Identification Of The Causative Genes For Three Chinese Families With Autosomal Dominant Retinitis Pigmentosa

Background: Retinitis pigmentosa is a heterogeneous group of inherited retinal diseases caused by the loss of photoreceptors,which is characterized by night blindness,progressive loss of peripheral vision,and complete loss of vision in the end stage.RP is the leading cause of blindness.The prevalence of RP is approximately 1 in 3,500 individuals.To date 70 causative genes for retinitis pigmentosa have been identified,which account for about 60-70% of all cases.New causative genes for RP still remain to be discovered.Subjects and methods:Three Chinese families with autosomal dominant retinitis pigmentosa were selected to identify causative genes using high-throughput sequencing technologies.The experiments were performed to study the function of candidate causative genes in vitro.The sequencing data were annotated and filtered against dbSNP138,1000 Genomes Project,Ex AC database.Sanger sequencing was performed to analysis whether any of the candidate variants cosegregated with the disease phenotype in the family.The wild type and mutant type plasmid were constructed.Cultured cells were transfected with WT and MUT plasmid.WB and ICC were performed to analysis expression and localization of protein in cultured cells.A Whole-genome linkage analysis was performed to identify the linkage interval using Humanmoni Chinese chip.In addition,Copy number variations(CNV)were detected by low-depth whole genome sequencing.Results:(1)Five novel heterozygous variants,located in ABCA4,TULP1,PRPF31,PRPH2,were discovered by whole-exome sequencing in the proband of RP pedigree 1.A splicing mutation in PRPF31(c.855+5G>A)cosegregated with the disease phenotype in the family by Sanger sequencing.Total RNA was isolated from blood of affected and unaffected cases and RT-PCR was performed to amplify the full cDNA of PRPF31.1.5-kb and 1.1-kb amplicons were observed in the RT-PCR product from affected case.However,only 1.5-kb amplicons were observed in the RT-PCR product from unaffected cases.Sequencing results of the 1.1-kb amplicons indicated loss of 409 nucleotides(c.996_c.1406),which located in the exons 10-14 of PRPF31.The PRPF31 localized in nucleus of Cos7 cells transfected with wild type PRPF31-pEGFP-N1 plasmid.However,the PRPF31 localized in cytoplasm of Cos7 cells transfected with mutation type PRPF31-pEGFP-N1 plasmid.(2)Seven candidate variants were identified by whole-exome sequencing and data analysisin the RP pedigree 2.A heterozygous mutation in ADAM15,c.713G>A(p.R238H),cosegregated with the disease phenotype in the family by Sanger sequencing.Compared with 239 T cells transfected with wild type ADAM15 plasmid,levels of ADAM15 were lower in 239 T cells transfected with mutation type ADAM15 plasmid(P=0.0298).Immunofluorescence assays show that mutation ADAM15 localized in cytomembrane,cytoplasm,and perinuclear of Cos7 cells as the same as wild ADAM15.(3)Thirty candidate variants were identified by whole-exome sequencing and data analysis in the RP pedigree 3.Seven variants cosegregated with the disease phenotype in the family by Sanger sequencing,which located in ENO4,FBXO18,NPBWR2,SLC7A11,UNC79,ZGPAT,LAMA5.Genetic linkage analysis shew that no linkage interval in this family was defined(LOD score<2.0).A total of 194 CNVs were detected by low-depth whole genome sequencing in the proband of the RP pedigree 3,whose sizes are 4.5-35.6kb.According to factors influencing the risk assessment of a CNV,a total of 13 CNVs were selected for candidate pathogenic CNVs,including eleven single-copy deletion CNVs and two duplication CNVs.Conclusion:(1)A heterozygous mutation c.855+5G>A in the PRPF31 gene was identified as causative mutation for the RP pedigree 1.The splicing mutation interferes in splicing of PRPF31 mRNA,which lead to a skipping of the exons 10-14 of PRPF31.The mutation decreased the level of PRPF31 and impaired the location of PRPF31 in cells.(2)A heterozygous mutation c.713G>A(p.R238H)in the ADAM15 gene was identified as causative mutation for the RP pedigree 2.The mutation decreased the level of ADAM15.However,no change of the location of ADAM15 in cells was observed.(3)Seven variants cosegregated with the disease phenotype in the RP pedigree 3,which located in ENO4,FBXO18,NPBWR2,SLC7A11,UNC79,ZGPAT,LAMA5.No linkage interval in this family was defined(LOD score<2.0).A total of 13 CNVs were selected for candidate pathogenic CNVs,including eleven single-copy deletion CNVs and two duplication CNVs.