Localization And Function Characterization Of A Disease-causative Gene Mutation In An Autosomal Dominant Congenital Nuclear Cataract Chinese Family

Purpose:Congenital cataract, an eye disease with the opacification of the lens, is the leading cause of childhood blindness worldwide. About one half cases of childhood blindness are caused by genetic alternations. We identified the genetic origin of an autosomal dominant congenital nuclear net-like cataract Chinese family and studied on the pathopoiesis mechanism of the mutation.Method:Family suffered from autosomal dominant congenital nuclear net-like cataract in three successive generations was recruited in the Eye Center of Second Affiliated Hospital, Medical College of Zhejiang University, Hangzhou, China. All members of the family underwent detailed family history taking and complete ophthalmic examination including visual acuity, slit-lamp examination, fundus examination, tonometer, keratometry, corneal topography and ultrasonic A/B scan. The genomic DNA samples were extracted from peripheral blood of all the members in the family and100normal control with the same genetic background. Mutation screening of coding regions of candidate genes was performed by bidirectional sequencing after polymerase chain reaction (PCR) amplification of exons and adjacent intronic regions. Sequence traces were analyzed with DNAMAN software. Computational analysis, including SIFT, PolyPhen2, Condel and conservative analysis are used to predict whether the amino acid substitution of the protein sequence is deleterious to the function of the protein.The coding region of the CRYAA gene was cloned from human lens cDNA library by PCR method. The c.161G>T change was generated by PCR site-directed mutagenesis. pET21b and pEGFP-C3plasmids were used to construct the wild-type and mutant expression plasmids of prokaryotic and eukaryotic cells (CRYAAwt-pET21b, CRYAAmut-pET21b, CRYAAwt-pEGFP-C3, CRYAAmut-pEGFP-C3) respectively. The biochemistry and biophysics analysis methods were used to perform functional study on the target protein. The localization and aggregation status of the target protein were observed after transfection of the above plasmids into the HeLa cells.Results:The phenotype of the cataract of the affected individuals in this family was characterized as nuclear net-like opacity without any other ophthalmic and systemic diseases. The inheritance pattern of the family is autosomal dominant. A heterozygous c.161G>T change in the coding region of Crystallin alpha A gene(CRYAA) was detected through mutation screening, leading to the substitution of a highly conserved arginine to leucine (p.R54L). The change cosegregated with the disease phenotype in the family. This mutation was not seen in200ethnically matched control chromosomes (100individuals). PolyPhen-2and Condel predicted the p.R54L change might be deleterious to the protein.Biophysical analysis indicated that the mutation modified the quaternary structures of aA-crystallin by increasing the oligomeric size. The mutation increased the solvent exposure of the Trp fluorophores, decreased the hydrophobic exposure, reduced the chaperone activity of aA-crystallin and promoted the formation of intracellular aggregates in the transfected HeLa cells. Our results reinforced the importance of the N-terminal region (NTR) in the regulation of aA-crystallin oligomeric size distributions and chaperone function.Conclusion:This study has identified a missense mutation (c.161G>T) in CRYAA, which resulted in a p.Arg54Leu mutation in NTR of aA-crystallin, in a Chinese family with autosomal dominant congenital nuclear net-like cataract. Biophysical studies indicated that the mutation impaired A-crystallin structure and function by increasing the oligomeric sizes and the exposure of Trp fluorophores to solvents, and decreasing the ANS-accessible hydrophobic sites and chaperone activity against insulin aggregation. Cellular studies further confirmed that the mutant was more prone to be trapped into aggregated in the HeLa cells although the mutation had no impact on aA-crystallin stability and folding. Our findings highlight the importance of Arg54in regulating aA-crystallin structure and function. The results herein also reinforced the genetic heterogeneity of congenital cataracts and the importance of NTR of aA-crystallin as a hot and key region for inherited mutations associated with congenital cataracts, provide theoretical basis for prevention and gene therapy of congenital cataract.