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Wen-Tao Deng, Ping Zhu, Frank M Dyka, Wolfgang Baehr, William W Hauswirth; Disease mechanisms of X-linked cone dystrophy caused by missense mutations in the red and green cone opsins. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1554.
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© ARVO (1962-2015); The Authors (2016-present)
Red/green cone opsin missense mutations N94K, W177R, P307L, R330Q and G338E have been identified in subjects with congenital blue cone monochromacy or color-vision deficiency. Studies on disease mechanisms due to these cone opsin mutations have been previously carried out exclusively in cell culture. Here we expressed these cone opsin mutants specifically in mouse cones via AAV in M-opsin knockout (Opn1mw-/-) mice to investigate their subcellular localization, the pathogenic effects on cone morphology, function, and cone viability.
AAV8-733 vectors expressing the above cone opsin mutants driven by the cone-specific PR2.1 promoter were injected subretinally into one eye of 1 month old Opn1mw-/- mice, while the contralateral eyes remained untreated. AAV-mediated transgene expression was analyzed by western blots. L/M-opsin mediated retinal function was analyzed 6 weeks post-injection by photopic electroretinography (ERG). Subcellular localizations of each cone opsin mutant and the other cone outer segment specific proteins were examined by immunohistochemistry. Cone viability in each mutant treated eyes was examined by retinal whole mounts stained with peanut agglutinin (PNA).
Expression of N94K and W177R was barely detectible while P307L, R330Q, and G338E showed expression levels comparable to that of endogenous M-opsin. N94K and W177R were misfolded and localized exclusively in cone inner segments and endoplasmic reticulum. In contrast, P307L, R330Q and G338E were detected exclusively in cone outer segments. Expression of R330Q and G338E, but not P307L, also partially restored expression and correct localization of the remaining cone outer segment proteins and resulted in partial rescue of M-cone mediated light responses. ERG amplitudes in R330Q and G338E injected eyes were 54.5 ± 15.5 µV and 56.3 ± 18.1 µV, respectively (average ± STD, n=8), significantly higher than the unrecordable M-cone ERGs from untreated contralateral control eyes (P < 0.0001). Expression of W117R and P307L caused severe cell toxicity resulting in rapid cone loss, while N94K, R330Q and G338E were only modestly toxic.
Although the underlying biochemical and cellular defects caused by these mutants are distinct, we predict they all present a gain-of-function phenotype, resembling autosomal dominant retinitis pigmentosa associated with the majority of rhodopsin missense mutations.
This is a 2021 ARVO Annual Meeting abstract.
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