Detection of mutations in the early onset breast and ovarian cancer susceptibility gene BRCA1 represents a considerable technical challenge. More than fifty such mutations have been identified since the gene was cloned in the fall of 1994, but these are extremely heterogeneous, stretching across 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 numbers 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, which consist of arrayed oligonucleotides synthesized onto the surface of silicone. In collaboration with Steve Foder of Affymetrix, we are investigating the application of this method to detection of mutations in BRCA1. Some chips have been synthesized which have various portions of the coding region of BRCA1 represented as a series of overlapping fourteen, seventeen, or twenty-mers representing both strands of BRCA1 exon eleven, which contains more than half of the coding region. In addition to the oligonucleotides representing the expected sequence, every possible single base change is also represented on the chip. PCR amplified patient material, either from genomic DNA or from RT-PCR amplification of RNA, is fluorescently labelled, chemically degraded to an average size of 50-60 base pairs, and hybridized against the dedicated BRCA1 chip in a carefully controlled environment in a hybridization chamber. After washing, the fluorescent signals on chip are read in a CCD camera. The pattern of hybridization to the grid of olignucleotides allows a determination of the sequence of the patient sample, revealing mutations which may be present. In order for this technique to be robust, it must be capable of detecting mutations for single nucleotide changes. A hybridization signal to one of the mutant oleonucleotides will appear which is not present in a normal control. In the case of frame shift mutations however, there will be no gain in signal but simply a loss of fifty percent of the signal for all oligonucleotides govering the frameshifts. We are currently testing the sensitivity of this method to detect frameshifts using two color hybridization, where normal samples are labelled in one color and a patient samples in another. Shifts in the fluorescence intensity for a particular set of oleonucleotides indicates that there has been a frameshift in patient sample. If successful, this form of molecular diagnosis may also be readily applied to detection of mutations in other genes. We are particularly interested in expanding this to the ATM gene, which is responsible for ataxia telangectasia, and for which heterozygotes are also at an increased risk for cancer.
