[The IC3D classification of the corneal dystrophies]. / IC3D-Klassifikation von Hornhautdystrophien.

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 well-defined 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 .

Genetic ablation in transgenic mice with an attenuated diphtheria toxin A gene.

We have previously generated microphthalmic mice lacking lens fiber cells by targeting the expression of the diphtheria toxin A (DT-A) gene in transgenic mice with regulatory sequences associated with the mouse gamma 2-crystallin gene. Because of the extreme toxicity of DT to animal cells and the potential leakiness of many tissue-specific regulatory regions, we investigated whether there might be an experimental advantage in using a mutant, attenuated form of the DT-A gene (tox-176) fused to gamma 2-crystallin regulatory sequences to ablate fiber cells in the ocular lens. In contrast to the microphthalmia observed in transgenic animals carrying the native DT-A gene, independent lines of mice transgenic for the gamma 2tox176 construct displayed predominantly cataracts or clinical anophthalmia. These contrasting phenotypes were transmitted within each pedigree, although for some lines some phenotypic heterogeneity among offspring was noted. The difference in phenotype between cataractous and clinically anophthalmic transgenic lines could not be ascribed to differences in the transgene copy number. Instead, the results suggest that transgene expression and hence the extent of genetic ablation are modulated by the site of chromosomal integration and, to a lesser extent, by epigenetic events. They also suggest that the attenuated gamma 2tox176 construct can integrate into chromosomal regions that are particularly favorable for expression without compromising embryological development and therefore that the tox-176 gene may be more versatile and effective than the wild-type DT-A gene for achieving genetic ablation with a broad range of cell- or tissue-specific regulatory sequences.

[Corneal macular dystrophy: clinical, histopathologic and ultrastructural features]. / Distrofia macular corneal: características clínicas, histopatológicas y ultraestructurales.

OBJECTIVE: To assess the main clinical, genetic, histopathological and ultrastructural features of Mexican patients with macular corneal dystrophy, and to compare the results with those previously reported. METHOD: We analyzed six cases where a histopathologic diagnosis of macular corneal dystrophy had been made between 1957 and 2004. RESULTS: Clinically, all corneas showed focal grayish-white stromal opacities with diffuse edges. Histopathologically, intrastromal granules stained strongly positive with Alcian blue and colloidal iron. Transmission electron microscopy showed enlargement of smooth endoplasmic reticulum and the presence of intracytoplasmic vacuoles that corresponded to glycosaminoglycans. Genetic analysis showed novel mutations in the CHST6 gene in 2 of the patients. CONCLUSIONS: Females were more affected than males and the mean age at the time of diagnosis was older than that reported previously, however the clinical, histopathological and ultrastructural features were similar to those of previous reports. As described in other cases in the literature, in some instances a disorder is found in CHST6 gene as a basis for this condition.

Bovine corneal protein 54K (BCP54) is a homologue of the tumor-associated (class 3) rat aldehyde dehydrogenase (RATALD).

Amino acid (aa) sequence data from Staphylococcus areas 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.

Calcification of basal ganglia and cerebellar roof nuclei in mentally defective patient with hidrotic ectodermal dysplasia. Analysis of intracranial concretions by electon microprobe.

This report describes, for the first time, an analysis by electron microprobe of concretions in the brain of an individual with striopallidodentate calcification. We also report the unique association of this intracranial syndrome with hidrotic ectodermal dysplasia. An institutionalized male with impaired intellectual function and hidrotic ectodermal dysplasia was known since the age of 3 years to have bilateral radiopaque densities in the region of the basal ganglia on skull roentgenogram. He died at age 29 in congestive heart failure from rheumatic pancarditis. At autopsy, concretions were identified in globus pallidus, caudate nuclei, thalamus, and dentate nuclei. Mineral deposits within the brain, analyzed by energy dispersive x-ray microanalysis, consisted predominately of calcium and phosphorus. Trace amounts of magnesium, iron, and silicon also were detected.

The contribution of morphology to our understanding of the pathogenesis of experimentally produced corneal vascularization.

In many disorders of the cornea, blood vessels invade this normally avascular tissue. Several theories have been proposed to explain the pathogenesis of corneal vascularization. Since none had received general acceptance, experimental studies of the phenomenon were begun several years ago in hamster cheek pouch chambers. These investigations which employed sequential morphologic studies during the process of corneal vascularization gave rise to a new hypothesis, namely, that corneal vascularization is usually a manifestation of the inflammatory response and mediated by leukocytes. This report briefly reviews the evolution, evidence, and current status of this theory.

Effect of inhibitors of arachidonic acid metabolism on corneal neovascularization in the rat.

We used computerized image analysis to evaluate quantitatively the ability of topically applied corticosteroids (dexamethasone sodium phosphate, prednisolone acetate), cyclooxygenase inhibitors (flurbiprofen, indomethacin, ketorolac), lipoxygenase inhibitors (REV 5901, esculetin, quercetin), and dual cyclooxygenase/lipoxygenase inhibitors (BW 755C, BW A540C) to reduce corneal neovascularization in the rat induced by silver/potassium nitrate cauterization. Significant decreases in the neovascular response were found with corticosteroids and cyclooxygenase inhibitors. A complete dose-response curve was performed for a representative compound from each class. Dexamethasone was found to be superior to flurbiprofen in its ability to reduce neovascularization in this model, while no significant inhibition was noted with either REV 5901 or BW 755C, even at high doses. We conclude that the corneal angiogenic response in this model can be reduced by inhibition of cyclooxygenase as well as by other mechanisms that are steroid-dependent but are, as yet, poorly defined.

Corneal neovascularization induced by xenografts or chemical cautery. Inhibition by cyclosporin A.

PURPOSE: Neovascularization of the cornea occurs in numerous pathologic states causing decreased visual acuity and blindness and is a major complication of corneal allotransplantation. The purpose of this study was to investigate the effect of topical and systemic cyclosporin A (CsA) on corneal angiogenesis induced by xenotransplantation or by chemical cauterization. The subcutaneous disc angiogenesis system (DAS) also was used to study the effects of CsA on angiogenesis in a nonocular site. METHODS: Corneal angiogenesis was provoked by either xenotransplantation or chemical cautery. Rats from experiments using both of these models were subdivided into four treatment groups. Topical treatment was administered by using 4% CsA eye drops or vehicle (castor oil) four times daily for 10 days. Systemic therapy consisted of daily (5 mg/kg per day) subcutaneous injections of CsA or vehicle. In the DAS experiments, rats received CsA or vehicle systemically or intradisc. The amount of neovascularization was quantitated by digital image analysis in corneal flat preparations and sections of discs. RESULTS: Rats that received xenografts or cautery manifested less corneal neovascularization than did control animals after topical of subcutaneous CsA treatment. CsA also enhanced the survival of corneal xenografts. A difference between CsA and vehicle-treated animals in the DAS experiments was not detected. CONCLUSIONS: CsA effectively retards the growth of new vessels in the cornea after xenotransplantation or chemical cauterization and prolongs xenograft survival. However, CsA does not suppress angiogenesis in all systems, because it was ineffective in blocking vessel growth in the subcutaneous DAS.

Neovascularization of the iris: an experimental model in cats.

Neovascularization of the iris was induced in cats by removing the vitreous and lens and creating a rhegmatogenous retinal detachment. The presence of new blood vessels on the anterior surface of the iris was verified from the second month onward by slit lamp examination, as well as by light microscopy six to twelve months after the operation. Control eyes undergoing vitrectomy and lensectomy, but without retinal detachment, did not develop rubeosis iridis. This model may allow investigation into causes and therapy of rubeosis iridis.

Calcification of soft contact lenses in patient with dry eye and elevated calcium concentration in tears.

Following radiotherapy for an extensive intraepithelial carcinoma of the conjunctiva and eyelid, a patient developed opacification of soft contact lenses used on the same side. Calcilm was demonstrated within the contact lenses by cytochemical methods and by energy-dispersive x-ray microanalysis. The phenomenon was associated with unilateral tear insufficiency and an elevated tear calcium level as determined by atomic absorption spectrophotometry.

Cholinergic stimulation of phosphatidic acid formation by rat cornea in vitro.

The cornea has one of the highest acetylcholine (ACh) concentrations of any tissue but the function of the ACh has remained enigmatic. During studies on corneal arachidonic acid metabolism, we observed that ACh stimulates formation of labeled phosphatidic acid in rat corneas whose phospholipids were prelabeled with [14C]arachidonate. ACh did not affect the metabolism of free [14C]arachidonate. [14C]Arachidonyl-phosphatidic acid formation was doubled after 10 min of incubation in the presence of ACh concentrations of 10(-4) M or greater. The stimulation by ACh could be completely blocked by atropine and scopolamine and partially blocked by d-tubocurarine. These studies suggest that intact rat cornea has muscarinic cholinergic receptors and that the enzymes of the inositol phospholipids pathway are present since phosphatidic acid is an obligatory intermediate in that cycle of reactions.

Effect of irradiation on vascularization of corneas grafted onto chorioallantoic membranes.

Several studies have shown that total body irradiation decreases the angiogenic response to corneal cauterization. This inhibition could be due to alterations in angiogenic stimuli within injured corneas and/or to a decreased ability of irradiated animals to respond to such stimuli. To determine whether total body irradiation specifically affects angiogenic stimuli within injured corneal tissue, cauterized corneas from mice exposed to 900 rads of total body irradiation and from non-irradiated controls were grafted onto the chorioallantoic membranes (CAM) of chick embryos and their abilities to stimulate the ingrowth of healthy embryonic blood vessels were compared. Cauterized corneas incorporated into CAM mesenchymal tissue were invaded by blood vessels in 34.6% of the irradiated group and in 75% of the non-irradiated controls. This difference in the two groups was statistically significant (P less than 0.03). Total body irradiation significantly decreased the frequency of vascular invasion of cauterized corneal tissues by healthy CAM blood vessels. This finding suggests that total body irradiation can specifically affect the stimulus for angiogenesis within cauterized corneas.

The effect of total-body irradiation on corneal neovascularization in the Fischer 344 rat after chemical cauterization.

Previous investigations of corneal neovascularization after irradiation yielded discordant results. Most studies indicated that new blood vessel formation in the cornea is inhibited by irradiation. However, others reported that angiogenesis after corneal cauterization is similar in both irradiated and nonirradiated animals. To assess the effect of total-body irradiation on neovascularization further, the amount of angiogenesis was determined in irradiated rats after chemically induced corneal injury. Corneal tissue was evaluated quantitatively with computerized image analysis 2, 3, or 4 days postcautery in rats perfused with India ink and gelatin immediately after death. The rats were exposed to a single dose (9 Gy) of total-body irradiation 6 days before corneal cauterization. In both the nonirradiated and irradiated rats, neovascularization increased with the duration of the postcautery interval. The amount of corneal neovascularization was not significantly different in the irradiated and nonirradiated rats at any of the postcautery intervals studied. This investigation suggests that endothelial cell migration plays a more important role than cell replication in the pathogenesis of corneal angiogenesis in the Fischer 344 rat. Moreover, the suppression of corneal angiogenesis by irradiation may be dependent on the experimental conditions and species examined.

The effect of oxygen on corneal neovascularization.

Since tissue oxygen levels are believed to play a pivotal role in new vessel growth in several situations, we studied the effect of several oxygen concentrations (0, 10, 21, 50, 75, or 100%) on corneal vascularization induced in the rat by chemical cautery. We achieved this by perfusing known concentrations of oxygen through goggles fitted over both eyes of the rat after corneal cauterization. Neovascularization was measured in flat corneal preparations with India ink-filled vessels 4 days postcautery using computerized image analysis. The angiogenic response of rats whose eyes were continuously exposed to 0-75% oxygen were not significantly different from each other. The mean response in corneas exposed to 100% oxygen was 10-21% lower than all of the other groups, and this difference was statistically significant when compared to oxygen concentrations of 0, 21 and 75%. The reason for the inhibitory effect of 100% oxygen remains to be determined, but it may represent a toxic effect of oxygen free radicals on the vascular endothelium.

Intercellular relationships in the synthesis of macromolecules by organ cultures of corneas.

Sepharose CL-4B chromatography of guanidine hydrochloride and aqueous extracts of 3H-glucosamine labeled intact corneal tissue reveals four peaks representing proteoglycans and glycoproteins. To evaluate the universality of the 4th peak, hereafter designated as Sepharose CL-4B (IV), its presence was investigated in rabbit, bovine, cat, rhesus monkey, and human corneal preparations. Following incubation in isotopically labeled medium, corneas were extracted with aqueous and/or 4M guanidine hydrochloride and subjected to Sepharose CL-4B chromatography. Sepharose CL-4B (IV) was detected in all species studied; 3H-glucosamine and 14C-amino acids, but not 35SO4, were incorporated into this peak which eluted in the range consistent with an apparent molecular weight of approximately 30,000 D. To determine which layers were involved in the synthesis of Sepharose CL-4B (IV) the layers of the rabbit cornea were incubated separately (stroma scraped of endothelium and/or epithelium, epithelium only, endothelium only). A distinct Sepharose CL-4B (IV) peak was not identified in the chromatographs obtained from organ cultures of corneal epithelium, endothelium, or from corneal stroma scraped of epithelium and/or endothelium. This decrease in Sepharose CL-4B (IV) synthesis occurred even if the scraped cornea was not allowed to expand in volume by compressing it beneath a membrane porous to the incubation medium. Thus, Sepharose CL-4B (IV) synthesis was enhanced significantly by the stroma being in conjunction with other corneal cells as they exist in vivo.

Familial subepithelial corneal amyloidosis--a lactoferrin-related amyloidosis.

PURPOSE: To isolate the protein that collects in increased amounts beneath the corneal epithelium in familial subepithelial corneal amyloidosis (FSCA), also known as gelatinous droplike corneal dystrophy, and to identify it by N-terminal amino acid sequencing. METHODS: Peptides resulting from pepsin digestion of a unique protein isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from frozen tissue from two corneas with FSCA were purified by high-pressure liquid chromatography followed by protein sequence analysis. The protein was identified by amino acid sequencing, Western blotting, and immunohistochemistry. RESULTS: A protein was identified in two corneas with FSCA that was not present in normal corneas or in corneas with other disorders. The amino acid sequences of two peptides derived from this protein were identical to portions of lactoferrin. The unique protein reacted with rabbit antihuman lactoferrin after Western blotting. The presence of lactoferrin in the amyloid within affected corneas was confirmed using the immunoperoxidase method on formalin-fixed, paraffin-embedded tissue sections and lactoferrin antiserum. CONCLUSIONS: Corneal tissue with FSCA contains lactoferrin, and this is the first form of amyloidosis found to be associated with this protein. Because lactoferrin is a product of lacrimal glands, the corneal lactoferrin may be derived from the tears. Because the gene for lactoferrin is on chromosome 3 (3q21-q23), this locus is a potential site for the FSCA gene.

Physical and genetic mapping of the macular corneal dystrophy locus on chromosome 16q and exclusion of TAT and LCAT as candidate genes.

PURPOSE: Macular corneal dystrophy (MCD) is an inherited autosomal recessive disorder that has been subdivided into three immunophenotypes, MCD types I, IA and II. We previously mapped the MCD type I gene to chromosome 16q22 and suggested that the MCD type II gene was linked to the same region. The purpose of this study was to construct a genomic contig spanning the MCD region and to narrow the MCD critical interval by haplotype analysis. The TAT and LCAT genes were mapped to determine if they might be the MCD gene. METHODS: The MCD contig was constructed by screening YAC, PAC, and BAC libraries with microsatellite, STS and EST markers, employing a systematic "DNA walking" technique. Polymorphic markers mapped and ordered on the contig were used to screen the MCD affected individuals and their family members for haplotype analysis. RESULTS: Twenty-two YAC, 30 PAC, and 17 BAC clones were mapped to form the MCD contig. Markers mapped on the contig include 19 microsatellite, 14 STS, and 15 EST markers. Moreover, 18 novel STS markers were generated. Using the mapped and ordered microsatellite markers, haplotype analysis on 21 individuals with MCD type I or type II and their family members from Iceland narrowed the MCD interval to 3 overlapping PAC clones. In addition, the TAT and LCAT genes were mapped outside the MCD region. CONCLUSIONS: We established a genomic contig for the MCD region and dramatically narrowed the MCD critical interval. Mapping data show that the TAT and LCAT genes are not the cause of MCD.

Mutations in corneal carbohydrate sulfotransferase 6 gene (CHST6) cause macular corneal dystrophy in Iceland.

PURPOSE: Macular corneal dystrophy (MCD) is subdivided into three immunophenotypes (MCD types I, IA and II). Recently, mutations in the carbohydrate sulfotransferase 6 gene (CHST6) were identified to cause MCD. The purpose of this study was to examine CHST6 for mutations in Icelandic patients with MCD type I. METHODS: Genomic DNA was extracted from leukocytes in the peripheral blood and the coding region of CHST6 was examined for mutations by polymerase chain reaction (PCR) and direct sequencing. RESULTS: Mutation analysis of the CHST6 coding region identified three different mutations in sixteen Icelandic patients with MCD type I. Eleven patients with MCD type I were homozygous for a C1075T mutation. One patient with MCD type I was found to be a compound heterozygous for C1075T and G1189C mutations. One family with MCD type I contained a 10 base pair insertion (ATGCTGTGCG) between nucleotides 707 and 708. In this family, two affected siblings had a homozygous insertion while both their affected mother and their affected maternal aunt had a heterozygous insertion and a heterozygous C1075T mutation. CONCLUSIONS: Three different nucleotide changes were identified in the coding region of CHST6 in sixteen Icelandic patients with MCD type I. All three of these alterations are predicted to affect the translated protein and each of them corresponded to a particular disease haplotype that we had previously reported in this population.

Familial subepithelial corneal amyloidosis (gelatinous drop-like corneal dystrophy): exclusion of linkage to lactoferrin gene.

PURPOSE: Because corneal tissue with familial subepithelial corneal amyloidosis (FSCA; gelatinous drop-like dystrophy of the cornea) contains lactoferrin the possibility that the FSCA gene was the human lactoferrin (hLF) gene was investigated. Due to contradictory published information we also mapped the hLF gene. METHODS: We mapped the hLF gene using a genomic clone of the entire hLF gene as a probe by fluorescence in situ hybridization (FISH). Utilizing PCR primers that are specific to the hLF gene, we also mapped the hLF via radiation somatic cell hybrid analysis. Linkage of the FSCA gene to the hLF gene was evaluated by genetic linkage analysis using polymorphic markers within and in the vicinity of the hLF gene. RESULTS: The hLF gene mapped to the short arm of chromosome 3 at 3p21. Linkage analysis using polymorphic markers for hLF and haplotype analysis of the 3p21 loci indicates that the FSCA gene is not linked to the 3p21 locus. CONCLUSIONS: The gene for FSCA is not the hLF gene in these families.

Senile scleral plaques: a histopathologic study using energy-dispersive x-ray microanalysis.

The investigators reviewed the pathologic findings of 21 senile scleral plaques in 17 enucleated globes. Eyes were fixed in formalin and routinely processed for light microscopic examination. Two representative calcified scleral plaques were subjected to energy-dispersive x-ray microanalysis. Plaques were located in the sclera just anterior to the insertion of the horizontal rectus muscles. Microscopic examination of the involved sclera disclosed a spectrum of histopathologic changes involving increased hematoxylinophilia of the scleral collagen, decreased stromal cellularity, the presence of scleral fibers with a unique corkscrew appearance, and calcium deposition. The calcified plaques contained calcium phosphate as indicated by histochemical methods and energy-dispersive x-ray microanalysis. Conjunctival elastosis was present in 12 of the 14 eyes in which sufficient conjunctiva was present for histologic evaluation. Accumulated actinic damage from solar irradiation may play a role in the pathogenesis of senile scleral plaques.