Identification and Characterization of the Genetic Basis of PPCD

描述（由申请人提供）：后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。基因座异质性已经被描述为PPCD，在大约1/3的受影响家系中，在10号染色体（PPCD3位点）上的锌指E-box结合同源盒1基因（ZEB1）中发现了切尾突变，在4个家庭的连锁分析中发现了与20号染色体（PPCD1位点）重叠区域的连锁。确定PPCD1的遗传基础，以及确定zeb1介导的COL4A3转录控制在PPCD3发病机制中的作用，将为了解PPCD的角膜内皮细胞转化和功能障碍提供重要见解，并揭示基于基因的治疗策略的潜在靶点。具体目的1是通过对29个尚未发现ZEB1突变的PPCD家族中受影响和未受影响个体的下一代（全外显子组）测序，确定后多形性角膜营养不良1的遗传基础。此外，将测量角膜内皮中可能在PPCD1小鼠中发挥作用的位置候选基因的表达。特异性目的II是表征ZEB1突变对后多形性角膜营养不良3中ZEB1蛋白功能、COL4A3表达和角膜内皮细胞表型的影响。这将通过：通过比较离体对照角膜内皮细胞、培养对照角膜内皮细胞、离体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来确定ZEB1依赖性基因调控；测定ZEB1突变对转化的角膜内皮细胞中突变体ZEB1蛋白的细胞定位的影响，以及ZEB1突变和减少的ZEB1蛋白对培养的角膜内皮细胞形态、增殖、活力和运动性的影响；并通过dna -蛋白结合试验和转染永活角膜内皮细胞系证明，ZEB1介导的COL4A3表达抑制依赖于ZEB1与COL4A3启动子中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.