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Updated: Jun 24, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Higher throughput assays for understanding the pathogenicity of variants of unknown significance (VUS) in the RPE65
Leila Azizzadeh Pormehr1, Kannan Vrindavan Manian1, Ha Eun Cho1
1Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Mass Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA.
Purpose:
RPE65 is a key enzyme in the visual cycle that regenerates 11-cis retinal. Mutations in RPE65 cause a retinal dystrophy that is treatable with an FDA-approved gene therapy. Variants of unknown significance (VUS) on genetic testing can prevent patients from obtaining a firm genetic diagnosis and accessing gene therapy. Since most RPE65 mutations have a low protein expression level, this study developed and validated multiple methods for assessing the expression level of RPE65 variants. This functional evidence is expected to aid in reclassifying RPE65 VUS as pathogenic, which in turn can broaden the application of gene therapy for RPE65 patients.
Methods:
30 different variants of RPE65 (12 pathogenic, 13 VUS, 5 benign) were cloned into lentiviral expression vectors. Protein expression levels were measured after transient transfection or in stable cell lines, using Western blots and immunostaining with flow cytometry. Then, a pooled, high throughput, fluorescence-activated cell sorting (FACS) assay with an NGS-based sequencing readout was used to assay pools of RPE65 variants.
Results:
There was a high correlation between protein levels measured by Western blot, flow cytometry, and the pooled FACS assay. Using these assays, we confirm and extend RPE65 variant data, including that Pro111Ser has a low, pathogenic expression level. There was a high correlation between RPE65 expression and previously reported enzyme activity levels; further development of a high throughput enzymatic activity assay would complement this expression data.
Conclusion:
This scalable approach can be used to solve patient pedigrees with VUS in RPE65, facilitating treatment and providing RPE65 structure-function information.
