Novel missense mutation E585K in retinitis pigmentosa leads to compromisedRPGRsplicing diversity

Abstract

23Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, are the major cause of 24 X-linked retinitis pigmentosa (RP). Herein we used whole-exome sequencing to screen 25 possible novel RPGR mutations in RP patients, and identified a novel missense mutation 26 E585K in a patient with early onset but slow disease progression, and a frameshift deletion 27 E998Gfs*78 in a patient with RP sine pigmento and high myopia. Intriguingly, bioinformatic 28 analysis indicated that E585K probably affected RPGR RNA splicing instead of the protein 29 sequence directly. Mini-gene assays in 293T cells revealed that splicing events of the E585K 30 mutant were found to be also exist in wildtype, but with a shifted pattern. In the E585K mini-31 gene usage of an upstream alternative 5′ splice site (5′ ss) of exon 14 was enhanced, and other 32 splicing events were suppressed, including the canonical 5′ ss of exon 14, skipping of exon 33 14/15 and retention of intron 14. As a result, RPGR splicing products of the E585K mini-gene 34 were predominated by transcripts containing a 4-bp deletion, with a small fraction of in-frame 35 transcripts containing a retended intron 14, which might explain the slow disease progression 36 in the patient carrying the mutation. RNA-Seq analysis further confirmed existence of these 37 splicing events in endogenous RPGR RNA in human retina, pointing to compromised splicing 38 diversity in the E585K mutant. Our findings thus added to the understanding of genotype-39 phenotype correlation in RP, and suggested that compromised RPGR splicing diversity might 40 play a role in molecular mechanism of the disease. 41