Fucosidosis: 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 Downloaded from iovs.arvojournals.org on 10/01/2021 Volume 15 Fucosidosis 627 Number 8 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 glycoproteins 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 Downloaded from iovs.arvojournals.org on 10/01/2021 628 Libert, Van Hoof, and Tondeur Investigative Ophthalmology August 1976 ^-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.