Using mouse embryonic stem cells, researchers at the National Cancer Institute (NCI), part of the National Institutes of Health, have developed a new method to evaluate which mutations, or changes, in a gene known to increase breast cancer susceptibility, may lead to cancer.

The new test, called a functional assay, is more comprehensive and reliable than most current methods. This new test could become a useful and viable tool for genetic counselors, and may have implications beyond cancer. The researchers believe that this test could also be useful for analyzing mutations found in other human disease-related genes.

According to a NIH press release, the results of this research will be published in the August 2008 issue of "Nature Medicine" and appear online July 6, 2008.

The proteins produced by the BRCA1 (BReast CAncer gene 1) and BRCA2 genes normally help to maintain the integrity of the cell's genetic material and function as tumour suppressors. For over a decade, it has been known that alterations or defects in BRCA1, BRCA2, and their associated proteins are linked to increased risks of early onset familial breast and ovarian cancers. Studies have shown that a woman who has a mutation in one of these genes has a 35 to 85 per cent risk of developing breast cancer by age 70, compared to the average American woman's lifetime risk of 12.3 per cent.

Some individuals who want to know if they have inherited a mutation in a gene such as BRCA1 or BRCA2 that will increase their risk of developing breast or ovarian cancer are now choosing to go for genetic testing. Such tests can provide reassuring information to those who do not have harmful mutations, and can be helpful to those with harmful BRCA1 or BRCA2 mutations because when they know that they are at risk, they can work with their physicians to find the course of action that is best for them.

"However, think about those individuals who are tested and find out that they have an unclassified [minor or previously unknown] change in these genes, and they do not know what it means," said senior author Shyam Sharan, Ph.D. of the NCI's Center for Cancer Research. "There is reason to believe that a significant number of women fall into this category, and our assay is likely to improve our understanding of unclassified mutations because it allows for analysis of all types of BRCA2 mutations."

Existing methods to distinguish between harmful and minor, or neutral, alterations in the BRCA1 and BRCA2 genes are based on data from segregation analysis, which looks at genetic alterations in families with a high incidence of cancer, and uses gene sequencing to detect the presence of a genetic variation. However, this approach has limitations. Most alterations, or variants, are rare, and familial data can be insufficient, resulting in a lack of a suitable test to assess more than 1,900 known BRCA1 and BRCA 2 variations. In addition, there is currently no suitable way to evaluate minor or less common mutations in the BRCA1 and BRCA2 genes. This new functional assay expands the ability to analyze mutations in the BRCA2 gene by examining the effect that these variations have on the cell.

The new test looks for the functional significance of variations in BRCA2 using mouse embryonic stem cells. Mouse Brca2 is essential for the viability of mouse embryonic stem cells, and the assay is based on the ability of the human BRCA2 gene to complement the loss of the Brca2 gene in the mouse cells. When introduced into a Brca2-deficient mouse embryonic stem cell in laboratory experiments, functionally normal human BRCA2 variants can compensate for the mouse Brca2 deficiency. On the other hand, harmful or deleterious variants of human BRCA2 do not have this ability, leading to their identification in this test.

Lead author of this paper, Sergey Kuznetsov, Ph.D., generated a set of cells in which one copy of the BRCA2 gene is inactivated or knocked out, and the other remains active but can be inactivated later. The researchers then introduced human genetic material, taking care to maintain all of the coding sequences and regulatory elements of the BRCA2 gene. When genetic material with neutral variants of human BRCA2 was introduced, the researchers were able to subsequently remove the remaining copy of the mouse BRCA2 without compromising the viability of the cell. The addition of genetic material with harmful variants, however, resulted in either cell death or deficient DNA repair. Therefore, the assay can be used to examine genetic material with minor or previously unknown variants. If a human variation does not alter the function of the mouse BRCA2, the risk of developing cancer is probably the same as that of the rest of the population, but if the change is disruptive, the risk of developing cancer increases significantly. The researchers are also working on development of a similar test for BRCA1 variants.

The researchers hope that other human disease gene functions may be evaluated in a similar fashion using this type of analysis. This represents an efficient method of analysis in which three to five gene variants can be analyzed in two to three months. The researchers have provided preliminary validation of the functional assay by testing 17 variants, and have established the reproducibility of the technique for BRCA2. They caution, however, that only when this technique is FDA approved for use in a clinical setting will it be available to patients for diagnostic testing.

The technology behind this new assay is available for further research, and Dr. Sharan's laboratory is interested in collaborating with commercial organizations to further develop it as a product under the appropriate NIH agreements.