| Background Cataract is a leading cause of blindness worldwide and congenital cataract is a common eye disease that a child is born with or develops soon after birth. Genetic factors as well as environmental factors play a significant part in cataract aetiology and about one third of all congenital cataracts are inherited. With the development of advanced molecular biological techniques, more and more genes causing congenital cataracts have been identified; however, the underlying mechanisms are not fully understood. The aim of this study was to identify the causative genetic mutation among the known cataract candidate genes in a Chinese family and to investigate the molecular mechanism of cataract that may develop due to the mutation.Methods Because of the small family size, we used the functional candidate gene analysis approach. We screened the Chinese family, clinically documented to have congenital nuclear cataracts, for mutation in the candidate genes CRYG (C & D), GJA8, CRY (AA & AB), CRYBA and CRY (BB1 & BB2) through polymerase chain reaction analyses and sequencing. Biophysical predictions of the altered protein were analyzed using the Bioinformatics tool of the Expasy Proteomics server (http://www.expasy.org). Recombinant wild-type and R11H mutant aB-crystallin were expressed in E.coli BL21 (DE3) and purified to homogeneity. The recombinant wild-type and mutant aB-crystallin were characterized by UV circular dichroism, bis-ANS fluorescence and thermal stability. The chaperone activities were measured using insulin and alcohol dehydrogenase (ADH) as substrates. Furthermore, wild-type and R11HαB-crystallin were expressed in HeLa and human lens epithelial (HLE) cells. The functional characteristics of mutant aB-crystallin were examined using confocal fluorescence microscopy and flow cytometry in comparison with wild-type aB-crystallin. The optimal condition for the expression of aA-crystallin in E.coli was studied. Results Sequencing of the exons of the CRYAB gene identified a sequence variation in exon 1 (32 G>A) with a substitution of Arg to His at position 11. All affected family members revealed this change but it was not observed in any of the unaffected members of the family or in the randomly selected 200 DNA samples from ophthalmologically normal individuals and in 40 unrelated senile cataract patients of the same ethnic background of the family members. Computer-assisted prediction suggested that this mutation affected the biochemical properties as well as structure of aB-crystallin. The R11H mutant exhibited altered structures, decreased surface hydrophobicity and enhanced chaperone-like activity compared with the wild-type aB-crystallin. It was remarkably similar to the wild-type protein in its subcellular distribution and thermal stability, but it resulted in increased cell apoptosis. After optimization of aA-crystallin expression in E.coli, recombinant aA-crystallin was purified by Ni-NTA sepharose resin affinity chromatography under natural conditions.Conclusions This study identified a novel mutation in CRYAB gene (R11H) in a Chinese family with autosomal dominant congenital cataract. The mutation in aB-crystallin resulted in altered folding, decrease in hydrophobicity and abnormal ability to induce cell death which could contribute to turbidity and loss of transparency of the lens. |
