Abstract
Purpose :
Mutations in RPGR (retinitis pigmentosa GTPase regulator) are a leading cause of retinitis pigmentosa (RP) and the most frequent cause of X-linked RP. There are two major RPGR isoforms: RPGRex1-18 (full length with 18 exons) and RPGRORF15 (exons 1-15 with part of intron 15 as the terminal exon and the UTR). Mutations in exon ORF15 account for >70% of XLRP cases, making it a a mutational hotspot in humans. However, the effect of loss of this domain is unclear. The purpose of this study is to understand the precise role of these RPGR isoforms in photoreceptors.
Methods :
Using a CRISPR/Cas9 strategy, we modeled a human RPGR disease-causing mutation in C57BL/6J mice by engineering a premature stop codon upstream of the mutational hotspot exon ORF15 (RpgrORF15-ko). We also specifically ablated the Rpgex1-18 isoform (Rpgrex1-18-ko) by introducing a frameshift mutation in exon 18. The 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 using OCT (optical coherence tomography) and fundus examinations. Retinal sections were examined by histology, transmission electron microscopy and immunofluorescence.
Results :
Injected founders were genotyped and germline transmission was confirmed in F1 mice. Mice were backcrossed to C57BL/6J for 5 generations to exclude off-target effects of the CRISPR/Cas9 targeting. ERG analysis of the RpgrORF15-ko mice revealed early and progressive loss of both rod and cone function starting at ~7 weeks of age. Consistently, the mutant mice showed early cone opsin and rhodopsin mislocalization and degeneration of the photoreceptor outer segments. The Rpgrex1-18-ko mice showed germline transmission of the mutation and are being analyzed using a similar strategy.
Conclusions :
Our studies assess a mouse model of Rpgr mutation that mimics human mutations that disrupt the mutational hotspot exon ORF15. In addition, our studies will assess, for the first time, the involvement of the Rpgrex1-18 isoform in retinal survival and function. These studies provide new knowledge about the role of RPGR isoforms in regulating photoreceptor function and associated pathogenesis.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.