: ultrastructural study of conjunctiva and skin and enzyme analysis of tears

/. Libert,9 F. Van Hoof," and M. Tondeur***

Conjunctival and skin biopsies from two new patients with fucosidosis were studied by electron microscopy. In both tissues, the connective tissue cells and the capillary endothelial cells were filled with single membrane limited inclusions of two types: (1) Clear inclusions containing a fibrillogranular reticulum. (2) Dark inclusions with a dense granular material. Specific stainings in ultrastructure suggest that these inclusions contain oligosaccharide chains. The ultrastructural aspect is characteristic for fucosidosis. Enzyme studies on tears realize an easy and secure technique for the diagnosis of the disease.

-Lhe interest of conjunctival biopsy in the io-i5 Most of the lysosomal acid hydrolases study of storage diseases is now abun- can be assayed in tears, and their defi- dantly documented. Pathologic changes ciency allows the diagnosis of several other have been demonstrated in the systemic inborn lysosomal diseases.16"18 mucopolysaccharidoses,1 several mucolip- Fucosidosis, an autosomal lysosomal dis- idoses,2"5 Fabry's disease,6'7 Niemann- ease, is characterized by the absence or the Pick's disease,8 and cystinosis.9 profound deficiency of a-L-fucosidase,19"21 The usefulness of tears for the enzymatic which results in the widespread accumula- screening of inborn lysosomal diseases was tion of fucose-containing glycosphingolip- recognized about two years ago and ap- ids, oligosaccharides, and polysaccharides, plied to Tay-Sachs and Fabry's diseases.7' together with an increased excretion of fucosides in urine.22 Clinically, a severe and a mild phenotype have been distin- From the Departments of Ophthalmology" and guished.23' 24 The patients suffering from Pediatrics,00" Hopital Saint-Pierre, Universite Libre de Bruxelles, and from the International the severe form present a progressive psy- Institute of Cellular and Molecular Patholo- chomotor retardation and a moderate gy,00 Universite Catholique de Louvain. chondrodystrophy somewhat reminiscent Supported by the Belgian Fonds de la Recherche of the mucopolysaccharidoses. Death oc- Scientifique N° 20,126 and N° 20,183, and by curs around the age of three to five years the United States Public Health Service (Re- search Grant N° AM 9235). in the severe phenotype. Survival to ado- Submitted for publication Jan. 6, 1976. lescence and beyond has been reported in Reprint requests: Dr. J. Libert, Department of the mild phenotype, which is characterized Ophthalmology, Hopital Saint-Pierre, 322 rue by the presence of angiokeratoma corporis 23 26 Haute, 1000 Bruxelles, Belgium. diffusum. " Ocular signs, as thin and 626

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tortuous vessels in the conjunctiva and conjunctiva. No suture was necessary, phenyl- the fundus have been described in this ephrine and an antibiotic ophthalmic ointment 7 were placed in loco as postoperative care. group.-"- - The skin biopsies were taken aseptically from No histopathological studies of the eyes the forearm after local anesthesia with an ethyl have been made, to our knowledge, in chloride spray. The specimens were cut with a fucosidosis. The purpose of the present curved forceps and a scalpel, divided into two paper is to describe the ultrastructural ab- fragments for fibroblast culture and ultrastructural study. normalities of the conjunctival and skin bi- The tissue fragments were immediately placed opsies, and the enzymatic findings in tears in 4 per cent glutaraldehyde (Millonig buffer, pH from two patients suffering from the severe 7.4) and divided into smaller fragments. After form of fucosidosis, and to stress the im- two hours in the fixative, the tissue was washed portance of these investigations in the di- in Millonig buffer containing 5.4 per cent glucose. Postfixation was conducted for 30 minutes with agnosis of the disease. 2 per cent osmium tetroxide (Millonig buffer, pH 7.4). After washing, the tissue was dehy- Brief case reports drated in ethanol and propylene oxide, and em- bedded in an epoxy resin (Epon). Case 1 is the younger sister of the patient 2S Semithin sections were performed in order to described by Loeb and co-workers in 1969, the orient the ultrathin sections perpendicularly to first patient in whom the deficiency of a-L-fucosi- the epithelial surface. Ultrathin sections were cut dase was evidenced. Their Italian parents are with diamond knives on a Reichert Om U 2 first cousins; one sibling is unaffected. The diag- microtome and stained as follows: (1) uranyl nosis was made at the age of five months by acetate and lead citrate according to Reynolds-9; enzymatic analysis of leukocytes, plasma, and (2) silver proteinate after periodic acid oxidation urine. A skin biopsy was performed at this time according to Thiery30; this latter reaction reveals for fibroblast culture and ultrastructural study. vic-glycol groups, thus mainly polysaccharides. She is now 2.5 years old. Somatic and psycho- Some conjunctival fragments were treated sepa- motor development are delayed, but there is no rately, dehydrated in acetone and embedded in other sign of neurological deterioration. The Vestopal W. The ultrathin sections were stained face has a mild gargoyle aspect. She . has a with phosphotungstic acid in aqueous solution lumbar hyperlordosis and a protruding abdomen (10 per cent weight per volume) at pH 1.5.31 with umbilical hernia. The spleen and the liver PTA stains and mucopolysaccharides. are slightly enlarged. Corneae and fundi are nor- For the colloidal iron staining, the specimen was mal. fixed in 6.25 per cent glutaraldehyde (cacodylate Case 2, a girl, is the first child of healthy non- buffer, pH 7.4). After one hour in the fixative, consanguineous Belgian parents. At the age of the tissue was washed in cacodylate buffer con- 2.3 years, the face has a mild gargoyle aspect; taining 5.4 per cent glucose. It was then prepared the thorax is broad; the spleen and the liver are and stained according to Hardin and Spicetv1'- slightly enlarged. Hypotrophy and psychomotor This technique is specific for acid mucopolysac- regression are evident. A spastic paraplegia of the charides and sialic acid-rich glycoproteins. lower legs is noted. X-ray examination shows a The sections were thereafter coated with a car- delayed skeletal maturation and abnormalities of bon film and examined with a Philips EM 300 the lumbar vertebral bodies. In the peripheral electron microscope. blood, 31 per cent of the lymphocytes are vacuo- For the enzymatic study, biopsies were imme- lized and foam cells are present in the bone mar- diately frozen. Homogenates were performed in row. The diagnosis was first made on typical conical sintered glass tissue grinders, in the pres- alterations in conjunctival biopsy and on the ence of 99 volumes of bidistilled water, and with- absence of a-L-fucosidase activity in tears. It out addition of detergent. Acid hydrolases were was later confirmed by liver biopsy and enzymatic 0 assayed as described earlier,- or with similar studies in leukocytes and plasma. A skin biopsy techniques using substrates derived of 4-methyl- was also performed for fibroblast culture and umbelliferone. The same techniques were applied ultrastructural study. to plasma and leukocytes. The latter were isolated either by collection of the buffy coat, after centrif- Material and methods ugation of heparinized blood, or by the dextran The conjunctival biopsies were obtained under sedimentation technique. topical anesthesia with oxybuprocaine hydrochlo- Tears were collected on strips of Whatman's ride (Novesine). They were taken with a smooth no. 44 filter paper, which were allowed to dry jaw forceps and scissors from the upper bulbar and kept as such for one to five days in the

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^-r Fig. 1. Patient 2. Conjunctival biopsy. Semi-thin section stained with toluidine blue. The epithelium displays an increasing number of cytoplasmic vacuoles from the basal to the apical layer. In the stroma, the capillary endothelial cells and the connective tissue cells also contain many clear vacuoles. * 1,000.

deep freeze before analysis. In the laboratory, the No metachromatic material could be paper strips were cut and put in 0.01 M or 0.1 demonstrated with toluidine blue in con- M citrate bufFer, pH 4.4. A vigorous mixing, fol- junctival and skin biopsies, except in the lowed by a centrifugation, allowed to recover the tear proteins in the supernatant. The latter was goblet cells of the conjunctival epithelium used for determination of a-L-fucosidase and and in the mastocytes of the stroma, as in seven other lysosomal hydrolases" as described normal controls. 1 earlier.-' Urine analysis was performed as reported Electron microscopy. in the same publication. Proteins were measured Xi CONJUNCTIVAL BIOPSY. by the method of Lowry and co-workers. In the epithelium, small vacuoles were observed in the basal Results layer. In the adjacent layers, all cells were Morphology. Biopsies of both patients filled with abnormal inclusions of greater displayed no evident differences in the ex- dimension (diameters between 0.3 and 4.0 tention of the storage process. microns). A marked tendency to conflu- Light microscopy. In the conjunctiva, ence was noted in the apical layer (Fig. the epithelial cells in the apical layers were 3). Goblet cells showed numerous clear distended by numerous clear vacuoles of inclusions aside from their normal mucous various sizes, while the basal layer seemed secretions (Fig. 4). The inclusions were unaffected (Fig. 1). The skin epithelium limited by a unit membrane, and contained had a normal aspect. In both conjunctiva a fine granular material and a few small and skin biopsies, a severe vacuolation was concentric lamellar structures, often or- evident in the endothelial cells of the capil- ganized in half rings in contact with the laries. The sweat glands of the skin were limiting membrane (Fig. 5). also heavily involved (Fig. 2). In the stroma, numerous cytoplasmic in- clusions distended the fibroblasts (Fig. 6), °ir- and /3-D-galaetosidase, n-glucosidase, (t-mannosidasc, the endothelial cells of the blood (Fig. 7), N-acetyl-/}-hexosnminidnse, methylumbelliferyl-phosphatase, and Ivsozvmr. and the lymphatic capillaries (Fig. 8), the

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J*

Fig. 2. Patient 2. Skin biopsy. Semi-thin section stained with toluidine blue. The secretory coils of the sweat glands are filled with clear intracytoplasmic vacuoles. The blood capillary endothelial cells are also severely involved. xl,000.

Fig. 3. Patient 1. Conjunctival epithelium. Uranyl acetate, lead citrate. The epithelial cells of the superficial layer are filled with clear inclusions, which contain a fine protein-like; reticulum and some lamellar profiles that appear at this magnification as dense half rings (arrows). x8,000.

perivascular cells and the perineural cells unit membrane, had two distinct aspects: (Fig. 9). The Schwann cells and the ax- clear inclusions were prominent and con- onal processes of the nerve fibers were tained a weakly osmiophilic reticulum. unaffected. The inclusions, limited by a Dense inclusions were less numerous and

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Fig. 4. Patient 1. Conjunctival epithelium. Uranyl acetate, lead citrate. The goblet cells show numerous clear inclusions aside from their normal mucous secretions; the latter appear as denser oval or ribbon-shaped striated bodies (arrows). xlO,000.

exhibited a moderately contrasted homog- enous material at low magnification, which appeared to be finely granular at high magnification. Some lamellar profiles .••*/'>:: V? (Fig. 7) and some dense aggregates (Fig. 6) were present in both types. Occasion- ally, the inclusions contained rather dense spherules whose ultrastructure resembles that of the neutral lipids (Fig. 8). Other cellular organelles were normal. SKIN BIOPSY. Epidermal cells seemed un- affected. Histiocytes and fibroblasts of the superficial dermis displayed clear and dense inclusions, identical to those observed in the conjunctival stroma. The endothelial cells of the blood and lymphatic capillaries, and the perineural cells were also involved, while the Schwann cells and the axonal processes appeared to be normal. In the sweat glands (Fig. 10), the myo- epithelial cells contained few small clear Fig. 5. Patient 1. Conjunctival epithelium. Uranyl inclusions. The clear and dark cells of the acetate, lead citrate. This higher magnification secretory coils were filled with numerous shows details of the lamellar structures and or the finely reticular content of the clear vacuoles, clear vacuoles containing a fine osmiophilic which are limited by a unit membrane. x60,000. reticulum and small lamellar lipid struc-

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Fig. 6. Patient 2. Conjunctival stroma. Uranyl acetate, lead citrate. The connective tissue cells have a characteristic aspect in fucosidosis: They contain both clear and dense inclusions with sometimes dense aggregates. x9,600.

tures. Some inclusions displayed a dense evident in the secretory coils and ducts of homogenous material and resembled the the sweat glands (Fig. 13). dense inclusions of the connective tissue Colloidal iron. Iron staining showed a cells. In the sweat ducts, the cells of the positive reaction in the periphery of the superficial and of the deep layers were dis- vacuoles of the conjunctival epithelial and tended by numerous clear and dense inclu- stromal cells. In the goblet cells, the mucus sions. was strongly contrasted with this technique SPECIFIC STAININGS. Silver proteinate. In while the inclusions had an aspect rather the conjunctival epithelial cells, the inclu- similar to that of the adjacent epithelial sions contained a fine granular material of cells (Fig. 14). variable density, which was markedly Phosphotungstic acid. PTA contrasted the stained with silver (Fig. 11). The lamellar collagen fibers and the tear film of the structures, observed with usual staining epithelium surface. The stored material techniques, were poorly contrasted. The and the inclusion's membrane were mark- mucus and the inclusions of the goblet edly stained. A variable density of the in- cells showed a similar contrast and were clusions was here also recognizable (Fig. only distinguishable by their shapes. 15). In both conjunctiva and skin, the con- Biochemistry. nective tissue cells and the endothelial cells PROPERTIES OF o-L-FUCOSIDASE IN NORMAL of the capillaries contained some inclusions TEARS. oi-L-fucosidase is relatively abundant that were strongly contrasted with silver in tears, where the enzyme displays a broad as a dense granular material, but many of pH activity curve, usually with 3 pH opti- the vacuoles remained clear with a faintly ma around 5.2, 6.3, and 7.4 (Fig. 16). The stained reticulum (Fig. 12). mean normal activity is 1.1 U per gram of Similar clear and dense inclusions were protein at the first two pH and 0.73 at pH

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Fig. 7. Patient 2. Conjunctival stroma. Uranyl acetate, lead citrate. The blood capillary endothelial cells are filled with both clear and dense inclusions, sometimes containing isolated lamellar profiles. (L: capillary lumen) x29,00O.

7.4, as well in children as in adults. The 7.6. In the liver, the Km is around 0.06 mM liver enzyme, for comparison, reaches its at pH 4.4. Thermostability (50° C.) of tear highest activity {1.6 U per gram of protein) a-fucosidase is similar at the three peaks of at pH 4.4, with a second peak around 6,4. the pH activity curve. The affinity of the tear enzyme for 4- ACID HYDROLASES IN TEARS FROM PATIENTS methylumbelliferyl-a-L-fucopyranoside is AND PARENTS. Fig. 16 illustrates the activity high, but depends on the pH. A Km value of a-fucosidase at various pH, in tears from of 0.04 mM has been measured at pH 5.1; a normal subject and from both patients 0.13 mM at pH 6.5, and 0.24 mM at pH and their parents. In the tears of the pa-

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Activity of this enzyme at pH 5.1 (peak of the pH curve), in leukocytes from the parents of case 1 was, respectively, 60 per cent of the control for the father and 54 per cent for the mother. Two other lyso- somal hydrolases (a-galactosidase and /?- hexosaminidase) were measured in these leukocytes and found to be normally active in patients and parents. In blood plasma from the patients, a- fucosidase was not completely absent but reduced to less than 3 per cent of the mean normal value of controls in patient 1 and to less than 1.2 per cent in patient 2. This deficiency extended all over the pH curve. Plasma a-fucosidase activity represented, respectively, 120 and 79 per cent of the normal value for the father and the mother of case 1. Other lysosomal enzymes assayed in patients and parents were normally active. Fig. 8. Patient 1. Conjunctival stroma. Urany] a-Fucosidase was assayed in urine of pa- acetate, lead citrate. The lymphatic vessels arc recognizable by their clear, acellular lumen and tient 1. The enzyme was barely detectable by the absence or basement membrane under between pH 5 and 6. Other lysosomal hy- their endothelinm. The two characteristic inclu- drolases (a- and /?-galactosidase, /S-glucu- sion types are evident in this lymphatic endo- ronidase,.^-hexosaminidase, and acid phos- thelial cell. Some dense aggregates and lipid phatase) were normal. spherules are also disclosed. (L: lumen) xl3,000. Discussion tients, there was no detectable activity at The two new cases of fucosidosis, de- any pH between 3.0 and 9.7. Seven other scribed here, obviously belong to the severe lysosomal enzymes (see methods) were phenotype of the disease. Mild Hurler-like assayed in the lacrymal fluid of the pa- dysmorphy without mucopolysacchariduria, tients and their parents and found to be psychomotor retardation, and neurologic normally active. disturbances were their symptoms. As the OTHER ENZYMATIC STUDIES. A liver needle other reported cases of severe phenotype biopsy was obtained from patient 2. There fucosidosis, they did not display angio- was no detectable activity of a-fucosidase, keratoma corporis diffusum nor vascular while the other lysosomal enzymes assayed tortuosities in the conjunctiva and in the were hyperactive: a-glucosidase displayed fundus. In Fabry's disease, where the vas- a 9-fold increase over the mean normal cular symptoms are barely similar, vascular activity, while a- and /?-galactosidase and abnormalities also are late symptoms, ap- N-acetyl-/?-glucosaminidase were six times pearing only during adolescence or adult- more active than normal. No liver biopsy hood.7 Patients with the severe phenotype was performed in patient 1 because of the of fucosidosis possibly do not survive long results already obtained in her brother. enough to develop them. Leukocytes were analyzed in both pa- The ultrastructural changes in our biop- tients. There was no detectable a-fucosidase sies resemble those reported in Hurler dis- activity at any pH between 3.1 and 8.6. ease. They consist mainly of vacuoles or

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Fig. 9. Patient 2. Conjunctival stioma. Uranyl acetate, lead citrate. The perineural cells (arrows) show many inclusions similar to those of the fibroblasts and endothelial cells in the vicinity, The myelin sheets, the axons and Schwann cells do not present any significant alteration. x6,000.

Fig. 10. Patient 2. Skin biopsy. Uranyl acetate, lead citrate. The myoepithelial cells of the sweat glands contain small clear inclusions (arrow). The cells in the secretory coil are dis- tended by numerous clear and dense inclusions. Little normal cytoplasm remains between the inclusions. Fibroblasts and endothelial cells in the vicinity show the characteristic lesions already described in the conjunctiva. x4,000.

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Fig. 11. Patient 2. Conjunctival epithelium. Silver proteinate. The inclusions contain a fine granular material of variable density, which is markedly stained with silver. The lamellar profiles are poorly stained. x21,000.

Fig. 12. Patient 2. Conjunctival stroma. Silver proteinate. The fibroblasts display two inclu- sion types. Dark inclusions contain a dense granular material which is strongly contrasted with silver. Clear inclusions show a faintly stained reticulum. x21,000.

inclusions resulting from the overloading Schwanii cells of the peripheral sensitive of the lysosomal system. The connective nerves. Contrary to the mucopolysacchari- tissue cells and the conjunctival epithelium doses with mucopolysacchariduria, the en- are heavily involved, while the pathological dothelial cells of the blood capillaries are process seems to spare the axones and the filled with abnormal inclusions in fucosido-

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Fig. 13. Patient 1. Skin biopsy. Silver proteinate. Fig, 14. Patient 2. Conjunctival epithelium. Col- The two inclusion types are also evident in the loidal iron contrasts strongly the mucus of the acinar cells of the sweat glands. Their aspect is goblet cells, while the inclusions remain clear similar to those of the connective tissue cells and with a fine positive reaction at their periphery, endothelial cells. x21,000. x 15,000.

sis. This storage in the vessels' walls is bodies. This heterogeneity is also evident probably responsible for the vascular tor- in the vessels of the liver, the rectal, and tuosities and the angiokeratoma in the mild the bronchial biopsies in our second case.31 phenotype of the disease. To our knowledge, the presence of these The fine structure of the stored material two inclusion types has not yet been de- in fibroblasts and endothelial cells is quite scribed in other lysosomal storage diseases. pathognomonic. It differs from what is ob- We assume that this electron microscopic served in the mucopolysaccharidoses, other appearance is characteristic of fucosidosis. forms of mucolipidoses, and sphingo- The staining properties with silver pro- lipidoses1*3 In the present study, two dis- teinate and phosphotungstic acid suggest tinct inclusion types are evident: clear that the major components of the inclu- vacuoles which contain a finely reticular sions are polysaccharides. The weakly posi- structure, similar to those seen in the muco- tive results obtained with colloidal iron polysaccharidoses, and less numerous dark could be due to the fact that these mole- inclusions exhibiting a dense almost ho- cules are poorly acidic. Alternatively, a loss mogenous content. The existence of these of water-soluble substances during the incu- two types of inclusions had already been bation in the staining solutions could also reported by Loeb and co-workers2S in astro- explain this result. Biochemically, the stor- cytes, macrophages, and endothelial cells age of fucose containing sphingolipids, in a brain biopsy taken from our first pa- oligosaccharides, and polysaccharides has tient's brother, while the neurones con- been reported in the liver, the brain, and tained only clear inclusions and a few zebra other tissues.-1' -'3a We believe that the

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2.0

1.5

10

0.5

3 A 5 6 7 8 9 10pH Fig. 15. Patient 2. Conjunctival stroma. Phospho- Fig. 16. pH activity curve of a-L-fucosidase in tungstic acid stains markedly the stored material tears. The test mixture contained 0.5 mM 4- which appears of heterogenous density in the methylumbelliferyl-a-fucopyranoside, 0.1 M ci- fibroblasts. Collagen (c) fibers are strongly con- trate (pH 3.0-6.9), phosphate (7.0-7.7), or gly- trasted while other structures are almost un- cine (8.4-9.7) buffer, and 10 to 50 jug of tear recognizable. x26,000. proteins, in a final volume of 0.1 ml. It was incubated for various time intervals up to one hour, and the course of the hydrolysis of the silver positive materials of the inclusions substrate was followed by the appearance of fluorescent free methyl umbelliferone. Enzyme could represent fucose-rich oligosaccharide activity of an adult control (A ) is compared with chains, either free or bound to polypep- that of the fathers (•,•) and mothers (#,O) tides. Variations in the proportions or in of patients 1 and 2, respectively. Activity in the the structure of these components might patients (•) was undetectable at all pH's. explain the occurrence of different inclu- sion types. Some lamellar lipid structures were also observed in the inclusions and itself, as in Durand's first patients,22 by an probably correspond to the multilayered increase in concentration of sweat electro- bodies described in the hepatocytes.21' "S) 3fi lytes. Moreover, in our second case, sweat This latter lipid material has been identified did not contain abnormal amounts of fuco- as fucosyl-glycosphingolipids.37 sides by comparison with normal secretor The ultrastructural changes of the sweat controls.31 coils and ducts resemble those reported in It must be pointed out that the histologic Hurler's disease/" except for the presence findings in skin biopsies are similar in the in our cases of some dark inclusions, similar second case aged 2.3 years and in the first to those of the connective tissue cells. The patient when she was five months old and extensive vacuolation of these glands is not did not, at this time, clearly manifest the surprising since sweat of secretor subjects disease clinically. It is likely that ultra- is known to contain fucosides as A, B, H, structural alterations are also present in the and Le active glycoproteins. This involve- conjunctiva before the appearance of the ment of the sweat glands did not express first clinical signs.

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The enzymatic study, besides of bringing tural observation of conjunctiva and skin, IN- the proof that the two children suffer from VEST. OPHTHALMOL. 10: 555, 1971. 3. Quigley, H. A., and Goldberg, M. F.: Con- fucosidosis, demonstrates the usefulness of junctival infrastructure in mucolipidosis III tear analysis in this disease. a-Fucosidase (pseudo-Hurler), INVEST. OPHTHALMOL. 10: activity is of the same magnitude in tears 568, 1971. as in liver, on a protein basis. To collect 4. Merin, S., Livni, N., Berman, E. R., et al.: tears is easy and harmless; it can be re- Mucolipidosis. IV. Ocular, systemic and ultra- structural findings, INVEST. OPHTHALMOL. 14: peated many times, even at the patient's 437, 1975. home, which could be of potential use in 5. Libert, J.: Conjunctival ultrastructure in man- therapeutic attempts. The enzyme is rela- nosidosis. In discussion of Linn-Murphree, A.: tively stable: 60 to 85 per cent of it re- Cataract with a-mannosidase deficiency, in mains active when tears are allowed to dry The Eye in Inborn Errors of Metabolism, on the paper strips at room temperature Birth Defects. In press. 6. Frost, P., Tanaka, Y., and Spaeth, G. L.: (10 to 20 minutes). Once dried, the enzyme Fabry's disease-glycolipid lipidosis: histo- is remarkably resistant. If the filter papers chemical and electron microscopic studies of are mailed in an envelope in temperate two cases, Am. J. Med. 40: 618, 1966. countries, over 50 per cent of the activity 7. Libert, J., Tondeur, M., and Van Hoof, F.: are still present after one week. If the paper The use of conjunctival biopsy and enzyme analysis in tears for the diagnosis of homo- strips are kept below -18° C, no detect- zygotes and heterozygotes with Fabry disease, able loss of activity occurs within one year. in The Eye in Inborn Errors of Metabolism, The method can, however, apparently not Birth Defects. In press. be used for the detection of heterozygotes 8. Libert, J., and Danis, P.: Diagnosis of type for fucosidosis (Fig. 16) and in this re- A Niemann-Pick disease by conjunctival biop- sy, Pathol. Europ. 10: 233, 1975. spect, is,thus not superior to the analysis 9. Wong, V. J., Kuwabara, T., Brubaker, R., of leukocytes. et al.: Intralysosomal cystine cristals in cysti- It is suggested that the complex pattern nosis, INVEST. OPHTHALMOL. 9: 83, 1970. 10. Carmody, P., Rattazzi, M., and Davidson, R.: of the pH activity curve in tears reflects Tay-Sachs disease. The use of tears for the the existence of multiple molecular associa- detection of heterozygotes, N. Engl. J. Med. tion forms of a single a-fucosidase, which 289: 1072, 1973. has been demonstrated in other tissues.30"43 11. Singer, J. D., Cotlier, E., and Krimmer, R.: The total absence of activity in patients Hexosaminidase A in tears and saliva for rapid identification of Tay-Sachs disease and indicates that all these enzyme forms are its carriers, Lancet 2: 1116, 1973. under the control of a single gene, which 12. Cotlier, E.: Tay-Sachs and Fabry's diseases: is the same that controls the lysosomal «- clinical and chemical diagnosis of two meta- fucosidase in all the tissues that were in- bolic eye diseases, Bull. N. Y. Acad. Med. 50: vestigated in fucosidosis patients. 777, 1974. 13. Del Monte, M. A., Johnson, D. L., Cotlier, Conjunctival biopsy and tear analysis are E., et al.: Diagnosis of Fabry's disease by easy to obtain and to send to the appropri- tear a-galactosidase A, N. Engl. J. Med. ate laboratory. These two methods allow 290: 57, 1974. the early and secure diagnosis of several 14. Goldberg, M. F.: The use of tears for hetero- lysosomal storage diseases and, among zygotes detection and genetic counseling, IN- VEST. OPHTHALMOL. 13 :159, 1974. them, of fucosidosis which is probably fre- 15. Del Monte, M. A.: a-Galactosidase in tears: 41 quently undetected. use in the diagnosis of homozygotes and heterozygotes with Fabry's disease, in The REFERENCES Eye in Inborn Errors of Metabolism, Birth 1. Kenyon, K. R., Quigley, H. A., Hussels, I. Defects. In press. E., et al.: The systemic mucopolysacchari- 16. Van Hoof, F.: Fucosidase and mannosidase doses, Am. J. Ophthalmol. 73: 811, 1972. in tears. In discussion of: Cotlier, E.: Identi- 2. Kenyon, K. R., and Sensenbrenner, J. A.: fication of homozygotes and heterozygotes by Mucolipidosis II (I cell disease). Ultrastruc- chemical analysis of tears, in The Eye in

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Inborn Errors of Metabolism, Birth Defects. en microscopie electronique: coloration de la In press. surface cellulaires et de l'appareil de Golgi 17. Van Hoof, F., Libert, J., Farriaux, J.-P., et par un melange d'acide chromique-phospho- al.: : tear analysis and conjunc- tungstique, C. R. Acad. Sci. 265: 1426, 1967. tival infrastructure. To be published. 32. Hardin, J. H., and Spicer, S. S.: Ultra- 18. Farriaux, J.-P., Legouis, I., Humbel, R., et structural localization of dialized iron reactive al.: La mannosidose, Nouv. Presse Med. 4: substances in rabbit neutrophils, basophils, 1867, 1975. and eosinophils, J. Cell. Biol. 48: 368, 1971. 19. Van Hoof, F., and Hers, H. G.: Mucopoly- 33. Lowry, O. H., Rosebrough, N. J., Farr, A. L., saccharidosis by absence of a-fucosidase, Lan- et al.: Protein measurement with the Folin- cet 1: 1198, 1968. phenol reagent, J. Biol. Chem. 193: 265, 20. Van Hoof, F., and Hers, H. G.: The ab- 1951. normalities of lysosomal enzymes in muco- 34. Tondeur, M., Vamos, E., and Libert, J.: polysaccharidoses, Eur. J. Biochem. 7: 34, Fucosidosis: biochemical and ultrastructural 1968. studies of two new cases. To be published. 21. Van Hoof, F.: Fucosidosis, in: Lysosomes 35. Philippart, M.: Fucosidosis: a novel neuro- and Storage Diseases, Hers, H. G., and Van visceral sphingolipidosis, Neurology 19: 304, Hoof, F., editors. New York, 1973, Academic 1969. Press. 36. Freitag, F., Kiichemann, K., Bliimke, S., et 22. Durand, P., Borrone, C., and Delia Cella, al.: Hepatic ultrastructure in Fucosidosis, G.: Fucosidosis, J. Pediatr. 75: 665, 1969. Virchows Arch. 7: 99, 1971. 23. Gatti, R., Borrone, R., Trias, X., et al.: 37. Dawson, G., and Spranger, J. W.: Fucosido- Genetic heterogenity in fucosidosis, Lancet sis: a glycosphingolipidosis, N. Engl. J. Med. 2: 1024, 1973. 285: 122, 1971. 24. Koussef, B. G., Beratis, B. G., Danesino, C., 38. Spicer, S. S., Garvin, A. J., Wohltmann, et al.: Genetic heterogenity in fucosidosis, H. J., et al.: The ultrastructure of the skin Lancet 2: 1387, 1973. in patients with mucopolysaccharidoses, Lab. 25. Patel, V., Watanabe, I., and Zeman, W.: Invest. 31: 488, 1974. Deficiency of a-L-fucosidase, Science 176: 39. Wiederschain, G. Y., and Rosenfeld, E. L.: 426, 1972. Two forms of a-L-fucosidase from pig kidney 26. Borrone, C., Gatti, R., Trias, X., et al.: and their action on natural oligosaccharides, Fucosidosis: clinical, biochemical, immuno- Biochem. Biophys. Res. Commun. 44: 1008, logic, and genetic studies in two new cases, 1971. J. Pediatr. 84: 727, 1974. 40. Carlsen, R. B., and Pierce, J. G.: Purifica- 27. Snyder, D.: Ocular manifestations of a-fuco- tion and properties of an a-L-fucosidase from sidosis, in The Eye in Inborn Errors of Me- rat epididymis, J. Biol. Chem. 247: 23, 1972. tabolism, Birth Defects. In press. 41. Robinson, D., and Thorpe, R.: Human liver 28. Loeb, H., Tondeur, M., Jonniaux, G., et al.: a-L-fucosidases, Clin. Chim. Acta 47: 403, Biochemical and ultrastructural studies in a 1973. case of "F" (fucosido- 42. Robinson, D., and Thorpe, R.: Affinity chro- sis), Helv. Pediatr. Acta 5: 519, 1969. matography of human liver a-L-fucosidase, 29. Reynolds, E. S.: The use of lead citrate at F. E. B. S. Lett. 45: 191, 1974. high pH as an electron opaque stain in elec- 43. Robinson, D.: Multiple forms of glycosidases tron microscopy, J. Cell Biol. 17: 208, 1963. in the normal and pathological states, En- 30. Thiery, J. P.: Mise en evidence des poly- zymes 18: 114, 1974. saccharides sur coupes fines en microscopic 44. MacPhee, G. B., Logan, R. W., and Prime- electronique, J. Micr. 6: 987, 1967. rose, D. A.: Fucosidosis: how many cases 31. Rambourg, A.: Detection des glycoproteines undetected? Lancet 2: 462, 1975.

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