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D. Burch, L. Richter, P. Flodman, M. Spence, F. Barría von–Bischhoffshausen, J. Bateman; Sequencing Analysis of Chromosome 22 in a Four–Generation Autosomal Dominant Cataract (ADC53) Chilean Family . Invest. Ophthalmol. Vis. Sci. 2005;46(13):826.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To map and identify the gene for ADC in a large Chilean family. Methods: ADC 53 is a four–generation Chilean family with 17 affected individuals. SIMLINK analyses were performed. We screened 41 family members with a panel of markers for known ADC loci using PCR primer sets. The products were resolved on an ABI 373 using Genescan 2.1 software [Applied Biosystems (ABI)]. Two point LOD scores were calculated using LIPED. The coding sequences for the candidate gene CRYBB2 were amplified using published primers (Litt et al., 1997) and newly–designed primers for exon 6. We sequenced PCR products using the ABI Dye Terminator Kit v1.1 with an ABI 3100Avant DNA sequencer (ABI, Foster City, CA.). Sequence analyses were performed using Consed, Phred, Phrap, and Polyphred (SeattleSNPs, UW–FHCRC, Seattle, WA). Results: SIMLINK analyses showed a maximum LOD score over 1000 simulations of 9.49. We calculated LOD scores between the ADC53 locus and known ADC loci and excluded all except for chromosome 22. D22S421 yielded evidence of linkage with a LOD score of 5.87. Sequence analyses of CRYBB2 demonstrated a C–T variant in exon 6 at nucleotide 475 and a second (no amino acid change) variant at 483. Conclusions: We demonstrated the presence of previously–reported variants (Litt et al., 1997, Vanita et al., 2001) in exon 6 of CRYBB2 in affected member of ADC53. The variant at 475 creates a premature stop codon which truncates the protein and is probably a mutation; it has been described in three apparently–unrelated families. (Litt et al., 1997; Vanita et al., 2001; Gill et al., 2000) Sequencing exon 6 with previously–reported primers yielded uninterpretable results due to the high homology (97.2%) between CRYBB2 and the 'pseudogene' CRYBB2P1 that are in close proximity. We designed primers more specific to CRYBB2, yielding high quality sequence. CRYBB2P1 is expressed in brain, hypothalamus, hippocampus, lymph and Burkitt lymphoma and is not a 'pseudogene'. This is the first report of this CRYBB2 mutation in a Chilean family. Haplotype analyses may clarify whether these variants represent a founder effect.
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