July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Functional genomics for inherited retinal diseases: characterizing variants of unknown significance in rhodopsin
Author Affiliations & Notes
  • Jason Comander
    Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts, United States
  • Aliete Wan
    Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts, United States
  • Keith Wu
    Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts, United States
  • Eric A Pierce
    Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Jason Comander, None; Aliete Wan, None; Keith Wu, None; Eric Pierce, None
  • Footnotes
    Support  NEI K12 EY16335, Research to Prevent Blindness Career Development Award, Foundation Fighting Blindness Enhanced Career Development Award, NIH P30EY014104 Core grant, Lions Eye Research Institute
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 6022. doi:
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    • Get Citation

      Jason Comander, Aliete Wan, Keith Wu, Eric A Pierce; Functional genomics for inherited retinal diseases: characterizing variants of unknown significance in rhodopsin. Invest. Ophthalmol. Vis. Sci. 2018;59(9):6022.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : With recent advances in gene augmentation therapy, providing an unambiguous genetic diagnosis to patients with inherited retinal diseases (IRDs) is increasingly important. However, genetic testing reports can be inconclusive due to the presence of DNA variants of unknown significance (VUS). A cell-based assay using 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. Optimizations to NGS data analysis improved the signal/noise ratio of low-frequency variant quantitation. Updated RHO variant classifications were generated based on these functional data, including 18% of known folding mutants which showed unexpectedly normal levels. Endocytosis mutants showed an intermediate phenotype, although a more general pathogenicity assay will be needed to detect all pathogenic mutation classes. Three retinitis pigmentosa pedigrees were solved by demonstrating that a VUS showed low or very low levels. Further experiments extend this assay to use low-multiplicity of infection, pooled transduction techniques in anticipation of larger library sizes.

Conclusions : A multiplexed assay efficiently identified pathogenic VUS in RHO based on surface expression, solving 3 pedigrees. This study demonstrates an optimized, 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 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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