Verkennen van het genetisch landschap van erfelijke borst- en eierstokkanker

The molecular basis of hereditary breast and ovarian cancer (HBOC) is very complex. This was illustrated in Chapter 1. Inherited BRCA1 and BRCA2 mutations are associated with a high risk for the disease. Other high, moderate and low penetrance genetic factors playing a role in HBOC are known. Nevertheless, for most of these genes, information is lacking to determine with accuracy their associated risk, which is a pitfall for genetic counselling. For this reason, most of these genes are not (yet) included in the diagnostics. This may change because recent research studies shown that the analysis of these genes definitely solve more families. In Chapter 2, we highlighted the operating workflow as it is performed at UZ Leuven. The genetic test included the analysis of BRCA1, BRCA2 and the detection of the c.1100delC CHEK2 mutation. This contributed to solve 15% of the families. Indeed, these mutations are deleterious as they lead to proteins that are expected to be non-functional, and therefore these mutations were reported to the physicians. The genetic basis of the HBOC phenotype thus remains unexplained in a great majority of the families. Besides the pathogenic mutations, DNA variations whose pathogenicity is uncertain (class 3 variants) were identified in 2% of the families. The associated risk of these variants to develop the disease thus is unknown. For this reason, more information is needed to further classify them (the underlying rationale here can be summarized as: clinical effect versus no clinical effect). We showed the difficulty to determine with certainty the classification. We also highlighted the fact that reporting variants of unknown pathogenicity can jeopardize the interpretation of the genetic report. A way to solve more families is for example to further analyse the functional effect of such variants. In Chapter 3, we studied the functional effect of BRCA2 variants with a complementation assay. Selected variants were introduced into a BAC containing the whole human BRCA2 gene by a double selection in SW102 E. coli cells. The mutated BAC was subsequently stably transfected into the Brca2-deficient V-C8 hamster cells. Our validation relied on variants with known functional effect and by testing them with two in vitro assays. Both types of variants (functional versus non-functional) were distinguished from each other by their number of nuclear centrosomes and RAD51 foci in response to irradiation. Although this method is labour intensive, we are quite confident that variants of unknown significance will be successfully analysed in the near future. In Chapter 4, we showed how the Next Generation Sequencing (NGS) method was evaluated with an amplicon-based strategy on the Roche GS-FLX platform, the first sequencer of second generation that was able to sequence relatively long reads. The coding regions of the BRCA1/2 genes were entirely covered and the variant calling was successful (analytical sensitivity of 100%). We also succeeded in analysing the raw data in repetitive, homopolymeric stretches, and we could thereby circumvent the lack of specificity of the pyrosequencing method. We showed that this technique allowed us to maintain a good balance between the accuracy of the test and the turn-around-time per analysis. NGS is still in use today on the Miseq platform (Illumina). In Chapter 5, the moderate penetrance genes RAD51D and RAD51C, both expected to increase the risk of ovarian cancer, were analysed in the context of an international collaboration. Classical mutational analysis allowed to identify one disease-causing mutation in RAD51D, while no deleterious mutation was found in RAD51C. This showed that the frequency of the pathogenic mutations in the RAD51C/D genes in our patients cohort was relatively low.List of abbreviations ... 1 Full description of gene names ... 2 Chapter 1: Introduction ... 3 Objectives ... 45 Chapter 2: Genetic testing for HBOC at UZ Leuven ... 47 Chapter 3: Functional analysis of BRCA2 variants of unknown significance ... 69 Chapter 4: Early implementation of Next Generation Sequencing ... 101 Chapter 5: Analysis of the moderate penetrance genes RAD51D & RAD51C ... 123 Chapter 6: General discussion and conclusions ... 139 Summary – Samenvatting - Résumé ... 149 Appendix ... 155 Curriculum vitae ... 161 Dankwoord, remerciements ... 165nrpages: 178status: publishe