Wednesday, July 9, 2008

NIH, Sanger Researchers Develop Assay for Assessing BRCA2 Variants

NIH, Sanger Researchers Develop Assay for Assessing BRCA2 Variants
[July 8, 2008]

By Andrea Anderson, a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – Scientists have developed a functional assay for distinguishing between dangerous and neutral mutations in a breast cancer susceptibility gene.

In a paper appearing online in
Nature Medicine this past Sunday, researchers from the National Cancer Institute and the Wellcome Trust Sanger Institute described how they used mouse embryonic stem cells to evaluate the functional implications of 17 BRCA2 sequence variants. They found that this assay could effectively and reliably categorize risky BRCA2 mutations — which can dramatically increase an individual’s risk of breast and ovarian cancer — as well as relatively innocuous mutations.

“Our assay is likely to improve our understanding of unclassified mutations because it allows for analysis of all types of BRCA2 mutations,” senior author Shyam Sharan, head of NCI’s Genetics of Cancer Susceptibility Section, said in a statement.

BRCA1 and BRCA2 mutations, which tend to be inherited in some families, can dramatically increase an individual’s cancer risk. For instance, those carrying a mutation in one of the genes have a 35 percent to 80 percent risk of developing breast cancer by their 70th birthday. In contrast, the average American woman has a 12.3 percent risk of developing breast cancer in that time.

But BRCA1 and BRCA2 sequence variants don’t all confer the same risk. Some are extremely deleterious (unfortunatly, that's what mine is...the complete Exon 19 is missing)
while others are neutral or low risk. Distinguishing between these extremes can be tricky, since mutations are often identified through genetic tests of families with a history of breast and ovarian cancer.

“Segregation analysis in cancer-afflicted families provides the most reliable information to distinguish between deleterious and neutral alterations identified in BRCA1 or BRCA2,” Sharan and his colleagues wrote. “However, there is an enormous need for a functional assay to classify variants for which such information is not available, because most mutations are rare, and familial data are often insufficient.”

That also makes it difficult to interpret BRCA1/BRCA2 genetic tests for those carrying unclassified or minor mutations — such as the 1,900 known BRCA1 or BRCA2 variations that don’t disrupt the gene product in an obvious way but may still affect gene function.

For this study, Sharan and his team exploited the properties of mouse embryonic stem cells to develop an assay for systematically testing BRCA2 variants. Specifically, the researchers took advantage of the fact that mouse stem cells need a functional copy of BRCA2 to survive.

The human BRCA2 gene can complement — that is, take over the function of — mouse BRCA2, but only if the human BRCA2 gene is functional. To test the functionality of various BRCA2 sequence variants, the team generated mouse embryonic stem cells that were missing one copy of BRCA2 and one conditional BRCA2 allele.

Then, they added bacterial artificial chromosomes containing individual human BRCA2 variants, inactivated the lone copy of mouse BRCA2, and looked at whether the cells could survive by relying on that human BRCA2 gene.

Variants that could not rescue the BRCA2 function in these mouse embryonic stem cells were classified as deleterious mutations, while those that could were considered neutral mutations. The researchers further characterized the variants using plating efficiency experiments and testing the cells’ sensitivity to DNA-damaging agents.

All told, the researchers tested 17 BRCA2 variants. As expected, mutations with known functional effects could not rescue the BRCA2 function while neutral mutations could. Among the previously unclassified variants, they found examples of both neutral and deleterious variants. Overall, the researchers classified eight of the 17 mutations as deleterious. The remaining nine appear to be either neutral or low risk.

While the researchers emphasized the assay’s promise for evaluating BRCA2 variants, they were quick to note that it will take time before the assay can be used as a clinical tool. “[U]ntil the assay is fully validated,” the authors warned, “caution must be exercised when using these data to make clinical decisions.”

According to a statement from the National Institutes of Health press office, Sharan is interested in collaborating with commercial organizations to develop the diagnostic test.

In the meantime, the authors are optimistic that such assays will eventually be used to characterize not only BRCA2 variants, but also variants in other disease-associated genes.

“We have established that this assay is accurate and is one of the most comprehensive among those available to date, allowing analysis of virtually any type of mutation,” they wrote, “and it may also serve as a model for investigating other human disease-associated genes that result in a phenotype detectable
in [embryonic stem] cells.”

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