Screening Disease-causing Mutations In Two Families With Hereditary Congenital Cataract And Study Of Function

Purpose:The aim of the study was to screen for the disease-causing mutations in two Chinese families with different phenotypes of bilateral hereditary congenital cataract and to dissect the molecular and cellular consequence of the novel mutation. Methods:All members of two families underwent a full ophthalmic and clinical examinations,Recording detailed family history and clinical data,After obtained informed consent of all family members,Normal and patients blood samples were collected.The genomic DNA was extracted from peripheral blood leukocytes,Family 1 choose a highly pathogenic gene of congenital cataract as candidate genes,Design specific primers(including exons and the flanking intronic regions),Candidate genes were amplified using PCR and screened for mutations using direct sequencing.We use the technique of recombinant DNA in vitro,Build CX50 wild-type,Then using site-directed mutagenesis techniques,complete construction of CX50 mutant gene expression vector,by transfection of connexin deficient 293 cells,recombinant proteins expression and distribution were assessed by Confocal microscopy.Family 2 choose the polymorphism of microsatellite genetic markers considered to be assoeiated with cataraets and Synthesis of primers.DNA fragments amplified by GStorm Gradient PCR.PCR products from each DNA sample were separated on a 8% Polyacrylamide gel and analyzed.Exclusion analysis was Performed by allele sharing analysis and gene sequencing. Results:According to the encoding regions of the candidate gene sequencing results showed that the proband was heterozygous c. 94T>A transversion at position 94 of the GJA8 in Family 1,which resulted in a Phe32-to-Ile(F32I) substitution at a highly conserved residue in the first transmembrane domains(TM1).This mutation co-segregated with all affected individuals in this pedigree and was not observed in unaffected family members and 100 normal controls.When expressed in 293 cells,CX50 and GFP form a fusion protein,Observed by confocal microscopy showed that both wild-type and mutant forms gap junction plaques,there is no difference in quantity,and in the cell membrane and cytoplasm can observe a punctate distribution.In Family 2 the c.130G>A transition of the GJA3 was predicted to result in a non-conservative substitution of valine-to-methionine at codon 44(p.V44M) with damaging effects on protein function. Conclusions:In Family 1 we have found a novel missense mutation identified in the first transmembrane domain(TM1) of GJA8. Our findings further extends the mutation spectrum of connexin 50 in association with congenital nuclear cataract,Identified mutation in this study involve substitutions of amino acids within the first transmembrane domain,Subcellular localization and the number of gap junction plaques that no change,The results of this study suggest that the CX50F32 I leads to cataract by other pathogenic mechanisms.The result of Family 2 confirmed p.V44 M can lead to congenital cortical cataract.