The Genetic Background And Mechanism Of Retinitis Pigmentosa

Retinitis pigmentosa(RP [MIM 268000]),the most common form of inherited retinal dystrophy(IRDs),displays a prevalence ranging from 1/3500 to 1/5000 among different populations.In the disease course of RP,rod photoreceptors will initially be affected followed by degeneration of cone photoreceptors and retinal pigment epithelium(RPE).RP exhibits significant genetic heterogeneity involving at least 63disease-causing genes(RetNet),among which 22 genes have been implicated in autosomal dominant RP(adRP).However,mutations in all identified RP genes can only account for 50% to 60% of all cases,leaving the heritability in nearly 40% of RP patients unknown.With the process of the next gernation screening(NGS)has been exceptionally productive in gene discovery and mutation detection for RP.Broadly,there are three NGS strategies: whole-exome NGS,whole-genome NGS and targeted-capture NGS.So how to explore the more effective and less expersive technology is the argue question.Here in our study,we employed a targeted sequence capture with NGS approach,the linkage mapping and the whole exome sequencing.And we find the two new caused gene of RP-PRPF4 and SPP2.HPrp4,encoded by PRPF4,and the gene we had reported previously SNRNP200 regulate the stability of U4/U6di-snRNP,which is essential for continuous splicing.SPP2 is considered as a member of the cystatin superfamily.However,the mechanism of these RP-caused genes is still underdetermined.In this study,we detected the mutations on the physiologic function of cellularand the morphology of the zebrafish model.Further,we investigated the probable mechanism of these mutations.For PRPF4,we found that the c.-114_-97 del,predicted to affect two transcription factor binding sites,was shown to down-regulate the promoter activity of PRPF4 by a luciferace assay.In fibroblasts from an affected individual with the p.Pro315 Leu variant,the expression levels of several tri-snRNP components,including PRPF4 itself,were up-regulated,with altered expression pattern of SC35,a spliceosome marker.The same alterations were also observed in cells over expressing hPrp4Pro315 Leu,suggesting that they arise as a compensatory response to a compromised splicing mechanism caused by hPrp4 dysfunction.Further,over expression of hPrp4Pro315 Leu,but not hPrp4 WT,triggered systemic deformities in wild-type zebrafish embryos with the retina primarily affected,and dramatically augmented death rates in morphant embryos,in which orthologous zebrafish prpf4 gene was silenced.We conclude that mutations of PRPF4 cause RP via haploinsufficiency and dominant-negative effects,and establish PRPF4 as a new U4/U6-U5 snRNP component associated with adRP.For SNRNP200,we found that Snrnp200 was ubiquitously expressed in zebrafish.Knocking down Snrnp200 in zebrafish triggered aberrant splicing of the cbln1 gene,up-regulation of other U4/U6-U5 tri-snRNP components,and down-regulation of a panel of retina-specific transcripts.Systemic defects were found correlated with knockdown of Snrnp200 in zebrafish.Only demorphogenesis of rod photoreceptors was detected in the initial stage,mimicking the disease characteristics of RP.For the SPP2,we found we identified a putative pathogenic variant,p.Gly97 Arg,in the gene SPP2,of which expression was detected in multiple tissues including retina.The p.Gly97 Argwas absent in 800 ethnically matchedchromosomes and 1400 in-house exome dataset,and was located in the first of the two highly conserved disulfide bonded loop of secreted phosphoprotein 2(Spp-24)encoded by SPP2.Overexpression of p.Gly97 Arg and another signal peptide mutation,p.Gly29 Asp,caused cellular retention of the exogenous mutants,ER stress in vitro,and primarily affected rod photoreceptors in zebrafish mimicking cardinal feature of RP.Screening for SPP2 mutations in RP patients with unknown genetic causes are also essential to gain better insights into the disease mechanism and genotype-phenotype correlations.