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Amanda-Jayne Francis Carr, William Letton, Lyndon da Cruz, Anthony T Moore, Pete Coffey; Autosomal Dominant Vitreoretinochoroidopathy: mislocalisation of BEST1 protein in iPSC-RPE cells.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1837.
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© ARVO (1962-2015); The Authors (2016-present)
Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare retinal dystrophy characterised by a band of circumferential pigmentary degeneration, retinal opacities, vitreous and vascular abnormalities, choroidal atrophy, neovascularisation, a reduced EOG light rise, and, in some instances nanophthalmos. ADVIRC is one of a number of clinically distinct diseases caused by mutations in the retinal pigment epithelium (RPE) protein, Bestrophin1 (BEST1). Previous studies, which have utilised in vitro splicing assays with minigene constructs, have suggested that the distinct ADVIRC phenotype may be a result of alternate splicing of the BEST1 protein. We have isolated fibroblast cells from a family diagnosed with ADVIRC expressing a heterozygous mutation in BEST1, p.V235A. This mutation has been shown to affect pre-mRNA splicing using in vitro splicing assays in non-RPE cells. We have used induced pluripotent stem cell (iPSC) technology to investigate the effects of this mutation in iPSC-RPE derived from ADVIRC patients.
Fibroblast cells isolated from the patient were reprogrammed using episomal vectors. iPSCs were encouraged to differentiate towards RPE cell lineage by removal of basic Fibroblast Growth Factor (FGF) from the culture medium. Pigmented foci were isolated, dissociated and seeded to form a monolayer of RPE. Patient iPSC-RPE were assessed by PCR, immunocytochemistry, and Western blot.
iPSCs from ADVIRC patients were pluripotent and readily formed pigmented foci approximately 5 weeks following bFGF removal. Foci were manually dissected, dissociated and seeded to form an RPE monolayer. ADVIRC iPSC-RPE expressed a panel of RPE cell markers, including BEST1, at the RNA and protein level. We found no evidence of alternate splicing of BEST1 as a result of the pV235A mutation, however, immunocytochemical analysis suggests that BEST1 is mislocalised within ADVIRC patient-derived iPSC-RPE.
These data suggest the distinct ADVIRC phenotype may be a result of mislocalisation of BEST1 protein in the RPE, and suggest that iPSC technology is a relevant platform with which to assess the affects of RPE cell protein mutations and their specific contributions to eye disease.
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