A functional genomics approach for characterizing variants of unknown significance: assaying known and novel rhodopsin variants

Abstract

Purpose : Characterizing the functional significance of DNA sequence variants of unknown significance (VUS) is currently a major bottleneck in interpreting human genetic variation. A library approach was developed to efficiently detect pathogenic rhodopsin (RHO) variants that fail to express on the cell surface. This approach, while initially focused on RHO, is meant to demonstrate a streamlined, general method for detecting pathogenic VUS.

Methods : A cDNA library was created with 210 known and novel RHO variants taken from the literature and from the results of genetic diagnostic testing for patients with inherited retinal degenerations performed in our department. To identify misfolding (class II) mutants, transfected cultured cells were sorted using flow cytometry based on RHO surface expression. Next generation sequencing and string-based bioinformatic methods were used to quantify the transfected DNA in high versus low RHO-expressing cell pools.

Results : The multiplexed assay successfully detected all pooled library variants and quantified their expression precisely (Z’=0.94, R²=0.92-0.95). Updated RHO variant classifications were generated based on these functional data. For example, 82% of known folding mutants were confirmed to show pathogenic surface expression, while 18% unexpectedly showed normal or slightly reduced levels. Endocytosis and stability/posttranslational modification mutants showed an intermediate phenotype, although a more general pathogenicity assay will be needed to detect all pathogenic mutation classes. Among 10 novel VUS, 3 (G18D, G101V, P180T) showed low or very low levels and were present in probands manifesting retinitis pigmentosa, confirming their pathogenicity in these patients.

Conclusions : A multiplexed assay efficiently identified pathogenic VUS in RHO based on surface expression, solving 3 pedigrees. The updated pathogenicity findings may be useful to investigators and genetic counselors in interpreting pathogenicity of RHO variants, and the surface expression data may inform RHO structure-function relationships. This study demonstrates a parallelized, higher-throughput cell-based assay for the functional characterization of VUS in rhodopsin, and can be applied more broadly in other genes and disorders.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Surface expression levels of 210 RHO variants as measured by single-plex and multiplex (NGS) assays were highly correlated, R²=0.91.