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A. R. Philp, M. Jin, S. Li, B. R. Roos, A. Iannaccone, R. G. Weleber, G. A. Fishman, S. G. Jacobson, G. H. Travis, E. M. Stone; Disease-Causing Mutations in the RPE65 Gene Abolish Retinoid Isomerase Activity. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2960.
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© ARVO (1962-2015); The Authors (2016-present)
We developed a robust functional assay to determine the pathogenic potential of mutations in the RPE65 gene and used this assay to evaluate mutations observed in patients with Leber congenital amaurosis (LCA).
A combination of SSCP and DNA sequencing was used to screen the RPE65 gene in a cohort of individuals diagnosed with LCA as well as panels of control samples with well-defined ethnicity. cDNA constructs were made for 13 of the putative disease causing variants we observed using site-directed mutagenesis . These were then expressed in 293T-LC cells that stably express LRAT and CRALBP. Expression of the mutant proteins in the cells was confirmed by immunoblot analysis. Isomerase activity was measured by monitoring 11-cis retinol formation from all-trans retinol substrate added to the media of the cultured cells.
We detected no isomerase activity in cells expressing RPE65 with the LCA-associated mutations R91W, Y239D and L408P, confirming our previous reports. In addition, mutations H182Y, E417Q, G40S, R44Q and R91Q, observed homozygously or as compound heterozygotes in patients diagnosed with LCA, showed no isomerase activity. Additionally, two novel mutations; Y318N and T101I, observed heterozygously in a patient diagnosed with LCA, resulted in significantly reduced isomerization activity. In contrast, the variations K294T, N321K and A434V that were in more than 5% of control individuals from at least one ethnic group, showed isomerization activity that did not significantly differ from normal.
We have developed a cell-culture expression and enzyme-assay system to help predict the pathogenic potential of mutations in the RPE65 gene. Our assay supports pathogenicity of the previously described mutations R91W, Y239D, L408P, H182Y, E417Q, G40S, R44Q, R91Q and of the novel mutations Y318N and T101I. The isomerization activity and allele frequency in control populations of the sequence variations K294T, N321K and A434V suggest that they are not pathogenic and may be ethnic polymorphisms. The ability to reliably differentiate between pathogenic and benign variants is timely as there is international interest in clinical trials of RPE65 gene replacement therapy.
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