Study Of Pathogenic Mutations In Families With Congenital Cataract

Purpose:Congenital cataract is the main cause of reversible blindness in infants.The prevalence is estimated about 1-6/10000,and more than half of them have family history.The study is mainly focused on the screening of pathogenic genes in two families with hereditary congenital cataract from different regions,and analyzed the mutation sites from the point of view of molecular Bioinformatics.Objects and methods:This study was conducted in two families with hereditary congenital cataract from different regions.One of them was three generations hereditary,and the other was four generations hereditary.1.A detailed medical history and eye examinations were performed on the above family members,and the family maps were drawn.2.Peripheral blood(3ml)was extracted from family members after consent was obtained.3.DNA was extracted from the nine members of two families and purified with Wizard Genomic DNA Purification kit.4.Based on the whole genome of probands,hot mutation spots in congenital cataract genes were amplified by PCR(polymerase chain reaction).The purified PCR products were detected by Sanger sequencing.5.The gene sequences were analyzed using Chromas 2.22 and CodonCode Aligner software to determine the pathogenic genes of the corresponding family.6.The mutations were pathogenically analyzed by bioinformatics software such as Kyte-Doolittle Hydropathy Plots,Mutation Taster,PolyPhen-2 and PROVEAN PROTEIN.7.The three-dimensional structure model of protein was established bySWISS-MODEL server and the corresponding structure was analyzed to predict and explore the pathogenesis of family 1 and family 2 from molecular bioinformatics.Results:1.PCR results showed that no missense or splice mutations were found in 18 candidate genes in family 1.That meant the pathogenic gene of this family has nothing to do with the candidate genes in this study.2.In the family 2,we found GJA3 gene mutation c.184G> A(p.E62K)through PCR,which meant the mutation led to the amino acid of glutamate encoded by the highly conserved sequence was substituted by Lysine.This mutation was not found in the 1000 genomes project and ExAC database.Simultaneously,it was also not found in the HGMD(The Human Gene Mutation Database)Professional Edition database,which meant that it was a novel mutation.3.GJA3-E62 K mutation was predicted as a pathogenic site in family 2 with Bioinformatics analysis.Conclusions:1.The genetic pattern of these 2 families in this study is autosomal dominant inheritance.2.The DNA sequencing results of family 1 are negative,and the pathogenicity genes of the family are not related to the candidate pathogenic genes we screened below(GJA8?GJA3?CRYGD?CRYAA?CRYBB2?CRYBA1?CRYAB?CRYGC?BSFP2?EPHA2?MAF?PITX3?HSF4?MIP?CRYGS?CRYBB1?CRYBB3?CRYBA4).There may be other pathogenic genes in family 1.3.The GJA3-E62 K mutation is a novel mutation,which contributes to the184th-guanine replaced by adenine(c.184g>a)in 2nd exon,resulting in the amino acid change from Glutamate to Lysine in the highly conserved region.This mutation in GJA3 will exert adverse effects on the structure and function of connexin(Cx),and thus yield the occurrence of congenital cataract.