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Purpose: We reported previously a 7-base intronic deletion close to the intron/exon junction of the SOD1 gene in 2 separate families with an autosomal-dominant form of keratoconus. The goal of this study was to determine if the 2 families (families A and H) shared a common haplotype by identifying closely linked new microsatellite markers flanking the SOD1 gene., Methods: Total genomic DNA was extracted from the blood of available members of families A and H. The DNA was amplified by polymerase chain reaction and digested with HpyCH4 III. A genomic contig was first constructed flanking the human SOD1 gene on chromosome 21q22.1-21q22.11. New polymorphic microsatellite markers were identified. All available individuals from the 2 families were genotyped using a set of 7 different markers (SOD1NU10, SOD1NU1, SOD1NU2, SOD1NU3, SOD1NU13, SOD1NU8, and SOD1NU9) to identify phase and the disease haplotype was constructed., Results: Five of the 7 markers are novel (SOD1NU1, SOD1NU2, SOD1NU3, SOD1NU8, and SOD1NU9). Family A is a 3-generation family and the disease haplotype was inferred based on segregation data for 7 different markers. Family H shared only 3 of the disease-associated alleles (SOD1NU1, SOD1NU2, and SOD1NU13) compared with family A., Conclusion: Based on the dissimilarity of disease-associated alleles, the 2 families do not appear to share the same haplotype and therefore are not closely related. This strongly supports the uniqueness of the 7-base deletion in intron 2 of the SOD1 gene to the keratoconus phenotype.

Purpose: To examine the distribution of extracellular matrix (ECM) and basement membrane (BM) components and of Na+,K+-ATPase in postcataract surgery (PCS) corneas. These corneas were from patients who never developed pseudophakic or aphakic bullous keratopathy (PBK/ABK) after cataract surgery. PCS corneas were compared with PBK/ABK and Fuchs' dystrophy corneas., Methods: The distribution of PBKIABK ECM and BM markers and of all three Na+,K+-ATPase alpha subunits was studied by immunofluorescence in 10 healthy, 11 PCS, 16 PBK/ABK, and 12 Fuchs' dystrophy corneas., Results: Fibrotic ECM proteins, tenascin-C and fibrillin-1, were found in only 1 of 10 healthy and in 2 of 11 PCS corneas. In contrast, these proteins were expressed in all PBK/ABK and more than half of the Fuchs' dystrophy corneas. BM components in PCS corneas were altered to a greater extent (40-60%), especially fibronectin and laminin-10. A decreased epithelial immunostaining for Na+,K+-ATPase alpha subunits was seen in approximately 40% of PCS corneas and in approximately two thirds of PBK/ABK and Fuchs' dystrophy corneas. However, the endothelial staining was normal in all groups., Conclusions: Because tenascin-C and fibrillin-1 were mostly found in diseased but not in PCS corneas, their expression may be related to later, clinical stages of corneal edema development. However, BM components abnormal in PBK/ABK and Fuchs' dystrophy corneas were also altered in PCS corneas without clinical evidence of ocular disease. This may result from subclinical corneal changes resulting from cataract surgery, lens removal, exposure to the intraocular lens, or endothelial cell damage. Alterations of epithelial Na+,K+-ATPase point to the importance of epithelial changes in the development of corneal edematous diseases.

Purpose: To describe the clinical course and alterations of the corneal extracellular matrix (ECM) and basement membrane (BM) in a cornea after hexagonal keratotomy, transverse keratotomies, and keratomileusis., Methods: Frozen sections of this cornea and of 12 normal corneas were studied by immunofluorescence with specific antibodies. The patient history was analyzed to allow a clinical correlation., Results: In the treated cornea, keratotomy scars and subepithelial fibrosis with neovascularization were seen. Around and beneath the epithelial plugs and along the keratotomy scars, deposits of types III, VI, VIII, and XIV collagen; fibrillin-1; fibronectin; and tenascin-C were found, together with short streaks of types IV (alpha 1-alpha 2) and VII collagen, laminin-1 and -5, entactin, and perlecan. alpha 3-alpha 4 Type IV collagen chains were abnormally absent from the BM around the epithelial plugs. At the edges of the keratomileusis flap, subepithelial fibrosis areas were found, with abnormal deposits of eight different collagen types, perlecan, fibronectin, fibrillin-1, and tenascin-C. The major part of the flap interface did not show ECM abnormalities. ECM alterations outside the scarred areas included the appearance of tenascin-C in the stroma and of alpha 1-alpha 2 type IV collagen in the epithelial BM, and the disappearance of fibronectin from Descemet's membrane., Conclusion: Five years after surgery, the treated cornea still presented BM abnormalities at sites of keratotomy scars and epithelial plugs. Several ECM components were abnormally expressed outside the scarred areas, consistent with an ongoing fibrosis in the treated cornea.

Purpose: To study alterations of the extracellular matrix (ECM) and basement membrane (BM) components in human keratoconus corneas., Methods: Fifteen normal and 13 keratoconus corneas were characterized by immunofluorescence with antibodies to 23 ECM and BM components., Results: Keratoconus staining patterns for posterior nonscarred regions and Descemet's membrane were normal. We focused on three areas of keratoconus corneas: (a) nonscarred anterior corneal regions, (b) scarred anterior and posterior corneal regions, and (c) gaps in Bowman's layer. In each of these areas, consistent ECM and BM changes could be found. Nonscarred regions showed decreased staining of the epithelial BM for entactin/nidogen, fibronectin, alpha 3-alpha 5 chains of type IV collagen, and chains of laminin-1. In contrast, scarred regions had greater than normal staining of the epithelial BM for these same components and also for laminin-5, perlecan, and type VII collagen. In the Bowman's layer gaps/breaks, focal fibrotic deposits containing type VIII collagen, fibrillin-1, tenascin-C, alpha 1-alpha 2 type IV collagen, and normal stromal ECM and epithelial BM components were seen. Fibrotic regions were largely restricted to areas where, because of the lack of Bowman's layer, the epithelium was in contact with the stroma., Conclusions: In a single keratoconus cornea, abnormalities in the ECM/BM patterns were not uniform. This may reflect locally increased protease activity (where few if any BM components are found) and ongoing wound healing (where more BM or ECM components or both are found).

Rabbits underwent implantation of either lyophilized or fresh porcine lenticules into the central or peripheral cornea. All animals were followed for up to four months by slit-lamp examination and macrophotography to determine implant rejection. Serum antibody levels to soluble porcine cornea extract were determined by an enzyme-linked immunosorbant assay. Only those animals receiving lenticules into the peripheral cornea experienced a rejection and developed an antibody response to the porcine cornea extract. The production of antibody preceded the appearance of vascularization of the implanted lenticules. Thus, the site of lenticule implantation, not the type of tissue preparation, determined the outcome of the graft.