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DS Mackay, OB Boskovska, HL S Knopf, WL Walker, UP Andley, A Shiels; A Novel Mutation in the Gene for A-crystallin Associated With Autosomal Dominant Cataract Linked to 21q . Invest. Ophthalmol. Vis. Sci. 2002;43(13):481.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify the genetic mutation underlying autosomal dominant nuclear cataract in a three generation Caucasian family. Methods: Leukocyte genomic DNA was prepared from family members using the QIAamp DNA blood maxi kit (Qiagen). PCR-based genotyping was preformed using Genethon microsatellite markers labeled with IRDye800 then visualized using a LiCor 4200 DNA analyzer. Lod scores were calculated using the Linkage package of programs. Mutation detection was performed by direct sequencing with dye-terminator chemistry on an ABI 310 DNA sequencer and confirmed by restriction fragment length analysis. Site-directed mutagenesis of wild type αA-crystallin was performed using the QuickChange kit (Stratagene). Results: Linkage analysis detected a positive two-point lod score (2.19 @ θ= 0) at marker D21S1885, which lies ∼0.225Mb distal to the gene for αA-crystallin (CRYAA). Haplotyping revealed that the cataract locus lay in the physical interval: D21S1260 - (1.77Mb) - CRYAA - (0.225Mb) - D21S1885 - (0.722Mb) - D21S1912. Sequencing of all three CRYAA exons identified a C≷T transition that introduced a novel AciI restriction enzyme site. Restriction analysis confirmed that this single nucleotide change was present only in affected members of the pedigree and was not detected in 50 unrelated normal chromosomes. The C≷T transition was predicted to result in a missense substitution of arginine to cysteine (R≷C) that lies outside the conserved 'α-crystallin' domain. Functional expression studies (Xi et al., ARVO 2002) showed that the R≷C mutant exhibits abnormal intracellular distribution and decreased chaperone-like activity in human lens epithelial cells. Conclusion: The results suggest that the novel C≷T transition in CRYAA is a causative missense mutation rather than a benign single nucleotide polymorphism. This study has identified the first dominant mutation in CRYAA located outside the conserved 'α-crystallin' domain associated with central/nuclear cataract in humans.
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