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Identification and Characterization of the Genetic Basis of PPCD

PPCD 遗传基础的鉴定和表征

基本信息

批准号：
9195093
负责人：
ANTHONY John ALDAVE
金额：
$ 38.5万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-12-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9195093
关键词：
Affect Binding Biological Assay Blindness COL4A3 gene Candidate Disease Gene Cell Line Chromosomes, Human, Pair 10 Chromosomes, Human, Pair 2 Chromosomes, Human, Pair 20 Code Copy Number Polymorphism Cornea Corneal Diseases Corneal Endothelium Corneal dystrophy Corneal edema DNA-Binding Proteins Development Endothelial Cells Family Fuchs&apos Endothelial Dystrophy Functional disorder Gene Expression Gene Expression Profile Gene Expression Regulation Gene Mutation Gene Proteins Gene Targeting Genes Genetic Glaucoma Heterogeneity Homeobox Human Human Chromosomes Inborn Genetic Diseases Individual Investigation Keratoplasty Lead Link Maps Measures Mediating Morphology Mus Mutation Nonsense Mutation Orthologous Gene Pain Pathogenesis Pathogenicity Phenotype Play Postoperative Period Repression Research Personnel Role Secondary to Therapeutic Transcriptional Regulation Transfection United States Variant Vision Visual impairment Zinc Fingers base cell motility cell transformation comparative genomic hybridization exome sequencing gene repression genetic linkage analysis genetic pedigree genome sequencing insight interest mouse model mutant next generation promoter protein expression protein function public health relevance screening transmission process vision science whole genome

项目摘要

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.

描述（由申请人提供）：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、青光眼和视力丧失的发生有关。已经描述了 PPCD 的基因座异质性，在约 1/3 受影响的家系中，在 10 号染色体（PPCD3 基因座）上的锌指 E-box 结合同源盒 1 基因 (ZEB1) 中发现了截短突变，4 个家系的连锁分析表明与 20 号染色体（PPCD1 基因座）上重叠区域的连锁。鉴定 PPCD1 的遗传基础和表征 PPCD3 发病机制中 ZEB1 介导的 COL4A3 转录控制将为了解角膜内皮细胞转化和功能障碍提供重要见解，从而表征 PPCD 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，对来自 29 个 PPCD 家族的受影响和未受影响的个体（其中尚未发现 ZEB1 突变）进行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。具体目标 II 是表征后部多形性角膜营养不良 3 中 ZEB1 突变对 ZEB1 蛋白功能、COL4A3 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、离体之间的基因表达谱来确定 ZEB1 依赖性基因调控 PPCD3角膜内皮细胞和ZEB1蛋白水平改变的培养角膜内皮细胞；确定ZEB1突变对突变型ZEB1蛋白在转化的角膜内皮细胞中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的形态、增殖、活力和运动性的影响；并使用 DNA-蛋白结合测定和永生化角膜内皮细胞系的转染证明，ZEB1 介导的 COL4A3 表达抑制依赖于 ZEB1 与 COL4A3 启动子中的 E2 盒基序的结合。