Unraveling the molecular pathology of retinal degeneration through single cell genomics

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.

Grant Number: 5R01EY031663-05
NIH Institute/Center: NIH
Principal Investigator: Radha Ayyagari