Connecting AMD SNPs to Functions Using Allele-specific Interactions

项目总结。 年龄相关性黄斑变性(AMD)是一种视网膜神经退行性疾病，是导致视力丧失的主要原因 在世界各地的老年人中。尽管抗血管内皮生长因子治疗对新生血管的治疗是有效的 对于更常见的萎缩性(干燥)型，目前还没有得到证实和批准的治疗方法。 这种疾病的危害。对AMD背后的遗传学和疾病机制的更多了解有可能有助于 在制定更有效的治疗策略方面。全基因组关联研究(GWAS)已经确定了一种 与AMD风险增加相关的大量单核苷酸多态(SNPs)。尽管这些 Gwas的研究引起了人们对补体系统在AMD中的作用的兴趣增加，分子机制通过 哪些AMD风险等位基因导致疾病风险增加，目前还知之甚少。了解AMD风险SNPs是 尤其具有挑战性，因为它们大多发生在基因组的非编码区。作为解决这个问题的一种方法 问题是，表达数量性状基因座(EQTL)的研究可以识别可能调节下游基因的SNPs 表情。然而，eQTL不提供SNP结合蛋白的信息。确定相交的GWA 具有转录因子结合位点的单核苷酸多态(SNPs)是另一种方法 识别功能SNPs及其相互作用的Tf的有用方法，但该方法需要先验知识 相关的TF。在本应用程序中，使用了一种据我们所知以前未应用于AMD的方法 研究，我们建议实施非蛋白质的蛋白质组疾病相关SNPs分析(PWAS)研究 编码区SNPs用于识别AMD中等位基因特异的蛋白质-DNA相互作用和调节活性的变化。这个 这种方法的基本原理是我们假设与AMD相关的功能性DNA SNP可能通过 与特定蛋白质的等位基因特异性相互作用。我们将研究完整的人类转铁蛋白和RNA结合蛋白 使用基于蛋白质阵列的方法使用携带SNP的DNA探针的曲目，其中超过1700人 对于每个探针，可以同时检测转录因子(TF)/DNA结合蛋白。已识别的等位基因特异性 蛋白质-DNA相互作用将使用一系列生物信息学分析进行优先排序，并使用人类视网膜进行验证 色素上皮(RPE)和光感受器(PR)细胞是从人类干细胞分化而来的。在目标1中，我们将确定 显示与等位基因特定的AMD相关SNPs的差异结合的TFS。目标2将在生物化学特征和 确定目标1中确定的TF的优先顺序。目标3将从功能上表征已确定的AMD-SNP等位基因特异性蛋白 AMD相关细胞类型的相互作用，并探索它们如何影响细胞行为和对AMD相关应激源的反应。 综上所述，我们希望这些研究将为治疗机制提供新的相关见解。 AMD的发展和进展的基础。

PROJECT SUMMARY. Age-related Macular Degeneration (AMD) is a retinal neurodegenerative disease that is a major cause of vision loss among the elderly worldwide. Although anti-VEGF treatments can be effective in the treatment of the neovascular (“wet”) form of the disease, there are no proven and approved treatments for the more common atrophic (“dry”) form of the disease. Greater understanding of the genetics and disease mechanisms underlying AMD has the potential to aid in the development of more effective treatment strategies. Genomewide association studies (GWAS) have identified a large number of single nucleotide polymorphisms (SNPs) that are associated with increased risk of AMD. Although these GWAS studies have led to increased interest in the role of the complement system in AMD, the molecular mechanisms by which AMD risk alleles lead to increased risk for the disease are poorly understood. Understanding AMD risk SNPs is particularly challenging because most of them occur in non-coding regions of the genome. As one approach to this problem, expression quantitative trait loci (eQTLs) studies can identify SNPs that are likely to modulate downstream gene expression. However, eQTLs do not provide information on SNP-binding proteins. Determining intersecting GWAS SNPs with transcription factor (TF) binding sites by chromosomal immunoprecipitation sequencing (ChIP-seq) is another useful approach to identify functional SNPs and their interacting TFs, but this approach requires a priori knowledge of the relevant TFs. In this application, using an approach that has not, to our knowledge, been previously applied to AMD research, we propose to implement a Proteome-Wide Analysis of disease-associated SNPs (PWAS) study of non-protein coding region SNPs to identify allele-specific protein-DNA interactions and alteration of regulatory activity in AMD. The rationale for this approach is our hypothesis that functional AMD-related DNA SNPs likely execute their function via allele-specific interactions with specific proteins. We will survey the entire human TF and RNA-binding protein repertoires with SNP-carrying DNA probes using a protein array-based approach in which greater than 1,700 human transcription factors (TFs)/DNA binding proteins can be simultaneously surveyed for each probe. Identified allele-specific protein-DNA interactions will be prioritized using a series of bioinformatics analyses and validated using human retinal pigment epithelial (RPE) and photoreceptor (PR) cells differentiated from human stem cells. In Aim 1 we will identify TFs that show differential binding to allele-specific AMD-associated SNPs. Aim 2 will biochemically characterize and prioritize the TFs identified in Aim 1. Aim 3 will functionally characterize the identified AMD-SNP allele-specific protein interactions in AMD-relevant cell types, and explore how they affect cell behavior and response to AMD-related stressors. Taken together, we hope that these studies will provide new therapeutically relevant insights in the mechanisms underlying the development and progression of AMD.