Screening And Identification Of Mutations Associated With Congenital Cataracts

Objective: Two Chinese families with congenital cataracts were recruited in northeast China.Affected individuals in Family 1 showed cataracts and microphthalmia,and affected individuals in Family 2 showed nuclear cataracts.The aim of this project is to detect the causative mutations for congenital cataracts in two Chinese families respectively,and further explore the potential pathogenesis of this disease.Methods: Genomic DNA of family members was extracted from peripheral blood.As to Family 1,coding exons,with flanking sequences of seven candidate genes,were screened using direct DNA sequencing.The identified mutations were confirmed by restriction fragment length polymorphism(RFLP)analysis.A full-length wild type or an R12 L mutant CRYAA was transfected into HEK293 T cells or Hela cells.Western blotting was performed to determine protein expression levels and protein solubility.Immunofluorescence was performed to determine protein sub-cellular localization.As to Family 2,proband-parents trio was subjected to targeted next-generation sequencing.Coding regions of 4813 disease-associated genes were captured.Enriched sample pool was added to flow cell and placed on Illumina Mi Seq system for sequencing.The candidate causative variants were validated by Sanger sequencing in all available family members.The interpretation of variants pathogenicity is following American College of Medical Genetics guidelines,and multiple algorithms,such as REVEL,SIFT and Poly Phen2,were also used to predict effect to protein damaging.We constructed MAF expression plasmids and luciferase reporter plasmids containing promoter regions of each 4 MAF target crystallin genes.A combination of pc DNA3.1-MAF expression plasmid and p GL3-crystallin promoter luciferase plasmid were transfected into the HEK 293 T cells.24 hrs after transfection,cells were harvested and lysed.The firefly and renilla luciferase activities for cell lysis were measured.Results: We identified a novel mutation in CRYAA(c.35G>T;p.R12L)that segregates with congenital cataract and microphthalmia in Family 1.This mutation altered a highly conserved arginine to a leucine(R12L)within CRYAA.When WT-CRYAA and R12L-CRYAA expression constructs were transfected into HEK 293 T cells,the mutant CRYAA had a much higher protein level in the supernatant compared to the wildtype CRYAA.What is more,the mutant protein aggregated in the precipitant where the wildtype was not detected.Immunofluorescence study showed the overexpressed mutant CRYAA formed large cytoplasmic aggregates and aggresomes.A missense variant in the MAF gene(c.812T>A,p.V271E)was identified in Family 2 with congenital nuclear cataract.This mutation altered a highly conserved valine(GUG)to glusate(GAG)within MAF.The variant is classified into “likely pathogenic” based on 28 criteria of the ACMG/AMP 2015 guideline,and its effect to protein was predicted damaging by multiple algorithms(deleterious by SIFT,Probably damaging by Poly Phen-2,and 0.941 by REVEL).Promoters of 4 crystallins that are responsive to wildtype MAF were cloned into p GL3-basic vector,and luciferase activities were measured to test their responses to mutant MAF.The luciferase activity was compared between wild type and V271 E mutant MAF after normalized to the empty group.The expression of the 4 crystalline constructs were significantly reduced by V271 E mutant,and the relative luciferase activities were reduced by 81.24%,42.97%,77.48% and 49.95% for CRYGA,CRYAA,CRYBA1 and CRYBA4 respectively.Conclusions: The novel mutation in the CRYAA gene((c.35G>T;p.R12L)identified in Family 1 may cause congenital cataracts and microphthalmia.The overexpressed mutant CRYAA formed large cytoplasmic aggregates and aggresomes,which may result in a cataract phenotype.The novel nonsense mutation in the MAF gene(c.812T>A,p.V271E)identified in Family 2 may cause nuclear cataracts.The mutation V271 E was associated with severe effect on the transactivation of the 4 crystallin expression constructs.The expression of the CRYGA,CRYAA and CRYBA4 constructs was significantly reduced,which may result in a cataract phenotype.Our data also expands the spectrum of known CRYAA and MAF mutations.