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Suresh B. Patil, Toby W. Hurd, Amiya K. Ghosh, Carlos A. Murga-Zamalloa, Hemant Khanna; Variable Effect Of Human Disease Causing Mutations In X-linked RP-associated Ciliary Protein RP2 On Its Function; Insights Into Associated Clinical Heterogeneity. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1831.
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Mutations in RP2, a ciliary protein, are associated with 8-10% of X-linked RP, which is a clinically and genetically heterogenic form of severe retinal degeneration. We carried out this study to delineate the mechanism by which mutations in Retinitis Pigmentosa 2 (RP2) result in photoreceptor degeneration and variable disease severity.
We performed immunofluorescence analysis of RP2 in cryosections of mouse and zebrafish retina using an anti-RP2 antibody. Morpholino (MO)-mediated suppression of rp2 in zebrafish embryos was performed using previously described procedures. To rescue the MO phenotype, we co-injected the mRNA encoding human RP2 (wild-type or mutant) along with rp2-MO into 2-cell stage embryos. The embryos were analyzed at 3-5 days post fertilization for retinal degeneration, using histology, immunogold and immunofluorescence microscopy.
We found that in mouse and zebrafish photoreceptors, RP2 primarily localizes to inner segment and base of the connecting cilium. Depletion of rp2 in zebrafish results in tail extension phenotype and microphthalmia. Further analysis revealed perturbations in photoreceptor development as evidenced by histological analysis and depletion of cone (Zpr1) and rod (Zpr3) photoreceptor staining. Injection of mRNA encoding wild type and some human disease-causing mutant proteins, such as R118H, E138G and L188P partly rescued rp2-knockdown phenotypes, indicating a hypomorphic effect of the mutation on its function. On the other hand, RP2 mutants C86Y, P95L and R282W were not able to efficiently rescue the abnormal tail defect and photoreceptor development phenotype compared to wild-type RP2, suggesting a loss of RP2 function due to these mutations.
Our results indicate that RP2 protein plays a key role in photoreceptor development and maintenance and that the clinical heterogeneity associated with RP2 mutations may, in part, result from the differential effects of the mutations on its function(s).
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