Identification and Characterization of the Mouse PPCD1 Gene

描述（由申请方提供）：PPCD 1小鼠由我们的小鼠群体中的自发突变引起，并表现出继发于角膜内皮化生的前房扩大和上皮化角膜内皮细胞堵塞虹膜角膜角。具有异常细胞角蛋白表达模式的分层多层角膜内皮细胞的存在与在人角膜内皮营养不良中观察到的那些非常相似，特别是后部多形性营养不良（PPD）和散发性条件虹膜角膜内皮综合征（ICE）。在PPCD 1小鼠中观察到的次要表型包括角膜新生血管形成、视网膜神经节细胞损失和感光细胞损失，这些都是人类失明的重要原因。小鼠PPCD 1表型表现出常染色体显性遗传模式，在敏感的DBA/2 J背景下具有完全的显性遗传，在C57 BL/6 J背景下具有显著降低的显性遗传。这项建议的目的是为小鼠PPCD 1表型提供分子解释，希望能为人类疾病提供线索。我们已经将小鼠PPCD 1基因（命名为“PPCD 1”）定位于2号染色体上的6.2 Mbp区间，并在该区间鉴定了87，000 bp的半合子重复。重复的终点位于破坏基因Csrp 2bp和6330439 K17 Rik的位置。位于序列中心的假基因LOC 100043552也可能是重复的。我们的Ppcd 1基因的主要候选者是Csrp 2bp。该预测基于PPCD 1动物中Csrp 2bp表达水平降低，Csrp 2bp诱导无效等位基因中异常眼睛大小和角膜混浊的初步证据，以及该基因产物与Zeb 1 locius（人类PPCD的另一种已知遗传原因）产物的已知物理相互作用。我们建议证实我们的假设，Csrp 2bp的突变是负责PPCD 1表型复制角膜内皮细胞化生在独立的重组小鼠模型。我们建议确定Csrp 2bp与Zeb 1相互作用导致PPCD 1表型的机制。我们将确定正常和异常Csrp 2bp基因表达的时间和空间定位，确定Csrp 2bp调控的基因启动子，并确定它们是否也受Zeb 1调控。我们将开发方法来测试Csrp 2bp的功能和调节，并证明其功能的破坏如何产生PPCD 1表型。我们将通过在PPCD 1患者中筛选CSRP 2BP和6330439 K17 Rik和LOC 100043552的直系同源物、C20 ORF 12和ZNF 133的突变，将我们的发现扩展到人类PPCD，其中ZEB 1已被排除为致病基因。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C57 BL/6 J修饰位点，影响PPCD 1的多态性。 公共卫生相关性：该建议的目的是确定负责“PPCD 1”的基因，PPCD 1是人类眼部疾病后多形角膜营养不良的小鼠模型。还将进行研究，以确定与PPCD 1相关的其他角膜和视网膜异常的基因和机制，这些异常是视力丧失的重要原因。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The purpose of this proposal is to determine the gene responsible for "PPCD1", a mouse model of the human ocular disorder posterior polymorphous corneal dystrophy. Studies will also be carried out to determine the genes and mechanisms responsible for other corneal and retinal abnormalities associated with PPCD1 that are a significant cause of vision loss.