It is well established that perturbations in high penetrance genes such as BRCA1 and BRCA2 predispose to breast cancer. However, low penetrance genes are still under investigation. Some apoptotic genes (for example, BIRC5, BCL2, DR4 and DR5) have been implicated, and we reported that a coding single nucleotide polymorphism (SNP) in the caspase 8 gene (CASP8 D302H) is associated with a reduced risk of breast cancer 1. We hypothesise that CASP8 and other apoptotic genes may play an important role in breast cancer susceptibility. The objectives were to study the functional effect of CASP8 D302H on apoptosis, and to perform a case-control analysis of other CASP8 variants to determine their effect on breast cancer susceptibility.

Apoptotic activity in peripheral blood lymphocytes (PBLs) was measured using Annexin-V FITC with propidium iodide and FACs analysis. Genotyping was conducted by TaqMan™ (ABI, UK).

We detected a 68% increase in apoptosis in PBLs after treatment with CD95 ligand (R & D Systems, UK) with anti-CD95 antibody (BioLegend, UK), and are currently optimising this assay as a functional screening tool. We identified 50 SNPs in CASP8 by database searching, and 15 more putative SNPs were sequenced, one of which is novel (T51087A in exon 13). Using data from 33 SNPs with a minor allele frequency >0.05 and various haplotype-tagging SNP (htSNP) selection programs, results suggested that 11 htSNPs (PCA method) need to be genotyped to adequately capture common genetic variation within CASP8. A case-control study of these 11 htSNPs is in progress.

These methods will be used to address the hypothesis that apoptotic genes are involved in breast cancer susceptibility and treatment outcome. In the future, this research will help us understand the role of the whole pathway and whether it will be amenable to manipulation by targeted treatments.