Gene regulation, genetic mechanisms and development of potential therapies for corneal endothelial dystrophies

研究背景和研究需要：角膜是位于眼睛前部的透明窗口。它保护眼睛不受外部环境的影响，并将光线聚焦到视网膜上。这一组织的最内层由一层特殊的角膜内皮细胞组成。这些细胞执行泵一样的机制从角膜外层清除水分，以确保组织获得最佳的水合，并调节关键营养物质进入角膜的其余部分。术语“角膜内皮营养不良(CED)”描述了一组导致这一特殊细胞层功能障碍的疾病。由于内皮细胞功能障碍，CED患者会出现角膜肿胀和混浊，导致视力受损。在一些患者中，青光眼的发展，进一步增加了视力障碍和/或失明的可能性。Fuchs内皮角膜营养不良(FECD)是一种与年龄相关的疾病，也是迄今为止最常见的CED，50岁的人中有4.5%受到影响。其他更罕见的CED类型包括一种称为后部多形性角膜营养不良(PPCD)的疾病。近年来，在了解CED的遗传原因方面取得了很大进展，我们现在知道几个不同基因上的某些遗传缺陷，称为突变，是导致疾病的原因。然而，我们仍然不完全了解这些疾病的潜在生物学基础，仍有20%的病例仍未从遗传学角度解释。角膜移植目前是CED患者视力丧失的唯一治疗选择，但移植物的长期存活率很低。手术还依赖于专业设施和健康的供体角膜，目前全球范围内存在短缺。只有了解突变如何改变角膜内皮的正常功能，才能开发适当的新治疗策略，以满足临床对替代治疗方案的迫切需求。目的：本研究旨在1)在未解决的CED病例中确定疾病的遗传原因，2)调查不同的CED相关突变如何和为什么导致内皮细胞功能障碍，3)利用这一知识开发新的治疗方法。方法：将使用一系列基因测序方法对CED患者的DNA样本进行分析，以确定疾病的遗传来源。同时，使用在计划中的角膜移植手术中取出的捐赠组织，我将使用我在实验室建立的技术来培养和维护角膜内皮细胞，以便能够研究特定突变如何以及为什么会导致细胞功能障碍和疾病。这些知识将被用来设计基因导向治疗策略。从疾病患者组织中建立的内皮细胞培养将作为理想的模型系统，在安全和与疾病相关的背景下测试不同治疗策略的有效性。这项研究的预期结果：这项研究将确定CED的遗传原因，在短期内有助于在受影响的家庭中更早地发现疾病的症状前，为遗传咨询提供信息，并可能改变疾病的临床管理。同时，将研究角膜疾病的细胞机制，以加强对疾病的生物学原因的了解，以便为这些威胁视力的疾病开发有效的非手术治疗。所获得的知识也将对个性化医学、与年龄相关的疾病和人类遗传学领域产生影响。

Background and need for the research: The cornea is the transparent window situated at the front of the eye. It protects the eye from the external environment and focuses light onto the retina. The inner most part of this tissue is comprised of a specialised layer of corneal endothelial cells. These cells perform a pump-like mechanism removing water from the outer layers of the cornea, to ensure that the tissue is optimally hydrated, and regulate the movement of crucial nutrients into the rest of the cornea. The term 'corneal endothelial dystrophies (CEDs)' describes a group of diseases that lead to dysfunction of this specialised cellular layer. Patients with CEDs experience corneal swelling and clouding, due to endothelial cell dysfunction, leading to impaired vision. In some patients, glaucoma develops, that further increases the likelihood of visual impairment and/or blindness. Fuchs endothelial corneal dystrophy (FECD) is an age-related disease and by far the most common CED, affecting 4.5% of people >50 years of age. Other much rarer types of CEDs include a disorder called posterior polymorphous corneal dystrophy (PPCD). In recent years, there has been much progress in understanding the genetic causes of CEDs and we now know certain genetic faults, known as mutations, in several different genes are responsible for disease. However, we still do not fully understand the underlying biological basis of these diseases, and >20% of cases still remain genetically unexplained. Corneal transplantation is currently the only treatment option available for CED patients experiencing visual loss, but the long-term survival of grafts is poor. Surgery also relies upon specialist facilities and healthy donor corneas, of which there is a currently a global shortage. Only by understanding how the mutations alter the normal functioning of the corneal endothelium, can appropriate new therapeutic strategies be developed to address the urgent clinical need for alternative treatment options. Aim: This research program aims to 1) identify the genetic cause of disease in unsolved CED cases and 2) investigate how and why different CED-associated mutations cause endothelial cell dysfunction and 3) use this knowledge to develop new therapies. Methods: DNA samples from CED patients will be analysed using a range of genetic sequencing methods to identify genetic origins of disease. In parallel, using donated tissue removed during planned corneal transplant surgery, I will use techniques that I have established in the laboratory to grow and maintain the corneal endothelial cells, to enable investigation of how and why particular mutations cause cellular dysfunction and disease. This knowledge will be harnessed to design gene-directed treatment strategies. Endothelial cell cultures established from diseased patients tissue will act as the ideal model system to test the efficacy of different treatment strategies in a safe and disease relevant context. Expected outcomes of the study: This study will identify genetic causes of CEDs that will in the short term facilitate earlier pre-symptomatic detection of disease in affected families, inform genetic counselling and may alter the clinical management of disease. In parallel, cellular mechanisms of corneal disease will be investigated to enhance understanding of the biological reasons for disease to enable effective non-surgical treatments to be developed for these sight threatening conditions. Knowledge gained will also impact upon the areas of personalised medicine, age-related disease, and human genetics.