aglogo.gif (1177 bytes)Development of a functional assay for the detection of BRCA1 and BRCA2 mutations


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 develop an assay which will test whether the protein function of BRCA1 and BRCA2 has been altered as a result of a DNA mutation.

Project description:

Several important biological roles for BRCA1 and BRCA2 have been demonstrated, and a number of observations indicate that they function in a similar cellular pathway. Both maintain genomic stability through their involvement in homologous recombination, transcription-coupled repair of oxidative DNA damage, and double-strand break repair. These roles are suggested by interactions of the Brca1 and/or Brca2 proteins with proteins known to be involved in DNA-repair, most notably RAD51 and RAD50. Furthermore, Brca1–/– and Brca2–/– murine stem cells and mice show repair deficiency and cell cycle checkpoint defects. This indicates that cells in which BRCA1/2 proteins are impaired, are more sensitive to DNA damage, which can be exploited to design a functional assay. More recently, lymphoblastoid cells carrying a heterozygous mutation in BRCA1 were shown to be slightly more radio-sensitive than wildtype controls. In this project, we shall investigate whether this is a consistent feature of all BRCA1/2 mutation carriers, and whether it is able to identify probable mutation-carriers in a blinded fashion.

Experimental design:

EBV-transformed lymphoblastoid cell lines from several carriers of a known deleterious mutation in either BRCA1 or BRCA2 are available. In addition, there are lines heterozygous for known harmless or neutral polymorphisms in BRCA1/2. Finally, we have EBV-transformed lymphoblastoid cell lines from several healthy controls available. A selection of these will be subjected to a range of different radio-sensitivity assays (see below). We will also include one or two cell lines in which the molecular defect in BRCA1/2 is unknown or uncertain, for example, from individuals requesting genetic counselling at the Department of Clinical Genetics of the LUMC (for these, the lymphocytes will need to be transformed with EBV).

Techniques to be applied:

Cell culturing, EBV-transformations, micronuclei test, the comet assay, cell survival analysis and S-phase fraction measurements (by flow cytometry) after g -irradiation

Statistical methods:

Fisher's exact test

Plan of work and time schedule:

The length of the project (24 or 36 weeks) determines the number of different cell lines that can be investigated, as well as the number of different assays that can be handled. A small project, for example, can be limited to BRCA1 only, and include only the micronuclei and comet assays. None of the proposed methodologies are technically very demanding. They are in fact routine procedures in the laboratories of Radiation Genetics, and Pathology (with whom we intimately collaborate in this project). The micronuclei test entails culturing the cells to a certain density, irradiating them, culture in the presence of cytochalasin B, fix the cells, spread the nuclei on microscopic slides, and count the number of binucleated cells with micronuclei. This is a measure for radiation-induced chromosomal damage. The comet assay is an electrophoresis-based assay: after irradiation, cells are mildly lysed, and double-stranded DNA is electrophoresed out of the nuclei into an agarose gel. The size of the resulting "tail" is a measure for the amount of double-strand breaks present.

Equipment to be used:

g -irradiation chamber; B&D Flow cytometer; light microscope; gel-electrophoresis

Patients involved

Yes, ~10

Laboratory animals involved

No

Clinical/Non-clinical

Non-Clinical

Approval required from the Committee on medical ethics

No

Approval required from the committee on the use of laboratory animals

No

Updated:  19-02-2003
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