Molecular Genetics Analysis And Pathogenesis Study Of Hereditary Retinal Diseases

1.Molecular Genetics Analysis of Hereditary Retinal DiseasesHereditary retinal diseases(HRDs)is a group of blindness diseases,which are common and clinically characterized by severly loss of vision and visual field.HRDs manifest with significant clinical heterogeneity,as variaties in the same disease,similarities and crossabilities across diferrent diseases,which bring some challenges to clinical diagnosis.Moreover,the disease is very heterogeneous genetically.By now,a total of 256 HRDs causative genes and 293 linkage loci have been revealed(www.RetNet.org)over the word.By far,no effective treatment has been developed for HRDs,and gene therapy is a research hotspot.Therefore,a clear molecular genetics diagnosis for patients with HRDs is the premise of radical treatment,has a very important practical significance.In this part of our study,we aim to investigate the molecular genetics causes for HRDs patients.(1).Four participants from a three-generation Chinese family with autosomal dominant familial exudative vitreoretinopathy(FEVR)was recruited.All coding and exon-intronic boundary regions of five targeted genes,including NDP,FZD4,LRP5,TSPAN12 and ZNF408 were amplified with polymerase chain reaction(PCR)and sequenced using Sanger sequencing to detect potential variants.Genetic investigations revealed FZD4 p.E160K as the disease-causing mutation for this family.Crystal structural analysis also indicated that this mutation could lead to the elimination of the hydrogen bond between residue 160 and asparagine at residue 152,thus altering the tertiary structure of the protein and further impairing the protein function.Our study demonstrates FZD4 p.E160K as a novel pathogenic mutation for FEVR.(2).We collected two families with autosomal dominant optic atrophy(ADOA).Targeted sequence capture microarrays that could capture the HRDs causative genes and loci were designed.We applied the targeted next-generation sequencing(NGS)approach to screen the disease-causing mutations for the two families.Two novel mutations in the OPA1 gene,p.Y305C and p.L65V were revealed as the disease-causing mutations for the two families respectively,after a series of screening and filtration,intrafamilal cosegregation analysis and bioinformatics analysis.Our results have helped with better clinical management and expanded the genetic spectrums for HRD.Taken together,both direct sequencing of targeted genes and targeted NGSapproach could offer rapid,efficiency and accuracy mutation screening in HRDs patients,so as to assist the clinical diagnosis,lay the foundation for the prenatal diagnosis and provide an important theoretical basis for follow-up gene replacement therapy.2.Study of Gene-Targeted Mice Model of SNRNP200 p.S1090L Implicated in Human Autosomal Dominant Retinitis Pigmentosa(RP)Retinitis Pigmentosa(RP)is one of the most common hereditary blindness leading disorders,characterized by degeneration of photoreceptor cells and retinal pigment epithelium(RPE).Night blindness and reduced visual field were the typical clinical features,accompanied with osteoid-like changes in the retinal pigment.RP,as a monogenic disease,it can be divided into four types:autosomal dominant retinis pigmentosa(ADRP),autosomal recessive retinis pigmentosa(ARRP)and X-linked retinis pigmentosa(XRRP)accorsing to the inheritance patterns.As reported,mutations in genes encoding components of the U4/U6-U5 triple small nuclear ribonucleoprotein(tri-snRNP)are disease causative for autosomal dominant RP(ADRP),including PRPF3,PRPF4,PRPF6,PRPF8,PRPF31,and SNRNP200.Protein encoded by the SNRNP200 gene,an component of the U5 snRNP plays a important role in the unwinding of the U4/U6 small nuclear RNA(snRNA).Previous studies of our and other research groups have confirmed that SNRNP200 p.S1090L is a pathogenic hotspot mutation of RP.To investigate the pathogenicity of SNRNP200 gene mutation,we genetated a SNRNP200 p.S1090L mice by a genomic engineering tool of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)system.Our present results revealed that protein encoded by the mutant allel was decaded,leading to the embryonic lethal in homozygous knockin mice(SNRNP200p’R1090L/p.R1090L).Half dosage of protein snrnp200 is sufficient for the maintenance of the development and visual function of mouse retina.Taken together,the preliminary study confirmed that haploinsufficiency is not the pathogenesis of the disease.