry-red spots, ataxia, myoclonus and gargoyle-like face have recently been reported [ 1 , 3 - - 5 , 7, 10, 11, 15, 20, 21,23]. In these cases fl-galactosidase was deficient in leukocytes or other tissues, but the pathogenesis of this disorder has been uncertain. Wenger et al. [21] reported sialidase as well as fl-galactosidase deficiency in fibroblasts, and suggested that sialidase deficiency was the primary defect. The clinical, morphological and biochemical studies in an adult case with macular cherry-red spot and other characteristic manifestations are described.
Case Report The patient, a woman aged 20, was born of healthy and unrelated Japanese parents. The family history was unremarkable. Her early growth and development were below the average. Generalized convulsive seizures occurred frequently after the age of 14, and 1 year later her extremities moved involuntarily on attempting any purposeful movement such as writing or
Fig. 1. Face of patient with gargoyle-like features Fig. 2. Fundoscopy manifestating cherry-red spot
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Fig. 3. Lymphocyte accompanying several vacuoles in cytoplasm. Giemsa x 1000 Fig. 4. Electron micrograph of lymphocyte with membrane-bound vacuoles containing floccular material. Some also contained membranous, granular and dense materials, x 15,000
reaching for objects. These symptoms were gradually progressive and she could not walk after the age of 17, so, the age of 19, was admitted to the National Akasaka Hospital. She had a coarse face with slight hypertelorism, saddle-shaped nose and thick eyebrows (Fig. 1). The liver and spleen were not palpable. She had numerous angiokeratomas on her soles and palms. On neurological examination, her mentation was retarded (IQ on WAIS was 58). Her vision, 20/50 on the right and 30/50 on the left, was uncorrectable. She had bilateral macular cherry-red spots (Fig. 2). Diffuse granular clouding of cornea was also seen with the slit lamp. Ocular movements were not smooth and horizontal nystagmus was observed on lateral gaze. She had difficulty in speaking, which was explosive and stuttering. There was limb and truncal ataxia with myoclonus and she could neither walk nor stand. Deep reflexes were hyperactive but there were no pathological reflexes and no sensory impairment. Peripheral blood counts and urinalysis were normal. About 30% of peripheral lymphocytes contained cytoplasmic vacuoles (Fig. 3). There were numerous foamy cells with positive PAS staining in the bone marrow. Serum transaminases, alkaline phosphatase, lactic dehydrogenase, total protein and its electrophoretic pattern, cholesterol, phospholipid, triglyceride and urea nitrogen were all normal. There was anterior beaking of L4 and L5 vertebral bodies on X-ray, hut there was no remarkable deformity elsewhere. The electroencephalographic studies showed moderate to marked abnormalities with disorganized background activity and occasional bilateral multiple spikes. The cerebrospinal fluid was normal. Electromyogram and motor and sensory nerve conduction studies were normal. CT scanning revealed no remarkable abnormalities.
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Anticonvulsant medication was effective in controlling myoclonus and generalized convulsions, but visual acuity and mentation deteriorated gradually.
Histological and Electron Microscopic Methods The preparations of peripheral lymphocytes were stained with Giemsa, PAS, toluidine blue, Sudan black B and Sudan III. Acid phosphatase staining was added. For electron microscopic study, 3% cacodylate buffered glutaraldehyde was layed over the buffy coat of heparinized blood and postfixed in 2% osmium tetroxide. A part of the surgically obtained vermiform appendix was immediately frozen in isopentane, cooled by dry ice, for routine histological and histochemical study. Another part was fixed with 3% glutaraldehyde and 1% osmium tetroxide. Each specimen of osmicated venous blood buffy coat, sural nerve and veriform appendix was dehydrated with alcohol and embeded in Epon 812. Thick and thin sections were obtained with Porter-Blum MT2 ultratome. Thin sections were stained with uranyl acetate and lead citrate and examined with an electron microscope, Hitachi HS-9.
Biochemical Methods
1. Analysis of Urinary Glycopeptide The analysis of urinary excretion of acid mucopolysaccharide and glycopeptide was performed according to the previous report with some modifications [17].
2. Biochemical Study of Peripheral Lymphocytes About 100 ml of venous blood with EDTA was obtained from the patient and six age- and sex-matched controls. Lymphocytes were separated by the Ficoll-Conray method. The lymphocytes, dried by lyophilization, were weighed out and delipidized with chloroform methanol (2 : 1 and 1 : 2, v/v), and then digested by in 0.1M tris buffer (pH 8.0) at 45°C for 48h. After deproteinization with 10% trichloroacetic acid, the digested solution, which was not precipitated with 5% cetylpyridinium chloride, was dialyzed against water for 48h. Hexose was estimated by the anthrome method using galactose as a standard. Sialic acid was determined by Warren's method [22] after hydrolysis with 0.1 N sulfuric acid at 80°C for I h. Hexosamine was determined by the indole-hydrochloric acid method after hydrolysis with 3 N hydrochloric acid at 100°C for 8 h in a sealed tube.
3. Leukocyte and Serum Lysosomal Enzymes The enzymes, except for sialidase, were measured according to Troost et al. [19]. Sialidase assay was carried out according to Thomas et al. [18] with some modifications. Substrates for the sialidase assay were fetuin, N-acetyl neuramin lactose and urinary glycopeptide (the first peak of column chromatogram, which was desalted and lyophilized (Fig. 10)). The incubation mixture was composed
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with 150 lal of leukolysate, 40 lal of substrate (fetuin: 25 m g / m l , N-acetyl neuramin lactose: 2.5 m g / m l or urinary glycopeptide: 25 m g / m l ) , 10 lal o f 1 M acetate buffer (pH 4.0). After incubation for 2 to 8 h at 37°C, the released free N-acetyl neuramic acid was determined by W a r r e n ' s m e t h o d [22]. After subtraction of the leukocyte and substrate blanks and correction of the interference o f 2 - - 3 deoxyribose, the enzyme activities were expressed as n m o l / m g protein/h. Protein was determined by the m e t h o d o f L o w r y et al. [8]. All assays were performed in duplicate.
Results
1. Histological and Ultrastructural Studies of Lymphocytes A b o u t 30 to 40% ofperipheral lymphocytes had several vacuoles in their cytoplasm (Fig. 3). Vacuoles were negative on Giemsa staining, PAS reaction, toluidine blue staining and Sudan black B and III, but positive on acid phosphatase staining (Table 1). Ultrastructurally the vacuoles were m e m b r a n e - b o u n d , usually 0.5 la to 1 la, very rarely up to 2 la in diameter and had moderately dense sparse floccular materials, small dense materials up to 0.2la in diameter and very occasionally glycogen granules in electron lucent matrix (Fig. 4). N o lamellated bodies or zebra bodies were encountered. N o abnormality was confirmed in the other cell organelles and in the nucleus.
2. Sural Nerve Examination Biopsied sural nerve contained two fascicles. M a n y vacuoles were seen in the epineurial and endoneurial fibroblasts (Fig. 5). There were no changes suggestive of the nerve fiber degeneration, which was also confirmed by quantitative studies. There were no a b n o r m a l deposits in the endoneurium. The vacuoles were also seen in the Schwarm cell (Fig. 6), and perineurial and endoneurial epithelium. Their ultrastructure was similar to those seen in the circulating lymphocytes. Scanty floccular materials, dense bodies and glycogen granules, especially rich in fibroblasts, were seen (Fig. 6), m a n y of which apposed each other and were frequently confluent. The cells with accumulated vacuoles did not show any other remarkable cytoplasmic changes.
Table 1,, Histochemical findings of various cells
PAS Sudan black B Sudan III Toluidine blue
Lymphocyte vacuoles
Foamy cells in bone marrow
Nerve cells in Auerbach's plexus
-
+ -
+++ + -
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Fig. 5. Transverse section of sural nerve. Vacuole-loaded fibroblasts in endoneurium (arrow). Loss and degenerative changes of myelinated fibers not evident. Toluidine blue x 650 Fig.6. Electron micrograph of sural nerve. Fibroblast and Schwann cell contained similar membrane-bound vacuoles. Many glycogen granules in fibroblast. × 15,000
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Fig. 7. Auerbach's plexus of vermiform appendix. Enlarged nerve cells, some loaded with PAS positive granules. PAS x 140 Fig. 8. Electron micrograph of cytoplasmic inclusions, myelin figure, in nerve cells of Auerbach's plexus, x 15,000
3. Examination of Vermiform Appendix Numerous balooned nerve cells were found in Auerbach's and Meissner's plexuses, which were positive with PAS staining (Fig. 7). Ultrastructurally the abnormally enlarged neurons contained numerous electron dense granules. These were zebra bodies or myelin figures (Fig. 8). Free ribosome, Golgi's complex and mitochondria were also prominent. The satellite cells were also loaded by numerous similar vacuoles and dense bodies. However, these vacuoles depicted somewhat different features from those in lymphocytes and fibroblasts; the content was more dense, more filamentous with frequent dense materials, membrane bodies and lipid droplets. Membranes of many vacuoles were occasionally continuous to the smooth endoplasmic reticulum (Fig. 9). In addition, very occasionally, a lamellar cupshaped wrapping structure was seen around the vacuole, suggesting sequesteration (Fig. 9). Golgi's complex, however, was not so hyperdeveloped in the satellite cells. Many vacuoles, similar to those in the lymphocytes were also present in the smooth muscle cells.
4. Urinary Glycopeptide The amount of sialic acid and hexose in the glycopeptide of the urine was increased 5 to 10 times, compared with those of controls (Fig. 10). However, the
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Fig. 9. Cytoplasmic vacuoles in satellite cells of Auerbach's plexus, membrane-bound. Their content was rauch more filamentous and frequent in membranous structures and lipid droplets. Vacuolar membranes were continuous in some part. Lamellar cup-shaped wrapping structure around vacuoles also seen (arrow). x 26,500
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Polysaccharide in urine
/Jglml
i
40(]
u
20C
os
....
--.%
0
6013
to
trol 0
20
;~ 40 60 tube number
80
~
Fig. 10. DEAE cellulose column chromatogram of urine. After application of 2000 ml urine to Bio-Gel P-6 column and elution with distilled water, eluates positive for cetylpyridinium chloride were applied. First 20 tubes (15 ml/tube) were eluted with 0.1 M tris buffer (pH 7.0) and linear gradient elution was conducted with NaC1 (0 to 1 M), in tris buffer. Hexose by orcinolsulfric acid method; sialic acid by Ehrlich method; uronic acid by carbazole method• First peak (*) was used as substrate for sialidase assay
uronic acid of mucopolysaccharide was not increased. The excretion of uronic acid was 1.56 mg/day, which was within normal range (normal control 0.8--9.3 m g / d a y , n = 15).
5. Biochemical Study of Lymphocytes (Table 2) Sialic acid and hexosamine in chloroform-methanol insoluble residue of lymphocyte were increased 2 to 3 times, which was evident in relation to protein content, dry weight or numbers of lymphocytes.
6. Lysosomal Enzyme Activities (Tables 3 and 4) The leukocyte fl-galactosidase was abnormally low; other lysosomal enzyme activities in leukocyte and serum fl-galactosidase level were normal.
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Table 2. Glycoprotein in lymphocytes Patient
Sialic acid Hexosamine Hexose
Controls
4.4 17.6 218
Mean + S.D.
(Range)
1.4_+ 0.4 5.4_+ 1.2 116 _+48
(1.0- 2.1) (3.5- 7.6) (73 -204)
Values are shown as gg/mg of chloroform-methanol insoluble residue
Table 3. Lysosomal enzyme activities Patient
Leukocyte B-galactosidase a-galactosidase N-acetylB-glucosaminidase B-glucuronidase a-mannosidase a-glucosidase B-glucosidase a-fucosidase Arylsulfatase A Serum B-galactosidase
Father
Mother
Controls (n = 17) Mean _+S.D.
Range
7.9 28.9 916.1
44.4 54.2 1066.7
35.7 41.8 855.9
71.6+ 15.3 36.3+ 11.6 864 _+ 174
(50.0- 92.8) (22.0- 62.2) (606 - 1934)
13.8 58.9 15.8 13.8 34.4 121.0
53.3 77.8 19.6 26.7 26.7 72.3
20.0 85.3 25.3 21.8 26.7 60.0
14.5_+ 6.6 84.8_+ 36.4 15.2_+ 5.0 12.8_+ 4.2 35.7_+ 5.4 68.9_+ 10.6
(1.7- 26.0) (45.5- 151.0) (8.2- 23.3) (5.419.6) (29.1- 43.0) (58.1- 117.0)
5.6
n.d.
n.d.
9.9_+
5.1
(4.0-
18.0)
Values are shown as nmol/mg protein/h in leukocytes and nmol/ml/h in serum. Substrates are p-nitrophenol derivatives except for arylsulfatase A (p-nitrocatechol sulfate) n.d.: not determined
Table 4. Sialidase activities in leukocytes Fetuin
N-acetylneuramin lactose
Urinary glycopeptide
Patient Father Mother
0 0.098 0.074
0.279 n.d. n.d.
0 n.d. n.d.
Controls Number Range
7 0.187--0.574
4 0.163--0.754
3 0.172--0.753
Values are shown as nmol/mg protein/h n.d.: not determined
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The leukocyte fl-galactosidase in the parents was slightly lower than the control ranges. Sialidase in leukocytes was deficient with fetuin and extracted urinary glycopeptide, but normal with N-acetyl neuramin lactose. Leukocyte sialidase activities in the parents were about 30 to 50% of the control values.
Discussion Our case has clinical features of both sphingolipidosis and mucopolysaccharidosis; macular cherry-red spots, angiokeratoma and progressive neurological symptoms as sphingolipidosis, and gargoyle-like face, corneal clouding and bony deformities such as mucopolysaccharidosis. Therefore, this case is compatible with mucolipidosis which Spranger et al. [12] proposed. Fucosidosis, mannosidosis and mucosulfatidosis were denied by enzyme studies. GMl-gangliosidosis and mucolipidosis I, II and III are unlikely because of the age of onset and clinical manifestations. Since Goldberg et al. [3] reported an adult case with macular cherry-red spot, corneal clouding and fl-galactosidase deficiency, several similar cases have been published. Our case had clinical manifestations similar to those previously reported. Most of these cases had vacuoles in lymphocytes, fibroblasts and other tissues. Therefore, we first paid attention to the characteristics of the vacuoles. The previous authors reported that the vacuoles were seen only in lymphocytes, fibroblasts or Kupffer cells in the liver, but in our study, the vacuoles were extensively present in the peripheral lymphocytes, fibroblasts, Schwann cells, perineurial epithelium, smooth muscle cells and endothelial cells, but not in the axons of the peripheral nerves. An essential derangement of the metabolic pathway common to these different kinds of cells might be reasonably considered in this disorder. These vacuoles were not stained with any dyes except for acid phosphatase staining, and electron microscopically, they had no special structures such as lamellated bodies or finger print structures, which are usually seen in ceroid lipofuscinosis or sphingolipidosis [6, 13]. Therefore, the accumulated substance in the vacuoles was obscure, but the vacuoles were positive with acid phosphatase staining. Ultrastructurally they were bound by a unit membrane. They had glycogen granules and dense substance, although very scant. Moreover, they seemed to be autophagiosized, which is compatible to the finding of the sequesteration suggested by Moe et al. [9]. These histochemical and ultrastructural findings suggest that the vacuoles are lysosomes [2]. The inclusions of the nerve cells of the vermiform appendix were stained with PAS and Sudan black B. Ultrastructurally there were many myelin-like figures or lamellated bodies, which had the same ultrastructure of inclusions in various conditions such as sphingolipidosis or mucopolysaccharidosis. These inclusions were found only in the nerve cells and satellite cells. Therefore, the accumulating substance in the neurons may be different from those in the other cells. Enzymatically some authors speculated that fl-galactosidase deficiency was not the primary but the secondary defect inhibited by abnormally accumulated substance, because of normal serum fl-galactosidase activities in the patients and
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n o r m a l leukocyte activities in the p a r e n t s [1, 16, 21]. O n the o t h e r hand, several cases h a d r e d u c e d fl-galactosidase activity in the serum or leukocytes o f the p a r e n t s [10, 23]. In o u r case,/~-galactosidase activity was very low in leukocytes a n d n o r m a l in serum. L e u k o c y t e / ~ - g a l a c t o s i d a s e activity in the p a r e n t s was lower than n o r m a l c o n t r o l values. Therefore, one can n o t conclude at present that flg a l a c t o s i d a s e is s e c o n d a r i l y inhibited by a n o t h e r m e t a b o l i c defect. W e m e a s u r e d sialidase in leukocytes instead o f f i b r o b l a s t s . L e u k o c y t e sialidase activities are lower t h a n those in fibroblasts, b u t they can be easily assayed with the i n c u b a t i o n time o f 2 to 8 h. These sialidase activities d e t e r m i n e d b y W a r r e n ' s m e t h o d are c o m p a t i b l e to those by the i s o t o p e m e t h o d o f Strecker et al. [14]. L e u k o c y t e sialidase o f o u r case was deficient with fetuin a n d extracted u r i n a r y g l y c o p e p t i d e , b u t n o r m a l with N-acetyl n e u r a m i n lactose. A l t h o u g h the exact cause o f this d i s c r e p a n c y is obscure, leukocyte sialidase m a y have enzyme specificity to substrates. T h e r e was an increase o f siliac acid in the g l y c o p r o t e i n o f urine a n d l y m p h o cytes, which suggests the cause to be sialidase deficiency. C o n s i d e r i n g the result o f low sialidase activities o f the p a r e n t s , sialidase deficiency is at least a f u n d a m e n t a l defect in o u r case.
Acknowledgement. The authors wish to thank Drs. M. Kawabuchi and H. Nagara for their cooperation.
References 1. Fukunaga, H., Hirose, K., Beppu, H., Uono, M., Suzuki, Y.: Two siblings with mucolipidosis. Clin. Neurol. (Tokyo) 16, 566--573 (t976) 2. Ghadially, F. N.: Lysosomes. In: Ultrastructural pathology of the cell, pp. 291--368. London, Boston: Butterworths 1975 3. Goldberg, M. F., Cotlier, E., Fichenscher, L. G., Kenyon, K., Enat, R., Borowsky, S. A.: Macular cherry-red spot, corneal clouding, and fl-galactosidase deficiency. Clinical, biochemical, and electron microscopic study of a new autosomal recessive storage disease. Arch. Intern. Med. 128, 387--398 (1971) 4. Goldstein, M., Kolodny, E. H., Gascon, G. G., Gilles, F. H.: Macular cherry-red spot, myoclonic epilepsy, and neurovisceral storage in a 17 year-old girl. Trans. Am. Neurol. Assoc. 99, 110--112 (1975) 5. Kuriyama, M., Umezaki, H., Okada, S., Tanaka, Y., Ishii, N.: Adult mucolipidosis with B-galactosidase deficiency: a clinical report, with studies ofurinary sialic acid-rich substance. Clin. Neurol. (Tokyo) 18, 358--363 (1978) 6. Lazarus, S. S., Vethamany, V. G., Schneck, L., Volk, B. W.: Fine structure and histochemistry of peripheral blood cells in N iemann-Pick disease. Lab. Invest. 17, 155--170 (1967) 7. Loonen, M. C. B., Lugt, L. v. d., Franke, C. L.: Angiokeratoma corporis diffusum and lysosomal enzyme deficiency. Lancet 1974 II, 785 8. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265--275 (1951) 9. Moe, H., Rostgaard, J., Behnke, O.: On the morphology and origin of virgin lysosomes in the intestinal epithelium of the rat. J. Ultrastruct. Res. 12,396--403 (1965) 10. Orii, T., Minami, R., Sukegawa, K., Sato, S., Tsugawa, S., Horino, K., Miura, R., Nakao, T.: A new type of mucolipidosis with fl-galactosidase deficiency and glycopeptiduria. Tohoku J. exp. Med. 107, 303--315 (1972) 11. Shibata, R., Yokota, K., Takashima, S., Mitsudome, A., Kurokawa, T.: A case of mucolipidosis. Brain Develop. (Tokyo) 7, 392--399 (1975)
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12. Spranger, J. W., Wiedemann, H. R.: The genetic mucolipidoses--diagnosis and differential diagnosis. Humangenetik 9, 113--139 (1970) 13. Stekhoven, J. H. S., van Haelst, U. J. G. M., Joosten, E. M. G., Loonen, C. B.: Ultrastructural study of the vacuoles in the peripheral lymphocytes in juvenile amaurotic idiocv. Juvenile form of generalized ceroid lipofuscinosis. Acta Neuropath. (Berl.) 38, 137--142 (1977) 14. Strecker, G., Michalski, J. C., Montreuil, J., Farriaux, J. P.: Deficit in neuraminidase associated with mucolipidosis II (I-cell disease). Biomedicine 25, 238--240 (1976) 15. Suzuki, Y., Nakamura, N.. Shimada, Y., Yotsumoto, H., Endo, H., Nagashima, K.: Macular cherry-red spots and fl-galactosidase deficiency in an adult. An autopsy casewith progressive cerebellar ataxia, myoclonus, thrombocytopathy and accumulation of polysaccharide in liver. Arch. Neurol. 34, 157--161 (1977) 16. Suzuki, Y., Nakamura, N., Fukuoka, K., Shimada, Y., Uono, M.: fl-galactosidase deficiency in juvenile and adult patients. Report of six Japanese cases and review of literature. Hum. Genet. 36,219--229 (1977) 17. Tanaka, Y., Takazono, I., Yasuoka, C., Iwatani, E., Gore, I.: The pattern of urinary chondroitin sulfate and chondroitin excretion with age. Kurume Med. J. 22, 153--157 (1975) 18. Thomas, G. H,, Tiller, G. E., Jr., Reynolds, L. W., Miller, C. S., Bace, J. W.: Increased levels of sialic acid associated with a sialidase deficiency in I-cell disease (mucolipidosis II) fibroblasts. Biochem. Biophys. Res. Commun. 71, 188--195 (1976) 19. Troost, J., van der Heijden, M. C. M., Staal, G. E. J.: Characterization of a-L-fucosidase from two different families with fucosidosis. Clin. Chim. Acta 73, 329--346 (1976) 20. Wenger, D. A., Goodman, S. I., Myers, G. G.: Beta-galactosidase deficiency in young adults. Lancet 1974II, 1319--1320 21. Wenger, D.A., Tarby, T. J., Wharton, C.: Macular cherry-red spots and myoclonus with dementia: Coexistent neuraminidase and fl-galactosidase deficiencies. Biochem. Biophys. Res. Commun. 82, 589--595 (1978) 22. Warren, L.: The thiobarbituric acid assay of siliac acids. J. Biol. Chem, 234, 1971--1975 (1959) 23. Yamamoto, A., Adachi, S., Kawamura, S., Takahashi. M.. Kitani, T., Ohtori, T., Shinji, Y., Nishikawa, M.: Localized fl-galactosidase deficiency. Occurrence in cerebellar ataxia with myoclonus epilepsy and macular cherry-red spot--a new variant of GM~-gangliosidosis.e Arch. Intern. Med. 134, 627--634 (1974) Received December 4, 1978
