Abstract
Purpose :
Currently, causal mutations in about 20% of patients with inherited retinal diseases (IRDs) remain undetermined upon whole exome or genome sequencing tests. One potential reason is that some of the mutations might affect non-coding cis-regulatory elements (CREs) that are poorly characterized. This study aims to systematically identify CREs associated with genes involved in IRDs.
Methods :
The recent advances in single-cell omics technologies allow the identification and evaluation of CREs in individual cell types. Single-cell transposase-accessible chromatin and RNA sequencing (scATAC-seq and scRNA-seq) analyses of the human retinas identify CREs linked to genes associated with IRDs in photoreceptor cells. High-throughput massively parallel reporter assays (MPRAs) in mouse retinas validate the predicted functionality of these CREs. Each CRE is labeled by a unique barcode and tested along with thousands of others for regulatory activities on a photoreceptor-specific promoter by DNA/RNA sequencing.
Results :
Integrative analysis of snRNA-seq and snATAC-seq on healthy human donor retinas identified 2929 candidate CREs associated with known IRD genes expressed by the photoreceptor cells. The CRE candidates are composed of non-coding regions within 500kbs of known IRD gene promoters, in which their chromatin accessibility is highly correlated with promoter accessibility and corresponding gene expression. A gene expression reporter library containing these candidate elements is constructed along with positive and negative controls, and the regulatory activity of each element is assessed in the neonatal mouse retina.
Conclusions :
Progress of our study will allow us to identify a list of functionally validated CREs linked to known IRD genes. Further combination with whole genome sequencing of unsolved IRD patients will enable us to systematically identify potential pathogenic mutations affecting gene regulation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.