II (I- disease): Ultrastructural observations of conjunctiva and skin

Kenneth R. Kenyon and Judith A. Sensenbrenner

Conjunctival and skin from a 25-monih-old patient with mucolipidosis II (I-cell disease) were studied by electron microscopy. In both tissues the subepithelial connective tissue was markedly hypercellular, as histiocytes and fbroblasts showed extensive vacuolation by single membrane-limited inclusions with fbrillogranular and membranous lamellar contents. The Schwann cells and axonal processes of peripheral nerves and the vascular perithelial cells were similarly affected. Histochemical and ultrastructural evidence indicates that these storage vacuoles may be derived from which contain excessive amounts of acid mucopoly- saccharide and . Biomicroscopically visible granularity of the corneal stroma suggests the accumulation of these storage substances also within the cornea. Symmetrically enlarged corneas were also present.

Key words: I-cell disease, mucolipidosis, , sphingolipidosis, acid mucopolysaccharide, glycolipid, , megalocornea, corneal clouding, ultrastructure, conjunctiva and skin, , fibrillogranular vacuole, membranous lamellar vacuole

T.he mucolipidoses (MLS's) are a newly an intermediate position between the muco- 1 categorizeJU d group of inherited metabolic polysaccharidoses and the . storage diseases which seemingly occupy The ten disorders currently included in this group are listed in Table I. Clinically, these disorders share many From the Eye Pathology Laboratory, Wilmer features of the mucopolysaccharidoses Ophthalmological Institute, and the Moore (MPS's), including coarse facial features, Clinic of The Johns Hopkins Hospital, Balti- more, Md. skeletal dysplasia, and mental retardation Supported in part by Public Health Service Post- (Table II). Biochemically, however, the doctoral Fellowship 1 F02 EY 48611-01 from MLS's resemble the sphingolipidoses (SLS's) the National Eye Institute (Dr. Kenyon). with respect to normal urinary excretion Presented at the Spring Meeting of the Associa- of acid mucopolysaccharide (AMP) and tion for Research in Vision and Ophthalmology, visceral storage of glycolipid and/or April 26 to 30, 1971, Sarasota, Fla. sphingolipid in addition to AMP. The sig- Reprint requests to: Dr. Kenneth R. Kenyon, Eye nificant ocular manifestations of the MLS's Pathology Laboratory, The Johns Hopkins are also common to both the mucopoly- Hospital, 601 North Broadway, Baltimore, Md. 21205. saccharide and sphingolipid disorders. Manuscript submitted June 11, 1971; manuscript These ocular features are variable and in- accepted June 29, 1971. clude corneal clouding (in generalized 555

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Fig. 1. Light photomicrograph of the conjunctiva shows connective tissue cells with vaciiolated cytoplasm (asterisks) to be most numerous in the collagenous stroma (C) which underlies the conjunctival epithelium (E). (Paraphenylenedianiine, phase contrast, x800.)

Table I. Classification of the mucopolysaccharidoses (MPS's), mucolipidoses (MLS's), and sphingolipidoses

Mucopolysaccharidoses Mucolipidoses Sphingolipidoses MPS I (Hurler) Generalized Tay-Sachs disease (G11 .-gangliosidosis MPS II (Hunter) (Gjii-gftngliosidosis I and II) I) MPS III (Sanfilippo) Sandhoff's disease (Gus-gangliosidosis MPS IV (Morquio) ID MPS V (Scheie) Juvenile sulfatidosis, Austin type Niemann-Pick disease MPS VI (Maroteaux-Lamy) MLS I (lipomucopolysaccharidosis) Gaucher's disease MLS II (I-cell disease) Fabry's disease MLS III (pseudo-Hurler polydys- Krabbe's disease trophy) Infantile metachromatic leukodys- Farber's disease trophy Sea-blue histiocyte syndrome Lactosyl ceramidosis '(chronic Niemann-Pick disease)

gangliosidosis, MLS I, and MLS III) and this disease. At present, only about ten macular cherry-red spot (in generalized cases have been reported.10 On the basis gangliosidosis, MLS I, and Farber's dis- of these reports, MLS II may be char- ease ). acterized as an autosomal recessive trait MLS II, the I-cell variant, is a rare dis- which is present at birth and is associated order which was first described in 1967 by with severe psychomotor retardation, early Leroy and DeMars,2 who found coarse cessation of growth, Hurler-like facies with cytoplasmic inclusions in cultured fibro- characteristic gingival hyperplasia, and ex- blasts (which they designated I-cells or treme skeletal dysplasia. Additional clinical inclusion cells) from two children with signs include mild hepatic enlargement,

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Fig, 2. Light photomicrograph of the skin reveals many vacuolated histiocytes (asterisks) in the superficial dermis. E, epidermal cells; C, collagenous dermis. (Paraphenylenediamine, phase contrast, xl,000.)

Table II. Comparative features of the mucopolysaccharidoses, mucolipidoses, and sphingolipidoses (after Spranger and Wiedemann1)

Mucopolysaccharidoses Mucolipidoses Sphingolipidoses Gargoyle-like facies Gargoyle-like facies No gargoyle-like facies Skeletal dysplasia Skeletal dysplasia No skeletal dysplasia Mental retardation (variable) Mental retardation (variable) Mental retardation Abnormal mucopolysacchariduria Normal mucopolysacchariduria Normal mucopolysacchariduria Visceral storage of AMP Visceral storage of AMP and gly- Visceral storage of colipids Ocular features (variable) Ocular features (variable) Ocular features (variable) Corneal clouding Corneal clouding Corneal clouding Retinal pigmentary degeneration Macular cherry-red spot Macular cherry-red spot Optic atrophy

restricted mobility, and unusually tight metachromatic with toluidine blue' thereby skin. Corneal opacities have been described indicating storage of both glycolipids and in only one case of probable MLS II,6 and AMP. macular cherry-red spot has never been The pathogenesis of MLS II is as yet reported. unclear. Biochemical studies of I-cell tis- Laboratory findings include vacuolated sues and cultured cells are not in complete lymphocytes and bone marrow cells and agreement; however, decreased activity of normal urinary excretion of AMP. The re- beta galactosidase is the most consistent fractile cytoplasmic inclusions in cultured finding to date.1"3' 5'7 fibroblasts are periodic acid-Schiff (PAS) To our knowledge, the histopathology of and Sudan black positive* and, following MLS II has not been previously reported. treatment with chloroform-methanol, are Thus, the present ultrastructural study of

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Fig. 3. In the superficial stroma of the conjunctiva, histiocytes and fibroblasts are numerous. The cytoplasm of these cells shows an alveolar reticulated appearance, as abnormal storage material (asterisks) has distended the cells, displaced their nuclei (N), and seemingly excluded other cytoplasmic organelles. E, epithelium; C, collagenous connective tissue. (x5,400.)

conjunctiva and skin from a patient with of I-cell disease. The was enlarged to 8 cm. I-cell disease is presumably the initial histo- below the right costal margin and a spleen tip was palpable. Everywhere on his body, the skin was pathologic description of this disorder. smooth, tight, and thickened. Case report Ophthalmic evaluation disclosed that the child followed objects and fixated well in all fields of The patient (JHH 140 25 75) was the 25- gaze. There was neither strabismus nor nystagmus. month-old son of unrelated Caucasian parents. Fol- The pupils were round, regular, and reactive to lowing an unremarkable pregnancy and delivery, light. External examination was normal except for coarse facies and inguinal hernias were noted, and an increased corneal diameter of 12.5 mm. bilater- subsequent psychomotor development was ex- ally. The corneas appeared grossly clear but tremely slow. In November, 1970, physical ex- showed diffuse fine stromal granularity by slit- amination revealed an alert but otherwise severely lamp examination. Intraocular pressures were 13.0 retarded child with the facial and skeletal stigmata mm. Hg (Schi^tz) bilaterally on two occasions.

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Fig. 4. At higher magnification, the cytoplasm of a conjunctival histiocyte (area indicated by large asterisk in Fig. 3) is filled by an alveolar rericulum of unit membranes (each — 60 A in thickness; arrows) interspersed with fibrillogramilar material (asterisk) and membranous material arranged as concentric (white circle) or parallel (black circle) lamellae. (*83,500.)

Dilated pupil funduscopy was entirely within acetate and lead citrate and were examined with normal limits; specifically, the disc and vessels ap- a JEM 100-B electron microscope. peared normal, and neither macular cherry-red spot nor retinal pigmentary change was evident. Results Laboratory studies showed metachromatically vacuolated lymphocytes, normal bone marrow, and Histologic examination of the conjunc- normal mucopolysacchariduria. Skeletal x-rays tiva revealed the accumulation of large showed severe dysostosis multiplex.1 mononuclear histiocytes with foamy cyto- plasm in the superficial stroma (Fig. 1). Materials and methods Similar cells in the deeper conjunctival Biopsies of inferior bulbar conjunctiva and an- connective tissue were also distended by terior abdominal skin were obtained under general fine cytoplasmic vacuoles. In the superficial anesthesia and immediately processed for histo- derm is of the skin, cellular vacuolation chemical study and electron microscopy. For histochemistry, frozen unfixed sections of was equally impressive (Fig. 2). skin were stained with toluidine blue, Alcian blue, Histochemical studies of the skin demon- PAS, oil red O, Sudan black, aldehyde fuchsin, strated connective-tissue cells which stained acid phosphatase, glucuronidase, and ribonuclease. positively for acid mucopolysaccharide by For electron microscopy, conjunctival and skin the Alcian blue technique. These cells, specimens were fixed in two per cent osmium tetroxide (buffered with 0.14M veronal acetate, however, did not show toluidine blue meta- pH 7.3, 280 milliosmolar) for 20 minutes at 0° chromosia (although chloroform-methanol C. and for one hour at room temperature. These extraction was not attempted) and did not tissues were then dehydrated in graded alcohols, stain with PAS, lipid. stains, or lysosomal embedded in Araldite epoxy resin, and sectioned stains. on a Porter-Blum MT-2 ultramicrotome. For orientation and light photomicrography, thick sec- By electron microscopy, numerous bal- tions (•—> 1 p) were stained with paraphenylene- looned histiocytes were especially striking diamine. Thin sections were stained with uranyl in the superficial stroma of the conjunctiva

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Fig. 5. In some conjunctival histiocytes, membranous lamellae form elaborate myelin-like con- figurations (circled) with — 55 A periodicity and characteristic splitting of the dense band (arrows). (x90,000.)

(Fig. 3). The cytoplasm of these cells was Frequently, these membranous lamellar packed with abnormal material which often inclusions assumed elaborate myelin-like excluded all other organelles except the configurations in which a characteristic nucleus. Discrete intracellular vacuoles periodicity of 50 to 60 A could be resolved were seldom evident; rather, rupture and (Fig. 5). In the deeper conjunctival stroma, coalescence of vacuoles had apparently connective tissue cells were also extensively produced an alveolar reticulum of mem- involved (Fig. 6), but there the abnormal branous material. At higher magnification, storage inclusions retained their integrity the storage material was seen to be com- as discrete, single membrane-limited vac- posed of sparse, fine, granular substance ules which contained predominantly fibril- and of membranous inclusions arranged as logranular material (Fig. 7). In these cells, parallel or concentric lamellae (Fig. 4). membranous lamellar vacuoles, lipoid

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Fig. 6. In the deeper conjunctival stroma, connective tissue cells are filled with discrete storage vacuoles (measuring ~ 0.5 to 1.0 M in diameter). N, nucleus; C, collagen. {x7,700.)

globules, and polymorphous electron-dense vascular endothelium of the lymphatics inclusions were less prominent than in the and capillaries was also not involved, but subepithelial connective tissue cells. perithelial cells of the capillaries consistent- In the skin, histicytes and fibroblasts of ly contained prominent storage vacuoles the superficial dermis displayed vacuolation (Fig. 11). which was identical to that of the con- In both tissues, neither extracellular junctival connective tissue cells (Fig. 8). vacuoles nor abnormal accumulations of In both conjunctiva and skin, the extracellular material were discernible by Schwann cells and axonal processes of electron microscopy. peripheral nerves showed these same vacuolar changes (Figs. 9 and 10). The Discussion epithelial cells of the conjunctiva and the In our patient with MLS II and in pre- epidermis were seemingly unaffected. The vious studies of the MLS's, the findings

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Fig. 7. At higher magnification, the cytoplasm of a conjunctival fibroblast, similar to those illustrated in Fig. 6, is seen to contain vacuoles which are limited by single unit membranes (each ~ 60 A in thickness; arrows) and which contain predominantly fine fibrillogranular material.

suggest that both AMP and glycolipids lipoid or membranous lamellar inclusions.5 are the abnormally accumulated substances In generalized gangliosidosis, visceral his- in these storage diseases. tiocytes have also been shown to contain Histochemical evidence for combined storage mater-ial which was PAS positive, storage of AMP and glycolipid in MLS II weakly sudanophilic, and weakly toluidine is afforded by the aforementioned work of blue metachromatic,9 whereas in the ocular Leroy and associates3 and of Matalon and tissues affected by this disorder, vacuolated co-workers.4 Both groups found positive histiocytes demonstrated an increased AMP staining with PAS and Sudan black and content by the colloidal iron reaction.10 metachromasia with toluidine blue in cul- Similarly, in mucolipidosis III (pseudo- tured I-cells, which also showed an in- Hurler polydystrophy), :Alcian blue stain- creased content of AMP and glycolipid by ing of skin fibroblasts11 and toluidine blue direct biochemical assay.4 These findings metachromasia of conjunctival connective are entirely consistent with observations of tissue cells12 have also indicated an ab- liver tissue from the present patient in normal accumulation of AMP. which hepatic reticuloendothelial cells held In the MPS's, wherein AMP storage pre- Sudan black-positive material, which by dominates, electron microscopy reveals electron microscopy proved to be large storage vacuoles with predominantly fibril-

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Fig. 8. In the skin, histiocytes of the superficial dermis show extensive vacuolation by single membrane—limited vacuoles which contain fibrillogranular material and membranous lamellae (asterisks). Inset, Two membranous lamellar vacuoles (extreme right of figure) are shown at higher magnification. E, epithelial cell; BM, basement membrane of epidermis; C, collagenous connective tissue; N, nucleus. (*8,750; inset, x40,000.) logranular contents (see Discussion, ref. metabolic disorders (including the MPS's 13), whereas in the SLS's, membranous and SLS's) in which storage substances are lamellar vacuoles have been shown to con- thought to accumulate within lysosomes, tain glycolipid (see Discussion, refs. 12 the impaired or deficient hydrolytic en- and 14). Ultrastructural studies of a variety zymes of which render these substances in- of tissues affected by MLS's have consist- digestible. Reports of significantly de- ently disclosed large numbers of both creased activity of one or more of the fibrillogranular and membranous lamellar acid hydrolases in tissues of MLS II pa- vacuoles.10'12'1517 And for MLS II, in par- tients2' 5"7 are consistent with the possibility ticular, the cellular defect appears to be of a lysosomal disorder in this disease. virtually a composite picture of the MPS's Additional histochemical studies of cul- and SLS's. Thus, the simultaneous occur- tured I-cells have also revealed increased rence of both vacuolar types in the MLS's activity of the lysosomal enzyme, acid constitutes strong morphologic evidence for phosphatase, an observation which suggests the accumulation of both AMP and glyco- that the I-cell inclusions may be activated lipid in these disorders and additionally lysosomes.2 This finding was also especially attests to their intermediate position be- striking in the liver specimen of the tween the MPS's and the SLS's. present patient, wherein focal epitheloid The concept of lysosomal diseases, first cell showed markedly increased introduced in 1964 by Van Hoof and Hers,18 histochemical staining for acid phosphatase, has subsequently been applied to those which by electron microscopy proved to

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Fig. 9. The Schwann cell of a conjunctival nerve contains numerous intracytoplasmic vacuoles. Inset, Higher magnification of vacuole (indicated by asterisk in figure) shows its single limiting membrane (arrow) which encloses granular and membranous contents. N, nucleus. (x27,000; inset, x855000.)

correspond to innumerable intracellular with the MPS's.21 In the absence of signs vacuoles with fibrillogranular and mem- of increased intraocular pressure, therefore, branous lamellar contents.8 Furthermore, the enlarged corneas of our patient may be in MLS P9 and in various MPS's and a connective tissue abnormality which is SLS's,20 ultrastructural localization of acid related to defective AMP metabolism. Cor- phosphatase activity has been specifically neal clouding, which is associated with associated with these storage vacuoles. On many of the metabolic storage diseases, the basis of such histochemical and electron presumably results from the light-scattering microscopic studies, therefore, it would effect of the abnormally accumulated stor- appear that in the MLS's, as well as in the age substances which perturb the lamellar MPS's and SLS's, the storage vacuoles may organization of the corneal stroma (see be derived from altered lysosomes which Discussion, refs. 14 and 22). Accordingly, have become engorged with accumulated several ultrastructural studies of corneas AMP and glycolipid substances. affected by various MPSV4> — and by Among the clinical features of the pres- generalized gangliosidosis10 have demon- ent patient, symmetrically enlarged corneas strated excessive intracellular and extra- and fine granularity of the corneal stroma cellular AMP in quantities which corre- are the only findings of direct ophthalmic lated with the extent of corneal clouding. interest. Both megalocornea and buphthal- In MLS II, therefore, it is probable that nios have been described in association AMP and glycolipid deposits throughout

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Fig. 10. The axonal process of a cutaneous nerve contains several membranous lamellar in- clusions (ML). A, axonal processes; S, Schwann cell cytoplasm. (x85,000.)

the cornea account for the fine stromal MLS variation,21 the cornea is clouded granularity observable by slit lamp in the (presumably by AMP and glycolipid ac- present patient. cumulation) even though the conjunctiva It is of additional interest, however, that shows essentially no cellular defect. And in many of these storage disorders, corneal finally, in comparing MLS II (I-cell dis- changes seem to occur independently of ease) and MLS III (pseudo-Hurler poly- pathologic alterations in adjacent tissues. dystrophy), we have determined that In particular, we have studied conjunctival the ultrastructural alteration of the con- ultrastructure in several of the MPS's and junctiva is far more severe in the former MLS's and have found that conjunctival disorder, whereas corneal clouding is clini- pathology is not always a reliable indicator cally significant only in the latter disease. of corneal opacification. In the systemic The physicochemical basis for these specific MPS's, for example, although the degree of reactions of the cornea to each disorder of vacuolation of conjunctival connective tis- AMP-glycolipid metabolism remains to be sue cells paralleled the extent of corneal determined. clouding in MPS types I, II, V, and VI, this relationship did not hold for MPS's III The authors gratefully acknowledge Dr. John E. and IV.13 Moreover, in macular corneal Dowling for use of laboratory facilities, Dr. Robert dystrophy (an inherited localized disorder G. Wyllie for histochemical studies, Mr. Kenneth 23 A. Linberg for technical assistance, Mr. Terry of AMP metabolism) and in Goldberg's George, R.B.P. for photomicrography, Mrs. Faye

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Fig. 11. The peripheral cell (P) of a dermal capillary contains many vacuoles with granular, membranous, and electron-dense contents. The endothelial cells (E) lining the capillary are apparently unaffected. N, nucleus; asterisk, capillary lumen; BM, basement membrane. (xl3,300.)

Snyder for secretarial assistance, and Drs. W. R. March of Dimes, 1969, pp. 174-185. Green, V. A. McKusick, H. A. Quigley, and C. I. 4. Matalon, R., Cifonelli, J. A., Zellweger, H., Scott for critical review of the manuscript. and Dorfman, A.: Lipid abnormalities in a variant of the Hurler syndrome, Proc. Nat. REFERENCES Acad. Sci. 59: 1097, 1968. 1. Spranger, J. W., and Wiedemann, H. R.: The 5. Leroy, J. G., and Spranger, J. W.: I-cell dis- genetic mucolipidoses: Diagnosis and differ- ease (Cont.), N. Engl. J. Med. 283: 598, ential diagnosis, Humangenetik. 9: 113, 1970. 1970. 2. Leroy, J. G., and DeMars, R. I.; Mutant 6. Luchsinger, U., Buhler, E. M., Menes, K., enzymatic and cytological phenotypes in cul- and Hirt, H. R.: I-cell disease, N, Engl. J. tured human fibroblasts, Science 157: 804, Med. 282: 1374, 1970. 1967. 7. Lightbocly, J., Wiesmann, U., Hadorn, B., and 3. Leroy, J. G., DeMars, R. I., and Opitz, J. M.: Herschkowitz, N.: I-cell disease: Multiple I-cell disease, in Clinical delineation of birth lysosomal-enzyme defect, Lancet 1: 451, defects, Vol. 6. IV. Skeletal dysplasias, Origi- 1971. nal Article Series, National Foundation— 8. Kenyon, K. R., Wyllie, R. G.3 and Senseii-

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bienner, J. A.: Hepatic infrastructure and 17. Loeb, H., Tondeur, M., Toppet, M., and histochemistry in mucolipidosis II (I-cell Cremer, W.: Clinical, biochemical and ultra- disease). In preparation. structural studies of an atypical form of muco- 9. O'Brien, J.: Generalized gangliosidosis, in polysaccharidosis, Acta Paediatr. Scand. 58: Clinical delineation of birth defects, Vol. 5. 220, 1969. IV. Skeletal dysplasias, Original Article Series, 18. Van Hoof, F., and Hers, H. G.: L'ultrastruc- National Foundation—March of Dimes, 1969, ture des cellules hepatiques dans la maladie pp. 190-206. de Hiirler (gargoylisme), C. R. Acad. Sci. 10. Emery, J. M., Green, W. R., Wyllie, R. G., Paris 259: 1281, 1964. and Howell, R. R.: Gin-gangliosidosis: Ocular 19. Freitag, F., Spranger, J., and Bliimcke, S.: and pathological manifestations, Arch. Oph- Die Bildung von Speicherelementen in Leber- thalmol. 85: 177, 1971. zellen bei Lipomucopolysaccharidose, Verh. 11. Scott, C. I.: Pseudo-Hurler poly dystrophy, in Dtsch. Ges. Pathol. 54: 545-550, 1970. Clinical delineation of birth defects, Vol. 6. 20. Wallace, B. J., Volk, B. W., Schneck, L., and VIII. The eye, Original Article Series, Na- Kaplan, H.: Fine structural localization of two tional Foundation—March of Dimes, 1971. hydrolytic in the cerebellum of chil- 12. Quigley, H. A., and Goldberg, M. F.: Con- dren with lipidoses, J. Neuropathol. Exp. junctival ultrastructure in mucolipidosis III Neurol. 25: 76, 1966. (pseudo-Hurler polydystrophy). In prepara- 21. McKusick, V. A.: Heritable disorders of con- tion. nective tissue, ed. 3, St. Louis, 1966, The C. 13. Kenyon, K. R., Quigley, H. A., Hussels, I., V. Mosby Company, p. 335. Goldberg, M. F., and Wyllie, R. G.: The 22. Kenyon, K. R., Topping, T. M., Green, W. R., systemic mucopolysaccharidoses: Ultrastruc- and Maumenee, A. E.: Ocular pathology of tural and histochemical studies of conjunctiva the Maroteaux-Lamy syndrome (systemic and skin. In preparation. mucopolysaccharidosis type VI): A histologic 14. Topping, T. M., Kenyon, K. R., Goldberg, and ultrastructural report of two cases, Am. M. F., and Maumenee, A. E.: Ultrastructural J. Ophthalmol. In press. ocular pathology of Hunter's syndrome: Sys- 23. Quigley, H. A., and Goldberg, M. F.: Scheie temic mucopolysaccharidosis type II, Arch. syndrome and macular corneal dystrophy: An Ophthalmol. In press. ultrastructural comparison of conjunctiva and 15. Freitag, F., Kiichemann, K., Bliimcke, S., and skin, Arch. Ophthalmol. 85: 553-564, 1971. Spranger, J.: Hepatic ultrastructure in fuco- 24. Goldberg, M. F., Cotlier, E., Fichenscher, L. " sidosis, Virchows Arch. (Zellpathol.) 7: 99- G., Kenyon, K. R., Enat, R., and Borowsky, 113, 1971. S. A.: A new autosomal recessive storage dis- 16. Freitag, F., Bliimcke, S., and Spranger, J.: ease with macular cherry-red spot, comeal Hepatic ultrastructure in mucolipidosis I clouding and beta-galactosidase deficiency: (lipomucopolysaccharidosis), Virchows Arch. A clinical, biochemical and electron micro- (Zellpathol.) 7: 189-204, 1971. scopic study. In preparation.

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