Deep mutational scanning reveals the pathogenicity and structural constraints of a comprehensive set of rhodopsin missense variants

Abstract

Purpose : In human genetic testing results for inherited retinal diseases such as retinitis pigmentosa (RP), the presence of variants of unknown significance (VUS) can be a major barrier to establishing a firm genetic diagnosis. VUS can even affect access to precision medicine clinical trials. Mutations in rhodopsin are the most common cause of dominant RP. The purpose of this work is to identify every potential RHO missense variant with a pathogenic surface expression level using deep mutational scanning and a high-throughput cell-based assay, with the goals of reclassifying a large number of VUS and obtaining structural insights into the rhodopsin protein.

Methods : A cDNA library with every possible RHO missense variant (N=7299) was designed, synthesized, cloned, produced in lentivirus, and transduced into HEK293T cells by spinfection at a low MOI, and selected by puromycin. Surface antibody staining and FACS was used to sort stained cells into low- and high-RHO-expressing populations. gDNA from each population was uncrosslinked, extracted, PCR amplified, and quantified using NGS deep sequencing of the cDNA region of the integrand. A second experiment assayed how expressed variants underwent selection during 25 days of passaging in terms of gain-of-function toxicity. ASM 1.0 software was used to derive a surface expression or fitness map of all amino acid variants present in the RHO library.

Results : Variant coverage in the final data was >99% of all missense variants. While <150 pathogenic or likely pathogenic missense mutations in RHO were previously known across all studies (ClinVar), this study identified hundreds of new missense mutations with pathogenic expression levels. Structural insights based on the variant heatmap included identification of a low constraint region at the C-terminus and in cytoplasmic loop 3, and mapped the extent of the constrained beta-plug region in extracellular loop 2. Tolerance of specific amino acid changes varied depending on their location within the structure (e.g. within transmembrane domains).

Conclusions : A simple surface protein expression assay, when multiplexed over nearly all potential RHO missense mutations, was able to greatly expand knowledge of medically-important RHO missense variants, as well as provide detailed structural insights into this archetypal G-protein coupled receptor.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.