Connecting AMD SNPs to Functions Using Allele-specific Interactions

使用等位基因特异性相互作用将 AMD SNP 连接到功能

• 批准号: 10322157
• 负责人: Jiang Qian
• 金额: $ 47.19万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10322157
• 关键词: ATAC-seq; Affect; Affinity; Age related macular degeneration; Alleles; American; Atrophic; Base Pairing; Binding; Binding Proteins; Binding Sites; Biochemical; Bioinformatics; Biological; Biological Assay; Blindness; CRISPR interference; Cell Differentiation process; Cell physiology; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Binding; DNA Probes; DNA-Binding Proteins; DNA-Protein Interaction; Data; Development; Disease; ES Cell Line; Effectiveness; Elderly; Electrophoretic Mobility Shift Assay; Engineering; Functional disorder; Gene Expression; Genetic Diseases; Genetic Risk; Genetic Transcription; Genetic study; Genome; Goals; Human; Immunoprecipitation; In Vitro; Individual; Interferometry; Investigation; Knowledge; Lead; Length; Luciferases; Maps; Molecular; Neurodegenerative Disorders; Phenotype; Photoreceptors; Population; Predisposition; Protein Analysis; Protein Array; Protein Microchips; Proteins; Proteome; Public Health; Quantitative Trait Loci; RNA-Binding Proteins; Reporter; Research; Resources; Retina; Retinal Pigments; Risk; Role; Series; Single Nucleotide Polymorphism; Site; Specificity; Stress; Structure of retinal pigment epithelium; Surveys; Technology; Testing; Transcriptional Regulation; Transposase; Untranslated RNA; Validation; XCL1 gene; base; behavioral response; bevacizumab; cell behavior; cell type; chromatin immunoprecipitation; complement system; disorder risk; effective therapy; genetic risk factor; genetic variant; genome editing; genome wide association study; genomic locus; high risk; high throughput screening; human embryonic stem cell; human stem cells; insight; interest; loss of function; neovascular; novel; novel therapeutics; predictive test; risk variant; screening; stressor; success; transcription factor; treatment strategy

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.

项目摘要。 视网膜相关性黄斑变性（AMD）是一种视网膜神经退行性疾病，是视力丧失的主要原因 在全世界的老年人中。尽管抗VEGF治疗在新生血管性肿瘤的治疗中是有效的， 虽然目前还没有针对更常见的萎缩性（“干性”）形式的经证实和批准的治疗方法， 的疾病。更深入地了解AMD的遗传学和疾病机制， 在开发更有效的治疗策略方面。全基因组关联研究（GWAS）已经确定了一个 大量的单核苷酸多态性（SNP）与AMD风险增加相关。虽然这些 GWAS研究已经引起了人们对补体系统在AMD中的作用的兴趣增加，这是通过免疫调节的分子机制。 哪些AMD风险等位基因导致该疾病风险增加还知之甚少。了解AMD风险SNP是 特别具有挑战性，因为它们中的大多数发生在基因组的非编码区。作为解决这个问题的一种方法 表达数量性状基因座（eQTL）研究可以识别可能调节下游基因表达的SNP。 表情然而，eQTL不提供SNP结合蛋白的信息。确定相交的GWAS 另一种是通过染色体免疫沉淀测序（ChIP-seq）获得的具有转录因子（TF）结合位点的SNP 这是一种识别功能性SNP及其相互作用TF的有用方法，但这种方法需要对功能性SNP及其相互作用TF的先验知识。 相关TF在本申请中，使用了一种据我们所知以前未应用于AMD的方法， 研究中，我们建议实施一项疾病相关SNP的蛋白质组全分析（PWAS）研究， 编码区SNP来鉴定AMD中等位基因特异性蛋白质-DNA相互作用和调节活性的改变。的 这种方法的基本原理是我们的假设，即功能性AMD相关DNA SNP可能通过以下途径执行其功能： 与特定蛋白质的等位基因特异性相互作用。我们将调查整个人类TF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探针的谱系，其中超过1，700人 转录因子（TF）/DNA结合蛋白可以针对每种探针同时检测。已识别等位基因特异性 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，并使用人类视网膜色素进行验证。 从人干细胞分化的色素上皮（RPE）和光感受器（PR）细胞。在目标1中，我们将确定 TF显示与等位基因特异性AMD相关SNP的差异结合。目标2将进行生物化学表征， 优先考虑目标1中确定的工作重点。目的3：对AMD-SNP等位基因特异性蛋白进行功能鉴定 研究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。 总之，我们希望这些研究将提供新的治疗相关的见解的机制， 是AMD发展和进展的基础。