The Mutation Screening And Functional Study Of Pathogenic Gene In Autosomal Dominant Congenital Nuclear Cataract

Purpose:Congenital cataract,one of the leading causes of treatable blindness in children worldwide.Genetic mutation is the most common cause for congenital cataract,we aim to identify the disease-causing gene in two families with autosomal dominat congenital nuclear cataract and explore the molecular mechanism of cataractogensis.Method:Two families affected with congenital cataracts were recruited in Zhongnan hospital,Wuhan university and Dongguan maternal and child care hospital.All the family members were determined by history records and ophthalmic examination including visual acuity,slit-lamp examination,fundus examination.The genomic DNA samples were extracted from peripheral blood of all the members.We applie the PCR-direct sequencing method to screen the candidate genes associated with the cataract family in Shishou,Hubei.The wild type and mutant type expression plasmids(pET-28a-WT and pET-28a-MU)were constructed for further Biophysical studies of the recombinant full-length βB1(WT)and mutant(MU)crystalline.We utilize targeted exome sequencing(TES)to screen all known cataract genes or locus for the disease-causing mutation in the proband and Sanger sequencing was used to confirm the potential pathogenic variants in the family in Dongguan,Guangdong.Computational analysis,including SIFT,PolyPhen2 and conservative analysis are used to predict whether the amino acid substitution of the protein sequence is deleterious to the function of the protein.Results:The phenotype of the cataract of the affected individuals in the family in Shishou was characterized as nuclear opacity with nystagmus.The inheritance pattern of the family is autosomal dominant.We identified a c.C749T(p.Q227X)transversion in exon 6 of CRYBB1,a cataract-causative gene.This nonsense mutation changes a phylogenetically conserved glutamine to a stop codon and is predicted to truncate the C-terminus of the wild-type protein by 26 amino acids.Comparison of the biophysical and biochemical properties of the recombinant full-length and truncated βB1 crystallins revealed that the mutation led to the insolubility and the phase separation phenomenon of the truncated protein with a changed conformation.Meanwhile,the thermal stability of the truncated βB1-crystallin was significantly decreased,and the mutation diminished the chaperoning ability of aA-crystallin with the mutant under heating stress.The phenotype of the cataract of the affected individuals in the family in Dongguan also was nuclear cataract.We successfully identified a novel c.C565G mutation in exon 2 of GJA8 gene.The mutation showed perfect co-segregation with the disease phenotype in the family but was not observed in 100 controls using Sanger sequencing.The heterozygous c.C565G mutation lead to the substitution of a highly conserved proline by alanine at codon 189(p.P189A)in the coding region of connexin50(Cx50).Moreover,the mutation was predicted be deleterious by both PolyPhen-2 and SIFT with consistent results.Conclusions:This study has identified two novel mutation(c.C679T,p.Q227X of CRYBB1 gene and c.C565G,p.P189A of GJA8 gene).Our findings highlight the importance of the C-terminus in βB1 crystallin in maintaining the crystalline function and stability,and provide a novel insight into the molecular mechanism underlying the pathogenesis of human autosomal dominant congenital cataract.