The long-term goal of this research is to understand the developmental and genetic basis for human X-linked juvenile RS. RS causes significant vision loss due to macular degeneration beginning in infancy and progressive changes in later age mimic age-related macular degeneration. Peripheral vision is also affected due to splitting of the retina through the nerve fiber layer which lies at the surface of the retina. Muller cells (retinal glial cells) are believed to be affected in RS, and this project will help elucidate some of the biology of these retinal glial cells. This proposal is to identify the molecular defect for RS by a positional cloning approach. The research plan encompasses five specific aims.
Aim one is to continue collecting RS families, to narrow the RS inclusion-interval. These will be used to find additional crossovers and potentially new deletions in the RS region.
Aim two is to clone the RS gene, which appears to be within a 950 kb critical region, contained on a single 2.2 Mb YAC clone (939h7). A cosmid sublibrary has been made from this clone, and the cosmids are being assembled into a contig. Transcribed segments are being isolated by exon-trapping, and selection-screening of cDNA. Candidate genes will be tested by amplifying exons from multiple affected and unaffected controls, and searching for deletions and/or point mutations by sequencing. The applicant is also evaluating connexin-33, which maps to the critical region, as a candidate gene.
Aim three is to determine the pattern of gene expression in retina, once the RS gene has been cloned. This will involve in situ hybridization and immunocytochemistry, using antibody against fusion proteins.
Aim four is to correlate the genotype with the phenotype in the different RS alleles. Phenotypic features to correlate include severity at birth, progression, and presence of macular degeneration features.
Aim five is to test the relationship between the ERG b-wave generator and the RS gene product.
