Uncovering novel biological processes and pathogenic mechanisms for glaucoma

项目总结/摘要 青光眼是影响全球6000万人的不可逆视力丧失的主要原因。尽管如此， 青光眼的预防和治疗是有限的。原发性开角型青光眼（POAG），最常见的形式， 其特征在于进行性视网膜神经节细胞死亡，导致视神经病变，通常由升高的 眼内压（IOP）。拟议研究的长期目标是确定基因组区域， 影响青光眼相关眼组织中的基因表达，并与大规模人类遗传学研究相结合， 关联研究，以揭示新的致病基因和关键的生物过程，可导致青光眼， 可以作为新疗法的有效药物靶点。全基因组关联研究（GWAS）已经导致了 发现了超过20个与POAG相关的基因组基因座和超过100个与IOP相关的基因座，然而提取 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，主要是因为相关的 变体位于非编码区。此外，青光眼的遗传性还有待检测。的 基因型-组织表达（GTEx）项目表明，与基因表达相关的遗传变异 然而，GTEx不包含眼组织的数据。 这项研究的工作假设得到了我们初步结果的支持， 调节作用将有助于青光眼的致病性，并将解释其遗传性， 受影响的基因会聚集在疾病相关的生物过程中。因此，我们在目标1中提出， 转录组和开放染色质区域的高质量图谱（指示转录活性），以及 确定与基因表达相关的遗传变异（eQTL）和选择性剪接（sQTL），在四个关键 青光眼的致病组织：流出道（小梁网和Schlemm管），睫状体， 视神经和黄斑视网膜，收集自100个死后供体。我们将使用RNA-seq，ATAC-seq， 我们将在大量组织上进行全基因组测序，并将进一步鉴定细胞类型特异性eQTL和sQTL， 我们可以从单细胞RNA-seq研究中获得细胞类型特异性基因表达谱。在目标2中， 将应用新的计算和统计方法，将Aim 1的数据与GWAS的高或低风险数据相结合， 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， 青光眼的潜在致病基因、途径和致病细胞类型。为了扩大我们的影响， 工作，我们将建立一个基于网络的平台，用于共享，浏览和检查基因表达和遗传 来自不同眼组织的调节数据与青光眼和其他复杂疾病的遗传相关性 眼睛特征（目标3）。这项拟议中的研究预计将大大提高我们对 青光眼的病理生理学，发现新的遗传风险因素，并开辟新的治疗途径， 发展我们的基因组学眼睛资源也将是研究其他眼睛的分子原因有价值的 用于视网膜变性等疾病的基因治疗，以及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.