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R. Koenekoop, I. Lopez, G.A. Fishman, S. Yzer, M.A. Musarella, J. Racine, A. Dorfman, P. Lachapelle, F.P. M. Cremers, R. Allikmets; Novel Expression of RPGRIP1 and Functional Analysis of Heterozygous Nonsense Mutations . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1705.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: RPGRIP1 encodes the retinitis pigmentosa GTPase interacting protein which is involved in anchoring the ciliary axoneme in outer segments of both photoreceptors, and recessive mutations cause Leber congenital amaurosis (LCA). To gain further insight into the functional deficits of RPGRIP1, we tested photopic hill responses of heterozygotes (htzs) with known mutations. We also developed a technique to express RPGRIP1 in peripheral white cells, to test the hypothesis that the htz phenotype is caused by a dominant negative mechanism. Methods: Photopic hill testing was done. Quantitative RT–PCR was performed to determine the expression levels of RPGRIP1 in normal controls, affecteds and carriers. Total RNA was extracted from peripheral human blood using PAXgene Blood RNA Validation Kit (Qiagen). Quantitative, real–time RT–PCR was performed using the QuantiTect SYBR Green RT–PCR Kit (Qiagen). Results: We found p.Arg768Ter and p.Gln483Ter mutations in LCA patients and their parents, predicting RPGRIP1 proteins with differently sized carboxy–terminal truncations. We expected the dysfunctions to be similar as both are presumed null alleles. The p.Arg768Ter htz had a photopic hill significantly shifted to the right, while the p. Gln483Ter htz had a completely different and abnormal photopic hill. We successfully amplified the expected RPGRIP1 mRNA fragment from peripheral blood and will test whether the truncated mRNA fragments act in a dominant negative manner. Conclusions: The htzs harboring p.Gln483Ter and p.Arg768Ter mutations have different phenotypes by photopic hill analysis. The dysfunction of the p.Gln483Ter htz appears to be considerably more severe. Differences between these nonsense mutations would not be expected if both are subject to nonsense mediated decay. RPGRIP1 mRNA can be found in peripheral blood allowing us to test the impact of mutations at the mRNA level and test if the truncated alleles act dominant negatively on the wild–type. Photopic hill analysis provides a functional assay of the effects of recessive RPGRIP1 mutations in heterozygous carriers whom do not exhibit overt retinal disease. Also, this relatively simple clinical test on parents with blind children may direct and fascilitate subsequent molecular diagnostics.
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