Higher Throughput Assays for Understanding the Pathogenicity of Variants of Unknown Significance (VUS) in the RPE65 Gene

Overview

Journal bioRxiv

Date 2025 Feb 20

PMID 39975398

Authors

Leila Azizzadeh Pormehr

Kannan Vrindavan Manian

Ha Eun Cho

Jason Comander

Affiliations

Soon will be listed here.

Abstract

Purpose: is a key enzyme in the visual cycle that regenerates 11-cis retinal. Mutations in 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 mutations have a low protein expression level, this study developed and validated multiple methods for assessing the expression level of variants. This functional evidence is expected to aid in reclassifying VUS as pathogenic, which in turn can broaden the application of gene therapy for patients.

Methods: 30 different variants of (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 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 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 , facilitating treatment and providing structure-function information.

References

Redmond T, Poliakov E, Yu S, Tsai J, Lu Z, Gentleman S . Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci U S A. 2005; 102(38):13658-63. PMC: 1224626. DOI: 10.1073/pnas.0504167102. View

Samardzija M, von Lintig J, Tanimoto N, Oberhauser V, Thiersch M, Reme C . R91W mutation in Rpe65 leads to milder early-onset retinal dystrophy due to the generation of low levels of 11-cis-retinal. Hum Mol Genet. 2007; 17(2):281-92. DOI: 10.1093/hmg/ddm304. View

Jin M, Li S, Moghrabi W, Sun H, Travis G . Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium. Cell. 2005; 122(3):449-59. PMC: 2748856. DOI: 10.1016/j.cell.2005.06.042. View

Feathers K, Jia L, Khan N, Smith A, Ma J, Ali R . Gene Supplementation in Mice Heterozygous for the D477G RPE65 Variant Implicated in Autosomal Dominant Retinitis Pigmentosa. Hum Gene Ther. 2023; 34(13-14):639-648. PMC: 10354729. DOI: 10.1089/hum.2022.240. View

Li Y, Yu S, Duncan T, Li Y, Liu P, Gene E . Mouse model of human RPE65 P25L hypomorph resembles wild type under normal light rearing but is fully resistant to acute light damage. Hum Mol Genet. 2015; 24(15):4417-28. PMC: 4492402. DOI: 10.1093/hmg/ddv178. View

Yue P, Li Z, Moult J . Loss of protein structure stability as a major causative factor in monogenic disease. J Mol Biol. 2005; 353(2):459-73. DOI: 10.1016/j.jmb.2005.08.020. View

Hanna R, Hegde M, Fagre C, DeWeirdt P, Sangree A, Szegletes Z . Massively parallel assessment of human variants with base editor screens. Cell. 2021; 184(4):1064-1080.e20. DOI: 10.1016/j.cell.2021.01.012. View

Rentzsch P, Witten D, Cooper G, Shendure J, Kircher M . CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 2018; 47(D1):D886-D894. PMC: 6323892. DOI: 10.1093/nar/gky1016. View

Ioannidis N, Rothstein J, Pejaver V, Middha S, McDonnell S, Baheti S . REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants. Am J Hum Genet. 2016; 99(4):877-885. PMC: 5065685. DOI: 10.1016/j.ajhg.2016.08.016. View

10.

Moiseyev G, Chen Y, Takahashi Y, Wu B, Ma J . RPE65 is the isomerohydrolase in the retinoid visual cycle. Proc Natl Acad Sci U S A. 2005; 102(35):12413-8. PMC: 1194921. DOI: 10.1073/pnas.0503460102. View

11.

Stanwyck L, Place E, Comander J, Huckfeldt R, Sobrin L . Predictive value of genetic testing for inherited retinal diseases in patients with suspected atypical autoimmune retinopathy. Am J Ophthalmol Case Rep. 2019; 15:100461. PMC: 6523031. DOI: 10.1016/j.ajoc.2019.100461. View

12.

Komarova E, Dontsova O, Pyshnyi D, Kabilov M, Sergiev P . Flow-Seq Method: Features and Application in Bacterial Translation Studies. Acta Naturae. 2023; 14(4):20-37. PMC: 9844084. DOI: 10.32607/actanaturae.11820. View

13.

Starita L, Young D, Islam M, Kitzman J, Gullingsrud J, Hause R . Massively Parallel Functional Analysis of BRCA1 RING Domain Variants. Genetics. 2015; 200(2):413-22. PMC: 4492368. DOI: 10.1534/genetics.115.175802. View

14.

Jones E, Lubock N, Venkatakrishnan A, Wang J, Tseng A, Paggi J . Structural and functional characterization of G protein-coupled receptors with deep mutational scanning. Elife. 2020; 9. PMC: 7707821. DOI: 10.7554/eLife.54895. View

15.

Philp A, Jin M, Li S, Schindler E, Iannaccone A, Lam B . Predicting the pathogenicity of RPE65 mutations. Hum Mutat. 2009; 30(8):1183-8. PMC: 2717180. DOI: 10.1002/humu.21033. View

16.

Motta F, Martin R, Porto F, Wohler E, Resende R, Gomes C . Pathogenicity Reclasssification of Missense Variants Related to Leber Congenital Amaurosis and Early-Onset Retinal Dystrophy. Genes (Basel). 2019; 11(1). PMC: 7016655. DOI: 10.3390/genes11010024. View

17.

Uppal S, Liu T, Poliakov E, Gentleman S, Redmond T . The dual roles of RPE65 S-palmitoylation in membrane association and visual cycle function. Sci Rep. 2019; 9(1):5218. PMC: 6435699. DOI: 10.1038/s41598-019-41501-w. View

18.

Kamiyama D, Sekine S, Barsi-Rhyne B, Hu J, Chen B, Gilbert L . Versatile protein tagging in cells with split fluorescent protein. Nat Commun. 2016; 7:11046. PMC: 4802074. DOI: 10.1038/ncomms11046. View

19.

Aoun M, Passerini I, Chiurazzi P, Karali M, De Rienzo I, Sartor G . Inherited Retinal Diseases Due to Variants: From Genetic Diagnostic Management to Therapy. Int J Mol Sci. 2021; 22(13). PMC: 8268668. DOI: 10.3390/ijms22137207. View

20.

Choi E, Suh S, Sander C, Hernandez C, Bulman E, Khadka N . Insights into the pathogenesis of dominant retinitis pigmentosa associated with a D477G mutation in RPE65. Hum Mol Genet. 2018; 27(13):2225-2243. PMC: 6005012. DOI: 10.1093/hmg/ddy128. View