Abstract
Purpose :
While there over 150 mutations in rhodopsin (RHO) that are known to cause retinitis pigmentosa, it is not well understood which mutations respond to pharmacologic chaperone therapy. Most mutations are associated with the impaired trafficking of the mutated RHO protein to the cell surface due to its misfolding and inability to bind to the native chromophore 11-cis retinal. Furthermore, some DNA variants in rhodopsin have uncertain pathological significance. We utilized a high-throughput, pooled, cell-based assay to quantitatively compare the cell surface expression of a library of rhodopsin variants in response to a variety of pharmacologic chaperone treatments.
Methods :
A pool of 211 rhodopsin variants was transfected into HEK293T cells, which were treated with 9-cis-retinal, 11-cis retinal, YC-001, or scriptaid. Using surface immunostaining and FACS, cells from each dose were sorted according to the quantity of rhodopsin that was successfully folded and transported to the cell surface. Residual transfected DNA inside the sorted cells was amplified and quantified using next-generation sequencing. Normalization, clustering, and variant classification methods are discussed.
Results :
Dose-response curves were determined for each of the 211 variants in response to the 4 different drugs. The retinals produced similar responses, while YC-001 and scriptaid produced distinct response patterns. The most retinal-responsive residues, including I179F, P180A, D190N were concentrated in the extracellular loop 2 / beta plug region (aa 175-200). This region, particularly residues 178 through 189, has been shown to form a disulfide bond with helix 3, creating a tight pocket for the native 11-cis retinal ligand. Selected strongly responsive variants (I179F, P180A, G182S, M39R, V20G, G18D) were successfully validated in unpooled assays. Hierarchical clustering and machine learning were used for data visualization and to classify variant pathogenicity.
Conclusions :
Using multiplexed assays allowed for a higher-throughput approach, efficiently producing quantitative information about variant pathogenicity and chaperone response profiles of rhodopsin variants. This approach, which can be extended to larger libraries and to other disease genes, is needed to understand, at scale, the diagnostic and therapeutic consequences of the diversity of human genetic variation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.