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NCI Team Develops Mouse Stem Cell Assay for Classifying BRCA1 Mutations

NEW YORK (GenomeWeb News) – Researchers from the National Cancer Institute have developed a mouse embryonic stem cell-based assay to distinguish deleterious from neutral mutations in the human BRCA1 gene.

The team used the assay to test more than a dozen mutations in the human breast cancer risk gene BRCA1. In each case, the assay appeared to accurately predict which mutations are deleterious in humans and which are neutral.

The findings, which appeared online last night in The Journal of Clinical Investigation, suggest that many, but not all, BRCA1 mutations in the National Institute of Health's Breast Cancer Information Core or BIC database are deleterious.

BRCA1 or BRCA2 can dramatically increase an individual's risk of breast and/or ovarian cancer, with mutations in the genes turning up in the majority of familial breast cancer cases.

"If you have a deleterious mutation, the risk of developing the disease is very high," senior author Shyam Sharan, head of the genetics of cancer susceptibility section at the NCI's mouse cancer genetics program, told GenomeWeb Daily News.

But the consequences of many of the roughly 800 BRCA1 and 1,100 BRCA2 mutations listed in the BIC database so far are unknown, and with a 2002 study by researchers at Myriad Genetics suggesting as many as 13 percent of individuals in the general population carry so-called "variants of unknown clinical significance," or VUSs, improved methods are needed for predicting which mutations pose a cancer risk and which don't, the NIH researchers noted.

At the moment, Sharan said, BRCA1 and BRCA2 mutations are usually classified as deleterious or neutral depending on how they segregate within families and/or within the overall population. But such information is not always available.

For the current study, he and his colleagues employed mouse embryonic stem cells to differentiate between deleterious and neutral BRCA1 mutations — a method similar to the one they described last year in Nature Medicine for determining the functional significance of various BRCA2 mutations.

The work stemmed from the team's effort to understand the functions of the BRCA1 and BRCA2 genes using a mouse model system, Sharan explained.

The team created mouse embryonic stem cells that were missing one BRCA1 allele and had a conditional copy of the other.

Because completely removing the gene is lethal to cells, the researchers could then add human BRCA1 constructs with bacterial artificial chromosomes and see which of these rescue mouse stem cell function. BRCA1 constructs that could restore normal mouse stem cell function were classified as having neutral mutations, whereas those that could not are thought to have deleterious mutations.

Indeed, when the team examined three known deleterious mutations previously shown to increase breast and/or ovarian cancer risk, they found that versions of BRCA1 carrying these changes could not rescue stem cell function in the absence of the mouse gene. On the other hand, a version of the human gene with a known neutral variation could.

The researchers also tested several BRCA1 constructs containing either VUSs, variants predicted to be deleterious, or variants affecting BRCA1 phosphorylation sites. Again, only versions of the gene with neutral mutations could rescue mouse stem cell function, results that the team verified in additional mouse and cell line experiments.

"Based on our findings, we believe that the [embryonic stem] cell-based assay is a reliable tool for understanding the functional significance of BRCA1 VUSs," Sharan and his co-authors concluded. "We also demonstrate that the mutant [embryonic stem] cells we generated are very useful to gain mechanistic insight into the role of BRCA1 in different biological processes."

Since publishing their BRCA2 assay, the team has generated around 25 to 30 more BRCA2 mutations and tested them in their mouse embryonic stem cell system. They are also in the process of generating and testing additional BRCA1 mutations.

"In every case, we are finding our results from this assay are exactly what the family linkage data suggests," Sharan said.

He noted that NCI is interested in licensing the BRCA1 and BRCA2 mouse stem cell assays. And although the results still have to be validated clinically, Sharan said, he believes the method is promising — not only for looking at the effects of BRCA1/BRCA2 mutations, but for other risk genes as well.

In a commentary also appearing in JCI, University of Pennsylvania researchers Susan Domchek and Roger Greenberg agreed that the method could pave the way to a better understanding of risk for these and other genes.

The pair also noted that coming up with a method for distinguishing between harmless and dangerous variants could eventually improve the level of risk information available to both patients and their physicians.

"The work … provides a new set of tools to tackle this very significant challenge," Domchek and Greenberg noted. "In addition, BAC reconstitution approaches have the potential for application in the study of VUSs of other inherited cancer susceptibility genes, including BRCA1, p53, and the colorectal cancer-associated genes MLH1 and MSH2."

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