Cellular and Genetic Defects in Keratoconus

圆锥角膜(KC)是一种常见的角膜营养不良症，影响年轻人，导致进行性变薄， 结疤和角膜形状丧失，最终会导致失明。胶原蛋白在人体内的交联 角膜可以变硬并延缓其变弱，但严重情况下需要进行角膜移植。虽然KC有一个 遗传成分强，病因复杂、多基因、多因素。有迫切的需要 了解其病因，有助于制定KC的早期诊断和治疗策略。为了解决这个问题，我们的 竞争性更新应用侧重于识别细胞缺陷、生物标记物和遗传原因 KC。除了明显的家族性KC外，绝大多数是孤立的，疾病可能是由罕见的病原体引起的。 编码序列变异体和全基因组常见的非编码变异体，它们会增加人的易感性。 阐明这些“孤立的KC”潜在的遗传缺陷需要一系列生物学证据。我们的 最近的研究和初步数据为目前的提议提供了这一生物学基础。首先，从整体上看- KC家族的外显子组测序，我们在与细胞应激相关的基因中发现了罕见的致病变异， 细胞骨架和细胞外基质(ECM)，它们现在被优先作为候选基因和网络 孤立的KC研究。第二，我们的KC和对照供体的转录和蛋白质组学特征 角膜在对角膜细胞至关重要的NRF2-抗氧化剂程序中发现了显著的失调 生存及其功能。最后，我们开发了角膜细胞培养模型，模拟关键的KC特征，来自 氧化应激对细胞外基质功能不全的影响，并对其进行检测。我们进一步开发了第一个角膜 来自人类诱导的多能干细胞的有机类化合物，将允许对基因和 生理性、角膜样环境中的治疗剂以及器官源性上皮和基质细胞中的治疗剂 文化。重要的是，这种方法将从基因定义的患者中产生细胞培养疾病模型 血细胞。这些细胞培养疾病的替代品特别重要，因为没有动物模型可以 可以捕捉到KC的多基因复杂性。在目标1中，我们将评估潜在的NRF2调节的抗氧化剂为 KC的泪液生物标记物，并在角膜细胞培养中研究这种网络。在目标2中，我们将确定 在分离的KC中增加疾病易感性的罕见致病变异和常见非编码变异 病例使用1000Genome和UK Biobank数据库作为对照。在目标3中，我们将从功能上测试 一种罕见的致病变异(例如，我们发表的C.G12982AHSPG2)会导致细胞疾病的概念 当CRISPR编辑成来自高多基因和非对照KC个体的细胞时的替代物 多基因得分低。我们的发现将导致潜在的抗氧化生物标志物，开发NRF2- KC治疗的激活剂，基因定义的KC细胞培养模型和对复杂基因的洞察 KC的体系结构。我们的研究与NEI在理解复杂性方面的目标高度相关 眼科疾病的遗传学、治疗和逆转视力丧失。

Keratoconus (KC), a common corneal dystrophy that affects young people, causes progressive thinning, scarring and loss of corneal shape, which can ultimately lead to loss of vision. Crosslinking of collagens in the cornea can stiffen and delay its weakening, but severe cases require corneal transplantation. Although KC has a strong genetic component, its etiology is complex, polygenic and multifactorial. There is an urgent need to understand its etiology for developing early diagnosis and treatment strategies for KC. To address this, our competitive renewal application focuses on identifying cellular defects, biomarkers and the genetic causes of KC. Beyond obvious familial KC, the vast majority are isolated where disease likely results from rare pathogenic coding sequence variants and genome-wide common noncoding variants that increase one's susceptibility. Elucidating the underlying genetic defects in these “isolated KC” requires a range of biological evidence. Our recent studies and preliminary data provide this biological foundation for the current proposal. First, by whole- exome sequencing of KC families, we identified rare pathogenic variants in genes related to cell stress, cytoskeleton and extracellular matrix (ECM), which are now prioritized as candidate genes and networks for the isolated KC studies. Second, our transcriptomic and proteomic characterizations of KC and control donor corneas identified significant dysregulation in the NRF2-antioxidant program that is crucial for corneal cell survival and its functions. Finally, we developed corneal cell culture models that mimic key KC features, from oxidative stress to ECM insufficiency, and assays to measure these. We further developed the first cornea organoids from human induced pluripotent stem cells that will allow functional studies of genes and therapeutic agents in a physiological, cornea-like setting and in organoid-derived epithelial and stromal cell cultures. Importantly, this approach will yield cell culture disease models from genetically defined patient blood cells. These cell culture disease surrogates are particularly important, as there are no animal models that can capture the polygenic complexity of KC. In Aim 1 we will assess potential NRF2-regulated antioxidants as tear fluid biomarkers for KC, and investigate this network in corneal cell cultures. In Aim 2 we will identify rare pathogenic variants and common noncoding variants that increase disease susceptibility in isolated KC cases using the 1000Genome and the UK Biobank databases as controls. In Aim 3 we will functionally test the concept that a rare pathogenic variant (e.g., our published c.G12982A HSPG2), will cause cellular disease surrogates when CRISPR-edited into cells derived from KC individuals with high polygenic and not controls with low polygenic scores. Our findings will lead to potential anti-oxidant biomarkers, development of NRF2- activators for KC treatments, genetically defined KC cell culture models and insights into the complex genetic architecture of KC. Our studies are highly relevant to the goals of the NEI in understanding the complex genetics of eye diseases, treatments and reversing vision loss.