Identification and Characterization of the Genetic Basis of PPCD

描述（由申请人提供）：后部多形角膜营养不良（PPCD）是一种遗传性角膜疾病，与角膜水肿、青光眼和视力丧失的发生有关。基因座异质性已被描述为PPCD，在大约1/3的受影响家系中，在10号染色体上的锌指E盒结合同源异型盒1基因（ZEB 1）（PPCD 3基因座）中鉴定出截短突变，在4个家族中进行的连锁分析表明与20号染色体上的重叠区域（PPCD 1基因座）连锁。PPCD 1的遗传基础的鉴定和PPCD 3发病机制中ZEB 1介导的COL 4A 3转录控制的表征将提供对表征PPCD的角膜内皮细胞转化和功能障碍的重要见解，并揭示基于基因的治疗策略的潜在靶点。具体目标I是通过下一代（全外显子组）测序，在29个PPCD家族中未发现ZEB 1突变的受影响和未受影响的个体中，确定后部多形性角膜营养不良1的遗传基础。此外，还将测量可能在PPCD 1小鼠中发挥作用的位置候选基因的角膜内皮表达。具体目标II是描述ZEB 1突变对后部多形性角膜营养不良3中ZEB 1蛋白功能、COL 4A 3表达和角膜内皮细胞表型的影响。这将通过以下方式实现：通过比较离体对照角膜内皮细胞、培养的对照角膜内皮细胞、离体PPCD 3角膜内皮细胞和具有改变的ZEB 1蛋白水平的培养的角膜内皮细胞之间的基因表达谱来确定ZEB 1依赖性基因调节;确定ZEB 1突变对转化的角膜内皮细胞中突变的ZEB 1蛋白的细胞定位的影响以及ZEB 1突变的影响和减少的ZEB 1蛋白对培养的角膜内皮细胞的形态、增殖、活力和运动性的影响;以及使用DNA-蛋白结合测定和永生化角膜内皮细胞系的转染证明ZEB 1介导的COL 4A 3表达的抑制依赖于ZEB 1与COL 4A 3启动子中的E2盒基序的结合。

DESCRIPTION (provided by applicant): Posterior polymorphous corneal dystrophy (PPCD) is an inherited disorder of the cornea that is associated with the development of corneal edema, glaucoma and loss of vision. Locus heterogeneity has been described for PPCD, with truncating mutations identified in the zinc finger E-box binding homeobox 1 gene (ZEB1) on chromosome 10 (the PPCD3 locus) in approximately 1/3 of affected pedigrees and linkage analysis in 4 families demonstrating linkage to overlapping regions on chromosome 20 (the PPCD1 locus). Identification of the genetic basis of PPCD1 and characterization of ZEB1-mediated transcriptional control of COL4A3 in the pathogenesis of PPCD3 will provide important insights into the corneal endothelial cell transformation and dysfunction that characterize PPCD and reveal potential targets for gene- based therapeutic strategies. Specific Aim I is to identify the genetic basis of posterior polymorphous corneal dystrophy 1 through next generation (whole exome) sequencing in affected and unaffected individuals from 29 PPCD families in which a ZEB1 mutation has not been identified. In addition, corneal endothelial expression of positional candidate genes implicated as potentially playing a role in the PPCD1 mouse will be measured. Specific Aim II is to characterize the effect of ZEB1 mutations on ZEB1 protein function, COL4A3 expression and corneal endothelial cell phenotype in posterior polymorphous corneal dystrophy 3. This will be accomplished by: determining ZEB1-dependent gene regulation through comparing the gene expression profile between ex vivo control corneal endothelial cells, cultured control corneal endothelial cells, ex vivo PPCD3 corneal endothelial cells and cultured corneal endothelial cells with altered ZEB1 protein levels; determining the effects of ZEB1 mutations on cellular localization of mutant ZEB1 protein in transformed corneal endothelial cells and the effect of ZEB1 mutations and reduced ZEB1 protein on the morphology, proliferation, viability and motility of cultured corneal endothelial cells; and demonstrating using DNA-protein binding assays and transfection of an immortalized corneal endothelial cell line that ZEB1-mediated repression of COL4A3 expression is dependent upon ZEB1 binding to the E2 box motifs in the COL4A3 promoter.