PROJECT SUMMARY/ABSTRACT The PPCD1 mouse arose from a spontaneous mutation in our mouse colony and exhibits an enlarged anterior chamber secondary to metaplasia of the corneal endothelium and blockage of the iridocorneal angle by the epithelialized corneal endothelial cells. The presence of stratified multilayered corneal endothelial cells with abnormal patterns of cytokeratin expression are remarkably similar to those observed in human corneal endothelial dystrophies, notably posterior polymorphous dystrophy (PPD), and the sporadic condition, iridocorneal endothelial syndrome (ICE). Secondary phenotypes observed in PPCD1 mice include corneal neovascularization, retinal ganglion cell loss, and photoreceptor loss, all significant causes of blindness in humans. The mouse PPCD1 phenotype exhibits an autosomal dominant pattern of inheritance, with complete penetrance on the sensitive DBA/2J background and significantly decreased penetrance on the C57BL/6J background. The objective of this proposal is to provide a molecular explanation for the murine PPCD1 phenotypes with hopes of shedding light on the human disease. We have mapped the mouse PPCD1 gene, designated Ppcd1, to a 6.2 Mbp interval on Chromosome 2, and identified a hemizygous 87,000 bp duplication in this interval. The endpoints of the duplication are located in positions which disrupt the genes Csrp2bp and 6330439K17Rik. LOC100043552, a pseudogene located in the center of the sequence, is also presumably duplicated. Our primary candidate for the Ppcd1 gene is Csrp2bp. This prediction is based on decreased Csrp2bp expression levels in PPCD1 animals, preliminary evidence for abnormal eye size and corneal opacities in induced null alleles of Csrp2bp and the known physical interaction of this gene product with the product of the Zeb1 locius (the other known hereditary cause of PPCD in humans). We propose to confirm our hypothesis that mutation of Csrp2bp is responsible for the PPCD1 phenotype by replicating corneal endothelial cell metaplasia in independent recombinant mouse models. We propose to identify mechanisms by which Csrp2bp interactions with Zeb1 cause the PPCD1 phenotype. We will determine the temporal and spatial localization of normal and abnormal Csrp2bp gene expression, identify Csrp2bp-regulated gene promoters and determine if they are also regulated by Zeb1. We will develop methods to test the function and regulation of Csrp2bp and demonstrate how disruption of its function produces the PPCD1 phenotype. We will extend our findings to human PPCD through screening for mutations in CSRP2BP and the orthologs of 6330439K17Rik and LOC100043552, respectively, C20ORF12, and ZNF 133 in PPCD1 patients in whom ZEB1 has been excluded as a causative gene. Putative causative mutations will be tested using in vitro functional assays. Finally, through linkage analysis, we will look for C57BL/6J modifier loci that influence the penetrance of PPCD1.