The Application Of 189-Target Capture Chip In Hereditary Retinal Diseases

Part Ⅰ The Establishment of Genetic Diagnostic Platform for Hereditary Retinal Diseases in Chinese PatientsPurpose This study aims to develop an efficient molecular diagnostic platform for hereditary retinal diseases（HRDs）,and to determine the genetic basis for 26 randomly collected Chinese families with a variety of HRDs.Methods Regular ocular examination was performed on the all participants in the cohort of Chinese families.We designed a high-throughput sequence capture microarray targeting 179 genes associated with HRDs and 10 candidate splice genes.We combined sequence capture with next-generation sequencing（NGS）to screen for mutations in the cohort of Chinese families.Variants detected by NGS were filtered,validated and prioritized by pathogenicity analysis.Genotypes and phenotypes were correlated.Results We identified 4 recurrent single mutations,2 compound mutations and 9 novel putative causative mutations including 5 putative pathogenic alleles（e.g.premature stop codons and frameshifts）and 4 novel missense variants that are very likely pathogenic.These findings provided specific genetic diagnoses in 15 of 26 families（57.7%）.Among these,identification of a mutation in VCAN in a family with a complicated phenotype helped to finalize the clinical diagnosis as Wagner syndrome.In another 5 families,11 potential novel pathogenic variants were identified.Conclusions A substantial number of potential new genes and new mutations associated with HRDs remain to be discovered.Identification of the novel HRDs-causing mutations in our study not only provide a better understanding of genotype-phenotype relationships in these diseases,but also demonstrate that the approach described herein is an effective method for large-scale mutation detection among diverse and complicated HRDs cases.Part Ⅱ Detection and Analysis of the Rhodopsin Gene in Autosomal Dominant Retinitis PigmentosaPurpose To investigate cytological mechanism for two consanguineous Chinese Han autosomal dominant retinitis pigmentosa（adRP）families caused by two missense mutations in rhodopsin（RHO）gene.Methods The wild-type RHO^WT,two mutants RHO^L95P and RHO^P53R were cloned into the eukaryotic plasmid vector pEGFP-N1 which is combined with green fluorescent protein（GFP）by the overlapping PCR method.Then the three plasmids were transfected into the human retinal pigment epithelial cell line（ARPE 19）and the human embryonic kidney 293 cell line（HEK 293）to observe the cellular localization of RHO-GFP fusion protein.And the expression of X box binding protein 1（XBP1）was detected by western blot,whcih is the critical factor of endoplasmic reticulum stress（ERS）.Results Wild type RHO-GFP fusion protein showed green fluorescence and distributed in endoplasmic reticulum（red fluorescence）and cell membrane,and both of two mutants RHO-GFP fusion protein were accumulated in the endoplasmic reticulum of the ARPE 19 cells.Compared with the wild type RHO^WT.empty plasmid pEGFP-Nl and untransfected cells,the HEK 293 cells transfected with RHO^L95P and RHO^P53R mutants were expressing mild upregulated XBP1.Furthermore,XBP1 was activted by specially spliced 26 bp intron in mRNA level in two mutants.Conclusions Heterozygous variant RHO p.L95P and RHO p.P53R are belong to the type of II mutation.The mutant rhodopsin protein in this type can not be delivery effectively from the endoplasmic reticulum to the cell membrane,and accumulated in the endoplasmic reticulum,causing ERS.Part Ⅲ Homozygous Mapping in a Chinese Han Hereditary Retinal Diseases Family with Complex Ocular PhenotypesPurpose To determine the genetic basis for a Chinese consanguineous family with complex ocular involvements,and to characterize the genotype-phenotype relationship in the family.Methods The family was clinically characterized by complete ophthalmologic examinations.NGS was performed to screen mutations in 179 genes associated with HRDs and 10 candidate splice genes.Variations detected by NGS were filtered,validated by Sanger sequencing and evaluated for pathogenicity.After a likely pathogenic mutation was found in BEST1 gene,homozygosity mapping on entire Chromosome 11 was performed in the kindred to further evaluate the casualty of the BEST1 mutation and to determine whether any additional gene within the homozygous region could be related to the complex phenotypes observed in the family.Results Targeted sequencing of the 189 genes followed by pathogenicity analysis identified a putative novel homozygous mutation,c.752G>A（p.C251 Y）in BEST1 gene,which fully co-segregated with the consistent phenotypes carried by all patients including autosomal-recessive bestrophinopathy（ARB）-like maculopathy,angle-closure glaucoma（ACG）and cataract.Individuals with heterozygous p.C251Y are clinically normal.p,C251Y affects a conserved amino acid that is located in the fifth transmembrane helix of Bestrophin-1,in which dominant mutations have been previously identified.The homozygous region shared by all patients encompasses BEST1 gene supporting the casualty of the mutation.Sanger sequencing of two additional candidate genes within the homozygous region did not reveal any pathogenic variation.Conclusions Homozygous p.C251Y of BEST,is very likely the causative mutation for the phenotypes observed in the family.The phenotypic consistency among all patients presumably defined a new panel of traits associated with BEST1 homozygous mutations,including ARB,ACG and cataract.The targeted sequencing approach is an effective molecular diagnostic method for complex HRDs cases.