Summary Age-related cataract (ARC) is a multifactorial disease and the leading cause of visual impairment and blindness worldwide. Genetic predisposition in association with other etiological factors may contribute to ARC. Although, there is some evidence for genetic influence for development of ARC, reports on gene mutations associated with ARC are scanty. In the present work, we identified a genetic variation (F71L) in the exon-2 of CRYAA gene in three unrelated female sporadic cases among 450 ARC patients but not in 144 normal non-cataractous controls. By comparing human recombinant wild-type and F71L-αA-crystallin, further we characterized the functional significance of this missense mutation. Size exclusion chromatography studies revealed that F71L mutation had no significant effect on the apparent molecular mass of αA-crystallin oligomeric complex. Intrinsic tryptophan fluorescence and far- and near-UV CD spectra indicated that F71L missense mutation did not significantly affect the secondary and tertiary structures of αA-crystallin. The ANS fluorescence emission spectra suggested no changes in surface hydrophobicity due to the F71L substitution. While the mutant αA-crystallin displayed almost complete loss (90%) of chaperone-like activity (CLA), in thermal aggregation of carbonic anhydrase, it showed 35-50% less protection in heat-induced aggregation of βL- and γ-crystallins. This is the first report of an αA-F71L mutation being associated with ARC. The results are consistent with the hypothesis that the mechanism of ARC in individuals carrying this mutation (F71L) might be due to the overall loss of in vivo chaperone activity due to interaction with other environmental factors.

Previously we identified a novel mutation (F71L) in the αA-crystallin gene associated with early onset of age-related cataract. However, it is not known how the missense substitution translates into reduced chaperone-like activity (CLA), and how the structural and functional changes lead to early onset of the disease. Herein, we show that under native conditions the F71L-mutant is not significantly different from wild-type with regard to secondary and tertiary structural organization, hydrophobicity and the apparent molecular mass of oligomer but has substantial differences in structural and functional properties following a heat treatment. Wild-type αA-crystallin demonstrated increased CLA, whereas the F71L-mutant substantially lost its CLA upon heat treatment. Further, unlike the wild-type αA-subunit, F71L-subunit did not protect the αB-subunit in hetero-oligomeric complex from heat-induced aggregation. Moreover, hetero-oligomer containing F71L and αB in 3:1 ratio had significantly lower CLA upon thermal treatment compared to its unheated control. These results indicate that α-crystallin complexes containing F71L-αA subunits are less stable and have reduced CLA. Therefore, F71L may lead to earlier onset of cataract due to interaction with several environmental factors (e.g., temperature in this case) along with the aging process.

AimTo identify the presence of a known or novel mutation/SNP in Exon-1 (ex-1) of alpha crystallin (CRYAA) gene in different types of age-related cataract (ARC) patients.Materials and MethodsSingle strand Conformation Polymorphism (SSCP) analysis was carried for the detection of single nucleotide polymorphism (SNP) in ex-1 of alpha crystallin (CRYAA) gene which was confirmed by sequencing.ResultsThe SSCP analysis of ex-1 of CRYAA gene revealed mobility shift in patients and controls, which was due to G>A transition at 6th position in exon-1 of CRYAA gene. All the three genotypes, GG, AA and GA, were detected in patients and controls indicating that G>A substitution is polymorphic. The analysis showed significant risk for heterozygotes (GA) as compared to pooled frequencies of homozygotes (GG + AA), which was 1.81 times for all the types of cataracts in general and 2.5 times for Nuclear Cataract and twice for Cortical Cataract.ConclusionThe GA heterozygotes were at higher risk for developing NC and CC types of cataracts, where as the GG homozygotes for MT and AA homozygotes for PSC types were at risk. To our knowledge, an association of G>A transition found in ex-1 of CRYAA gene with ARC, with differential risk of genotypes for individual type of cataracts has not been reported previously.