Search for new breast cancer susceptibility genes

Background of research project:

A number of genes have been identified which - when mutated - predispose strongly to the development of breast cancer. These include BRCA1, BRCA2, and TP53. Mutations in these genes can be found to be inherited in families with a typical clustering of cancer. However, in certain families, primarily those without a case of ovarian cancer or male breast cancer, few mutations in these genes are found, despite the fact that some of them show multiple cases of early onset breast cancer. This has been taken as evidence that still other breast cancer predisposition genes must exist.

Research questions:

To search for genetic linkage with breast cancer in families in which the involvement of BRCA1 and BRCA2 have been excluded.

Project description:

Linkage analysis is a statistical analysis (maximum likelihood method) in which the inheritance of alleles at polymorphic DNA-markers is compared with the inheritance of the disease. In a search for linkage, polymorphic DNA markers are selected randomly, and all individuals from the families, for which a DNA-sample is available, are genotyped for these markers in the laboratory. The frequency and penetrance of the gene is modelled according to available epidemiological data. The likelihood that the marker is linked to the gene is expressed as the log of odds (or: lod score). Typically, one or a few families do not provide sufficient information to find significant evidence for linkage. A further limitation is that the trait may be caused by more than one gene, so that the chance of finding a gene is determined to a large extent by the proportion of families linked to it.

Experimental design:

Previous work has identified 22 breast cancer families without ovarian cancer and without male breast cancer, but with at least 3 cases of breast cancer diagnosed under the age of 60 for whom a DNA-sample is available. This DNA was isolated from peripheral blood lymphocytes. We have also collected paraffin-embedded tumor tissues from at least one breast cancer patient per family, and have isolated DNA from tumor cells. The DNA-samples will be genotyped with several polymorphic markers, for chromosomes regions harboring candidate breast cancer genes. With the same markers, tumor DNA will be examined for the presence of loss of heterozygosity (LOH). Many cancer predisposition genes show loss of the wildtype allele in the tumor cells of an individual carrying a mutation in the gene, and this may also be true for a new breast cancer susceptibility gene. This information can significantly improve the power of linkage analysis.

Techniques to be applied:

Polymorphic marker typing (PCR, gel-electrophoresis); software-assisted marker analysis; haplotyping; loss of heterozygosity analysis

Statistical methods:

Genetic linkage analysis; linkage under heterogeneity; linkage incoporating LOH data; automated data-processing; database maintenance (relational; Access)

Plan of work and time schedule:

The length of the project determines the number of markers that can be analyzed. After genotyping the markers, and processing of allele scoring, the chromosome region under investigation will be haplotyped. These data will be combined with results from LOH analysis with the same markers. All data will then be integrated for linkage analysis, testing for linkage to breast cancer. Results will be analyzed under the assumption of homogeneity (a single gene causes breast cancer in all 22 families) and heterogeneity (assuming two or more genes).

Equipment to be used:

an automated DNA sequencer (ABI377) for genotyping

Updated:  19-02-2003
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