A novel αB-crystallin mutation associated with autosomal dominant congenital lamellar cataract

PURPOSE. To identify the mutation and the underlying mechanism of cataractogenesis in a five-generation autosomal dominant congenital lamellar cataract family. METHODS. Nineteen mutation hot spots associated with autosomal dominant congenital cataract have been screened by PCR-based DNA sequencing. Recombinant wild-type and mutant human alpha B-crystallin were expressed in Escherichia coli and purified to homogeneity. The recombinant proteins were characterized by far UV circular dichroism, intrinsic tryptophan fluorescence, Bis-ANS fluorescence, multiangle light-scattering, and the measurement of chaperone activity. RESULTS. A novel missense mutation in the third exon of the ali-crystallin gene (CRYAB) was found to cosegregate with the disease phenotype in a five-generation autosomal dominant congenital lamellar cataract family. The single-base substitution (G -> A) results in the replacement of the aspartic acid residue by asparagine at codon 140. Far UV circular dichroism spectra indicated that the mutation did not significantly alter the secondary structure. However, intrinsic tryptophan fluorescence spectra and Bis-ANS fluorescence spectra indicated that the mutation resulted in alterations in tertiary and/or quaternary structures and surface hydrophobicity of alpha B-crystallin. Multiangle light-scattering measurement showed that the mutant aB-crystallin tended to aggregate into a larger complex than did the wild-type. The mutant all-crystallin was more susceptible than wild-type to thermal denaturation. Furthermore, the mutant aB-crystallin not only lost its chaperone-like activity, it also behaved as a dominant negative which inhibited the chaperone-like activity of wild-type aB-crystallin. CONCLUSIONS. These data indicate that the altered tertiary and/or quaternary structures and the dominant negative effect of D140N mutant alpha B-crystallin underlie the molecular mechanism of cataractogenesis of this pedigree.