Programme: The Genetics of Breast Cancer 
DCS-project RUL1999-2021

 

A linkage search for new breast cancer susceptibility genes

Purpose

Hereditary breast cancer has a heterogeneous genetic basis. Two genes, BRCA1 and BRCA2, jointly explain the large majority of families with either the breast-ovarian cancer syndrome, with at least one case of male breast cancer, or with at least 6 cases of breast cancer diagnosed under 60. Roughly 60% of the families with 4 or 5 cases of breast cancer diagnosed under 60, and no cases of ovarian cancer, are not accounted for by these genes. The aim of this proposal is to map and identify additional breast cancer predisposition genes by means of linkage analysis in this particular group of families. A secondary objective is the characterization, at the genetic, histological, and immunohistochemical level, of non-BRCA1/2 breast tumours, in an attempt to identify phenotypic markers associated with new breast cancer loci.

Plan of investigation

A crucial prerequisite is the availability of a sufficient number of families suitable for linkage analysis. We intend to select 150 breast cancer-only families complying with the following criteria:

  1. no ovarian cancer in the family
  2. no male breast cancer in the family
  3. BRCA1 and BRCA2 excluded by comprehensive mutation-analysis and haplotype-sharing
  4. At least 3 female breast cancer patients diagnosed under 60, of whom at least 3 cases can be genotyped (i.e., blood-sampled).

Through our previous work on hereditary breast cancer, we currently have 16 such families available for analysis. We intend to capitalize on the infrastructure created by the 7 Clinical Genetics Centres (CGCs) or Family Cancer Clinics (FCCs) in the Netherlands, and the Foundation for the Detection of Hereditary Tumours (FDHT). The FCCs have seen over 2,000 families for clinical diagnostic purposes since the cloning of BRCA1 and BRCA2 (i.e., 1995-1997) and on average, each are still seeing ~100 new families annually. We estimate that about 5% will meet the criteria set forth above. Paraffin-embedded tissues will be retrieved from all breast cancer patients if available, while all patients alive at the time of ascertainment will be blood sampled. All families meeting the clinical criteria will be subjected to comprehensive BRCA1/2 mutation-testing and haplotype analysis. The linkage search will be performed with those families in which both genes could be excluded with high certainty, by genotyping 400 microsatellite markers. A 60-marker LOH-allelotype will be compiled from 75 tumours from these families to search for candidate loci. Promising hints for linkage will be further investigated by typing closely flanking markers to reconstruct linked haplotypes, and by analysing the tumour material for LOH. This haplotype-data will be used in linkage disequilibrium analysis based on precedent with BRCA1 that a large proportion of gene carriers will share a small number of different haplotypes. Should definitive linkage evidence be obtained, then this project can achieve considerable fine-mapping of the locus to mount a gene cloning effort along standard positional cloning strategies.

Results obtained so far...

Since 1991, we have collected 197 breast cancer families, with various levels of blood sampling, in order to characterize them at the genetic level. Of these, 169 were collected as "research families". These families have been genetically characterized as part of a previous DCS-project. were invited to participate in our research on the basis that they would be informed only about the results if these were immediately relevant to them (e.g. in the event a mutation was detected). The Foundation for the Detection of Hereditary Tumours (FDHT) collected 118 families, who were broadly blood-sampled by the Department of Human Genetics in Leiden. The Clinical Genetic Centre (CGC) in Rotterdam submitted 51 families, mainly in the period 1991-1994, during which BRCA1 was mapped but not yet identified. Other CGCs submitted 28 families, mostly for diagnostic purposes. When BRCA1 and BRCA2 were identified, all 7 CGCs in the Netherlands started to offer mutation-testing in their own laboratories as part of counselling procedures, supported by National Health Insurance. No new families entered our studies since then through this routing. Initially, the FDHT collected families when they contained at least 3 first-degree relatives with breast cancer diagnosed any age, but since 1995 only when they contain at least 4 cases diagnosed under 60. The FDHT stopped to approach families independently in 1996, forwarding all newly reported families directly to the CGCs. Thus only when mediated by the CGCs did new families enter their registration, and only 17 new kindreds were collected in the period 1995-1997 under the research agreement. The distribution of the various levels of breast cancer clustering in the present collection of 169 research families is shown in the table.