The purpose of the study was to identify genes that are differentially expressed in normal versus keratoconus corneas. Total RNA isolated from corneal stromal cell cultures was reverse-transcribed and then amplified by the polymerase chain reaction (PCR) using defined, arbitrary primers. The products were displayed on polyacrylamide gels and bands that were differentially expressed were excised, re-amplified and subcloned. The resulting clones were sequenced and utilized as probes for Northern blots with cultured cell RNA or Southern blots of corneal cDNA. One of the products that appeared to be more highly expressed in keratoconus cultures and corneas displayed 100% homology with leukocyte common antigen related protein (LAR), a transmembrane phosphotyrosine phosphatase. Western analyses and immunohistochemistry with monoclonal and/or polyclonal antibodies to LAR were used to examine keratocyte cultures and fresh frozen normal, keratoconus and pseudophakic bullous corneas. We identified a gene product with 100% homology to LAR that is expressed at the RNA level in keratoconus corneas and cell cultures but is found only at low or undetectable levels in normal cultures and normal and pseudophakic bullous keratopathy (PBK) corneas. By Western blotting and immunofluorescence with specific LAR antibodies, the protein was identified in keratoconus stromal cell cultures but not in normal cultures. When fresh frozen tissue was examined, LAR protein was localized to numerous stromal cells throughout central keratoconus corneas, while no central staining was seen in normal or bullous keratopathy corneas. LAR, a transmembrane phosphotyrosine phosphatase, is more highly expressed in keratoconus corneas and stromal cell cultures as demonstrated by differential display, Northern analyses, immunohistochemistry and Western blotting.
Copyright 1999 Academic Press.