Molecular Diagnosis And Mechanistic Study Of Hereditary Retinal Degeneration

1.Molecular diagnosis of hereditary retinal degenerationHereditary retinal degeneration(HRD)is a group of diverse retinal degenerative diseases caused by genetic mutations and is clinically characterized by vision loss and primary retinal degeneration.HRD is becoming one of the most common and severest eye diseases that cause blindness.It's also a primary cause for refractory blindness.By far,no effective and applicable treatment has been developed for HRD.Gene therapy is turning into a research hotspot for HRD treatments,while its fundamental basis is precise genetic diagnosis.HRD,as a group of monogenic diseases,presents significant genetic heterogeneities.A total of 240 HRD causative genes have been revealed,and the number of mapped loci is even higher,which has reached 279.Thus,developing a novel and economic diagnostic platform with high efficiency,accuracy and feasibility will help with the molecular diagnosis of HRD.In this part of our study,we aim to investigate the molecular disease causes for HRD patients.Herein,we have developed an effective genetic diagnostic tool for HRD based on targeted next-generation sequencing(NGS)approach.We have further applied this approach in the investigation of a cohort of multiple Chinese families with HRD,and have identified the disease-causing mutations in different types of HRD.Our results have expanded the genetic and clinical spectrums for HRD.Novel genotype-phenotype correlations have been defined.We have,for the first time,revealed USH2A and EYS mutations in patients with retinitis pigmentosa sino pigmento(RPSP),identified the association between MYO7A mutations and Usher syndrome type 2,and found LCA5 mutations in patients with cone dystrophy.Taken together,by means of targeted NGS approach,we can perform mutation screening in HRD patients with high efficiency and accuracy.2.Identification of PRPF4 as a novel disease causative gene for retinitis pigmentosa,and mechanistic study of precursor messenger RNA splicing genes,deficiencyRP shows progressive loss of photoreceptors with heterogeneous genetic background.Reportedly,mutations in genes encoding components of the U4/U6-U5 triple small nuclear ribonucleoprotein(tri-snRNP)are disease causative for autosomal dominant RP(ADRP),which plays crucial roles in precursor messenger RNA(pre-mRNA)splicing.Known RP-causing splicing genes include PRPF3,PRPF6,PRPF8,PRPF31,and SNRNP200.Brr2,protein encoded by the SNRNP200 gene,is an important component of the U5 snRNP and functions in the unwinding of the U4/U6 small nuclear RNA(snRNA).Prp4,protein encoded by the PRPF4 gene,is a crucial factor for the U4/U6 di-snRNP and modulates its stability,while mutations in the PRPF4 gene have never been found in HRD patients.In the second part of our study,we aim to study the pathogenesis of RP caused by defects in PRPF4 and SNRNP200 genes.We have,for the first time,identified the association between PRPF4 mutations and RP,and thus recognized PRPF4 gene as a novel RP causing gene.Two mutations,PRPF4 c.-114_-97del and p.Pro315Leu,are found as disease causative in a sporadic RP patient and an ADRP family,respectively.Pathogenicity of the two mutations is further confirmed by functional studies in cellular and zebrafish models.We have found that PRPF4 c.-114_-97del and p.Pro315Leu could cause RP via haploinsufficiency and dominant negative,respectively.Furthermore,two recurrent SNRNP200 mutations are further revealed as disease causative in two separate RP families.Retinal phenotypes and splicing abnormalities caused by SNRNP200 deficiency are studied to illustrate its pathogenesis.3.Identification of SPP2 as a novel disease causative gene for retinitis pigmentosa and analysis of it pathogenesisRP shows progressive loss of photoreceptors with heterogeneous genetic background.Whole-exome sequencing(WES)captures and sequences all exons of 18134 human genes.This region covers all the important information required by protein synthesis together with most functional variations.WES has tremendous advantages,and is regarded as an efficient sequencing technology with high specificity and coverage.Herein,by using WES in a Chinese family with ADRP,we have identified a putative pathogenic variant,p.Gly97Arg,in the SPP2 gene,whose expression is detected in multiple tissues including retina.The p.Gly97Arg variant is absent in 800 ethnically matched chromosomes and 1400 in-house exome dataset,and is located in the first of the two highly conserved disulfide bonded loop of secreted phosphoprotein 2(Spp-24)encoded by SPP2.Overexpression of p.Gly97Arg and another signal peptide mutation,p.Gly29Asp,cause cellular retention of both endogenous wild type and exogenous mutants in vitro,and primarily affect rod photoreceptors in zebrafish mimicking cardinal feature of RP.Taken together,our data indicate that the two mutations of SPP2 have dominant negative effects and cellular accumulation of Spp-24 may be particularly toxic to photoreceptors and/or retinal pigment epithelium.SPP2 has a new role in retinal degeneration.Conclusions:Herein,by means of multiple genetic and mechanistic tests,we have studied the genetic causes and pathogenesis for HRD.We have modified genetic diagnosis for HRD patients,assisted their clinical diagnosis,revealed novel genotype-phenotype correlations for HRD,identified new HRD causative genes,and defined their relevant pathogenesis.