Mutation detection in the early onset breast and ovarian cancer susceptibility gene BRCA1 represents a considerable technical challenge. More than one hundred fifty extremely heterogeneous mutations have been identified to date, stretching over virtually the entire coding region of this large gene. While methods for mutation detection such as SSCP or direct sequencing have been successful in the research environment for studying small numbers of samples, the potential need to scale up mutation detection to much larger number of DNA samples for clinical purposes will rapidly outstrip the throughput capabilities of these more laborious methods. An attractive alternative is the use of DNA chips, high density oligonucleotide arrays synthesized on a silicon surface. In collaboration with Steve Fodor of Affymetrix, we are investigating the application of this method to BRCA1 mutation detection. Chips have been synthesized in which more than half of the BRCA1 coding region is represented as a series of overlapping twenty-mers encompassing both strands of exon eleven. Over 96,600 oligonucleotides were designed to detect the expected sequence along with all possible single base substitutions, single base insertions, and 1-5 bp deletions. PCR amplified patient material from genomic DNA is fluorescently labelled, chemically degraded to an average size of 50-100 nucleotides and hybridized against the dedicated BRCA1 chip in an environmentally controlled hybridization chamber. After washing, fluorescent signals on the chip are read through scanning confocal microscopy. The hybridization pattern to the grid reveals mutations which may be present. There is expected to be a loss of fifty percent of the signal for all oligonucleotides covering mutated regions. Normal reference and patient test samples are cohybridized to the arrays and differences in hybridization signals quantitated by two-color analysis. Evaluation of both gain of signal data to mutant oligonucleotides as well as loss of signal from wild type oligonucleotides led to the development of an algorithm for mutation detection. Fourteen of fifteen patient samples with known exon 11 mutations were accurately diagnosed, and no false positive mutations were identified in 20 control samples. We are particularly interested in expanding this analysis to the BRCA2 gene and the ATM gene, which is responsible for ataxia telangectasia, and for which heterozygotes are also at an increased risk for various cancers.
