Genetic Analysis And Preliminary Functional Study For Hereditary Congenital Cataract Due To MIP Gene

Background and Purpose Congenital cataracts,one of the most common causes of visual impairment,are responsible for approximately 10% of blindness cases in children.Genetic factors play an important role in the development of congenital cataracts.This study aimed to identify the genetic defects associated with autosomal dominant congenital punctate cataracts in a Chinese family and to explore the potential pathogenesis.Methods A three-generation Chinese Han family including 16 members with congenital cataracts was recruited for our study.All members of the pedigree were given complete ophthalmologic examinations and did not have any other eye disease.After written informed consents were obtained from subjects,5 ml of venous blood from each subject was collected and genomic DNA was extracted.Linkage analysis was performed by microsatellite markers that are associated with congenital cataracts,and logarithm(base10)of odds(LOD)scores were calculated using the LINKAGE program.After positive two-point LOD scores were obtained,the coding exons and the flanking regions of the candidate gene were amplified by polymerase chain reaction(PCR)with the primers.The functional consequences of the mutations were analyzed with biology software.The wild type and G215 D mutant MIP were transfected separately into Hela cells to determine the subcellular localization and by applying Western blot analysis to determine the expression.ResultsThe study confirmed a three-generation congenital autosomal dominant family with punctate cataracts.By linkage and haplotype analysis,positive two-point LOD scores were obtained at markers D12S1622(Zmax = 2.71 at h = 0.0),D12S1724(Zmax = 2.71 at h = 0.0),and D12S90(Zmax = 2.71 at h = 0.0),which flank the major intrinsic protein of lens fiber(MIP)gene on chromosomal region 12q13.Direct sequencing of the encoding region of the MIP gene revealed a novel mutation(G.D)in exon 4 at nucleotide 644,which caused a substitution of glycine to aspartic acid at codon 215(p.G215D)for the MIP protein.The mutation cosegregated with all patients with congenital punctate cataracts,but it was absent in the healthy members.Bioinformatics analysis predicted that the mutation affects the function of the MIP protein.The wild type(WT)and G215 D mutant of MIP were transfected with green fluorescent protein(GFP)into Hela cells separately,and it was found that the G215 D mutant was aberrantly located in the cytoplasm instead of in the plasma membrane.Western blot analysis revealed that WT-MIP and G215D-MIP had similar protein levels in western blot analysis of the cell lysates,indicating that mutation did not result in the expression or instability of the protein.Conclusions Our study presented genetic and functional evidence linking the new MIP mutation of G215 D to autosomal dominant congenital cataracts.The G215 DMIP had a lower hydrophobicity and was aberrantly located in the cytoplasm of Hela cells instead of the plasma membrane.We speculate that G215D-MIP would be unable to exert channel functions which further explore the pathogenesis of cataracts.