Genomic study and mutation functional analysis of autosomal dominant congenital cataract

Purpose. Cataract is a leading cause of blindness worldwide. Although congenital cataract (CC), with a prevalence of 3 to 15 per 10,000 children, is less common than age-related cataract, genetic and molecular studies on CC provide information that contributes to understanding the mechanism of cataract formation, which helps to develop non-surgical treatments to slow down or prevent lens opacity. Among CC, autosomal dominant congenital cataract (ADCC) is a major form. This study aimed to identify disease-causing mutations for ADCC and to characterize their biological effects for the better understanding of cataractogenesis.; Methods. Six Chinese ADCC families with diverse phenotypes were recruited. Each study subject was given complete ophthalmic examination. A gene exclusion program using linkage analysis with 39 SNPs and 28 microsatellite markers indicating 15 ADCC genes and sequencing was conducted. For families with no detectable association with these genes, whole genome scan with 512 microsatellite markers was performed. Two-point linkage analysis was carried out by MLINK of FASTLINKAGE version 4.1p. Disease-causing mutations were detected by candidate gene sequencing. Structure-function predictions of gene variants were performed by ExPASy proteomics software. Specific mutant gene functions were investigated by creating recombinant constructs, site-directed mutagenesis, and in vitro expressions after transfections in COS-7 cells. For alphaA-crystallin (CRYAA) and gammaD-crystallin (CRYGD) mutants, the expression level, solubility in Triton X-100, subcellular distribution and heat shock response were examined by western blotting, confocal double immunofluorescence staining and quantitative reverse transcription-polymerase chain reaction.; Results. Five sequence variations in 3 crystallin genes were identified to associate with cataract independently in 5 families. No mutation was found for the remaining family. Three of the variations were novel mutations which completely segregated with disease, namely R12C CRYAA identified in a family with lamellar cataract, G165fs CRYGD with nuclear cataract, R15S CRYGD with coralliform cataract. There were a novel cataract-associated polymorphism S31W betaB2-crystallin (CRYBB2) with coronary cataract and a reported mutation P24T CRYGD with coralliform cataract. Three novel substitutions of R12C CRYAA, RISS CRYGD and S31W CRYBB2 were predicted to enhance the protein hydrophobicity. After in vitro expressions, R12C CRYAA behaved similarly as wildtype in detergent solubility and subcellular distribution. However, the mutant delayed expressions of heat-shock protein 70s. This altered heat shock response indicated a different chaperoning ability of R12C mutant on the misfolded substrate proteins, which is likely to be involved in cataract formation. Moreover, G165fs CRYGD showed a dramatic loss of detergent solubility. The mutant protein was redistributed to the nuclear envelope. This was expected to affect the nuclear degradation during fiber cell differentiation and result in lens opacity. G165fs CRYGD is the first characterized deletion mutation of CRYGD that causes ADCC and also the first CRYGD mutation that causes the protein mislocalization.; Conclusions. Novel variations of crystallins were identified to associate with ADCC. Our data supported the crucial role of crystallins in lens fiber cell maturation and lens transparency maintenance. Solubility change, mislocalization and altered functional response of mutant crystallins could lead to opacity formation. These results contribute to a better understanding of the pathogenesis of cataract.