Characterizing variants of unknown significance in rhodopsin: A functional genomics approach

Abstract

Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in-house genetic diagnostic testing were created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next-generation sequencing-based readout was developed so that a flow cytometry-based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized the surface expression of every RHO library variant with a high degree of reproducibility (r 2 = 0.92-0.95), recategorizing 37 variants. For example, three retinitis pigmentosa pedigrees were solved by identifying VUS which showed low expression levels (p.G18D, p.G101V, and p.P180T). Results were validated across multiple assays and correlated with clinical disease severity. This study presents a parallelized, higher-throughput cell-based assay for the functional characterization of VUS in RHO, and can be applied more broadly to other inherited retinal disease genes and other disorders. K E Y W O R D S functional genomics, inherited retinal diseases, next-generation sequencing, retinitis pigmentosa, rhodopsin, variant library, variant pathogenicity, variant of unknown significance, VUS

Molecular basis for variations in the sensitivity of pathogenic rhodopsin variants to 9-cis-retinal

“…Though the effects of these mutations may differ in the native context of the rod outer segment, we show that the observed PME of previously characterized variants are highly consistent with previous classifications ( Fig. 2 A ) and with the results of another recent high throughput investigation of retinopathy variant expression ( 9 ). Our measurements also identify 13 previously uncharacterized variants with severely deficient PME and seven that exhibit moderately deficient PME ( Table S1 ).…”

“…(7) Most experimentally characterized adRP variants accumulate in the endoplasmic reticulum (ER) in a manner that compromises their maturation within the secretory pathway. (8)(9)(10) In contrast, CSNB variants are typically well expressed, but exhibit constitutive activation. (11) Nevertheless, there are wide variations in the age of onset and severity of the retinopathies that likely reflect differences in the molecular effects of these mutations and other uncharacterized variants.…”

“…There are currently over 100 known mutations in the rhodopsin G protein-coupled receptor that cause a spectrum of visual retinopathies including autosomal dominant retinitis pigmentosa (adRP) and congenital stationary night blindness (CSNB) ( 7 ). Most experimentally characterized adRP variants accumulate in the endoplasmic reticulum in a manner that compromises their maturation within the secretory pathway ( 8 , 9 , 10 ). In contrast, CSNB variants are typically well expressed but exhibit constitutive activation ( 11 ).…”

Beyond Sector Retinitis Pigmentosa: Expanding the Phenotype and Natural History of the Rhodopsin Gene Codon 106 Mutation (Gly-to-Arg) in Autosomal Dominant Retinitis Pigmentosa

“…All of the mutations listed above (including in codon 106) as described in the Human Gene Mutation Database (HGMD; www.hgmd.cf.ac.uk), based on their experimentally studied biochemical and cellular characteristics, as class 2 mutations [40]. That is, they are mutations that cause protein misfolding and instability, leading to protein retention in the endoplasmic reticulum (ER) [32].…”

Combinatorial clinically driven blood biomarker functional genomics significantly enhances genotype-phenotype resolution and diagnostics in neuromuscular disease

“…Rigorous VUS-reclassification and resolution of genotype-combinations per American College of Medical Genetics and Genomics (ACMG) guidelines[9] requires understanding disease mechanisms using an integrative approach combining functional-assays with phenotype-correlation[10-12]. Gene-based or other biomarker testing from easily-accessible tissue (eg: blood/urine) is needed since muscle-biopsies or skin-derived-transdifferentiated myotubes are invasive, costly, and adipocyte contamination can compromise quality.…”