Identification And Molecular Consequence Of A Disease Causing Gene In An Autosomal Dominant Congenital Cataract Family

Congenital cataract is caused by many factors in the process of embryonic development of lens,which is significantly clinical and genetic heterogeneous.Congenital cataract leads to the abnormal appearance and dysfunction.There are various clinical features and pathogenic genes of congenital cataract.Hence,the precise and efficient genetic screening is necessary.Targeted exome sequencing is a rapid and efficient tool for screening mutations in genetic defects.In this study,a targeted exome sequencing screening and bioinformatics analysis were applied in a large Chinese family affected with congenital cataract.Vectors and cell lines were established according to the wild type and mutant sequence of human GJA3.Finally,the molecular mechanisms of GJA3/p.T148I mutation were studied.This novel mutation expanded the spectrum of GJA3 mutations and demonstrated the pathogenic mechanisms of the gap junction channels and apoptosis in congenital cataract.Chapter 1.Genetic screening in a Chinese family affected with congenital cataractObjective:Congenital cataract is inherited in a variety of ways,and autosomal dominant congenital cataract is the commonest one.With the development of molecular genetic technologies,there are seventy pathogenic genes or candidate loci associated with congenital cataract.In this study,a family with the autosomal dominant congenital cataract was recruited in Xiamen,Fujian province,China.Genetic screening was performed to identify the disease causing genes.Methods:The family members were recruited from the ophthalmology center of the First Affiliated Hospital of Fujian Medical University,and they were enrolled from their living area and collected family data.All family members received medical histories,general physical examinations and ophthalmologic examinations.According to family histories and physical examinations,the family pedigree was mapped and genetic analysis was carried out to determine the genetic model.Genomic DNA was isolated from peripheral blood,and high quality genome libraries were established for exons and introns adjacent regions of the autosomal dominant congenital cataract related genes by targeted exome sequencing.Captured and enriched data were used for high-throughput sequencing and analysis.Results:There were four generations in this family and the clinical data of thirty-four family members were collected,which was in line with autosomal dominant congenital cataract.Five patients were affected with congenital cataract and twenty-nine members were unaffected,therein.The proband and the other patients were diagnosed with bilateral pulverulent nuclear cataracts.The quality and sequencing depth of targeted exome sequencing data can be used for further genetic analysis.Conclusions:A large family from Fujian province was recruited which was affected with bilateral pulverulent nuclear cataracts.A targeted exome sequencing analysis which covering 134 related genes of congenital cataract can be used for the detection of disease-causing genes and pathogenesis in this family.Chapter 2.The screening of disease-causing genes, bioinformatic analysis and vectors constructions.Objective:The results of targeted exome sequencing in this family were analyzed,and the pathogenic mutations were verified by Sanger sequencing.The eukaryotic expression vectors of the HLEC and HEK-293 cells were established to explore the molecular mechanisms of mutation pathogenesis.Methods:The high quality data were selected for bioinformatic analysis,and compared with the GRCh37/hg19 human genome sequence.The SNP and InDel were detected by BWA and GATK softwares,clear data were annotated according to the mutation database.The results of Sanger sequencing were used for validation,and DNAMAN software was used to analyze the genetic features.A novel mutation GJA3/p.T148I was revealed in this family,and was carried out to be analyzed by the pathogenicity prediction,hydrophilicity analysis and protein homology modeling.Wild type and mutant vectors expressing Cx46/EGFP and Cx46/Flag were constructed through DNA cloning and recombinant methods.Results:The mutation was located in No.443 base pair of GJA3 gene,which lead to a convertion from cytosine C to thymine T?ACC?ATC?and presented separation in this family.The predicted results of PolyPhen-2,SIFT and Mutation Taster of GJA3/p.T148I indicated that the mutation was pathogenic.The hydrophobicity of the CL domain in GJA3/p.T148I mutantion protein was significantly higher than that in the wild type in Hydrophobic analysis.Results of homologous modeling showed an?-helix deletion in the terminal of C domain.With the human GJA3 as a template,the vectors of pEGFP-N1-Cx46-WT,pEGFP-N1-Cx46-T148I,pSin-cx46/flag-WT,pSin-cx46/flag-T148I and pEGFP-N1-Cx46-G143R were successfully constructed.Conclusions:A novel GJA3/p.T148I mutaion was revealed in this autosomal dominant congenital cataract family,and pathogenicity prediction,hydrophobicity analysis,homologous modeling were all predicted to be pathogenic.The sequence of GJA3 wild type was used as template for the vectors and further analysis of the pathogenic mechanism.Chapter 3.The molecular mechanisms of GJA3/p.T148I mutation.Objective:The expression vectors were transfected into the HLEC and HEK-293cells,and the molecular mechanisms of GJA3/p.T148I mutation were studied by analyzing the changes of the mutant protein locations,cell proliferation,protein expression level and gap junction function.Methods:The vectors were transfected into the HLEC and HEK-293 cells.Protein locations and gap junction channels were observed in the fluorescence microscope.The stable cell lines contained the wild type and the mutation were uesed for the detections of the protein expression and the hemichannel function.The CCK-8 assay was used to detect the cell proliferation.Oxidative damages were induced by H₂O₂ in HLEC and HEK-293 cell lines.Annexin V-FITC/PI was used to test the cell apoptosis and protein changes in MAPK pathway.Results:The intracellular localization of Cx46 protein was affected by the GJA3/p.T148I,so that the mutant Cx46 were accumulated and were in line with the expression level of Cx46 protein,while the Cx43 was not affected.The GJA3/p.T148I weakened the hemichannel formed by Cx46 and accelerated cell proliferation activity.Oxidative damages can induce the down-regulation of GJA3 gene expression in HLEC,while the GJA3/p.T148I can induce cell apoptosis and decreased the ability against oxidative damage.The abnormal expression of MAPK pathway was also detected in mutant cells.Conclusions:In the study of the functional analysis,GJA3/p.T148I mutation can not only increase the protein expression of Cx46 and abnormal accumulation in cells,but also can reduce the Cx46 hemichannel function,accelerate cell proliferation activity.The induction of apoptosis and decreasing the ability against oxidative damage,as well as an accommodative disorder in MAPK pathway were detected in mutant cells.As a result,this novel mutation not only expanded the spectrum of GJA3 mutations,but also verified the pathogenic mechanisms of the gap junction channels and apoptosis in congenital cataract.