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Wei Zhang, Lijing li, Ramesh Periasamy, Laura Moreno Leon, Manisha Anand, Michael Brodsky, Hemant Khanna; Isoform-specific Rpgrmutant mice depict distinct roles of the RPGR isoforms in photoreceptors. Invest. Ophthalmol. Vis. Sci. 2019;60(9):462. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
X-linked retinitis pigmentosa (XLRP) is the most serious type Retinitis pigmentosa (RP). Mutations in RP GTPase regulator (RPGR) are due to most of patients with XLRP. RPGR has two major isoforms: RPGRconst(all 19 exons) and RPGRORF15(exons 1-15 + part of intron 15) in human. There is considerable phenotypic heterogeneity in RPGR patients, which can be attributed to the distinct effects of the mutations on specific isoforms. The purpose of this study is to understand the precise role of the major RPGR isoforms in photoreceptors.
Using CRISPR/Cas9 strategy, we introduced a human RPGR disease-causing mutation in C57BL/6J mice, which results in a premature stop codon upstream of mouse RPGR exon ORF14/15(RpgrORF14/15-ko). We also specifically ablated the Rpgrconstisoform (Rpgrconst-ko) by introducing a frame-shift mutation in exon 18 using CRISPR/Cas9 strategy. RNA and protein expression from the mutant gene was confirmed by RT-PCR and immunoblotting. Photoreceptor function was determined by electroretinography (ERG) and retinal morphology was examined by histology, transmission electron microscopy and immunofluorescence. Localization of RPGR and its partner proteins were detected on non-fixed retina and dissociated photoreceptor cells.
The founder mice were backcrossed to C57BL/6J for 5 generations to exclude off-target effects of the CRISPR/Cas9 targeting. In addition to earlier onset rod and cone dysfunction (~7 weeks of age), mis-localization of opsins and degeneration of the photoreceptor outer segments, the RpgrORF14/15-ko retinas did not express glutamylated RPGR although the polyglutamylation of the axoneme was unaltered. The Rpgrconst-komice, on the other hand, exhibited delayed onset cone dysfunction (~5 months of age) and mislocalization of opsins. TEM analysis revealed minor disruption of the outer segment disc arrangement.
Our results suggest that dysfunction of distinct RPGR isoforms can manifest as an earlier or a delayed onset photoreceptor degeneration, and open new avenues of research to delineate the mode of clinical variability observed in patients with RPGR mutations.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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