Uncovering novel biological processes and pathogenic mechanisms for glaucoma

项目摘要/摘要 青光眼是影响全球6000万人不可逆转视力丧失的主要原因。尽管如此， 预防和治疗青光眼是有限的。原发性开角型青光眼(POAG)是最常见的形式， 以进行性视网膜神经节细胞死亡为特征，导致视神经病变，通常由 眼压(IOP)。这项拟议研究的长期目标是确定 影响青光眼相关眼组织的基因表达，并结合大规模的人类基因 协会研究，以揭示可能导致青光眼的新致病基因和关键生物学过程，这 可作为新疗法的有效药物靶点。全基因组关联研究(GWAS)导致了 发现了20多个与POAG相关的基因组基因座和100多个与眼压相关的基因座，但提取 从这些遗传关联中获得的生物学见解一直是具有挑战性的，这主要是因为相关的 变异体位于非编码区。此外，青光眼的许多遗传性尚未被检测到。这个 基因-组织表达(GTEx)项目表明，基因变异与基因表达有关 变异(EQTL)极大地增加了疾病风险，然而GTEx不包含眼组织的数据。 由我们的初步结果支持的拟议研究的工作假设是，数百 调节效应将有助于青光眼的致病性，并将解释其遗传性的大部分原因，以及 受影响的基因将聚集在与疾病相关的生物过程中。因此，我们在目标1中提议，创造 高质量的转录组和开放染色质区域图(指示转录活性)，以及 识别四个关键基因中与基因表达(EQTL)和选择性剪接(SQTL)相关的遗传变异 青光眼的致病组织：流出途径(小梁网和Schlemm管)、睫状体、 视神经和黄斑视网膜，取自100名尸检捐献者。我们将使用RNA-seq、atac-seq和 对大块组织进行全基因组测序，我们将进一步识别特定细胞类型的eQTL和sQTL 可供我们使用的单细胞RNA-SEQ研究中的细胞类型特异性基因表达谱。在目标2中，我们 将应用新的计算和统计方法，将来自目标1的数据与高或 正常眼压性青光眼和几个危险因素特征，目的是确定新的遗传关联，以及 青光眼的潜在致病基因、途径和致病细胞类型。扩大我们的影响 为此，我们将建立一个基于网络的共享、浏览和查看基因表达和基因的平台 来自不同眼睛组织的调节数据与青光眼和其他复合体的遗传关联 眼睛特征(目标3)。预计拟议的研究将显著提高我们对 青光眼的病理生理学，发现新的遗传危险因素，开辟治疗的新途径 发展。我们的基因组学眼睛资源也将对研究其他眼睛的分子病因具有价值 疾病，如视网膜变性疾病，并用于组织靶向基因治疗设计。

Project Summary/Abstract Glaucoma is a leading cause of irreversible vision loss affecting 60 million people world-wide. Despite this, prevention and treatment of glaucoma are limited. Primary open angle glaucoma (POAG), the most common form, is characterized by progressive retinal ganglion cell death leading to optic neuropathy, often caused by elevated intraocular pressure (IOP). The long-term goals of the proposed research are to identify genomic regions that affect gene expression in glaucoma-relevant eye tissues, and in combination with large-scale human genetic association studies, to uncover novel causal genes and key biological processes that can lead to glaucoma, which may serve as effective drug targets for novel therapies. Genome-wide association studies (GWAS) have led to the discovery of over 20 genomic loci associated with POAG and over 100 loci associated with IOP, however extracting biological insights from these genetic associations has been challenging, largely due to the fact that the associated variants lie in noncoding regions. Furthermore, much of the heritability of glaucoma has yet to be detected. The Genotype-Tissue Expression (GTEx) project has shown that genetic variants associated with gene expression variation (eQTLs) substantially contribute to disease risk, however GTEx does not contain data for ocular tissues. The working hypothesis of the proposed research, supported by our preliminary results, is that hundreds of regulatory effects will contribute to the pathogenicity of glaucoma and will explain much of its heritability, and that the affected genes will cluster in disease-relevant biological processes. We thus propose in Aim 1, to create a high-quality map of the transcriptome and open chromatin regions (indicative of transcriptional activity), and to identify genetic variants associated with gene expression (eQTLs) and alternative splicing (sQTLs) in four key pathogenic tissues for glaucoma: outflow pathway (trabecular meshwork and Schlemm’s canal), ciliary body, optic nerve, and macular retina, collected from 100 postmortem donors. We will use RNA-seq, ATAC-seq, and whole genome sequencing on bulk tissue and we will further identify cell type-specific eQTLs and sQTLs using cell type-specific gene expression profiles from single cell RNA-seq studies that are available to us. In Aim 2, we will apply novel computational and statistical methods that integrate data from Aim 1 with GWAS data of high or normal-tension glaucoma and several risk factor traits, with the goal of identifying new genetic associations, and the underlying causal genes, pathways, and pathogenic cell types for glaucoma. To broaden the impact of our work, we will build a web-based platform for sharing, browsing, and inspecting gene expression and genetic regulation data from the different eye tissues in relation to genetic associations with glaucoma and other complex eye traits (Aim 3). The proposed research is expected to significantly improve our understanding of the pathophysiology of glaucoma, uncover novel genetic risk factors, and open new avenues for therapeutic development. Our genomics eye resource will also be valuable for studying the molecular causes of other eye diseases, such as retinal degeneration diseases, and for tissue-targeted gene therapy design.