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The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (P, Fuchs endothelial corneal dystrophy (FECD) is one of the most common reasons for corneal transplantation, and is known to cluster in families. Here, the authors discover new genetic loci associated with FECD with sex-specific effects and implications for disease mechanism.

BACKGROUND: The recent availability of genetic analyses has demonstrated the shortcomings of the current phenotypic method of corneal dystrophy classification. Abnormalities in different genes can cause a single phenotype, whereas different defects in a single gene can cause different phenotypes. Some disorders termed corneal dystrophies do not appear to have a genetic basis. PURPOSE: The purpose of this study was to develop a new classification system for corneal dystrophies, integrating up-to-date information on phenotypic description, pathologic examination, and genetic analysis. METHODS: The International Committee for Classification of Corneal Dystrophies (IC3D) was created to devise a current and accurate nomenclature. RESULTS: This anatomic classification continues to organize dystrophies according to the level chiefly affected. Each dystrophy has a template summarizing genetic, clinical, and pathologic information. A category number from 1 through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy. The most defined dystrophies belong to category 1 (a welldefined corneal dystrophy in which a gene has been mapped and identified and specific mutations are known) and the least defined belong to category 4 (a suspected dystrophy where the clinical and genetic evidence is not yet convincing). The nomenclature may be updated over time as new information regarding the dystrophies becomes available. CONCLUSIONS: The IC3D Classification of Corneal Dystrophies is a new classification system that incorporates many aspects of the traditional definitions of corneal dystrophies with new genetic, clinical, and pathologic information. Standardized templates provide key information that includes a level of evidence for there being a corneal dystrophy. The system is user-friendly and upgradeable and can be retrieved on the website www.corneasociety.org/ic3d . KEY WORDS: corneal dystrophy, inherited corneal disease, genetic corneal disease, corneal histopathology, gene, mutation, key reference, eponym, epithelial basement membrane dystrophy, epithelial recurrent erosion dystrophy, subepithelial mucinous corneal dystrophy, Meesmann corneal dystrophy, Lisch epithelial corneal dystrophy, gelatinous drop-like corneal dystrophy, Grayson-Wilbrandt corneal dystrophy, lattice corneal dystrophy, lattice gelsolin type dystrophy, granular corneal dystrophy 1, granular corneal dystrophy 2, Avellino corneal dystrophy, Reis-Bucklers corneal dystrophy, Thiel-Behnke corneal dystrophy, macular corneal dystrophy, Schnyder corneal dystrophy, Schnyder crystalline corneal dystrophy, congenital stromal corneal dystrophy, fleck corneal dystrophy, posterior amorphous corneal dystrophy, central cloudy dystrophy of Francxois, pre-Descemet corneal dystrophy, Fuchs endothelial corneal dystrophy, posterior polymorphous corneal dystrophy, congenital hereditary endothelial dystrophy 1, congenital hereditary endothelial dystrophy 2, X-linked endothelial corneal dystrophy

The current paper reviews the genetic basis of diseases affecting the cornea, including corneal dystrophies and systemic diseases with corneal manifestations (e.g. the mucopolysaccharidoses), discusses the mechanisms by which corneal injury and disease can affect corneal transparency and discusses stem cell therapy as a potential therapy for corneal injury and disease. Several diseases of the cornea have been described, many of which are inheritable. The identification of the genetic mutations responsible for these diseases has led to a better understanding of the mechanisms underlying their corneal manifestations. Corneal disease and injury can affect corneal transparency. Stromal keratocytes play an important role in cellular and extracellular transparency of the cornea. They synthesize type I and V collagen and keratan sulfate proteoglycans that together regulate collagen fibril size and spacing important for extracellular matrix transparency. There are strong indications that cellular transparency of keratocytes relies on the presence of corneal crystallins, which provide a uniform density of proteins in the cytoplasm that increase the spatial order necessary for transparency. After injury, activated keratocytes show heterogeneity of cytoplasmic proteins, decreased spatial ordering and increased light scattering. Stem cell transplantation is regarded as a potential new therapy that can replace keratocytes and restore corneal transparency and normal structure after injury or corneal disease.

Udgivelsesdato: 2010-Jan Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging. TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 microg TGFBIp per mg wet tissue. Two-dimensional (2-D) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (6 years). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) data of TGFBIp isoforms separated on large 2-D gels show that TGFBIp is proteolytically processed from the N-terminus. This observation was supported by in silico 2-D gel electrophoresis showing that sequential proteolytical trimming events from the N-terminus of mature TGFBIp generate TGFBIp isoforms which form a similar "zig-zag" pattern when separated by 2-D polyacrylamide gel electrophoresis (PAGE). This study shows that in humans TGFBIp is more abundant in mature corneas than in the developing cornea and that the processing of TGFBIp changes during postnatal development of the cornea. In addition, TGFBIp appears to be degraded in a highly orchestrated manner in the normal human cornea with the resulting C-terminal fragments being retained in the cornea. The age-related changes in the expression and processing of corneal TGFBIp suggests that TGFBIp may play a role in the postnatal development and maturation of the cornea. Furthermore, these observations may be relevant to the age at which mutant TGFBIp deposits in the cornea in those dystrophies caused by mutations in the transforming growth factor beta induced gene (TGFBI) as well as the mechanisms of corneal protein deposition.

Transforming growth factor β-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported. © 2009 International Union of Crystallography All rights reserved., This work was supported by National Eye Institute Grant R01 EY 12712, the Danish National Research Foundation, the Danish Natural Science Research Council, the Danish Association for Prevention of Eye Diseases and Blindness, the Synoptik Foundation, Aarhus University Research Foundation and the Danish Medical Research Council. Further support was provided by BIO2006-02668, PSE-010000-2007-1 and the CONSOLIDER-INGENIO 2010 Project 'La Factoría de Cristalización' (CSD2006-00015) from Spanish public agencies, FP6 Strep Project LSHG-2006-018830 'CAMP' and FPT Collaborative Project 223101 'AntiPathoGN' from the European Union and 2005SGR00280 from the National Catalan Government

Context.—Marginal zone lymphomas (MZLs) are the most common lymphomas encountered in the orbit and ocular adnexa. The accurate categorization of these lymphomas is critical to avoid undertreatment or overtreatment. Objective.—To identify features of orbital MZLs that distinguish them from other lymphomas and reactive lymphoid infiltrates and support the categorization of orbital MZL as mucosa-associated lymphoid tissue (MALT)–type MZLs. Design.—Biopsies from 149 patients with lymphoid lesions of ocular adnexa were examined. Additional immunohistochemical stains and fluorescence in situ hybridization study for the MALT1 locus were performed in selected cases, and patient charts were reviewed. Results.—A total of 115 lymphomas and 34 reactive infiltrates were identified, of which B-cell lymphomas constituted 92% and MZLs constituted 54% of all lymphomas. Certain clinical features (young age, race, bilaterality) favored a reactive infiltrate, but none were diagnostic. Histologic features, such as infiltrative lesions, reactive B-cell follicles, and lymphoepithelial lesions, overlapped between reactive infiltrates and conjunctival MZL. In contrast to conjuctival MZL, orbital MZL infrequently showed reactive follicles, rarely showed epithelial tissue, and did not show lymphoepithelial lesions. Cytogenetic abnormality involving the MALT1 locus was demonstrated in only 15% of ocular adnexal MZLs. Conclusion.—Many MZLs of orbital soft tissue lack key features associated with MALT-type MZL, and the designation MALT lymphoma should be avoided in their diagnosis.

Aims: To evaluate mutations in the transforming-growth-factor-β-induced (TGFBI) gene in patients of Czech origin with autosomal dominant corneal dystrophies. Methods: The coding sequence of the TGFBI gene was analysed in 22 affected Czech individuals from 7 apparently unrelated families. Comparison of phenotype to genotype was performed. Results: A H626P mutation, previously only described in a family with a variant of lattice corneal dystrophy (LCD), was detected in one family with superficial geographic corneal opacities. Light microscopy of 2 samples obtained following either a prior superficial keratectomy or keratoplasty showed amyloid but no fuchsinophilic deposits. In a family with LCD type I, an R124C mutation was identified. The R124L mutation was shown to be causative of Reis-Bücklers corneal dystrophy in 2 families. A family with Thiel-Behnke corneal dystrophy exhibited an R555Q mutation. In 2 families with granular corneal dystrophy type I, the typical R555W change was identified. Conclusion: The phenotype of the family with the H626P mutation differed from the phenotype previously reported for this change.

Purpose To locate the mildest and/or earliest changes in the retina and/or choroid in Sveinsson chorioretinal atrophy (SCRA), using more advanced techniques than previous studies. Methods We used fundus photography, intravenous fluorescein angiography (IVFA) enhanced ocular coherence tomography (OCT) scans, microperimetry and multifocal electroretinography (mfERG) in an attempt to locate the mildest changes in SCRA. Eight patients with SCRA were examined. To improve the resolution of OCT scans, several consecutive recorded B-scans were retrieved for each location of interest. The scans were processed off-line with an averaging algorithm developed to maximally reduce laser speckle (noise). Static microperimetry was performed using the Rodenstock scanning laser ophthalmoscope (SLO). Results Biomicroscopy and fundus photographs disclosed an apparent thinning of the retinal pigment epithelium (RPE) in the areas minimally affected, where possible changes in the transparent sensory retina were not visible. In minimally affected areas a choriocapillaris filling defect was evident on IVFA, but some choroidal blood vessels remained open. High-resolution OCT scans in normal eyes showed three highly reflective outer layers, probably representing the junction of the inner and outer photoreceptor segments in the case of the innermost layer, the interdigitizing outer photoreceptors and RPE villi in the case of the middle layer, and the outer RPE in the case of the outermost layer. The middle layer was absent in the transition between affected and unaffected areas in all eyes with SCRA. In the more severely affected areas, the innermost layer was discontinuous and associated with increasing thinning of the outermost layer. Microperimetry of the transition areas sometimes showed clearly defined lesions that were non-responsive to stimuli. It also revealed elevated thresholds (10-15 dB) at the margins and normal thresholds in apparently unaffected areas. Conclusions The mildest changes seen in SCRA on OCT are found at the outer photoreceptor/RPE junction; the changes in the outer RPE, choriocapillaris and inner photoreceptor segments may be secondary. Corresponding functional deficits are confirmed on microperimetry and mfERG.

Purpose To report the histopathologic features in an eye with Sveinsson chorioretinal atrophy (SCRA), also termed helicoid peripapillary chorioretinal degeneration , for the first time. Participant An 82-year-old woman clinically and genetically confirmed to have SCRA. Design Examination of an eye obtained after death. Method Light microscopic examination of an eye of an 82-year-old woman documented to have SCRA since the age of 10 years. Main Outcome Measure The findings in ocular tissues were determined by light microscopy. Results In the most advanced areas of chorioretinal atrophy, the sensory retina, retinal pigment epithelium (RPE), choriocapillaris, and choroid were absent. In the transition between affected and unaffected areas, the RPE and the outer segments of the photoreceptors only were affected. The optic nerve was smaller than normal, but well myelinated. Other ocular tissues retained a relatively normal appearance for a patient who had died at this age. Conclusions The mildest and presumably earliest morphologic changes involved the photoreceptor outer segments, the RPE, and choriocapillaris in this progressive degenerative disease of the retina and choroid.

The lattice corneal dystrophies (LCD) and granular corneal dystrophies (GCD) are autosomal dominant disorders of the corneal stroma. They are bilateral, progressive conditions characterized by the formation of opacities arising due to the deposition of insoluble material in the corneal stroma leading to visual impairment. The LCDs and GCDs are distinguished from each other and are divided into subtypes on the basis of the clinical appearance of the opacities, clinical features of the disease, and on histopathological staining properties of the deposits. The GCDs and most types of LCD arise from mutations in the transforming growth factor beta-induced (TGFBI) gene on chromosome 5q31. Over 30 mutations causing LCD and GCD have been identified so far in the TGFBI. There are two mutation hotspots corresponding to arginine residues at positions 124 and 555 of the transforming growth factor beta induced protein (TGFBIp) and they are the most frequent sites of mutation in various populations. Mutations at either of these two hotspots result in specific types of LCD or GCD. The majority of identified mutations involve residues in the fourth fasciclin-like domain of TGFBIp.

Purpose To characterize the clinicopathologic phenotype as well as the molecular genetic basis of an autosomal dominant form of corneal amyloidosis. Design Clinicopathologic and molecular genetic study of a family with a form of corneal amyloidosis. Participants Forty-nine individuals from one family were studied. Methods The medical records of affected family members were reviewed, and corneal tissue from those who had undergone penetrating keratoplasty (PK) was examined. Several family members were examined clinically, and corneas were photographed. Deoxyribonucleic acid from blood or buccal swabs was extracted from each consenting family member to determine the status of their transforming growth factor β–induced (TGFBI) gene. The coding region of the TGFBI gene was analyzed for mutations in the proband's DNA, and compared with the nucleotide sequences of normal individuals. This was performed by amplifying and sequencing all exons of the TGFBI gene. In all other family members, only exons 4, 8, 11, and 12 of the gene were amplified, sequenced, and analyzed for mutations. Main outcome measures Clinicopathologic manifestations in relation to mutational status of the TGFBI gene. Results Slit-lamp biomicroscopy revealed bilateral multiple polymorphic, polygonal, refractile, chipped ice–appearing gray and white opacities. There were also occasional deep filamentous lines that did not form a distinct lattice pattern. Corneal tissue of affected individuals who underwent PK contained widespread deposits of amyloid within the corneal stroma, particularly in the deep central stroma. Twelve members of the family were found to have a heterozygous single mutation in the TGFBI gene leading to a predicted amino acid substitution of aspartic acid for alanine (A546D). Nine of these individuals had ophthalmologist-documented corneal disease. The remaining 3, who were 11, 14, and 15 years old, were asymptomatic. In addition, 4 inconsequential polymorphisms with the nucleotide changes 387 G/A (R129R), 981 G/A (V327V), 1416 T/C (L472L), and 1620 C/T (F540F) were found. Conclusion A distinct, progressive form of corneal amyloidosis with an autosomal dominant mode of inheritance is characterized clinically by the presence of refractile polymorphic corneal opacities. We have designated this entity, which is caused by an A546D mutation in the TGFBI gene, polymorphic corneal amyloidosis .

Aims: To study the long term toxic effects of intraocular benzalkonium chloride (BAC). Methods: 19 patients exposed to intraocular BAC preserved viscoelastic during cataract surgery in February 1999 developed severe striate keratopathy immediately postoperatively. 16 patients, including two who underwent penetrating keratoplasty, were studied in the period April to June 2000. Ocular symptoms, visual acuity, biomicroscopy, intraocular pressure, dilated funduscopy, specular endothelial microscopy, and corneal pachymetry findings were recorded. The corneal and iris specimens of the two patients who underwent keratoplasty were studied by light, transmission, and scanning electron microscopy. Results: Six males and 10 females, aged 64–98 years, were studied 14–16 months postoperatively. All patients were symptomatic. 12 patients had best corrected visual acuity of 6/12 or better and four patients of between 6/18 and 6/60. Five patients had corneal epithelial oedema and 11 had Descemet's membrane folds. The central corneal thickness, 620 (SD 71) μm, in affected eyes was significantly higher (p

Purpose We investigated whether mitochondrial DNA (mtDNA) variants affect the susceptibility of Fuchs endothelial corneal dystrophy (FECD). Methods Ten mtDNA variants defining European haplogroups were genotyped in a discovery dataset consisting of 530 cases and 498 controls of European descent from the Duke FECD cohort. Association tests for mtDNA markers and haplogroups were performed using logistic regression models with adjustment of age and sex. Subset analyses included controlling for additional effects of either the TCF4 SNP rs613872 or cigarette smoking. Our replication dataset was derived from the genome-wide association study (GWAS) of the FECD Genetics Consortium, where genotypes for three of 10 mtDNA markers were available. Replication analyses were performed to compare non-Duke cases to all GWAS controls (GWAS1, N = 3200), and to non-Duke controls (GWAS2, N = 3043). Results The variant A10398G was significantly associated with FECD (odds ratio [OR] = 0.72; 95% confidence interval [CI] = [0.53, 0.98]; P = 0.034), and remains significant after adjusting for smoking status (min P = 0.012). This variant was replicated in GWAS1 (P = 0.019) and GWAS2 (P = 0.036). Haplogroup I was significantly associated with FECD (OR = 0.46; 95% CI = [0.22, 0.97]; P = 0.041) and remains significant after adjusting for the effect of smoking (min P = 0.008) or rs613872 (P = 0.034). Conclusions The 10398G allele and Haplogroup I appear to confer significant protective effects for FECD. The effect of A10398G and Haplogroup I to FECD is likely independent of the known TCF4 variant. More data are needed to decipher the interaction between smoking and mtDNA haplogroups.

Amyloidosis is a disease characterized by the formation of extracellular amyloid deposits. Immunoglobulin light-chain amyloidosis can appear as a local disorder presenting with mild symptoms or as a life threatening systemic disease. The systemic form of immunoglobulin light-chain amyloidosis is the most common type of amyloidosis in western countries although it is a rare disease. Identification of the proteins forming amyloid fibrils is essential for the diagnosis of the disease and knowledge about the overall protein composition of the deposits may lead to a larger understanding of the deposition events thereby facilitating a more detailed picture of the molecular pathology. In this pilot study, we investigated the protein composition of amyloid deposits isolated from human specimens of the eyelid, conjunctiva, and orbit. Deposits and internal control tissue (patient tissue without apparent deposits) were procured by laser capture microdissection. Proteins in the captured amyloid and control samples were quantified by liquid chromatography tandem mass spectrometry using the label-free exponential modified Protein Abundance Index (emPAI) method. Immunoglobulin light chain kappa or lambda was found to be the most predominant protein in the amyloid deposits from the eyelid, conjunctiva, and orbit. Five proteins, apolipoprotein A-I, carboxypeptidase B2 (TAFI), complement component C9, fibulin-1 and plasminogen were found solely across all amyloid but not in the control tissue. In addition, the protein profiles identified apolipoprotein E and serum amyloid P component to be associated with the immunoglobulin light chain deposits across all three tissues analyzed. The method used in this study provided high sensitivity and specificity for the type of amyloid and may provide additional information on the pathology of the amyloid deposits in the ocular tissues studied.

Purpose In this study, we investigated whether the phenotypic difference observed between two lattice corneal dystrophy type 1 (LCD type 1) cases caused by either a single A546D substitution or an A546D/P551Q double substitution in TGFBIp (transforming growth factor beta induced protein) can be ascribed to (i) a difference in the proteomes of corneal amyloid deposits, (ii) altered proteolysis of TGFBIp, or (iii) structural changes of TGFBIp introduced by the P551Q amino acid substitution. Experimental design Amyloid deposits were isolated from the corneas of two siblings with LCD type 1 resulting from A546D/P551Q mutations in the TGFBI gene using laser capture microdissection and subsequently analyzed by LC-MS/MS. Proteolytic processing of TGFBIp was addressed by counting peptide spectra. Lastly, to study the possible effect of the P551Q substitution, recombinant FAS1–4 domain variants were subjected to in vitro stability assays. Results The amyloid proteomes and TGFBIp processing of the two A546D/P551Q LCD type 1 cases were similar to each other as well as to the A546D amyloid proteome previously reported by us. The stability assays revealed a minor destabilization of the FAS1–4 domain upon the addition of the P551Q mutation, moreover, it resulted in different accessibility to tryptic cleavage sites between the A546D and A546D/P551Q mutant FAS1–4 domain variants. Conclusion and clinical relevance The difference in A546D and A546D/P551Q LCD type 1 phenotypes cannot be ascribed to altered corneal amyloid composition or altered in vivo proteolytic processing of TGFBIp. Instead, a small difference in thermodynamic stability introduced by the P551Q mutation most likely causes structural changes of TGFBIp. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000307 (http://proteomecentral.proteomexchange.org/dataset/PXD000307).

Purpose: To report the onset of malignant peripheral nerve sheath tumor of the orbit 8 years after irradiation in a patient with neurofibromatosis type-1. Methods: Case report of a young man with neurofibromatosis type-1 who received irradiation for presumed bilateral optic nerve and chiasmal gliomas and in whom a malignant peripheral nerve sheath tumor later developed. Exenteration with extirpation of the entire contents of the orbit was performed 6 times. Results: Complete recurrence of the tumor occurred after each surgical procedure until the patient died of malignancy. Conclusions: Our case underscores the risk of irradiation, especially in children with neurofibromatosis type-1, and emphasizes that radiotherapy should never be given as an empirical therapy. The authors believe that irradiation and neurofibromatosis type-1 may, in combination, pose a significant risk for the development of malignancies. Clear-cut indications and a precise tissue diagnosis are desirable before the initiation of radiotherapy, particularly in the pediatric population. We recommend that if irradiation is necessary in persons with neurofibromatosis type-1, regular follow-up is imperative. In view of the hostile nature of malignant peripheral nerve sheath tumor, early aggressive treatment appears to be the only viable alternative at present.

Background Glaucoma is a group of chronic eye diseases often associated with an elevated intraocular pressure (IOP). If not controlled, the condition leads to blindness. The eye tissue responsible for maintaining aqueous humor resistance and thus normal IOP is the trabecular meshwork (TM). Adenoviral vectors are capable of transducing the TM in several rodent species. Because of the relevance of the non-human primate model in the study of glaucoma, gene transfer to the eyes of cynomolgus monkeys was investigated. Methods Four cynomolgus monkeys were injected with AdenoGFP into the anterior chamber: two monkeys received 109 pfu and the other two 107 pfu. One monkey received four consecutive injections into the same eye (107 pfu in each injection) over a 7-month period. In vivo gene transfer (fluorescence) and IOP were evaluated by standard clinical ophthalmic instruments (slit lamp biomicroscopy, gonioscopy and tonometry). Histopathology and cellular distribution were assessed postmortem. Results The first injection of the lower viral dose resulted in marked TM-preferred gene transfer visible non-invasively by in vivo gonioscopy. The expression of the transgene lasted for 3–4 weeks with little or no signs of clinical inflammation. Gene transfer was achieved on three sequential occasions (3–4 weeks each) but failed and induced substantial, albeit reversible, corneal abnormalities on the fourth occasion. Conclusions Gene transfer to the TM and cornea can be monitored non-invasively in non-human primates, allowing correlation of gene transfer with physiological parameters. Because of ocular immune privilege, repeated anterior chamber administrations of adenoviral vectors expressing appropriate genes may have therapeutic potential for glaucoma. Copyright © 2001 John Wiley & Sons, Ltd.

We describe a case of focal argyrosis of the conjunctiva clinically simulating a melanoma. An 82-year-old woman was referred for an asymptomatic pigmented conjunctival lesion. Her only significant past ocular history was strabismus surgery 76 years earlier. Biopsy of the conjunctiva and lateral rectus muscle revealed the discoloration was pigment granules. Energy-dispersive x-ray microanalysis revealed the pigmentation to be silver deposits. The patient had strabismus surgery probably using a silver clip. Argyrosis should be considered in the differential diagnosis of focal pigmented conjunctival lesions. [Ophthalmic Surg Lasers 2000;31:495-498]

PURPOSE: To improve our understanding of the role of specific genes on corneal transparency through a review of linkage to specific chromosomal loci and the identification of the mutant genes dealing with the corneal dystrophies. METHOD: Relevant recent literature on the corneal dystrophies is reviewed. RESULTS: Molecular genetic studies of the corneal dystrophies suggest that genes on at least 10 human chromosomes are involved in the maintenance of corneal transparency (chromosomes 1, 5, 9, 10, 12, 16, 17, 20, 21, and X). Within the 10 chromosomes to which corneal dystrophies have been mapped, specific genetic mutations in seven genes (GSN, BIGH3, KRT3, KRT12, MSS1, GLA, and ARSC1) have been identified in 15 corneal dystrophies. Some corneal dystrophies that are considered distinct clinicopathologic entities are actually caused by different mutations in the same gene. For example, lattice dystrophy types I and IIIA, granular corneal dystrophy types I, II (Avellino dystrophy), and III (Reis-Bucklers dystrophy), and Thiel-Behnke corneal dystrophy are the result of mutations in BIGH3. Mutations in three genes (GSN, BIGH3, MSS1) are associated with amyloid deposition in the cornea. A gene for keratoconus has been mapped to chromosome 21, which is noteworthy because of the established association of keratoconus in Down syndrome (trisomy 21). CONCLUSION: Recent genetic studies on the corneal dystrophies provide insight into some of these disorders at a basic molecular level. Some corneal dystrophies that were previously believed to be distinct clinicopathologic entities are closely related at the molecular level with the different phenotypes resulting from distinct mutations in the same gene. This new knowledge is leading to a revised classification of the corneal dystrophies and to the development of animal models of corneal dystrophies. The latter will lead to a better understanding of the pathogenesis of the disorders and hence to novel therapeutic approaches to those dystrophies that cause significant visual impairment. Research of this nature is only in its infancy.

Objective To review the cytologic findings of vitreous specimens and propose a simplified approach to them. Study design Seventy-four vitreous specimens from 60 patients obtained either during a pars plana vitrectomy or by vitreous aspiration were reviewed. Clinical correlation was obtained on all patients. Results Findings suggestive of a specific disorder were present in 30 specimens (41%); cytologic examination of the remaining 44 showed nonspecific changes. A lymphoproliferative disorder, the intraocular malignancy suspected most often in this series, was identified in eight specimens (11%). Large cell lymphomas were evident in 5 specimens, 2 specimens were suspicious for lymphoma, and 1 specimen was consistent with plasmacytoma. Twelve specimens (16%) contained hemorrhage. In rare instances, specific infectious agents, such as parasites (5%), bacteria (1%) and fungi (3%), could be identified. The diagnosis of viral infections required ancillary studies. Lens fragments were identified in four cases (5%), and a diagnosis of lens-induced endophthalmitis could be rendered in one case (1%). Changes consistent with sarcoidosis were present in 3% of cases. Conclusion Based on this experience with vitreous specimens submitted for clinical reasons, we found that they could be divided into three broad diagnostic categories: inflammation/infection (54 specimens/41 patients), hemorrhage (12 specimens/12 patients) and malignancy (8 specimens/7 patients).

BACKGROUND Second primary malignancies are common after bilateral retinoblastoma; their estimated incidence has been as high as 51% 50 years after diagnosis. Fifteen patients who developed sebaceous gland carcinoma after radiation therapy have been reported in the literature, five of whom were treated for bilateral retinoblastoma. METHODS The authors conducted a retrospective chart review of patients treated for bilateral retinoblastoma at Duke University Medical Center who later developed sebaceous gland carcinoma. RESULTS This article reports two patients who developed sebaceous gland carcinoma after radiation therapy for bilateral retinoblastoma. CONCLUSIONS Delay in diagnosis is often associated with sebaceous gland carcinoma. Because high mortality is observed with metastatic disease, the recognition of this association is important for anyone who follows patients with a history of bilateral retinoblastoma or prior cranial radiation therapy. Cancer 1998;83:767-771. © 1998 American Cancer Society.

Multicentric melanomas in the same eye are rare, with few cases substantiated by histology and serial sectioning. We report a patient with two documented choroidal malignant melanomas in one eye. The initial tumor spontaneously decreased in size for over 2 years before a second tumor appeared in a noncontiguous location in the same eye. After enucleation, serial sections showed that the two lesions were independent choroidal melanomas. We review the literature regarding multiple, independent intraocular choroidal malignant melanomas. ( Surv Ophthalmol 42 :441–448, 1998.

• Background: We characterized the neovascularization that follows the intracorneal injection of bovine albumin (BA) in rabbits as a model of corneal angiogenesis. • Methods: New Zealand white rabbits received intracorneal injections of phosphate-buffered saline with and without various amounts of BA. The rabbits were co-sensitized or presensitized by intramuscular BA or were not sensitized. The corneal vascular response was quantified by ranking photographs taken periodically after the injection. • Results: In pre-sensitized animals, blood vessels were apparent within 4 days and reached maximum intensity 14 days after the intracorneal injection. Corneas also vascularized in nonsensitized rabbits, but a larger dose (>0.2 mg BA) was required than in pre-sensitized animals (>0.02 mg BA). Vascularization began later in non-sensitized animals and was less extensive than in pre-sensitized animals. • Conclusion: The intracorneal injection of BA is a reproducible model of corneal angiogenesis in rabbits and should allow the involved immunological mechanisms to be elucidated.

To determine the immunophenotype or immunophenotypes of macular corneal dystrophy in Saudi Arabia.We studied 56 cases of macular corneal dystrophy. Tissue from 60 corneal transplant buttons was stained by the avidin-biotin complex method using an anti-keratan sulfate monoclonal antibody. The serum antigenic keratan sulfate was measured in 23 of the 56 patients, four unaffected relatives, and 13 individuals with chronic actinic keratopathy. Serum and corneal tissue were studied in 17 of the 50 affected individuals with corneal transplant material.Thirty-five corneas (58.3%) of 29 of 50 patients did not react with anti-keratan sulfate monoclonal antibody. The stroma and abnormal intracellular and extracellular corneal accumulations reacted with anti-keratan sulfate monoclonal antibody in seven corneas (11.7%). The stroma in the other 18 corneas (30.0%) from 15 patients did not react with the anti-keratan sulfate monoclonal antibody, but corneal fibroblasts did. Twenty-one of the 23 patients with macular corneal dystrophy had no detectable serum antigenic keratan sulfate (9 ng/ml); two had values of 12 and 51 ng/ml, respectively, and their corneal stroma and abnormal accumulations reacted with anti-keratan sulfate monoclonal antibody.We detected macular corneal dystrophy type IA, a new immunophenotype characterized by the lack of detectable antigenic keratan sulfate in the serum (9 ng/ml), and a corneal stroma that did not react with the keratan sulfate monoclonal antibody but in which corneal fibroblasts did react with keratan sulfate monoclonal antibody (in 15 of 50 patients).

To study the leading indications and changing trends for penetrating keratoplasty (PK) over the past 3 decades.This is a retrospective review of 696 cases of PK. The indications for PKs performed at the Duke University Eye Center during the years 1980-1981, 1990- 1991, and 2000-2001 were tabulated to determine trends over the past 3 decades. The main outcome measures were indications for PK.During this study, 696 PKs were performed. The leading indications for PK and their respective frequencies during 1980-1981, 1990-1991, and 2000-2001 were failed grafts (10.8%, 19.0%, 27.0%, respectively), pseudophakic bullous keratopathy (PBK)/aphakic bullous keratopathy (ABK) (19.4%, 20.6%, 16.7%, respectively), Fuchs dystrophy (15.6%, 13.0%, 23.8%, respectively), keratoconus (13.4%, 8.2%, 11.8%, respectively), and corneal scar (7.0%, 8.9%, 10.7%, respectively). The number of PKs for failed grafts and Fuchs dystrophy increased over time.In this study, failed graft has gradually become the leading indication for PK, whereas most other studies have reported PBK as the leading indication. Unlike many other studies, Fuchs dystrophy was a common indication for PK.

TGFBIp, also known as keratoepithelin and βig-h3, is among the most abundant proteins in the human cornea and approximately 60% is associated with the insoluble fraction following extraction in sodium dodecyl sulfate (SDS) sample buffer. TGFBIp is of particular interest because a wide range of mutations causes amyloid or fuchsinophilic crystalloid deposits in the cornea leading to visual impairment. In the present study, we show that the SDS-insoluble fraction of TGFBIp from porcine and human corneas is covalently linked to the NC3 domain of type XII collagen in a TGFBIp:type XII collagen stoichiometric ratio of 2:1 via a reducible bond. Since type XII collagen is anchored to striated collagen fibers of the extracellular matrix, its interaction with TGFBIp is likely to provide anchoring for cells to the extracellular matrix through the integrin-binding capability of TGFBIp. Furthermore, the TGFBIp-type XII collagen molecule will affect our understanding of the molecular pathogenesis of the TGFBI-linked corneal dystrophies.

Purpose Fuchs endothelial corneal dystrophy (FECD) is a genetically heterogeneous disorder that has been primarily studied in patients of European or Asian ancestry. Given the sparse literature on African Americans with FECD, we sought to characterize the genetic variation in three known FECD candidate genes in African American patients with FECD. Methods Over an 8-year period, we enrolled 47 African American probands with FECD. All participants were clinically examined with slit-lamp biomicroscopy, and when corneal tissue specimens were available, histopathologic confirmation of the clinical diagnosis was obtained. The coding regions of known FECD susceptibility genes collagen, type VIII, alpha 2 (COL8A2); solute carrier family 4, sodium borate transporter, member 11 (SLC4A11); and zinc finger E-box binding homeobox 1 (ZEB1 [also known as TCF8]) were Sanger sequenced in the 47 probands using DNA isolated from blood samples. Results Twenty-two coding variants were detected across the COL8A2, SLC4A11, and ZEB1 genes; six were nonsynonymous variants. Three novel coding variants were detected: a synonymous variant each in COL8A2 and SLC4A11 and one nonsynonymous variant in ZEB1 (p.P559S), which is predicted to be benign and tolerated, thus making its physiologic consequence uncertain. Conclusions Variation in the COL8A2, SLC4A11, and ZEB1 genes is present in only a small fraction of our African American cases and as such does not appear to significantly contribute to the genetic risk of FECD in African Americans. This observation is on par with findings from previous sequencing studies involving European or Asian ancestry patients with FECD.

Knowledge about keratoconus (KC) and the corneal dystrophies continues to advance at a steady pace in view of the wide interest in these topics to basic scientists and clinical ophthalmologists. As in the past the articles on KC outnumber those on all of the other corneal dystrophies combined. Major advances have been made mainly regarding several specific corneal dystrophies, but KC remains a perplexing disorder with many unanswered questions. As we learn more and more about the corneal dystrophies from genetic linkage analyses and molecular biological techniques it will eventually be possible to understand the fundamental defect that causes these conditions.

Purpose Specific mutations in the transforming growth factor beta induced (TGFBI) gene are associated with lattice corneal dystrophy (LCD) type 1 and its variants. In this study, we performed an in-depth proteomic analysis of human corneal amyloid deposits associated with the heterozygous A546D mutation in TGFBI. Methods Corneal amyloid deposits and the surrounding corneal stroma were procured by laser capture microdissection from a patient with an A546D mutation in TGFBI. Proteins in the captured corneal samples and healthy corneal stroma were identified with liquid chromatography-tandem mass spectrometry and quantified by calculating exponentially modified Protein Abundance Index values. Mass spectrometry data were further compared for identifying enriched regions of transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3) and detecting proteolytic cleavage sites in TGFBIp. Results A C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin 1 domain (FAS1–4), serum amyloid P-component, apolipoprotein A-IV, clusterin, and serine protease HtrA1 were significantly enriched in the amyloid deposits compared to the healthy cornea. The proteolytic cleavage sites in TGFBIp from the diseased cornea are in accordance with the activity of serine protease HtrA1. We also identified small amounts of the serine protease kallikrein-14 in the amyloid deposits. Conclusions Corneal amyloid caused by the A546D mutation in TGFBI involves several proteins associated with other varieties of amyloidosis. The proteomic data suggest that the sequence 571-YHIGDEILVSGGIGALVR-588 contains the amyloid core of the FAS1–4 domain of TGFBIp and point at serine protease HtrA1 as the most likely candidate responsible for the proteolytic processing of amyloidogenic and aggregated TGFBIp, which explains the accumulation of HtrA1 in the amyloid deposits. With relevance to identifying serine proteases, we also found glia-derived nexin (protease-nexin 1) in the amyloid deposits, making this serine protease inhibitor a good candidate for the physiologically relevant inhibitor of one of the amyloid-associated serine proteases in the cornea and probably in other tissues. Noteworthy, the present results are in accordance with our findings from a previous study of corneal amyloid deposits caused by the V624M mutation in TGFBI, suggesting a common mechanism for lattice corneal dystrophies (LCDs) associated with mutations in the TGFBIp FAS1–4 domain.

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

Human transforming growth factor β induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compromise transparency. The complete three-dimensional structure of TGFBIp is not yet available, with the exception of a partial X-ray structure of the archetype FAS1 domain derived from Drosophila fasciclin-1. In this study, small-angle X-ray scattering (SAXS) models of intact wild-type (WT) human TGFBIp and a mutant (R124H) are presented. The mutation R124H leads to a variant of granular CD. The deduced structure of the TGFBIp monomer consists of four FAS1 domains in a simple “beads-on-a-string” arrangement, constructed by the superimposition of four consecutive Drosophila fasciclin domains. The SAXS-based model of the TGFBIp R124H mutant displayed no structural differences from WT. Both WT TGFBIp and the R124H mutant formed trimers at higher protein concentrations. The similar association properties and three-dimensional shape of the two proteins suggest that the mutation does not induce any major structural rearrangements, but points towards the role of other corneal-specific factors in the formation of corneal R124H deposits.

Fuchs endothelial corneal dystrophy (FECD) is a common, late-onset disorder of the corneal endothelium. Although progress has been made in understanding the genetic basis of FECD by studying large families in which the phenotype is transmitted in an autosomal dominant fashion, a recently reported genome-wide association study identified common alleles at a locus on chromosome 18 near TCF4 which confer susceptibility to FECD. Here, we report the findings of our independent validation study for TCF4 using the largest FECD dataset to date (450 FECD cases and 340 normal controls). Logistic regression with sex as a covariate was performed for three genetic models: dominant (DOM), additive (ADD), and recessive (REC). We found significant association with rs613872, the target marker reported by Baratz et al.(2010), for all three genetic models (DOM: P = 9.33×10(-35); ADD: P = 7.48×10(-30); REC: P = 5.27×10(-6)). To strengthen the association study, we also conducted a genome-wide linkage scan on 64 multiplex families, composed primarily of affected sibling pairs (ASPs), using both parametric and non-parametric two-point and multipoint analyses. The most significant linkage region localizes to chromosome 18 from 69.94cM to 85.29cM, with a peak multipoint HLOD = 2.5 at rs1145315 (75.58cM) under the DOM model, mapping 1.5 Mb proximal to rs613872. In summary, our study presents evidence to support the role of the intronic TCF4 single nucleotide polymorphism rs613872 in late-onset FECD through both association and linkage studies.

Retinoic acid receptors (RARs) modulate gene expression following association with retinoic acid (RA). In transient transfection, an RARα-β-galactosidase fusion protein (RAR-LacZ) was able to transactivate expression in the absence of RA. When expressed in the ocular lens of transgenic mice, this constitutively active RAR-LacZ fusion gene resulted in founder and progeny animals that exhibited cataracts and microphthalmia, both being characteristics of retinoid-in-duced teratogenesis. The transgenic phenotypes indicate that retinoid teratogenesis can be mimicked by expression of a constitutively active RAR-LacZ fusion protein in retinoid-sensitive tissues.

Amino acid (aa) sequence data from Staphylococcus aureus V8 protease-digested bovine corneal 54-kDa protein (BCP54) fragments were utilized to derive mixed oligodeoxyribonucleotide (oligo) primers complementary to the reverse translation products of these sequences. These degenerate oligo primers were used to prime the amplification of BCP54 sequence from bovine corneal epithelial cell cDNA. The cDNA probe generated by this mixed oligo-primed amplification of cDNA was cloned and dideoxy-sequenced. A search of the GenBank database (version 63.0) revealed extensive sequence similarity to the cDNA encoding tumor-associated rat liver (class 3) aldehyde dehydrogenase (RATALD). Nucleotide (nt) and aa sequence alignment of the BCP54 translation product reveals it is 78% and 84% homologous with RATALD at the nt and aa levels, respectively. Conservation of aa sequence elements common to the aldehyde dehydrogenase family thought to be of structural/functional significance is further substantiated by this analysis. Included in the discussion is the likelihood that gene sharing (genes encoding metabolic enzymes and other stable proteins) may extend to the cornea.

Corneal endothelial decompensation is a frequent complication when silicone oil is used as a tamponade following vitrectomy for treatment of proliferative vitreoretinopathy in an aphakic eye. We evaluated the clinical, histopathological, and ultrastructural features of silicone oil-induced keratopathy in 10 patients who developed corneal complications requiring penetrating keratoplasty. Clinically, some cases showed corneal edema, corneal hypesthesia, endothelial opacification, band keratopathy, and peripheral corneal vascularization. Histopathologically, retrocorneal membranes were present, and different degrees of stromal hypercellularity, superficial stromal calcification, and vascularization were noted. The ultrastructural features of endothelial cell loss and retrocorneal membrane formation are consistent with changes previously reported in rabbits and cats receiving intracameral silicone oil injections.

Background: The recent availability of genetic analyses has demonstrated the shortcomings of the current phenotypic method of corneal dystrophy classification. Abnormalities in different genes call Cause a single phenotype, whereas different defects in a single gene can Cause different phenotypes. Sonic disorders termed corneal dystrophies do not appear to have a genetic basis. Purpose: The purpose Of this Study was to develop a new classification system for corneal dystrophies, integrating up-to-date information on phenotypic description, pathologic examination, and genetic analysis. Methods: The International Committee for Classification of Corneal Dystrophies (IC3D) was created to devise a current and accurate nomenclature. Results: This anatomic classification continues to Organize dystrophies according to the level chiefly affected. Each dystrophy has a template summarizing genetic, clinical, and pathologic information. A category number from I through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy. The most defined dystrophies belong to category 1 (a well-defined corneal dystrophy in which a gene has been mapped and identified and specific imitations are known) and the least defined belong to category 4 (a suspected dystrophy where the clinical and genetic evidence is not yet convincing). The nomenclature may be updated over time as new information regarding the dystrophies becomes available. Conclusions: The IC3D Classification of Corneal Dystrophies is a new classification system that incorporates many aspects of the traditional definitions of corneal dystrophies with new genetic, clinical, and pathologic information. Standardized templates provide key information that includes a level of evidence for there being a corneal dystrophy. The system is User-friendly and upgradeable and call be retrieved on the website www.corneasociety.org/ic3d.