The Genetic And Functional Analysis Of Congenital Cataract Induced By Connexin Mutations

BackgroundCataract is a visual disorder caused by opacity of the lens and reduced light transmission.It is one of the major causes of blindness in humans.Cataract can be divided into congenital cataracts and senile cataracts depending on the time of cataract.Congenital cataract is a common cause of blindness in children.There are many extra gene mutations in congenital cataract.According to the different functions of the causative genes,they can be roughly divided into six categories:lens protein gene,gap junction protein gene,development regulatory factor gene,Cytoskeletal protein genes,tyrosine kinase receptor genes,and chromatin repair protein genes.Gap junction protein,as an important cell membrane protein,is an important subunit that constitutes a connecting channel between cell gaps and has the function of mediating the transport of small molecules between cells,such as metabolites,ions,and second messengers.Gap junction plays an important role in maintaining cell homeostasis and lens differentiation and its homeostasis.The subunits that make up the gap junction channels in the human lens can be divided into the following molecular weights:Cx43,Cx46,and Cx50,which are encoded by the genes:GJAl,GJA3,and GJA8,respectively.Studies have shown that the abnormal function of GJA3 and GJA8 leads to the occurrence of congenital cataract.At present,it has been found that more than 20 GJA3 mutations and 23 GJA8 mutations are associated with human congenital cataracts,and mutations in the GJA3 or GJA8 gene may cause alterations in the structure,function,etc.of the encoded Connexin,which in turn affect the intercellular spaces.The function of the connecting channel is abnormal and eventually induces cataracts.However,the specific molecular mechanisms that cause congenital cataracts by mutations in the gap junction protein gene are not completely understood.Objectives1.Identification of the causative genes of two autosomal dominant congenital cataract families.2.To investigate the molecular mechanism of congenital cataract in two families by mutation of GJA3 and GJA8 genes.Methods1.Ethical authorizationPrior to the study of cataract families,we submitted research applications for clinical specimens to the Ethics Committee of Henan University and the Ethics Committee of Kaifeng Central Hospital and passed the review of the ethics committee.2.Clinical diagnosis and family analysisWe cooperated with Kaifeng Eye Hospital to collect cataract families and,with the informed consent of family members,professional ophthalmologists performed detailed ophthalmologic examinations?vision examinations,slit lamp examinations?and related medical history of family members.The ancestral spectrum of cataracts inherited from the family is tentatively determined by drawing pedigree maps.3.Whole blood genomic DNA extraction and candidate gene screening and identificationAfter determining that the cataract of the family is not a complication of other diseases,we took peripheral blood from family members,then extracted peripheral blood genomic DNA,and used PCR to pair 11 candidate genes?cryaa,cryab,crybbl,crybb2,crygc,Crygd,GJA3,GJA8,Hsf4,Mip,and Pitx3?were amplified,and the PCR products were subjected to exon sequencing?Jin Zhizhi?and gene sequence analysis to identify pathogenic genes.4.Western blot analysis of expression of wild and mutant gap junction proteins in lens tissue of patientsThe anterior lens capsule epithelium obtained from cataract surgery was collected from pedigree members.The expression of gap junction protein in lens of senile cataract patients in the cataract and the control group was detected by Western blot.5.Immunofluorescence Assay for Subcellular Localization of Gap Junction Protein and Expression and Distribution of Cx50 in Mouse LensThe GFP-WT-Cx50 and GFP-Insert-Cx50 were constructed and transfected into HLE cells.Immunofluorescence technique was used to observe the localization of Cx50 in the cells.Expression and Distribution of CX50 in Mouse LensIn order to detect the distribution of Cx50 in the mouse lens,mice of different weeks of age?age?were selected and the lens was taken for paraffin sectioning.Subsequently immunofluorescence staining was performed and the distribution of Cx50 in mouse lens was observed by laser confocal microscopy.6.Semi-channel function detection of gap junction proteinsThe GFP-WT-Cx50 and GFP-Insert-Cx50 plasmids were transiently transfected into HLE and the ability of the PI dyes to pass through the wild-type Cx50 and the mutant Cx50 was detected using a dye uptake assay to show whether the gap junction protein hemichannel function abnormalities..7.MTT assay to determine the effect of mutant gap junction proteins on cell proliferationThe HLE cells were transiently transfected with GFP-WT-Cx50 and GFP-Insert-Cx50 plasmids,and the proliferation rate of HLE cells was detected by MTT assay to show the effect of the mutant Cx50 on the cell proliferation.Result1.CX46 frameshift mutations are associated with the occurrence of a dominant-dominant hereditary cataract.Using genetic map analysis techniques,we analyzed a family with congenital cataract and determined that the family is a family dominant hereditary cataract family.The DNA of peripheral blood of normal people,probands and other patients in the family was extracted and 11 pairs of exons of congenital cataract candidate genes were amplified by PCR and sequenced.The results showed that the insertion of base C?c.1194₁195ins C?after the 1195 site of the GJA3 gene cDNA in the proband resulted in a frameshift mutation?Cx46fs400?after the C-terminal 400 amino acids of Cx46,and the mutation was co-segregated in the family..To investigate the effect of Cx46fs400 on Cx46 protein,we used bioinformatics to predict the biochemical properties of Cx46 protein.It was found that this mutation may cause the increase of hydrophobicity of Cx46 and may cause the secondary structure of Cx46 to change.To determine whether this mutation affects the protein stability of Cx46 in the patient's lens,we examined the expression level of Cx46 in the lens tissue of the proband in this family.The results show that Cx46fs400 causes a significant decrease in Cx46 expression levels?Cui XK,Zhu KK,Zhou Z,et al.A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family.[J].International Journal of Ophthalmology,2017,10?5?:684-690.?.2.A new GJA8 gene insertion mutation?c.285ins?has been identified that is associated with the occurrence of familial dominant hereditary cataracts.By analyzing the pedigree genetic maps of congenital cataract family 2?a total of 10 people in 3 generations and 6 patients?,it was determined that the inheritance pattern of this family was an autosomal dominant inheritance.DNA from the peripheral blood of normal people,probands,and other patients in the family was extracted and 11 exons of congenital cataract candidate genes were amplified by PCR and sequenced.The results indicated that the prokaryotic GJA8 cDNA inserted 15 bases?tacgcggtgcactac??c.285ins?after the 285 site,resulting in the insertion of five amino acid residues?YAVHY?into the second transmembrane region of Cx50?p.H95_A96insYAVHY?.The result of immunofluorescence detection showed that this mutation caused Cx50 to change from membrane localization to cytoplasm localization.Using the dye uptake assay technique to detect half-channel function of Cx50,the results showed that this mutation caused an impairment of the half-channel function of Cx50.Subsequently,we used immunofluorescence to observe whether Cx50 colocalized with the endoplasmic reticulum marker protein GRP78 and the lysosomal marker protein lamp1.The results showed that this mutation can cause co-localization of Cx50 with endoplasmic reticulum and lysosomes.MTT assay was used to detect the proliferation rate of HLE cells transiently transfected with GFP-WT-Cx50 and GFP-Insert-Cx50.It was found that this mutation does not affect cell proliferation.Finally,in order to explore the expression profile of Cx50 in mouse lens development.We selected a 4-week old mouse lens for paraffin sectioning and tissue immunofluorescence staining.The results showed that Cx50 expressed more in the equatorial region of mouse lens.?Cui X,Zhou Z,Zhu K,et al.A Novel Cx50 Insert Mutation from a Chinese Congenital Cataract Family Impairs Its Cellular Membrane Localization and Function[J].Dna&Cell Biology,2018,37?5?:449-456.?Conclusion1.A new GJA3 frameshift mutation was found in a family dominant genetic cataract family DNA.The GJA3 cDNA inserts one base C after the 1195 site,causing the Cx46 protein to start shifting from 400 amino acids.This frameshift mutation alters the stability of the CX46 protein.2.In another family of autosomal dominant cataract pedigrees,we found that 15 amino acids were inserted after the GJA8 gene cDNA 285 site.This new mutant results in insertion of five amino acid residues in the second transmembrane region of Cx50?YAVHY?This mutation makes the cell membrane localization pattern of Cx50 abnormal and impairs the half-channel function of Cx50.