Genetic Diagnosis Of Congenital Cateracts Using Target Enrichment Technology And Function Characterization Of Disease-causative Mutation(EPHA2 P.G668D)

Purpose:To identify the disease-caustive mutations in twenty-eight Chinese congenital cataracts famlilies.And to elucidate the functional basis of a novel EPHA2 mutant,EPHA2 p.G668D,by determining its protein distribution and cellular behavior.Methods:Twenty-eight families suffered from congenital cataracts were recruited in the Eye Center of Second Affiliated Hospital,Medical College of Zhejiang University.The genomic DNA samples were extracted from peripheral blood of all family members and 100 normal controls.Angilent SureSeleet Target Enrichment System and next-generation sequencing technology were used to identify potentially disease-caustive SNVs within forty-eight candidate genes.Computational analysis,including SIFT,PolyPhen2,MutationTaster were used to predict whether the amino acid substitution is deleterious or benign.The potentially disease-caustive SNVs were then confirmed by PCR and Sanger sequencing.All members of the FAMILY 12 with EPHA2 p.G668D mutation underwent detailed family history taking and complete ophthalmic examination including slit-lamp examination,visual acuity testing and fudus examinations.To understand the pathologic role of EPHA2 during the onset of cataract,we first examined the expression of ephrins in human anterior lens capsule tissues.EPHA2 gene cDNA was synthesis in vitro.EPHA2 G668D mutant was constructed by PCR site-directed mutagenesis.To investigate the role of mutant EPHA2 proteins in cell migration and apoptosis,GV358-EPHA2WT GV358-EPHA2G668D and GV358 were transfected into human lens epithelial cell line(HLE B3).To further investigate the locolization and proteins level of mutant,pEGFP-N1-EPHA2WT,pEGFP-N1-EPHA2G668D and pEGFP-N1 were transfected into Hek293T cell line.Results:Fourteen disease-caustive mutations were detected in fourty-eight candidate genes using targeted exome sequencing and then confirmed by Sanger sequencing.Among them,ten mutations are novel,four have been linked to congenital cataracts.These mutations were co-segregated with the affected individuals in fourteen families,but were not observed in unaffected family members or in the 100 unrelated healthy controls.One novel heterozygous c.2003G>A(p.G668D)in the coding region of the EPHA2 gene was found in FAMILY 12,and result in congenital posterior polar cataracts.We detected mRNA expression of ephrins in human anterior lens capsule tissue.EPHA2 G668D mutant protein promoteed cell migration in human lens epithelial cell line,while did not affect cell apotosis.Mutation also promoted the formation of intracellular aggregates of EphA2 protein in the transfected Hek293T cells.Western blot showed a obviously lower level of mutant proteins compared to wild type after transfection.Meanwhile,a pretreatment of proteasomal inhibitor(MG-13 2)of transfected Hek293T cell increased the level of mutant protein.Conclusions:Angilent SureSeleet Target Enrichment System and next-generation sequencing technology are efficient and feasible way in genetic diagnosis of congenital cataracts.EphA2 mutant proteins are susceptible to degradation,and this result in a common pathogenic mechanism which called "loss-of-function effect".This alternation may promote lens epithelial cells migration and result in congenital posterior polar cataract.Our data suggested that Eph/ephrin siganaling plays an important role in lens function and cataract genisis.