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Univ. of Washington Team Uses Capture Method to Sequence Breast, Ovarian Cancer Risk Genes

By Andrea Anderson

NEW YORK (GenomeWeb News) – A University of Washington research team has come up with a genomic capture-based sequencing method for identifying mutations in a set of genes linked to breast and/or ovarian cancer.

In a proof-of-principle study appearing online yesterday in the Proceedings of the National Academy of Sciences, the researchers demonstrated that they could specifically capture and sequence 21 breast and ovarian cancer-associated genes, including BRCA1 and BRCA2, for less than $1,500 per sample. Using this approach, they were able to detect a range of changes in these genes — from SNPs to small and large insertions and deletions — without picking up false-positive mutations.

"Our approach evaluates the 21 known genes that predispose to moderate/high risks of developing breast and/or ovarian cancer simultaneously," lead author Tom Walsh, a researcher with the University of Washington's department of medicine and genome sciences, told GenomeWeb Daily News in an e-mail message. "It detects small mutations (point mutations and indels) as well as large mutations (exon deletions and duplications)."

Walsh noted that current approaches typically rely on PCR amplification followed by Sanger sequencing of individual exons for finding point mutations and indels, followed by another test to detect larger exonic deletions and duplications.

Although inherited mutations in several genes have been linked to elevated breast and ovarian cancer risk, the researchers explained, much of the breast and ovarian cancer-related genetic testing that's currently done is based on family history and tends to focus on the BRCA1 and BRCA2 genes — a strategy that they say may miss some women with paternally-inherited BRCA1/2 mutations or mutations in other cancer-related genes.

"To identify as many mutations as possible that are responsible for inherited predisposition to breast and ovarian cancer, it is useful to analyze multiple genes, not only BRCA1 and BRCA2," the team wrote.

With that goal in mind, the researchers went about testing a high-throughput approach for detecting SNPs, small indels, and larger rearrangements in nearly two-dozen breast and/or ovarian cancer-associated genes.

The team first generated custom Agilent oligonucleotides corresponding to the complete coding, non-coding, and flanking sequences for 21 genes implicated in inherited cancer risk. Together, these sequences account for roughly a million bases of DNA per individual.

New moderate- to high-risk breast and/or ovarian cancer-associated genes will likely be incorporated into the method as they are discovered, Walsh explained.

The researchers then used the Illumina Genome Analyzer IIX to do paired-end sequencing of target gene-enriched DNA for 20 women with breast or ovarian cancer who carried known, inherited cancer-related mutations.

To do this, the team made DNA libraries from genomic DNA in patient blood samples and hybridized these libraries to the cancer gene-targeting oligonucleotides.

To keep the cost per sample low, the team ran one sample per flow cell lane. They noted that it will be possible to further decrease costs in the future by incorporating a barcoding or indexing approach that allows for more samples per sequencing lane.

After sequencing the targeted regions to an average of 1,286 fold coverage per nucleotide, the team aligned the sequences to the human reference genome, identifying mutations present on both strands of DNA and in at least 15 percent of sequence reads. They then verified variants using Sanger sequencing and characterized each potential mutation based on its location and predicted consequences.

In the process, the researchers identified a range of point mutations as well as several small insertions and deletions, with no false-positive mutations turning up.

Based on the number of reads at each base, they were also able to obtain information on larger duplications or deletions, identifying five deletions and one duplication in the samples tested.

Those involved in the study say such risk-gene sequencing approaches may find more widespread applicability in the wake of a March ruling by a US District Court that came down against Myriad Genetics' BRCA1 and BRCA2 patents — a decision Myriad is appealing.

If such decisions are upheld in future court battles, the researchers noted that "it may be that tools for more efficient genetic testing for cancer susceptibility genes will be developed and clinically applied." That, in turn, could lead to more widespread cancer gene testing, the researchers explained.

"By allowing comprehensive parallel testing of multiple cancer susceptibility genes, we will be able to confidently identify the fraction of women with breast or ovarian cancer who carry a germline alteration in a cancer susceptibility allele and the characteristics of the tumors of patients' inherited mutations in various genes," they wrote.

For their part, the researchers are planning to use their method to test a large group of patients from families severely affected by breast cancer, Walsh said. And, he added, the team hopes their study will spur the development of guidelines that support and guide the use of high-throughput sequencing in a diagnostic setting.

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