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Tao Xu, Shao-Bin Wang, Ron A Adelman, Shaomin Peng, Lawrence J Rizzolo; Disease-causing mutations of CLDN19 alter gene expression in retinal pigment epithelia (RPE) and diminish visual function. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3591. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations of CLDN16 or CLDN19 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), but only CLDN19 mutations cause visual impairment. To explore mechanisms whereby claudin-19 mutations might affect on ocular function, naturally occurring mutations were expressed in mice and in human RPE cultures.
Claudin-19 mutations, G20D and R81W, were constructed by site directed mutagenesis. Wild type and mutant CLDN19 plasmids were transiently expressed human fetal RPE (hfRPE) and ARPE-19. AAV viral vectors encoding wide type and mutant CLDN19 were injected in to the subretinal space of 1-month old C57/BL6 mice. The localization of claudins was visualized with immunofluorescent staining. Protein degradation pathways were inhibited and the effects on steady-state levels of claudin-19 were measured with immunoblots. The expression of RPE signature genes was evaluated with Quantitative-PCR. For functional studies, multifocal ERG (mfERG) recordings were obtained, 2-weeks post-transfection.
Two-weeks after transfection, immunostaining of mouse RPE cells revealed wild-type Claudin-19 mainly localized to the cell membrane, but G20D and R81W mutations were distributed in distinct cytoplasmic locations. The mislocalized protein aggregates were ubiquitin positive and their degradation was retarded by inhibitors of proteasomes. Mice injected with wide type CLDN19 show normal mfERG recordings (P1 wave amplitude = 1.1±0.3 μV). In contrast, mice transduced with mutant CLDN19 showed impaired light response (P1 wave amplitude, G20D = 0.48±0.25 μV, and R81W = 0.61±0.26 μV). In primary cultures of human fetal RPE, overexpression of wide type CLDN19 upregulated melansome-associated genes MITF, PMEL and TYR, as well as a tight junction related gene, MMP2. Expression of the G20D and R81W mutations dramatically downregulated MITF, PMEL, TYR, MMP2 and PEDF.
FHHNC mutations of claudin-19 disrupt the trafficking of claudin-19 and adversely affect the expression of genes for melanogenesis. Claudin-19 mutants impaired the light response measured by mfERG.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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