RPGR isoform imbalance causes ciliary defects due to exon ORF15 mutations in X-linked retinitis pigmentosa (XLRP)

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGR(ex1-19) and RPGR(ORF15), are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGR(ORF15) messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGR(ORF15) mRNA also exhibited significantly higher levels of RPGR(ex1-19) mRNA. Four samples with unaltered RPGR(ex1-19) levels carried mutations in RPGR(ORF15) that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGR(ex1-19)/RPGR(ORF15) isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGR(ex1-19) (mimicking the increase in RPGR(ex1-19) to RPGR(ORF15) isoform ratio) or RPGR(ORF15) (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGR(ORF15) protein and to the higher levels of the RPGR(ex1-19) isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGR(ex1-9) to RPGR(ORF15) ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGR(ex1-19)/RPGR(ORF15) ratio may lead to better therapeutic outcomes.