Unraveling the molecular pathology of retinal degeneration through single cell genomics

通过单细胞基因组学揭示视网膜变性的分子病理学

基本信息

批准号：
10624311
负责人：
Radha Ayyagari
金额：
$ 68.66万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10624311
关键词：
Ablation Affect Animal Model Architecture Biological Assay Blindness CRISPR/Cas technology Cell Culture Techniques Cell Nucleus Cell model Cells Choroid Chromatin Code Collaborations Computer Analysis DNA DNA methylation profiling Data Data Set Development Disease Disease model Enhancers Epigenetic Process Female Gene Mutation Genes Genetic Genetic Models Genome Genomics Goals Human Human Genome Individual Inherited Japan Joints Left Maps Methodology Modeling Molecular Multiomic Data Mutation Mutation Detection Netherlands Nucleic Acid Regulatory Sequences Nucleotides Outcome Outcome Study Pakistan Pathologic Pathology Patients Peripheral Physiological Positioning Attribute RNA RNA Splicing Recording of previous events Regulatory Element Research Research Personnel Resolution Resources Retina Retinal Degeneration Retinal Diseases Retinal Dystrophy Sequence Analysis Site Structure Structure of retinal pigment epithelium Switzerland System Testing Time Tissue Sample Tissues Transcript Untranslated RNA Validation Variant base editing candidate validation causal variant cell type epigenomics genetic pedigree genome sciences genome sequencing human tissue improved in vivo induced pluripotent stem cell inherited retinal degeneration innovation insight invention macula male methylome molecular pathology mouse model multidisciplinary multiple omics novel therapeutics stable cell line tool transcriptome whole genome

项目摘要

Abstract: The overarching goal of this proposal is to understand the molecular pathology of inherited retinal degeneration (IRD) by (a) generating maps of human retinal cell type-specific regulatory elements, (b) utilizing these maps to identify non-coding IRD causative mutations within retinal regulatory elements, and (c) gaining insight into the molecular underpinnings of pathological non-coding IRD mutations using cellular and animal models. IRDs are the most common cause of irreversible blindness in young individuals affecting 1 in 3000 individuals. Mutations in coding and splice site sequences in known IRD associated genes contribute to about 60%-65% of cases while the remaining 40%-35% of cases are currently unresolved. Mutations in non-coding or regulatory sequences are suggested to be responsible for a large proportion of these unresolved cases. Although the ENCODE and Roadmap Epigenomics projects have generated detailed maps of regulatory elements for the majority of body tissues, retina is left out. Lack of these maps is a major limitation in identifying IRD causative mutations involving regulatory sequences in retinal cells. We have analyzed the whole genome sequence (WGS) of 125 pedigrees with IRD; of these, 49 remain unresolved with no candidate causative nucleotide changes or structural variants (SVs) in coding or splice site sequences. This leads us to hypothesize the involvement of non-coding variants in pathology. We also have access to more than 391 additional IRD pedigrees that remained unresolved after WGS analysis. In this application we propose to test the hypothesis that non-coding sequence changes are involved in IRD pathology for the majority of these unresolved pedigrees. We will conduct the following studies: Aim 1, establish human retinal cell type specific maps of regulatory elements using innovative single cell genomics methodologies we developed, Aim 2, rank prioritize candidate causative variants using the retinal cell type-specific regulatory element maps and WGS of unresolved pedigrees, Aim 3, validate the impact of high ranking non-coding candidate disease causing variants in the context of the genome architecture of retinal cell types by developing patient iPSC-derived retinal cell models and mouse models. These studies will result in the establishment of retinal cell type-specific high-resolution multi-omic maps and will potentially identify, for the first time, non-coding variants involved in the pathology of IRD. The outcomes of these studies will (1) significantly enhance our understanding of the architecture of retinal cell type-specific regulatory networks, (2) reveal the molecular pathology underlying IRD, (3) establish a highly valuable, publicly-available data set of cis-regulatory elements relevant to retinal degenerative diseases as a resource for retinal disease research, (4) improve mutation detection in patients, and (5) facilitate discovery and development of novel therapies for IRD. We have assembled a multidisciplinary team of outstanding investigators with expertise in epigenetics (Ren), genome sciences (Frazer) and IRD genetics and disease modeling (Ayyagari) who are well positioned to complete this ambitious project.

抽象的：该提案的首要目标是了解遗传性视网膜变性的分子病理学（IRD）通过（a）生成人类视网膜细胞类型特异性调节元件的图谱，（b）利用这些图谱识别视网膜调控元件内的非编码 IRD 致病突变，以及 (c) 深入了解使用细胞和动物模型研究病理性非编码 IRD 突变的分子基础。 IRD 是导致年轻人不可逆转失明的最常见原因，影响三千分之一。突变已知 IRD 相关基因的编码和剪接位点序列导致约 60%-65% 的病例而其余40%-35%的案件目前尚未解决。非编码或监管突变序列被认为是造成这些未解决案件的很大一部分的原因。虽然 ENCODE 和路线图表观基因组学项目已经生成了详细的监管要素图谱大多数身体组织，视网膜被排除在外。缺乏这些地图是识别 IRD 病因的主要限制涉及视网膜细胞调节序列的突变。我们分析了整个基因组序列 (WGS) 125个谱系的IRD；其中，49 个仍未解决，没有候选致病核苷酸编码或剪接位点序列的变化或结构变异（SV）。这导致我们假设非编码变异参与病理学。我们还可以访问超过 391 个额外的 IRD WGS 分析后仍未解决的谱系问题。在此应用中，我们建议测试假设大多数 IRD 病理学都涉及非编码序列变化未解决的血统。我们将进行以下研究：目标1，建立人类视网膜细胞类型特异性使用我们开发的创新单细胞基因组学方法绘制的调控元件图谱，目标 2，排名使用视网膜细胞类型特异性调控元件图和全基因组测序（WGS）对候选致病变异进行优先排序未解决的谱系，目标 3，验证高排名非编码候选致病因素的影响通过开发源自患者 iPSC 的视网膜细胞类型基因组结构中的变异视网膜细胞模型和小鼠模型。这些研究将导致建立视网膜细胞类型特异性高分辨率多组学图谱，并将有可能首次识别涉及的非编码变异 IRD 的病理学。这些研究的结果将（1）显着增强我们对视网膜细胞类型特异性调节网络的结构，(2) 揭示 IRD 背后的分子病理学， (3) 建立一个非常有价值的、公开的与视网膜相关的顺式监管要素数据集退行性疾病作为视网膜疾病研究的资源，(4) 改进患者的突变检测， (5) 促进 IRD 新疗法的发现和开发。我们汇集了多学科由具有表观遗传学 (Ren)、基因组科学 (Frazer) 和 IRD 专业知识的杰出研究人员组成的团队遗传学和疾病建模（Ayyagari）有能力完成这个雄心勃勃的项目。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Single cell RNA sequencing confirms retinal microglia activation associated with early onset retinal degeneration.

DOI：
10.1038/s41598-022-19351-w
发表时间：
2022-09-10
期刊：
SCIENTIFIC REPORTS
影响因子：
4.6
作者：
Kumari, Asha;Ayala-Ramirez, Raul;Zenteno, Juan Carlos;Huffman, Kristyn;Sasik, Roman;Ayyagari, Radha;Borooah, Shyamanga
通讯作者：
Borooah, Shyamanga

Role of H3K4 monomethylation in gene regulation.