Wednesday, April 2, 2025
4/2/2025
The role of non-coding variants in Usher disease - PROJECT SUMMARY/ABSTRACT Primary cilia, or non-motile cilia, are present in almost every mammalian cell. Ciliopathies cause a spectrum of diseases in multiple organs, including the brain, kidney and liver. The most debilitating, however, are sensory neuropathies leading to deafness and blindness. Retinal ciliopathies account for one third of all inherited retinal diseases (IRDs), and are a major cause of visual impairment and blindness in the pediatric population. Usher syndrome is the most common retinal ciliopathy and presents a tremendous health burden due to congenital hearing impairment and progressive decline in vision. Usher type 2 (USH2) is the most common subtype with the USH2A gene accounting for the majority of cases. USH2 is inherited in an autosomal recessive manner; however, in approximately 20% of patients with clinical features typical for USH2, only one mutation of USH2A has been identified by exome sequencing, thereby precluding a definitive diagnosis. The second disease variant may reside in non-coding regions of the USH2A gene. Whereas the protein coding variants of IRDs have been studied in human and other model systems, the role of non-protein coding variants contributing to monoallelic disease is much less understood. Non-coding variants are difficult to classify, especially in gene products as large and complex as USH2A (spans 800 kilobases and contains 72 exons), and thus remain under-diagnosed. Genome sequencing of IRD patients can identify pathogenic non-coding variations in regulatory regions to explain causative changes and is increasingly being used to genetically diagnose patients suffering from suspected monogenic disease. Phasing genetic variation is also critical for human disease studies. We hypothesize that genomic sequencing and haplotype phasing of the USH2A locus and its surrounding regulatory regions will provide a more accurate detection of pathogenic variants in monoallelic patients. This will be accomplished by combining the base-level accuracy of Illumina short-read sequencing with the longer read lengths obtained from Nanopore based amplification-free targeted sequencing. Identification of non-coding variants and their phase information are essential first steps towards understanding their pathogenic effects in patient- derived stem cells. The pathogenicity of non-coding variants will be explored using luciferase knock-in cell culture systems as well as patient-derived induced pluripotent stem cells (iPSCs) differentiated in vitro to form retinal organoids (ROs). Use of ROs will provide clinically relevant tissue from patients that can be edited using CRISPR-Cas9 technology to determine variant pathogenicity and mutational burden. This proposal has tremendous therapeutic potential as non-coding deep-intronic variants have been the focus of gene targeting and gene editing technologies in various IRDs.
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