Abstract
Purpose :
The diverse DNA variants found in human patients can differ not only in their pathogenicity but also in their response to therapeutic compounds. Both of these issues can prevent inherited retinal disease patients from receiving treatment. Our goal is to create high-throughput cell-based assays to categorize variants and their differential sensitivity to potential therapeutic compounds. For example, 9-cis retinal, a form of vitamin A, is a known molecular chaperone for misfolded mutant rhodopsin (RHO), and mutations in RHO are a major cause of autosomal dominant retinitis pigmentosa.
Methods :
A library of 211 rhodopsin variants was created and transfected into HEK293 cells. After transfection, all variant-expressing cells were pooled. Cells were treated with 5 different doses of 9-cis retinal for 24 hours. The cells were fixed and stained with a monoclonal anti-rhodopsin antibody to detect rhodopsin that was successfully folded and transported to the cell surface. Using FACS, cells from each dose were sorted according to their rhodopsin surface expression. Residual transfected DNA inside the cells was extracted and the RHO amplicon amplified via PCR. The relative rhodopsin expression of each library variant for each dose was quantified by next-generation sequencing. Clustering algorithms partitioned the variants into clusters which behaved similarly across the experimental conditions.
Results :
Sorted cells from 3 biological replicates were sequenced across 7 conditions using 4*106 total sequencing reads. Library coverage was 99-100%. Replicate experimental conditions showed low dispersion within clusters. Biologically, previously unclassified RHO mutations partitioned into either retinal repsonsive groups containing class II and III mutants, or a non-retinal responsive groups containing class I, IV, and VI mutants.
Conclusions :
Functional genomics approaches can be used to classify the pathogenicity and the response to potential therapeutic compounds of a large number of RHO variants. Multiple mutations with no previously-known mechanism were identified that could be particularly amenable to chaperone treatment. This pharmacogenomic approach helps to better understand therapeutic options in RHO, and can also be applied to mutations in other genes.
This is a 2021 ARVO Annual Meeting abstract.