The research in this proposal involves identifying the genetic defect in the autosomal recessive disease macular corneal dystrophy (MCD). The abnormalities involved in MCD are mainly related to defects in posttranslational modifications which occur in the Golgi apparatus of corneal fibroblasts. We will test the hypothesis that these defects in MCD are all related to a mutation in a single gene which result in a defective protein that alters the functions of the Golgi apparatus in MCD cells. Two distinct approaches will be used to identify this defective protein. 1) A new PCR technique called differential display, that identifies alterations in mRNA between two similar yet different cell populations, will be used to compare mRNA levels from MCD fibroblasts with those in normal corneal fibroblasts. If the genetic defect in MCD involves a Golgi bound protein then this technique will most likely identify this abnormality. 2) The 8F1-3 antigen is an intermediate filament associated protein which has been shown to be abnormally localized to lysosomal vesicles in MCD. This is the only intracellular abnormality which has been reported in MCD. This protein will be cloned and the nucleotide sequence determined in order to identify this protein and to determine its intracellular function. It is likely that the 8F1-3 antigen contains the genetic defect in MCD and this will be determined by nucleotide sequencing the 8F1-3 gene from MCD cells as well. Together, these two experimental designs are highly likely to identify the genetic defect in MCD. They will also yield valuable information on intermediate filaments and their associated proteins.
| Dunlevy, J R; Berryhill, B L; Vergnes, J P et al. (1999) Cloning, chromosomal localization, and characterization of cDNA from a novel gene, SH3BP4, expressed by human corneal fibroblasts. Genomics 62:519-24 |
