The functional Role of CASP8 D302H and Other Apoptosis Gene Variants In Breast Cancer

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. We recently reported that a coding single nucleotide polymorphism (SNP) in the caspase 8 gene (CASP8 D302H) is associated with a reduced risk of breast cancer. More recently we identified a CASP8 4-SNP haplotype associated with an increased risk of breast cancer. Our hypothesis is that CASP8 and other apoptotic genes influence breast cancer susceptibility via effects on the apoptotic response. Our objectives are to study the functional effects of CASP8 D302H and the 4-SNP haplotype on apoptosis induction in peripheral blood lymphocytes (PBLs). We have recruited women attending mammography screening and measured the ability of their PBLs to undergo drug-induced apoptosis. Levels of apoptosis and caspase-8 activity were determined by FACS analysis. Based on data from 61 samples, apoptosis levels range from 50% to 92% (median 78%, SD 9.5) and CASP8 protein levels 44% to 94% (median 68%, SD 13.2). We have successfully detected variations in apoptotic/CASP8 response and aim to determine whether these variations correlate with CASP8 genotype, to help us understand the mechanism of the association with breast cancer

Abstract 2844: Association of genetic variants in TNFRSF10B and breast cancer

Susceptibility to non-Mendelian forms of breast cancer likely involves many low penetrance variants from multiple genes. Recent associations between genetic variants in CASP8 and breast cancer suggest that CASP8 is one such gene contributing to breast cancer risk (Cox et al. 2007; Shephard et al. 2009). Here we investigate TNFRSF10B for associations with breast cancer. TNFRSF10B is a death receptor gene whose protein signals via caspase-8 in the extrinsic apoptosis pathway. We selected 11 tagging-SNPs (tSNPs) to represent the majority of the common genetic variation in the TNFTRSF10B gene. These tSNPs were genotyped on 1,992 independent cases and controls from Sheffield, UK. We performed 11 single marker association analyses and a haplotype-mining analysis using hapConstructor (Abo et al. 2008). HapConstructor implements a stepwise forward-backward procedure to search for haplotypes associated with disease. The algorithm allows for automatic consideration of non-contiguous SNP sets and the construction of numerous different genetic models that consider haplotypes, composite genotypes, and both monotype (chromosome-based) and diplotype (individual-based) tests. Briefly, all single locus tests are performed, then loci that surpass predefined significance thresholds are considered in all two-locus combinations, and so on. For the single marker association testing, dominant, recessive, and trend tests were performed. For the hapConstructor analysis, monotype haplotype tests and diplotype dominant and recessive tests were considered. HapConstructor is implemented within a Monte Carlo testing framework, with all p-values estimated empirically from 100,000 simulations under the null. Seven SNPs reached nominal significance in at least one of the association tests performed in the single SNP tests. Five of these were based on a recessive model (most significant single SNP yielded p = 0.002). Using hapConstructor, the most significant finding was a 4-SNP haplotype that was associated with an increase of risk (p=0.0006; OR=1.75). Four haplotypes were also identified that were associated with a decrease in risk and each obtained the same level of significance and risk (p=0.00096; OR=0.35). These results for reduced risk were obtained using the diplotype recessive model, similar to the single SNP findings. These results provide initial evidence that further common genetic variants in genes in the extrinsic apoptosis pathway, beyond CASP8, are involved in breast cancer susceptibility

The <i>CASP8</i> -652 6N del promoter polymorphism and breast cancer risk: a multicenter study

A recent study on an Asian population reported a six-nucleotide insertion-deletion polymorphism (-652 6N del) in the CASP8 promoter region to be strongly associated with a decreased risk of multiple types of cancer, including breast cancer (BC). Here, we investigate the effect of this deletion in four independent large European BC case-control studies, including data from a total of 7,753 cases and 7,921 controls. The combined per allele odds ratio (OR) was 0.97 (95% confidence interval (CI), 95% CI = 0.93-1.02). The present result indicates that the CASP8 -652 6N del variant has no significant effect on BC risk in Europeans

Frequent p16-Independent Inactivation of p14^ARF in Human Melanoma

BackgroundThe tumor suppressors p14ARF (ARF) and p16INK4A (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. MethodsWe examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. ResultsWe observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. ConclusionGenetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16