A new variant of mucolipidosis was recently described and named mucolipidosis IV (ML IV).''2 A prominent feature in four patients with this disease was a moderate to severe corneal clouding seen at birth or in early infancy.2 Other ocular abnormalities included squint and amblyopia, both probably secondary to the corneal clouding. Systemically, these patients suffered from mental and motor retardation which was mild to moderate.
Electronmicroscopy of the conjunctiva * revealed, in all cases, the same two types of cytoplasmic inclusion bodies which can be found in all other mucolipidoses: singlemembrane limited vacuoles filled with fibrillogranular material and lamellar cytoplasmic bodies. The disease seems to be inherited as an autosomal recessive disorder and was described, until now, only in Jewish people. ML IV can be easily diagnosed when the combination of the clinical, ocular and systemic findings with the typical ultrastructural conjunctival changes are discovered.
The corneal histopathology of ML IV was not previously described. We had the opportunity to study the changes in corneal bottons removed by keratoplasty in two patients and the changes in the corneal epithelium removed by biopsy in another patient, and to compare them to the conjunctival ultrastructure. The conjunctival changes of patients two and three were previously described.2 The histologic and ultrastructural abnormalities in the cornea and their therapeutic application are the subject of the present paper.
M. G., a girl, was the first child born to healthy unrelated, young Jewish parents of Polish origin. At age of three months the child was referred to an ophthalmologist because of a squint. The eye examination revealed mild vertical nystagmus, esotropia and corneal clouding consisting of punctate opacities in the anterior stroma (Fig. 1). The corneal clouding was more pronounced in the center. The corneal diameter was 11.5 mm in each eye and the intraocular pressure was normal. The fundus could be seen with difficulty but the disc, the macula, and the posterior pole seemed normal. At the age of 20 months lamellar keratoplasty was performed in the right eye. This was initially successful but four months later a graft opacity appeared. The child has now a right concomitant esotropia.
Mild psychomotor retardation was first noticed at the age of four months. A medical examination at two years revealed a thin little girl whose head circumference and height were at the 25th percentile. Her live edge was firm and palpable, three cm below the costal margin. The spleen was not palpable. She was generally hypotonic, but her tendon reflexes were markedly increased. There were athetoid movements of her fingers.
Biochemical screening of urine for reducing substances, altered amino-acid pattern, and excess acid mucopolysaccharides was negative. Chromosome studies on lymphocytes and on cultured skin fibroblasts were essentially normal. Dermatoglyphics were within the range of normal as were the full blood count, serum glucose, calcium, phosphorous, alkaline phosphatase and electrolytes. There were no antibodies to rubella or toxoplasmosis in the serum. Radiolabeled sulfate uptake and lysosomal enzyme assays in cultured skin fibroblasts yielded normal results. Intracellular enzymes examined were N-acetyW3glucosaminidase, /3-galactosidase, N-acetyl-/3galactosaminidase, a-L-fucosidase, agalactosidase, ß -glucuronidase a-mannosidase, 0-glucosidase, acid phosphatase and arylsulfatase A. An electroencephalogram was suggestive of a widespread disturbance of brain function (high potential delta waves with a tendency to paroxysmal activity).
Fig. 1. The cloudy cornea in a patient with ML IV, Slit lamp biomicroscopy showed the opacity to be diffuse from limbus to limbus but essentially concentrated in the epithelial layer.
The early case history of this patient, M.S., a girl, was previously described (patient four in reference two).
At the age of two years there was an obvious clouding of both cornea, more pronounced in the central area, fading toward the periphery. Slit lamp examination showed a diffuse dusty opacification of the corneal epithelium. The deep corneal layers seemed normal. The corneal diameter was 11 mm .in each eye and the intraocular pressure was normal. Corneal epithelial scraping resulted in a clearing of the cornea enough to enable fundoscopy and retinoscopy. A normal fundus and a refractive error of -6.50 were found. The cloudiness of the cornea returned later but the exact time of its recurrence was not determined. Gonioscopy showed a wide angle without anomalies. Electroreîinography was performed and showed reduced photopic and scotopic responses.
The case history was previously described (K. V., a girl, patient two in reference two).
Corneal cloudiness and squint were apparent at the age of eight months. The clouding of the cornea seemed, by slit-lamp examination, to be greatest at the level of the epithelium but also to a lesser extent to involve the stroma. The intensity of the clouding seemed to vary with time, at some times the right cornea and at others the left cornea being the more opaque. The squint was concomittant and convergent, initially unilateral right, then alternating, and finally permanently unilateral left. Retinoscopy revealed a myopia of about -12.0 diopters in both eyes. When treatment by patching failed to relieve the left unilateral esotropia and it seemed that the left cornea is much more opaque than the right it was decided to do a left keratoplasty. The operation and the postoperative course were uneventful; but four months later some haziness was seen in the superficial corneal layers. Laboratory tests as performed in patient one yielded similar results.
MATERIALS AND METHODS
Lamellar keratoplasty was performed in patient one. A corneal button of 7 mm containing about 2/3 of the corneal thickness was removed and replaced by similar donor material. In patient two the corneal epithelium was removed by scraping with a Bard-Parker knife. In patient three, a 7 mm perforating corneal graft was performed. In all three patients, a conjunctival biopsy was performed at the same time by removing about three mm of bulbar conjunctiva.
The materials were immediately fixed in phosphate buffered five percent glutaraldehyde at a pH of 7.4 for two hours. The cornea was fixed during a prolonged period 20 hours), washed several times in buffer phosphate, and post-fixed in osmium tetroxide two percent for one hour.
All three patients were diagnosed as mucolipidosis IV on the basis of their clinical findings and the ultrastructure of the conjunctiva. The conjunctival epithelial cells contained two types of storage bodies in their cytoplasm (Fig. 2). In the stroma, mainly, laminated structures were found, both in the fibroblasts and in the vascular endothelial cells.
Light microscopy of the cornea (Fig. 3). The epithelium was, by far, the most involved of all corneal layers. Most epithelial cells were swollen and contained a vacuolar and foamy cytoplasm. Bowman's membrane seemed intact. Many keratocytes contained small vacuoles in their cytoplasm. Descemet' s membrane was normal. The endothelium could not be evaluated.
Electromicroscopy of the cornea. Corneal epithelial cells (Figs. 4-5) were distended by multiple single-membrane limited vacuoles. These vacuoles were clear or contained small, poorly developed laminated bodies. In case two (Fig. 6) well developed laminated bodies, rather than vacuoles, dominated the picture, in contrast to the other two cases.
Fig. 2. Conjunctival epithelium: some vacuoles (arrows) show a mosaic pattern consisting of lamellar material at one end, a myelin-like pattern at the other end and an optically clear area in between. (Case 1; original magnification ? 13500; M-mucus cell; vi-microvilli; N-nucleus; mmitochondria.)
Bowman's membrane was normal. Most keratocytes (Fig. 7) were swollen and contained in their cytoplasm two types of storage bodies: single-membrane limited vacuoles filled by fibrillogranular substance and laminated structures. In some keratocytes the cytoplasm was rather mildly affected (Fig. 8) while in others the fibrillogranular material seemed to be spilled out from the vacuoles into the surrounding stroma (Fig. 9).
Fig. 3. Corneal epithelial cells (Ep) and keratocytes (k) are ballooned containing a foamy cytoplasm. Bowman's membrane (Bo) is intact. (Case 1, Toluidine blue, original magnification x 1000; Epon section 1µ; St-stroma.)
Fig. 4. Corneal epithelial cells are filled by multiple cytoplasmic single-membrane limited vacuoles. Some small vesicles coalesce into a larger vacuole (V). (Case 1; original magnification ? 5000; N-nucleus; D-desmosomal junctions between epithelial cells.)
Fig. 5. Corneal epithelial cells. Higher magnification shows that many vacuoles contain several small laminated bodies (arrowheads) and are otherwise optically empty. (Case 3; original magnification ? 11400; N-nucleus.)
The conjunctival ultrastructural changes found in our patients are identical to those described in other cases of ML IV2 and some other mucolipidoses.8 Corneal clouding is a distinct and prominent feature of the clinical picture of most mucopolysaccharidoses, some mucolipidoses and one or two sphingolipidoses. The severe and early corneal involvement distinguishes ML IV from the other mucolipidoses, and the absence of excessive mucopolysacchariduria distinguishes this disease from the group of mucopolysaccharidoses.
In the group of mucopolysaccharidoses, numerous reports on the histological changes and ultrastructure of the cornea of patients with Hurler's disease (MPS l-H) indicated that a consistent picture is found: abnormal storage material in the form of intracytoplasmic vacuoles are seen in epithelial cells, in subepithelial histiocytes interrupting Bowman's membrane, in keratocytes and in endothelial cells. >e The subepithelial region seems to be most affected.3'4'7 The vacuoles are singlemembrane limited and are filled by fibrillogranular substance or are optically clear but some keratocytes contain also lamellar ("myelinated") cytoplasmic bodies.5'6 There is histochemical evidence that the fibrillogranular material in the vacuoles is acid mucopolysaccharide while the lamellar bodies consist of glycolipids.8 The materials are probably stored in the lysosomes.9
Fig. 6. Epithelial cells from corneal biopsy. The cytoplasm contains many more laminated bodies and fewer clear vesicles than the other two cases. (Case 2; original magnification ? 16400.)
Fig. 7. Corneal stroma: the keratocyte contains separately both types of storage bodies: optically dense membranous cytoplasmic bodies (MCB) and vacuoles filled by fibrillogranular material (V). Note the discontinuity of the cell membrane (arrows). Ccollagen fibers. (Case 1; original magnification ? 32000.)
Fig. 8. Corneal stroma: the keratocyte is distended by a large vacuole. Some of the fibrillogranular material is extracellular, possibly spilled out from the cell through breaks in the cell membrane (arrowhead); (Col-collagen; V-singlemembrane limited vacuole). (Case 3; original magnification ? 20000.)
The pathology of the cornea was found to be essentially similar in other mucopolysaccharidoes such as Hunter's disease (MPS II) even in cases of clinically clear cornea,10'11 in Scheie's disease (MPS l-S),12 in Sanfilippo's syndrome (MPS III),13 and in both the mild and severe forms of Metroteaux-Lamy disease (MPS Vl A and B).14"5
Membranous or lamellar cytoplasmic inclusions were also found in Fabry's disease, one of the sphingolipidoses. However, here they are concentrated in the basal layer of the epithelium, while the stroma and endothelium are normal.16·17
Of all mucolipidoses, only the cornea of GM1 -gangliosidosis was, to the best of our knowledge, described histologically. Both by light-microscopy18 and by electronmicroscopy,19 this cornea resembled the cornea of Hurler's disease (MPS l-H). The cornea epithelium and the keratocytes were affected and Bowman's membrane was interrupted by multiple foamy histiocytes. In another case20 the epithelium was normal and no subepithelial histiocyte deposits were found.
Fig. 9. Corneal stroma: two keratocytes with small vacuoles in the cytoplasm. The cellular membrane is not seen. The structure of the stromal collagen fibers seems normal. (Case 3; original magnification ? 25000.)
The present study shows that in MLIV the corneal intracytoplasmic storagE materials! as revealed b) electronmicroscopy, are similar to thos found in generalized gangliosidosis (OMI gangliosidosis type I) and resemble thosE found in all mucopolysacoharidoses. Th distribution of the deposits in the cornea is however, different The most affectec tissue! by far, is the epithelium - thc keratocytes being also affected bu Bowmar's membrane being well preservec without any histiocytes. Found in boff examined patients this particulai intracorneal distribution seems specific foi mucolipidosis IV and distinguishes thi5 disease from all other previously describec mucolipidoses and mucopolysacchari doses!
It seems conceivable that a certair sequence of the progress of the disease ir the keratocytes exists. Initially! thE cytoplasm contains one or more smal vacuoles filled by fibrillogranular materia while the cellular organelles seem normal Later the keratocyte is distended by Iarg~ vacuoles, contains lamellar bodies and thE cell organelles show destruction. In th final stage the cell is degenerating and th fibrogranular substance is spilled ou through breaks in the cell membrane.
The distension of the keratocytes and 01 the epithelial cells may explain thE cloudiness of the cornea Keratoplasty foi surgical treatment of a cornea opaque duE to a storage disease is accepted and gooc results were claimed.' However! we havE now doubts about keratoplasty being thc procedure of choice in mucolipidosis IV The practical failure of the grafts in oui cases could be a chance occurrence bu conceivably could result from the fact tha the epithelium is the most severely affectec corneal tissue in this disease. So, when thE recipienIs corneal epithelium regrowth~ over the donor cornea! the disease proces~ is re-established. Follow-up of these anc other cases of ML IV impressed us that the corneal cloudiness sometimes increases with time but more often is fluctuating - at some times being more, at others less severe. This raises the possibility of treatment by repeated epithelial scrapings, especially to prevent amblyopia in the more affected eye.
As all our patients were still young, it is not clear whether the corneal clouding becomes worse later in life. Recently, Newell et al 2' described a 23-year-old patient with a mucolipidosis and corneal clouding which could be an adult suffering of ML IV. The corneal clouding did not increase with time.
The exact storage material in ML IV is not yet known. In a recent study" a high intracellular level of two gang I ios ides was found in cultured fibroblasts of three patients with ML IV. These two gangliosides were GM3 and G03 and they possibly represent the lipid portion of the storage material of ML IV. The mucopolysaccharide portion was not yet defined.
The cornea of three patients with corneal cloudiness due to mucolipidosis IV (ML IV) was examined by light and electronmicroscopy. Light microscopy of the cornea showed swollen epithelial cells, which contained a foamy cytoplasm and vacuolated keratocytes. By electronmicroscopy, intracytoplasmic single-membrane limited vacuoles filled by fibrillogranular substance and laminated structures were discovered. Bowman's membrane was intact Compared to the cornea in other storage diseases, the severely affected epithelium together with intact Bowman's membrane distinguishes ML IV from the group of mucopolysaccharidoses and from GM1gangliosidosis.
We wish to thank Dr. C. Legum, Department of Genetics, Tel-Aviv Medical School, for referring a patient; Prof. Elaine R. Berman, for the biochemical studies; Prof. I.S. Levij, Department of Pathology, Hadassah Hebrew University Medical School; and Dr. Kenneth R. Kenyon, Wilmer Eye Institute, for critical reading of the manuscript.
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