Identification and Characterization of the Mouse PPCD1 Gene

小鼠 PPCD1 基因的鉴定和表征

• 批准号: 8309049
• 负责人: CHRISTOPHER A BRADFIELD
• 金额: $ 33.86万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8309049
• 关键词: Address; Alleles; Animals; Anterior; Biochemical; Biological Assay; Blindness; Candidate Disease Gene; Cells; Characteristics; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 20; Collaborations; Complex; Cornea; Corneal Endothelium; Corneal Neovascularization; Corneal Opacity; Corneal dystrophy; Cytokeratin; DNA; DNA Sequence Rearrangement; Data; Development; Disease; Endothelial Cells; Exhibits; Eye; Family member; Gene Expression; Gene Expression Profile; Generations; Genes; Genetic; Glaucoma; Goals; Heterogeneity; Heterozygote; Human; Human Chromosomes; In Vitro; Inheritance Patterns; Inherited; Iris Diseases; Laboratories; Light; Maps; Metaplasia; Methods; Michigan; Molecular; Mus; Mutation; Orthologous Gene; Pathogenesis; Patients; Pattern; Penetrance; Phenotype; Photoreceptors; Positioning Attribute; Proteins; Pseudogenes; Recombinants; Regulation; Retinal; Retinal Ganglion Cells; Role; Sampling; Sampling Studies; Screening procedure; Secondary to; Signal Pathway; Structure; Syndrome; System; Testing; Transcript; Universities; XCL1 gene; anterior chamber; base; cell type; chromatin immunoprecipitation; design; genetic linkage analysis; histone acetyltransferase; human disease; in vitro Assay; insight; mouse model; promoter

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.

项目摘要/摘要 PPCD1小鼠是从我们的小鼠群体中自发突变而来的，并表现出一种扩增的 继发于角膜内皮化生和虹膜角膜阻塞的前房 角度由上皮化的角膜内皮细胞决定。分层多层角膜的存在 具有异常细胞角蛋白表达模式的内皮细胞与观察到的非常相似 在人类角膜内皮营养不良中，特别是后部多形性营养不良(PPD)，以及 散发性，虹膜角膜内皮综合征(ICE)。在PPCD1中观察到的次级表型 小鼠包括角膜新生血管、视网膜神经节细胞丢失和光感受器丢失，所有这些都是显著的 人类失明的原因。小鼠PPCD1表型表现为常染色体显性遗传模式 遗传，在敏感的DBA/2J背景上完全外显，并显著降低 在C57BL/6J背景上的透视度。这项提议的目标是提供一种分子 对小鼠PPCD1表型的解释，希望能揭示人类疾病。我们 已将小鼠PPCD1基因定位于2号染色体上一个6.2 Mbp的区间，命名为“Ppcd1”，并 在这个区间内发现了一个87,000个碱基对的半合子重复。定位复制的终结点 在干扰基因Csrp2BP和6330439K17Rik的位置。LOC100043552，一个定位于 在序列的中心，也可能是重复的。我们的Ppcd1基因的主要候选基因是 Csrp2个基点。这一预测是基于PPCD1动物中Csrp2BP表达水平的降低，初步 Csrp2BP诱导零等位基因异常眼大小和角膜混浊的证据 该基因产物与ZEB1locius(另一已知遗传型)产物的物理相互作用 人类PPCD的原因)。我们建议确认我们的假设，即Csrp2BP突变是 通过在独立的重组细胞中复制角膜内皮细胞化生对PPCD1表型的影响 老鼠模型。我们建议确定Csrp2BP与ZEB1相互作用导致 PPCD1表型。确定正常和异常的时间和空间定位 Csrp2BP基因表达，鉴定Csrp2BP调控的基因启动子并确定它们是否也 受ZEB1监管。我们将开发方法来测试Csrp2BP的功能和调节，并演示 其功能的破坏如何产生PPCD1的表型。我们将把我们的发现扩展到人类PPCD 通过对CSRP2BP以及6330439K17Rik和LOC100043552的同源基因突变的筛查， 在排除了ZEB1的PPCD1患者中，C20ORF12和ZNF133分别是 致病基因。推测的致病突变将使用体外功能分析进行测试。最后， 通过连锁分析，我们将寻找影响PPCD1外显性的C57BL/6J修饰基因座。