| Purpose:Congenital cataract is a common cause for childhood blindness worldwide,approximately one third of congenital cataracts has inheritable.Until now,at least70 genes or loci have been mapped in the congenital cataracts.The genetic research of congenital cataract not only enhance our understanding of the disease,but also help us to elucidate the pathogenic mechanisms of congenital cataract,lay a solid foundation for prenatal diagnosis and treatment of this disease.We collected two congenital cataract families in Henan province,Chinese,and screened disease-associated genes,predicted pathogenicity of gene mutations using bioinformatics methods,and studied the molecular results of novel mutation via cell transfection at last.Methods:Two autosomal dominant congenital cataract(ADCC)families in Chinese were recruited from the Zhengzhou Eye hospital,Zhengzhou,Henan,China.All members of the two families underwent detailed medical histories and ophthalmologic and clinical examinations in Zhengzhou Eye hospital.Family trees were drawn based on informed consent.The genomic DNA samples were extracted from peripheral blood following obtained informed consent of all family members.According to the relationship of genotype and phenotype,we established a primary primers library of congenital cataract gene based on NCBI,ENSEMBLE and HGMD database(including exons and flanking intronic regions).All the exons and flanking intronic regions were amplified by polymerase chain reaction(PCR)and screened for mutation by Sanger sequencing.In bioinformatics,SIFT,PolyPhen-2,Mutation Taster,homology modeling and hydrophobicity analysis software wereused to predict whether the mutation is deleterious or neutral to the functions of the protein.In the study of molecular mechanism,PCR high fidelity primers were designed to amplify the target encoding sequence from genomic DNA eukaryotic expression vectors(pSIN-CRYGS/Flag-WT)were constructed by recombinant technology,and then mutant(pSIN-CRYGS/Flag-S82P)was generated by PCR site-directed mutagenesis.The wild and mutant expression vectors were transfected into human lens epithelial cells(HLEC)and HEK-293 cells.Immunofluorescence was used to observe the sublocation of the target fusion protein.Western Blot was used to detecte the expression of the target fusion protein,flow cytometry was used to detecte cell apoptosis.Results:1.Genetic analysis of the two families: the first family was identified as nuclear opacity after the detailed history investigation and physical examination,the second pedigree was identified as posterior polar opacity without any other ophthalmic and systemic disease.Both the inheritance patterns of the two families are autosomal dominant.The Sanger sequencing showed the first family carried a heterozygous c.244T>C change in the coding region of CRYGS gene,resulting in the substitution of a highly conserved serine to proline(p.82S>P).Sequence comparisons and literatures review were performed baesd on NCBI,ENSEMBLE and HGMD database.Site mutation was cosegregated with the phenotype in this family and bioinformatic prediction showed CRYGS(c.244T>C)is a novel mutation associated with congenital cataract.This novel mutation exists in all patients and was not seen in other family members or one hundred healthy controls individuals.The second family carried a deletion c.279-281 delGAG change in the coding region of CRYBA1 gene,causing the deletion of a highly conserved glycine(?G91).This is a known mutation according to the database and literature.This mutation cosegregated in all affected individuals in the family,and was not detected in unaffected individuals or 100 healthy control subjects from the same ethnic background.2.Further bioinformatic analysis and molecular mechanism of the biological information were performed on the CRYGS(c.244T>C)mutation in the first family.The analysis results showed the missense mutation of CRYGS(c.244T>C)can affect protein structure and function based on softwares of SIFT,PolyPhen-2 and Mutation Taster.Homology modeling and hydrophobic analysis showed that the differences of wild-type and mutant proteins in structure and hydrophobicity were not obvious.Eukaryotic expression vectors of pSIN-CRYGS/Flag-WT and pSIN-CRYGS/Flag-S82 P were transfected into HLEC and HEK-293 cells to over expression wild-type and mutant fusion proteins.Results of cell immunofluorescence suggested that the mutant protein showed no obvious in aggregation,but significantly reduction can been detected in the cell filamentous skeleton structure.Western Blot showed that the expression of mutant protein was significantly lower than wild-type.The results of flow cytometry showed that the apoptosis rate of mutant cells was higher than wild type in early stage.Conclusions:1.A novel mutation c.244T>C of CRYGS gene was identified in the first family,which expand the mutation spectrum of autosomal dominant congenital cataract.The molecular consuquences of CRYGS(c.244T>C)suggested this mutation can cause reduction of the CRYGS protein,disruption of the cytoskeleton structure,inducing early cell apoptosis,and leading to ADCC at last.2.The results of genetic analysis in the second family confirmed the deletion(c.279-281delGAG)in the CRYBA1 can lead to congenital posterior polar cataract. |
