Programme: The Genetics of Breast Cancer
Protein truncation mutations in the BRCA1 and BRCA2 genes strongly predispose to the development of breast and ovarian cancer. Two unresolved issues preclude accurate risk assessment in mutation carriers. Firstly, there is good evidence that a substantial proportion of true deleterious mutations are either technically missed 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 limited. Secondly, despite high overall cumulative risks, disease outcome in carriers is highly variable and probably co-determined by other factors, both genetic and environmental. The purpose of this project is thus twofold: 1) To improve the specificity and sensitivity of current genetic testing; 2) To identify genetic factors that may modify the risk conferred by BRCA1 and BRCA2.
We intend to address these issues by a combination of population genetic and cell biological analyses, for which we have already collected a variety of resources. A small number of families have been identified with suggestive evidence of being linked to BRCA1 or BRCA2, but in which mutation-testing has thus far remained negative. These families might be caused by hitherto unknown mutation mechanisms, which will be investigated by comprehensive sequence and fiber-FISH analyses. The functional relevance of the missense and other UVs in BRCA1 and BRCA2 will be investigated by clinical validation (i.e., co-segregation with disease, pathology and LOH in tumors), and determination of the allele-frequencies of remaining candidates in patients and controls (healthy women aged 85 or over). The resulting "likely disease-causing mutations" will then be assayed in vitro to assess their capability to affect the biochemical function of the Brca1 and Brca2 proteins. A validated functional assay is not yet available and will be developed on the basis of the involvement of both proteins in DNA damage repair. Through our previous mutation-screening work, we already have identified a number of UVs in both BRCA1 and BRCA2, while several others have been made available through the national BRCA1/2 mutation database of the Dutch DNA Diagnostic Laboratories. Finally, we have identified 307 women with a deleterious mutation in a BRCA gene for which we have DNA stored. Within the framework of the ongoing, DCS-supported GEO-project, we intend to increase this to >1,200 samples, which will serve as a national BRCA-carrier DNA-bank, accessible for all project-participants. This collaboration shall determine the frequency of polymorphic variants in candidate modifier genes in BRCA gene carriers in a case-case association study-design. The genotype data will be stored centrally to allow joint analysis with environmental factors as collected in the GEO-project. In the current proposal, candidates will include genes known to be involved the cellular BRCA- and DNA damage repair pathways, some of which will be identified through our own microarray analysis of the radiosensitive phenotype of EBV-transformed lymphoblasts from heterozygous mutation carriers.
This study will provide insight into the population genetic characteristics of BRCA1 and BRCA2 genetic variation among the Dutch. It could reveal new mutation mechanisms, and will unmask missense mutations as deleterious variants. A functional assay as a pre-screening modality will greatly direct diagnostic mutation-testing. Further insight into the existence of genetic modifiers will be obtained. All are crucial pieces of information to optimise BRCA DNA-testing in the Netherlands, as well as individual risk assessment in the clinical setting.