aglogo.gif (1177 bytes)Clinical validation of BRCA1 and BRCA2 sequence variants of uncertain clinical significance

Background of research project:

Mutations in the BRCA1 and BRCA2 genes strongly predispose to the development of breast and ovarian cancer. For this reason, DNA-testing is offered to women at high familial risk for breast cancer. However, there is good evidence that a substantial proportion of true deleterious mutations are either technically missed by the applied mutation-detection methodology, or being misclassified as being of unknown clinical significance. The latter category in particular includes missense mutations and intronic changes with unknown effect on mRNA-processing ("unclassified variants" or UVs). The exclusion power of a negative test-result of current mutation-detection technology is therefore still limited.

Research questions:

To validate the disease-association of unclassified variants in BRCA1 and BRCA2 by analysis of linkage, mRNA-processing, loss of heterozygosity and protein staining in tumors.

Project description:

In this project we want to find evidence that either supports or refutes the possibility that an unclassified sequence variant is causally related to disease outcome in carriers. A genetic variant in BRCA1 or BRCA2 could either be an irrelevant polymorphism, a low-risk variant, or a previously unrecognized high-risk variant. It is impossible to distinguish between these possibilities on the basis of a single test-result. Only a comprehensive approach, incorporating data on mutation type, clinical validation, population genetics, and functional consequences of the DNA change on the protein, can provide a meaningful classification of genetic variation. We shall here focus on the first three aspects.

Experimental design:

During previous mutation analysis in breast cancer families, we already have identified 25 missense and 16 unclassified variants of which the relevance to disease is currently unknown. The inheritance of the variant will be followed in the family where it was discovered, to see if it co-segregates with disease (linkage analysis). The mRNA from a carrier will be investigated, by RT-PCR, to investigate whether the variant causes any aberrant splicing. We will examine one or two breast or ovarian cancers for the presence of loss of heterozygosity. Tumors that develop in carriers of true deleterious mutations generally show loss of the wildtype allele. Finally, we shall perform immunostaining with antibodies directed against the BRCA1 and BRCA2 proteins in these tumors. Most tumors in which BRCA1/2 play a significant role have lost protein expression. At any stage along this series of experiments, a DNA variant can be unmasked as unrelated to disease (e.g., no co-segregation, or loss of "mutant" allele in tumors). To verify an alledged polymorphism about 200 healthy controls will be genotyped to establish its allele frequency.

Techniques to be applied:

PCR; RT-PCR; DNA sequencing; loss of heterozygosity analysis (genotyping on an automated DNA sequencer); immunohistochemistry (in collaboration with the Department of Pathology).

Statistical methods:

Fishers's exact test

Plan of work and time schedule:

The length of the project (12, 24, or 36 weeks) will determine how many variants can be investigated. All aspects of the variant (family analysis, mRNA, and tumor studies) will need to be completed in order to arrive at a classification.

Equipment to be used:

Thermocycler (PCR); automated DNA sequencer; light microscope

Patients involved

Yes, 10-50

Laboratory animals involved




Approval required from the Committee on medical ethics


Approval required from the committee on the use of laboratory animals


Updated:  19-02-2003
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