British Journal of Ophthalmology, 1989, 73, 665-668

Corneal thickness in nephropathic cystinosisBARRETT KATZ,'2 RONALD B MELLES,', JERRY A SCHNEIDER,3AND NARSING A RAO4

From the University of California, San Diego, Departments of'Ophthalmology, 2Neurosciences, and'Pediatrics, and 4Estelle Doheny Eye Foundation, University ofSouthern California, USA

SUMMARY Cystinosis is a rare autosomal recessive metabolic disorder in which non-protein cystineaccumulates within cellular lysosomes owing to a defect in lysosomal cystine transport. Thepathognomonic ocular manifestation of cystinosis is the deposition of distinctive iridescent crystalsin the cornea, not associated with any inflammatory response or recognised change in cornealfunction. We measured corneal thickness in nine patients with infantile nephropathic cystinosis.We also studied a corneal button from one of these patients who underwent corneal transplanta-tion. All nine patients had increased corneal thickness in comparison with an age matched controlpopulation. Electron microscopy analysis of the cystinotic button revealed structural changes ofboth epithelium and endothelium layers. Increased corneal thickness in patients with nephropathiccystinosis may reflect subclinical corneal oedema.

Cystinosis is an autosomal recessive metabolicdisorder characterized by the accumulation of non-protein cystine within lysosomes of cells owing to adefect in lysosomal cystine transport.' It occurs inthree phenotypical forms: a nephropathic childhoodform, a benign adult form, and an intermediateadolescent form. The childhood form is the mostcommon and the most devastating. Systemic compli-cations include the renal Fanconi syndrome withrickets and growth retardation, and renal failure thatnecessitates transplantation by an average age of10 years.2 The adolescent and adult forms are associ-ated with milder disease but share the patho-gnomonic ocular manifestation of cystinosis: distinc-tive iridescent crystals in the cornea and conjunctiva.3The crystals are birefringent, intracellular, and ofvarying form. Needle shaped or fusiform crystalsare seen in the cornea, whereas polymorphous,rectangular, or rhomboidal crystals infiltrate theconjunctiva.' Ultrastructural analysis has shownthese crystals to be intralysosomal.6 I Crystallo-graphic analysis has shown the rectangular forms tobe composed of L-cystine.? Patients with cystinosiscommonly complain of photophobia and occasion-

Correspondence to Barrett Katz, MD. Pacific Presbyterian MedicalCenter and the Smith-Kettlewell Eye Research Institute, 2340 ClayStreet, San Francisco, CA 94115, USA.

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ally develop corneal erosions.9 A pigmentary retino-pathy affecting the periphery has been described inthe nephropathic form of the disease' but not in thebenign form." Crystals are also seen in the iris, ciliarybody, choroid, retinal pigment epithelium, sclera,episclera, extraocular muscles, and optic nervesheath.- 12The formation of corneal cystine crystals has a

recognisable spatial as well as temporal sequence.'3Anterior deposition begins early in life and proceedsposteriorly as the patient ages; deposition advancesmore rapidly at the periphery.'3 We measuredcorneal thickness in a group of patients with infantile-onset nephropathic cystinosis to determine whetherthe accumulation of corneal intralysosomal cystine isassociated with recognisable changes. We alsoexamined a corneal button from one of our patientsundergoing corneal transplantation and correlated itsultrastructure with our clinical observations.

Subjects and methods

We studied nine patients between the ages of 3 and 28(mean age=12-7 years) with infantile onset nephro-pathic cystinosis. Three were male and six female.Three were taking oral cysteamine (a free thiol withcystine-depleting capacity). Five of the nine had hadsuccessful renal transplantation and three of these

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Barrett Katz, Ronald B Melles, Jerry A Schneider, and Narsing A Rao

were taking steroids. The patients had a completeneuro-ophthalmological examination by one of us(BK). All were essentially emmetropic and hadabundant crystal deposition in their conjunctiva andcornea. Pupillary responses were normal, withoutevidence of afferent pupillary defect. Ophthalmo-scopic examination revealed mild depigmentation ofthe peripheral retina in all the patients. The fundus inall of them was patchy and mottled from the midequator to the ora serrata. The optic nerves were ofnormal colour, contour, and capillarity. No abnor-mality of nerve fibre layer could be detected.A control group consisted of nine normal young-

sters without corneal abnormalities or history ofocular disease. Their ages ranged from 3 to 16 years(mean 10-4 years). Four were male and five female.None of the subjects in either group were taking oralcontraceptives or wore contact lenses-factors whichmay cause an increase in corneal thickness.'4We used a commercially available ultrasonic

pachymeter (DGH-2000 Ultrasonic Pachymeter,DGH Technology Inc., Pennsylvania) to determinecorneal thickness.' The pachymeter has a 1-5 mmdiameter solid probe tip and a transducer frequencyof 20 mega hertz. The instrument has a measurementrange of 0-20 to 1-3 mm, a resolution of 1 pm, and anaccuracy of±5 ptm.

All measurements were made by one of us (BK).The pachymeter probe was applied to the centreof the cornea after topical application of 0*5%proxymetacaine (proparacaine). Three measure-ments were recorded for each eye; the average of thethree measurements was calculated and recorded.We examined a corneal button obtained from

patient 8, who (after our study) underwent cornealtransplantation at age 20. The button was taken fromthe operating room and immediately fixed in half-strength Karnovsky's solution (2-5% glutaraldehydeand 2-0% paraformaldehyde in 0-1 M phosphatebuffer, pH 7-2). The tissue was processed for trans-mission electron microscopy by post-fixation in 2.0%osmium tetroxide, and dehydrated through a seriesof graded concentrations of ethyl alcohol and inpropylene oxide. The tissue was embedded inPolybed resin (Polysciences, Warrington, PA).Selected areas were examined by transmissionelectron microscopy. Sections 1 ptm thick wereprepared for light microscopy by toluidine bluestaining.

Results

The mean corneal thickness in the patients withcystinosis was 611-2 ptm (n= 18, SD=42-4, SE= 10-0)(Table 1). The mean corneal thickness of the normalsubjects was 548-4 ptm (n=18, SD=28-4, SE=6-7)

Table 1 Corneal thickness in patients wit/i nephropathliccystinosis

Patient no. Age (years) Central corneal thickness (am)

Right eye Left eye

1 3 623 6272 4 647 5993 7 605 5734 9 628 6485 9 572 5586 15 644 6477 19 562 5628 20 665 6759 28 590 549Mean (SD) 12 7 (8-4) 611-2 (42-4)

Table 2 Corneal thickness in control group

Patient no. Age (years) Central corneal thickness (pan)

Right eye Left eye

1 3 555 5892 6 576 5753 8 552 5574 9 547 5565 11 517 5046 13 559 5447 13 563 5718 15 564 5589 16 494 491Mean (SD) 10-4 (4-3) 548 4 (28.4)

(Table 2). Corneal thickness was significantly higherin the cystinosis patients (Student's t test, p<0.001).Simple linear regression analysis showed poor corre-lation of the corneal thickness in cystinosis withpatient age (r=0-2).

This increase in corneal thickness was not accom-panied by obvious abnormality of epithelium orendothelium, nor was it reflected in decreased acuityor clinically recognisable oedema. It was not accom-panied by raised intraocular pressure. No abnor-mality recognisable at the slit-lamp (other thanstromal infiltration with crystalline material) could beseen.

Histological examination of the corneal buttonrevealed an intact epithelium with normal Bowman'slayer and Descemet's membrane. Basal cells of theepithelium showed intracellular oedema. Superficialkeratocytes showed vacuolated cytoplasm. Therewas degeneration of the endothelium with focal cellloss. Ultrastructural examination of the corneashowed the presence of rectangular intracytoplasmiccrystalline inclusions throughout the entire cornealstroma, within keratocytes. Deposition of crystallineinclusions was not associated with any recognisableinflammatory reaction or change of extracellular

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667Corneal thickness in nephropathic cystinosis

Fig. 1 Electron microscopy ofendothelial cell (E) reveals swollen mitochondria with disarray ofcytoplasmic organelles.Legend: E=endothelial cell. B (arrow) =banded basement membrane. D= Descemet's membrane. N=nucleus of endothelialcell (x4800).

matrix. The endothelial cells showed disorganisedorganelles within their cytoplasm. Mitochondriawere swollen and cristae were indistinct (Fig. 1).

Discussion

Corneal thickness varies little either betweenpatients or between eyes of the same patient.' Theultrasonic pachymetric determination of cornealthickness used in this study has been shown to be ahighly reliable method which eliminates errors due toobserver interpretation inherent in the opticalmeasurement of corneal thickness.'7 The meancorneal thickness of our control patients is similar tothat found by previous investigators,' 9 whoreported the average thickness of the normal humancornea (measured centrally) to be between 050 and057 mm.

Corneal thickness depends on the water content ofthe cornea; there is a linear relationship betweencorneal thickness and hydration.'9 For the humancornea Mishima found the relation between the twomeasurements to be defined by the equation:H=7-0 q-0-64, where H is the hydration expressedin water weight per dry weight of tissue and q is the

corneal thickness in millimetres.'9 The thickenedcystinotic cornea may therefore reflect increasedwater content.

Absorption of water into the polysaccharidematrix of the cornea leads to increased cornealthickness. Yet the cornea swells only in a directionperpendicular to its plane of curvature, a result of theunique arrangement of collagen fibrils parallel to theplane of the corneal curvature. '9 The expansion of thestroma along this corneal plane is restricted by theplanar arrangement of the collagen fibrils; there arefew fibrils running in the anterior to posterior direc-tion to restrain swelling.The maintenance of corneal hydration is depend-

ent on metabolic activity within the corneal endo-thelium. Ouabain, an inhibitor of Na+-K'-ATPase(an enzyme necessary for active transport), causescorneal swelling when applied directly to endo-thelium. Oxygen is necessary for the maintenance ofcorneal hydration," which indicates that the aerobicphase of the carbohydrate metabolism providesenergy for the maintenance of the corneal thickness.Langham and Taylor found an inhibitory effect of 2,4dinitrophenol on the fluid excretion of the cornea2"and concluded that the dehydration of the cornea is

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Barrett Katz, Ronald B Melles, Jerry A Schneider, and NarsingA Rao

linked to oxidative phosphorylation. Since thecorneal endothelium is rich in mitochondria, the siteof the electron transport system, it is assumed thatthe active trichloracetic acid cycle in the cornealendothelium supplies the energy necessary for themaintenance of normal corneal thickness.The cornea swells when damaged. Increased

corneal thickness may be an early sign of cornealdystrophy, keratitis, or trauma, when each interfereswith corneal endothelial function; the degree ofswelling generally parallels the severity of the under-lying disease.'9 Epithelial damage also results inswelling of the cornea, though it is less pronounced.

Patients with nephropathic cystinosis have exces-sive amounts of cystine in cellular lysosomes. Cystineis not known to be increased in collagen fibrils of thecornea, but it accumulates within keratocytes andepithelial cells.2" An osmotic gradient might resultfrom increased cystine within the corneal stroma andaccount for some element of increased stromalhydration. The intralysosomal site of cystine deposi-tion, however, would argue against the crystalsthemselves generating any change in tissue oncoticpressure. We think it more likely that the alteredcorneal thickness observed in our cystinotic patientsis a reflection of subclinical dysfunction of epithelialand/or endothelial cells leading to the occurrence ofclinically unrecognised stromal oedema. Thishypothesis is supported by the ultrastructuralchanges we observed in the epithelial and endotheliallayers of the cornea in our cystinosis patients. Wespeculate that increased amounts of cystine interferewith fluid transporting mechanisms of the cornealendothelium and epithelium, leading to altered watercontent of the cystinotic cornea. There is experi-mental support for striking similarities betweentransport related characteristics of the corneal endo-thelium and the kidney proximal tubule epithelium.2The alterations of corneal physiology which we

believe are caused by epithelial and endothelialdysfunction may be analogous to the disturbancesin the proximal tubule epithelium that culminate inthe Fanconi syndrome. Interference with activetransport across epithelial surfaces may be themechanism whereby accumulation of cystine causes

both the renal Fanconi syndrome and increasedwater content of the cornea manifest as increasedcorneal thickness. Whether this shared epithelialanomaly and ensuing increase in corneal thicknessoccur in association with non-cystinotic causes of theFanconi syndrome require further study; such studiesare now in progress.

This work was supported by grants DK 18434 and RR 00827 from theNational Institutes of Health, and by gifts from the Bernard L MassFoundation and the Cystinosis Foundation.The authors thank Dr Stuart Winthrop for invaluable assistance in

obtaining the corneal button.

References

I Jonas AJ, Smith ML, Schneider JA. ATP-dependent lysosomalcystine efflux is defective in cystinosis. J Biol Chem 1982; 257:13185-8.

2 Gahl WA, Schneider JA, Thoene JG, Chesney R. Course ofnephropathic cystinosis after age 10 years. J Pediatr 1986; 109:605-8.

3 Katz B, Melles RB, Schneider JA. Corneal sensitivity innephropathic cystinosis. Am J Ophthalmol 1987; 104: 413-6.

4 Katz B, Melles RB, Schneider JA. Contrast sensitivity functionin nephropathic cystinosis. Arch Ophthalmol 1987; 105: 1667-9.

5 Katz B, Melles RB, Schneider JA. Glare disability in nephro-pathic cystinosis. Arch Ophthalmol 1987; 105: 1670-1.

6 Schulman JD, Wong VG, Olson WH, Seegmiller JE. Lysosomalsite of crystalline deposits in cystinosis as shown by ferritinuptake. Arch Pathol 1970: 90: 259-64.

7 Wong VG, Kuwabara T, Brubaker R, Olson W, Schuman J,Seegmiller JE. Intralysosomal cystine crystals in cystinosis.Invest Ophthalmol Vis Sci 1970; 9: 83-8.

8 Frazier PD, Wong VG. Cystinosis: histologic and crystallo-graphic examination of crystals in eye tissues. Arch Ophthalmol1968; 80:87-91.

9 Kaiser-Kupfer MI, Fujikawa L, Kuwabara T, Jain S, Gahl WA.Removal of corneal crystals by topical cysteamine in nephro-pathic cystinosis. N Engl J Med 1987; 316: 775-9.

10 Wong VG, Lietman PS, Seegmiller JE. Alterations of pigmentepithelium in cystinosis. Arch Ophthalmol 1967; 77: 361-79.

11 Lietman PS, Frazier PD, Wong VO, Shotton D, Seegmiller JE.Adult cystinosis-a benign disorder. Am J Med 1966; 40: 511-7.

12 Frangois J, Hanssens M, Coppieters R, Evens L. Cystinosis. Aclinical and histopathologic study. Am J Ophthalmol 1972; 73:643-50.

13 Melles RB, Schneider JA, Rao NA, Katz B. Spatial andtemporal sequence of corneal crystal deposition in nephropathiccystinosis. Am J Ophthalmol 1987; 104: 598-604.

14 Soni PS. Effects of oral contraceptive steroids on the thickness ofhuman cornea. Am J Optom Physiol Opt 1980; 57: 825-34.

15 Thornton SP. A guide to pachymeters. Ophthalmic Surg 1984;15:993-5.

16 Ehlers N, Bramsen T. Central thickness in corneal disorders.Acta Ophthalmol (Kbh) 1978; 56: 412-6.

17 Salz JJ, Azen SP, Berstein J, Caroline P, Villasenor RA,Schanzlin DJ. Evaluation and comparison of sources of vari-ability in the measurement -of corneal thickness with ultrasonicand optical pachymeters. Ophthalmic Surg 1983: 14: 750-4.

18 Murrell WJ, Shigab Z, Lmberts DW, Avera B. The cornealendothelium central corneal thickness in pigmentary dispersionsyndrome. Arch Ophthalmol 1986;104: 845-6.

19 Mishima S. Corneal thickness. Surv Ophthalmol 1969;13:57-96.20 Langham ME, Taylor IS. Factors affecting the hydration of the

cornea in the excised eye and the living animal. Br J Ophthalmol1956;40: 321-40.

21 Kenyon KR, Sensenbrenner JA. Electron microscopy of corneaand conjunctiva in childhood cystinosis. Am J Ophthalmol 1974;78: 68-76.

22 Fischbarg J, Lim JJ. Role of cations, anions and carbonicanhydrase in fluid transport across rabbit corneal endothelium.J Physiol (Lond) 1974; 241: 647-76.

Acceptedfor publication 3 February 1989.

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