Genetics & Genomics of Fuchs Endothelial Corneal Dystrophy

 描述(申请人提供)：Fuchs内皮性角膜营养不良(FECD)是一种与年龄相关的退行性疾病，导致角膜水肿和视力丧失。FECD在40岁以上的白人中占5%，是美国角膜移植的主要适应症。TCF4基因内含子CTG三联重复扩增是大多数患有FECD的高加索人FECD的常见遗传原因。突变的扩展CUG转录本(CUGexp)在FECD受试者的血管内皮细胞中以离散的核糖核灶的形式聚集。基于其他罕见的神经退行性疾病，如由扩展重复序列引起的强直性肌营养不良，这些病灶通过隔离正常mRNA调节所需的RNA结合蛋白(RBPs)而产生毒性。剪接因子MBNL1与FECD中的CUGexp焦点共定位，导致MBNL1调控的基因错剪。在这项建议中，我们将研究CTG18.1在使用多代FECD家庭的亲子传播中的代际不稳定性。将注意CTG18.1的扩张和收缩，并注意起源的父母和临床影响。我们将检查FECD内皮细胞中扩展的重复序列在躯体上的不稳定性和扩展，以解释为什么这一组织层如此容易发生与年龄相关的退化。使用裂隙灯、共焦显微镜和镜面显微镜，我们将检查FECD受试者扩大重复的早期角膜表现以及亚临床疾病受试者的异常。我们希望通过对具有中等和较大扩增的FECD组织进行RNA测序，并与没有扩展重复序列的FECD样本进行比较，来批判性地检查CUGexp焦点对转录组的影响。我们将研究其剪接受限制性商业惯例的MBNL和CELF家族调控的转录本，包括与细胞外基质蛋白和上皮-间充质转化相关的基因。MBNL1和MBNL2与TCF4的CUGexp转录本的物理相互作用将用RNA免疫沉淀进行检测。我们将确定这些限制性商业惯例中的哪些是通过siRNA介导的下调在FECD细胞系中形成病灶的关键决定因素。蛋白质质谱仪将被用来鉴定与CUGexp有亲和力的其他限制性商业惯例。我们将在细胞培养模型系统中测试CUGexp焦点形成的双链RNA和单链反义寡核苷酸抑制剂。我们的目标是识别用于治疗开发的先导化合物。

 DESCRIPTION (provided by applicant): Fuchs' endothelial corneal dystrophy (FECD) is an age-related degenerative disorder resulting in corneal edema and loss of vision. FECD occurs in 5% of whites greater than 40 years of age and is the leading indication for corneal transplantation in the U.S. Intronic CTG triplet repeat expansions at CTG18.1 locus of TCF4 are a common genetic cause of FECD in a majority of Caucasians with FECD. Mutant expanded CUG transcripts (CUGexp) accumulate as discrete ribonuclear foci in the endothelium of FECD subjects. Based on other rare neurodegenerative disorders such as Myotonic Dystrophy caused by expanded repeats, these foci exert toxicity by sequestering RNA binding proteins (RBPs) required for normal mRNA regulation. The splicing factor MBNL1 has been shown to co-localize with the CUGexp foci in FECD resulting is missplicing of genes regulated by MBNL1. In this proposal, we will examine intergenerational instability of CTG18.1 in parent-child transmissions using multi-generational FECD families. Expansions and contractions of CTG18.1 will be noted with attention to parent of origin and clinical impact. Somatic instability and expansion of the expanded repeats in FECD endothelium will be examined to account for why this tissue layer is so prone to age-related degeneration. Using slit lamp, confocal, and specular microscopy, we will examine for early corneal findings specific for the expanded repeats in FECD subjects as well as abnormalities in subjects with subclinical disease. We want to critically examine the impact of the CUGexp foci on the transcriptome by performing RNA sequencing of FECD tissue with intermediate and large expansions compared to FECD samples without the expanded repeats. We will examine the transcripts whose splicing is regulated by the MBNL and CELF family of RBPs including genes involved with extracellular matrix proteins and epithelial-mesenchymal transition. The physical interaction of MBNL1 and MBNL2 with the CUGexp transcripts of TCF4 will be examined with RNA immunoprecipitation. We will determine which of these RBPs are critical determinants for foci formation by siRNA mediated down regulation in a FECD cell line. Protein mass spectrometry will be utilized to identify additional RBPs with an affinity for CUGexp. We will test duplex RNA and single-stranded antisense oligonucleotide inhibitors of CUGexp foci formation in a cell culture model systems. We aim to identify lead compounds for therapeutic development.