Pathogenesis of age-related Fuchs Endothelial Corneal Dystrophy

 描述(申请人提供)：这是一个多PI R01应用程序，用于测试三核苷酸重复扩增(TNR)是Fuchs内皮性角膜营养不良(FECD)发病机制中的因果事件的假设。FECD是一种常见的角膜内皮细胞单层退行性疾病。不幸的是，目前还没有阻止疾病进展的药物，其发病机制也不是很清楚。因此，手术角膜移植是唯一可行的治疗方法。事实上，终末期FECD是美国同种异体角膜移植最常见的原因，每年有超过14,000例移植。最近，我们在转录因子4(TCF4)基因的第三内含子中发现了一个CTG TNR扩展序列，它与79%的FECD病例相关。到目前为止，这是FECD中发现的最具预测性的遗传异常。在FECD患者中，CTG TNR扩增范围为50-2000个重复，显著高于非FECD患者，后者通常有12-18个CTG重复。TCF4 CTG TNR扩增区如何促进FECD表型尚不清楚。类似的不稳定TNR序列的扩展被认为是几种罕见和衰弱的神经疾病的致病机制，如脊髓小脑性共济失调、亨廷顿病、脆性X综合征和1型强直性肌营养不良。这些TNR扩展区域被认为干扰了正常的蛋白质功能、含有TNR的基因的表达和/或顺式和反式的RNA剪接。此外，有毒物质的翻译 来自TNR扩展序列的正义和反义转录本的重复相关非ATG(RAN)翻译产物也被认为是导致疾病进展的一个因素。我们的初步研究表明，类似的事件也发生在FECD细胞中。根据我们的数据，来自TCF4 CTG TNR扩张区的转录本保留在细胞核中，并在FECD组织和原代FECD单层细胞中形成称为RNA灶的核糖核包涵体。这些RNA焦点隔离剪接因子MBNL1，导致许多转录本的剪接改变。我们也有证据表明TCF4 CTG TNR扩展RNA的RAN翻译，在FECD患者的角膜内皮细胞中产生了小的同聚蛋白聚集体。我们的中心假设是，来自TCF4 CTG TNR扩张区的RNA焦点和RAN翻译蛋白产物通过改变基因表达和扰乱关键细胞功能而导致FECD。我们的前提是压力因素(例如 氧化应激)促进TCF4 CTG TNR扩展序列的不稳定性，导致包含RNA焦点的CUG重复序列的形成和RAN翻译产物的积累。这些产物的存在导致不同的mRNA剪接和关键基因的表达改变，刺激内质网应激介导的未折叠蛋白反应(UPR)、内皮细胞向间充质转化(EMT)和无序的细胞外基质(ECM)沉积。我们将通过两个特定的目标来验证我们的假设-1)表征与FECD中TCF4 CTG TNR扩张相关的分子异常；2)确定TCF4 CTG TNR扩张在FECD发病机制中的作用。这两个特定目标的完成将提供对TCF4、CTG、TNR扩增如何参与FECD发病机制的机制理解。了解FECD发病机制背后的分子事件对于未来制定针对FECD的靶向治疗策略至关重要。

 DESCRIPTION (provided by applicant): This is a multiple PI R01 application to test the hypothesis that trinucleotide repeat expansion (TNR) is a causal event in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD). FECD is a common, degenerative disease of the corneal endothelial cell monolayer. Unfortunately, there are no medical therapies to halt disease progression, and the pathogenesis is not well understood. As a result, surgical transplantation of the cornea is the only viable treatment. In fact, end-stage FECD is the most common cause of allogeneic corneal transplantation in the U.S., responsible for greater than 14,000 grafts annually. Recently, we have identified a CTG TNR expansion sequence within the third intron of the transcription factor 4 (TCF4) gene that associates with 79% of FECD cases. To date, this is the most predictive genetic anomaly identified for FECD. In FECD, CTG TNR expansions range from 50-2000 repeats, which are significantly higher than non-FECD patients who typically have 12-18 CTG repeats. How this TCF4 CTG TNR expansion region promotes the FECD phenotype is not clear. Similar expansion of unstable TNR sequences have been identified as the causative pathogenic mechanism in several rare and debilitating neurologic disorders, such as spin cerebellar ataxias, Huntington disease, fragile X syndrome and myotonic dystrophy, type 1. These TNR expansion regions are thought to interfere with normal protein function, expression of the TNR containing gene, and/or RNA splicing in cis and trans. In addition, translation of toxic Repeat-Associated Non-ATG (RAN) translation products from the sense and antisense transcripts of the TNR expansion sequences have also been identified as a contributing factor towards disease progression. Our preliminary studies suggest similar events are occurring in FECD cells. According to our data, transcripts from the TCF4 CTG TNR expansion region are retained in the nucleus and form ribonuclear inclusions called RNA foci in FECD tissue and primary FECD monolayer cells. These RNA foci sequester the splicing factor MBNL1, leading to altered splicing of numerous transcripts. We also have evidence for RAN translation of TCF4 CTG TNR expansion RNA, producing small homopolymeric protein aggregates in corneal endothelial cells of FECD patients. Our central hypothesis is that RNA foci and RAN translation protein products arising from the TCF4 CTG TNR expansion region causes FECD by altering gene expression and disrupting critical cell functions. It is our premise that stress factors (e.g. oxidative stress) promote instability in the TCF4 CTG TNR expansion sequence resulting in the formation of CUG repeat containing RNA foci and accumulation of RAN translation products. The presence of these products leads to differential mRNA splicing and altered expression of critical genes, stimulating ER stress-mediated activation of unfolded protein response (UPR), endothelial to mesenchymal transformation (EMT) and disordered extracellular matrix (ECM) deposition. We will test our hypothesis with two specific aims - 1) Characterize the molecular anomalies associated with TCF4 CTG TNR expansion in FECD; 2) Determine the role of TCF4 CTG TNR expansion in pathogenesis of FECD. Completion of these two specific aims will provide a mechanistic understanding of how TCF4 CTG TNR expansion is involved in FECD pathogenesis. Understanding the molecular events underlying the pathogenesis of FECD will be essential for future development of targeted therapeutic strategies for management of FECD.