Identification and Characterization of the Mouse PPCD1 Gene

项目概要/摘要 PPCD 1小鼠是由我们的小鼠群体中的自发突变产生的， 继发于角膜内皮化生和虹膜角膜阻塞的前房 角上皮化的角膜内皮细胞。存在分层的多层角膜 具有细胞角蛋白表达异常模式的内皮细胞与观察到的那些非常相似 在人角膜内皮营养不良，特别是后部多形性营养不良（PPD）中， 散发性条件，虹膜角膜内皮综合征（ICE）。PPCD中观察到的次要表型1 小鼠包括角膜新生血管形成、视网膜神经节细胞损失和光感受器损失，所有这些都是显著的 人类失明的原因。小鼠PPCD 1表型表现出常染色体显性模式， 遗传，在敏感DBA/2J背景上具有完全不敏感性， 在C57BL/6J背景上的反射率。本提案的目的是提供一种分子 解释小鼠PPCD 1表型，希望能揭示人类疾病。我们 已将小鼠PPCD 1基因（命名为"PPCD 1"）定位于2号染色体上的6.2 Mbp区间， 在该区间鉴定了87，000bp的半合子重复。复制的端点位于 在破坏基因Csrp2bp和6330439K17Rik的位置。LOC100043552是一个假基因， 在序列的中心，也可能是重复的。Ppcd1基因的主要候选者是 Csrp2bp。该预测基于PPCD 1动物中Csrp2bp表达水平降低，初步 在Csrp2bp的诱导无效等位基因中存在异常眼睛大小和角膜混浊的证据， 该基因产物与Zeb1 locius（另一种已知的遗传性 人类PPCD的原因）。我们建议证实我们的假设，即Csrp 2bp突变负责 通过在独立的重组体中复制角膜内皮细胞化生， 小鼠模型。我们建议确定Csrp2bp与Zeb1相互作用导致Csrp2bp与Zeb1相互作用的机制。 PPCD 1表型。我们将确定正常和异常的时间和空间定位 Csrp2bp基因表达，鉴定Csrp2bp调节的基因启动子，并确定它们是否也 由Zeb1调节。我们将开发方法来测试Csrp2bp的功能和调节，并证明 其功能的破坏如何产生PPCD 1表型。我们将把我们的发现扩展到人类PPCD 通过筛选CSRP2BP中的突变以及6330439K17Rik和LOC 100043552的直系同源物， 在PPCD 1患者中，C20 ORF 12和ZNF 133分别被排除，其中ZEB 1被排除作为PPCD 1患者的一个基因。 致病基因将使用体外功能试验检测推定的致病突变。最后， 通过连锁分析，我们将寻找影响PPCD 1基因多态性的C57BL/6J修饰位点。

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.