Identification and Characterization of the Genetic Basis of PPCD

描述(申请人提供)：后部多形性角膜营养不良(PPCD)是一种遗传性角膜疾病，与角膜水肿、青光眼和视力丧失有关。PPCD的基因座异质性已被描述，在大约1/3的患病家系中发现了位于10号染色体上的锌指E盒结合同源框1基因(PPCD3位点)的截短突变，并在4个家系中发现了与20号染色体重叠区域(PPCD1基因座)连锁的连锁分析。对PPCD3发病机制中PPCD1基因基础的鉴定和ZEB1介导的COL4A3转录调控的研究将为PPCD的角膜内皮细胞转化和功能障碍提供重要的认识，并为基于基因的治疗策略提供潜在的靶点。具体目的1是通过对29个PPCD家系中未发现ZEB1突变的患者和非患者进行下一代(全外显子)测序，以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PPCD1小鼠中发挥作用的位置候选基因的角膜内皮表达。目的二是研究ZEB1基因突变对后发性多形性角膜营养不良中ZEB1蛋白功能、COL4A3表达和角膜内皮细胞表型的影响3.通过比较体外对照角膜内皮细胞、培养对照角膜内皮细胞、体外培养的PPCD3角膜内皮细胞和培养的ZEB1蛋白水平改变的角膜内皮细胞的基因表达谱，确定ZEB1突变对ZEB1蛋白在转化的角膜内皮细胞中定位的影响，以及ZEB1突变和ZEB1蛋白降低对培养的角膜内皮细胞的形态、增殖、活力和运动能力的影响；并通过DNA-蛋白质结合分析和永生化角膜内皮细胞系的转染证明，ZEB1介导的对COL4A3表达的抑制依赖于ZEB1与COL4A3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.