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Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, are the major cause of X-linked retinitis pigmentosa (RP), in which exon open reading frame 15 (ORF15) of RPGR has been implicated to play a substantial role. We identified a novel hemizygous missense mutation E585K of RPGR from whole-exome sequencing of RP. RNA-Seq analysis and functional study were conducted to investigate the underlying pathogenic mechanism of the mutation. Our results showed that the mutation actually affected RPGR ORF15 splicing. RNA-Seq analysis of the human retina followed by validation in cells revealed a complex splicing pattern near the 3′ boundary of RPGR exon 14 in the ORF15 region, resulting from a variety of alternative splicing events (ASEs). The wildtype RPGR mini-gene expressed in human 293T cells confirmed these ASEs in vitro. In contrast, without new RNA species detected, the mutant mini-gene disrupted the splicing pattern of the ORF15 region, and caused loss of RPGR transcript heterogeneity. The RNA species derived from the mutant mini-gene were predominated by a minor out-of-frame transcript that was also observed in wildtype RPGR, resulting from an upstream alternative 5′ splice site in exon 14. Our findings therefore provide insights into the influence of RPGR exonic mutations on alternative splicing of the ORF15 region, and the underlying molecular mechanism of RP.

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, are the major cause of X-linked retinitis pigmentosa (RP). Herein we used whole-exome sequencing to screen possible novel RPGR mutations in RP patients, and identified a novel missense mutation E585K in a patient with early onset but slow disease progression, and a frameshift deletion E998Gfs*78 in a patient with RP sine pigmento and high myopia. Intriguingly, bioinformatic analysis indicated that E585K probably affected RPGR RNA splicing instead of the protein sequence directly. Mini-gene assays in 293T cells revealed that splicing events of the E585K mutant were found to be also exist in wildtype, but with a shifted pattern. In the E585K mini-gene usage of an upstream alternative 5′ splice site (5′ ss) of exon 14 was enhanced, and other splicing events were suppressed, including the canonical 5′ ss of exon 14, skipping of exon 14/15 and retention of intron 14. As a result, RPGR splicing products of the E585K mini-gene were predominated by transcripts containing a 4-bp deletion, with a small fraction of in-frame transcripts containing a retended intron 14, which might explain the slow disease progression in the patient carrying the mutation. RNA-Seq analysis further confirmed existence of these splicing events in endogenous RPGR RNA in human retina, pointing to compromised splicing diversity in the E585K mutant. Our findings thus added to the understanding of genotype-phenotype correlation in RP, and suggested that compromised RPGR splicing diversity might play a role in molecular mechanism of the disease.