BRCA2 missense mutations and breast cancer

Investigators: Fergus Couch, Ph.D.; Noralene Lindor, M.D.

Interpretation of results from mutation screening of the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. In most cases, this is quite straightforward, but classification of rare missense variants in these genes presents a difficult problem because it is not known whether these subtle changes in the proteins alter function sufficiently to predispose cells to cancer development. A missense mutation is one in which there is only one site in the entire gene that is diiferent from normal. As missense mutations account for approximately 43 percent of all known variants detected by clinical mutation screening in BRCA1 and BRCA2 this has become a significant clinical genetics issue. Clinical utility of BRCA testing would be greatly increased if such missense mutations could be reliably classified in terms of their predisposition to disease.

The goal of Project #2 is to study a large group of these missense mutations, also called variants of uncertain significance (VUS), to determine which mutations increase breast and ovarian cancer risk and which mutations have no effect.

There are three specific aims in this project:

1. Predict which BRCA2 missense mutations predispose to cancer using a likelihood model of disease causality
2. Evaluate the influence of BRCA2 missense mutations on BRCA2 function
3. Construct a comprehensive model for estimating disease association of BRCA2 missense mutations that incorporates multiple data sources

To answer this question, the researchers have identified families that possess these mutations and recruited many family members into their study. By learning which family members (those with or without cancer) have the mutations, they will calculate the likelihood that each mutation contributes to cancer development. The investigators are also conducting laboratory studies that measure the effect of the mutations on the ability of BRCA2 to repair of DNA damage. By combining the two approaches, the researchers aim to identify the mutations that contribute to cancer and to develop an established method for investigating the many other similar mutations in BRCA1 and BRCA2.

Recently the investigators reported on the assessment of the clinical significance of 196 BRCA1 and 351 BRCA2 variants of uncertain significance identified through genetic testing. The classification of these variants was achieved using a combination of genetic and functional studies. The genetic studies involved determination of the odds of cancer causality of each mutation using a combined likelihood model encompassing information on co-occurrence of variants with other known deleterious (cancer causing) mutations, detailed analysis of personal and family history of cancer in carriers of variants and analysis of co-segregation of variants with disease in pedigrees.

Genetic data were derived from the Myriad Genetics Laboratories database of nearly 70,000 full-sequence tests of the entire BRCA1 and BRCA2 genes and additional families ascertained as part of an ongoing study of breast cancer families carrying missense BRCA1 and BRCA2 mutations at the Mayo Clinic. A total of 133 variants had odds of at least 100:1 in favor of neutrality (not causing cancer) whereas 12 had odds of 1000:1 in favor of being deleterious (cancer causing) and almost certainly predispose to breast and ovarian cancer. Another 43 had odds of at least 20:1 in favor of being deleterious and have the potential to be classified as deleterious if additional family data can be obtained. Of these, three BRCA2 and five BRCA1 variants were also defined as cancer causing because they promote aberrant RNA splicing. Thus, eight BRCA2 variants and 12 BRCA1 variants can now be considered cancer predisposing for clinical purposes. These results will prove useful for improved genetic counseling of patients and their families and for validation of other approaches to mutation classification.

As the genetic approach to classification is limited by availability of family data, the investigators further characterized a series of BRCA2 missense mutations using functional assays that measure the ability of normal and mutant forms of BRCA2 to repair DNA damage in cells. Analysis of eight missense mutations classified as deleterious using the genetic data described above showed that all of the variants disrupted the ability of BRCA2 to repair DNA damage. Likewise seven known neutral variants had no effect on BRCA2 recombination activity. The strong correlation between the functional assay and the genetic studies suggest that it may be possible to classify missense mutations in BRCA2 by functional assays alone in the absence of genetic data. This finding has important implications for classification of the many variants in BRCA2 that have been found in small numbers of families.

In the next year the research team plans to:

1. Collect additional families with BRCA1 and BRCA2 variants and combine the data with the Myriad Genetics Laboratories results to classify additional missense mutations
2. Conduct splicing analysis of 60 cell lines derived from individuals carrying intronic BRCA2 mutations in order to identify deleterious mutations without a need for genetic studies
3. Characterize additional BRCA2 mutations using the functional assays.

The researchers hope that the knowledge gained during this study will improve risk assessment and counseling for carriers of missense mutations in the BRCA2 gene; and lead to a better understanding of the BRCA2 tumor suppressor gene mutations that, in turn, might lead to better and more specific forms of treatment for people with any BRCA2 mutation.

Collaboration
Mayo Clinic Cancer Center researchers are also working with many other research groups around the world that are helping recruit families which possess the genetic mutations being studied. Major contributors include Sean Tavtigian, Ph.D., International Agency for Research on Cancer (IARC); Alvaro Monteiro, Ph.D., H. Lee Moffitt Cancer Center; David Goldgar, Ph.D., University of Utah; and Edwin Iversen, Ph.D., Duke University.