Unraveling the molecular pathology of retinal degeneration through single cell genomics

点击翻译按钮获取中文摘要

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.