Genetics & Genomics of Fuchs Endothelial Corneal Dystrophy

 描述（由申请人提供）：Fuchs角膜内皮营养不良（FECD）是一种年龄相关性退行性疾病，导致角膜水肿和视力丧失。FECD发生在5%的40岁以上白人中，并且是美国角膜移植的主要适应症。TCF 4的CTG18.1位点处的Intronic CTG三联体重复扩增是大多数患有FECD的高加索人中FECD的常见遗传原因。突变扩增的CUG转录物（CUGexp）在FECD受试者的内皮中积累为离散的核糖核灶。基于其他罕见的神经退行性疾病，如由扩增重复序列引起的强直性肌营养不良症，这些病灶通过隔离正常mRNA调控所需的RNA结合蛋白（RBP）发挥毒性。已显示剪接因子MBNL1与FECD中的CUGexp病灶共定位，导致由MBNL1调节的基因的错误剪接。在本提案中，我们将使用多代FECD家庭研究CTG18.1在亲子传播中的代际不稳定性。将注意CTG18.1的扩张和收缩，并注意起源和临床影响。将检查FECD内皮中扩增重复序列的体细胞不稳定性和扩增，以解释为什么该组织层如此容易发生年龄相关性变性。我们将使用裂隙灯、共聚焦和镜面反射显微镜检查FECD受试者中扩增重复序列特异性的早期角膜发现以及亚临床疾病受试者中的异常。我们希望通过对具有中等和大扩增的FECD组织进行RNA测序，与没有扩增重复序列的FECD样品进行比较，来严格检查CUGexp病灶对转录组的影响。我们将研究转录本的剪接是由MBNL和CELF家族的RBP，包括涉及细胞外基质蛋白和上皮间质转化的基因。MBNL1和MBNL2与TCF 4的CUGexp转录物的物理相互作用将用RNA免疫沉淀法检查。我们将确定这些RBP中的哪些是通过siRNA介导的FECD细胞系下调形成病灶的关键决定因素。将使用蛋白质质谱法鉴定对CUGexp具有亲和力的其他RBP。我们将在细胞培养模型系统中测试CUGexp病灶形成的双链RNA和单链反义寡核苷酸抑制剂。我们的目标是确定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.