Researchers Develop a Method to Evaluate
Variations Identified in Breast Cancer Susceptibility Genes
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. The results of this research will be published in the August
2008 issue of Nature Medicine and appear online July 6,
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 tumor 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 percent risk of developing breast cancer by age 70,
compared to the average American woman’s lifetime risk of 12.3
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 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.
For more information on Dr. Sharan’s laboratory, please go to http://ccr.cancer.gov/Staff/staff.asp?profileid=5567.
For more information on obtaining the technology for research
and development, please visit the NCI-Technology Transfer Center
website at http://ttc.nci.nih.gov/.
For more information on genetic testing for BRCA1 and BRCA2,
please visit http://www.cancer.gov/cancertopics/factsheet/Risk/BRCA .
For more information about cancer, please visit the NCI website
at http://www.cancer.gov, or
call NCI’s Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).
The National Institutes of Health (NIH) — The Nation's
Medical Research Agency — includes 27 Institutes and Centers
and is a component of the U.S. Department of Health and Human Services.
It is the primary federal agency for conducting and supporting basic,
clinical and translational medical research, and it investigates
the causes, treatments, and cures for both common and rare diseases.
For more information about NIH and its programs, visit www.nih.gov.
Reference: Sharan SK, Liu P, Kuznetsov SG. Mouse
ES-cell-based functional assay to evaluate mutations in BRCA2. Nature
Medicine, August 2008, Vol. 14, No. 8.