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Lance P. Doucette, alina radziwon, Ian M MacDonald; Molecular analysis of the p.D477G in RPE65 as a cause of Choroideremia. Invest. Ophthalmol. Vis. Sci. 2018;59(9):6058.
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RPE65 is a gene best known to cause recessive Leber’s Congenital Amaurosis, and is the subject of one of the first successful gene therapies. One variant, c.1430A>G:p.D477G, has been shown to cause autosomal dominant choroideremia-like disease in two studies. As little is known about how this variation causes disease in human patients, and we aimed to explore the frequency of this variant in our CHM cohort, and the effects of this variant in a cell-based system.
As part of a larger study, we have performed Whole Exome Sequencing (WES) analyses on a patient diagnosed with choroideremia. We also examined a cohort of 90 patients with a choroideremia diagnosis and were negative for coding region CHM mutations. To examine the effects of this variant, we have developed expression vectors to express both wildtype (pcDNA3.1-RPE65(WT)) and the p.D477G variant (pcDNA3.1-RPE65(D477G)). HeLa and ARPE19 cells were transfected with these constructs and Western blot protein analysis, as well as immunofluorescence are being carried out.
WES screening identified a single patient clinically diagnosed with choroideremia with the p.D477G variant within RPE65. We also identified another patient heterozygous for the p.D477G through screening of our CHM-negative patient cohort. Transfection of the D477G vector into both HeLa and aRPE19 cells showed a band of lower molecular weight on Western analysis compared to the wildtype.
The p.D477G variant is a cause of a choroideremia-like phenotype though seems to be rare in occurrence. The presence of this variant in two of our patients suggest a common pathology between LCA, and CHM. The p.D477G variant shows a lower molecular weight band on Western analysis suggesting a change in post-translational modification. Experiments are currently underway to elucidate the nature of this post-translational modification, and how this may affect cellular localization of the p.D477G variant, and it’s effect on WT RPE65 protein.
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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