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Association of a Common Variant of the CASP8 Gene With Reduced Risk of Breast Cancer

Gordon MacPherson, Catherine S. Healey, M. Dawn Teare, Sabapathy P. Balasubramanian, Malcolm W. R. Reed, Paul D. P. Pharoah, Bruce A. J. Ponder, Mark Meuth, Nitai P. Bhattacharyya, Angela Cox

Affiliations of authors: Division of Genomic Medicine, University of Sheffield Medical School, Sheffield, U.K. (GM, MDT, MM, AC); Department of Oncology, University of Cambridge, Strangeways Research Laboratory, Cambridge, U.K. (CSH, PDPP, BAJP); Academic Surgical Oncology Unit, University of Sheffield Medical School, Sheffield, U.K. (SPB, MWRR); Saha Institute of Nuclear Physics, Calcutta, India (NPB)

Correspondence to: Angela Cox, PhD, Institute for Cancer Studies, University of Sheffield Medical School, Beech Hill Rd., Sheffield S10 2RX, U.K. (e-mail: a.cox{at}shef.ac.uk)


    ABSTRACT
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Apoptosis, or programmed cell death, is perturbed in many cancers. We tested the hypothesis that coding polymorphisms of the death receptor 4 (DR4), caspase-8 (CASP8), and caspase-10 (CASP10) genes might act as low-penetrance breast cancer genes. Single-nucleotide polymorphisms (SNPs) of these genes were genotyped in a series of 999 breast cancer case patients and 996 control subjects from Sheffield, U.K., and in a second, independent U.K. population of 2192 case patients and 2262 control subjects from East Anglia. In the Sheffield study, the rare H allele of CASP8, D302H, was associated with a reduced risk of breast cancer in a dose-dependent manner (Ptrend = .007). Furthermore, the CASP8 D302H association, but not that of the other CASP8 SNPs examined (T21914C, G50121A, and G50358A), was replicated in the East Anglian study. The combined adjusted odds ratios for breast cancer were 0.83 (95% confidence interval [CI] = 0.74 to 0.94) for the DH heterozygote and 0.58 (95% CI = 0.39 to 0.88) for the HH homozygote (Ptrend = .0002, adjusted for study). The reproducible, dose-dependent association of CASP8 D302H with breast cancer indicates the potential importance of inherited variation in the apoptosis pathway in breast cancer susceptibility.


The known breast cancer susceptibility genes account for only a minority of breast cancer cases, and it has been estimated that there is a 40-fold range of genetic risk for breast cancer in the general population (1,2). Single-nucleotide polymorphisms (SNPs) of genes from a number of plausible etiologic pathways are under investigation to determine their contribution to this variation in susceptibility.

Whereas genes involved in DNA repair have been shown to act as low-penetrance breast cancer genes (35), the role of genetic variation in the apoptosis pathway has been less well investigated. Apoptosis, or programmed cell death, is an essential defense against hyperproliferation and cancer (6). Apoptosis results from a cascade of protease reactions carried out by caspases (aspartate-specific cysteine proteases) and regulated by the B-cell CLL/lymphoma 2 (BCL2) family of proteins (7). External death signals are received by cell surface receptors such as death receptors 4 and 5 (DR4 and DR5, also known as TNFRSF10A and TNFRSF10B), leading to the activation of initiator caspases 8 and 10 (8). Mutations in DR4 and DR5 have been implicated in metastatic breast cancer (9). We tested the hypothesis that SNPs in key genes involved in the initiation of apoptosis—caspase-8 (CASP8), caspase-10, (CASP10), and DR4—can act as low-penetrance breast cancer susceptibility genes, using two large U.K. series of breast cancer case patients and control subjects.

The first study population comprised patients with histopathologically confirmed breast cancer recruited from surgical outpatient clinics at the Royal Hallamshire Hospital, Sheffield, between November 1998 and June 2002. Ninety-four percent of the case patients had invasive cancer. Healthy control subjects were recruited from those attending the Sheffield Breast Screening Service between September 2000 and August 2002 if their mammograms showed no evidence of a breast lesion. All case patients and control subjects were residents of Sheffield and were white Anglo-Saxon. These patients and control subjects were described previously (4). There is no statistically significant difference in median age of diagnosis/recruitment between the case patients and control subjects (control subjects median = 57, range = 45–80 years; case patients median = 58, range = 28–91 years; P = .85, two-sided Wilcoxon rank sum test).

The second study population comprised case patients with invasive breast cancer drawn from the Anglian Breast Cancer (ABC) Study, an ongoing population-based study of breast cancer case patients ascertained through the East Anglian Cancer Registry (10). Patients diagnosed before age 55 years since 1991 and still alive in 1996 (prevalent case patients, median age = 48 years), together with all patients under 70 years at recruitment who were diagnosed between 1996 and 2000 (incident case patients, median age = 52 years), were eligible. Female control subjects were randomly selected from EPIC-Norfolk, a component of the European Prospective Investigation of Cancer (EPIC), which comprises 25 000 individuals resident in Norfolk (East Anglia), aged 45–74 years (11). More than 98% of case patients and control subjects are of white ethnicity. The median age of diagnosis of case patients is lower than the median age of recruitment of the control subjects (control subjects median = 65, range = 45–81 years; case patients median = 51, range = 25–65 years; P<.0001, two-sided Wilcoxon rank sum test). The study was approved by the South Sheffield local research ethics committee, the Norwich local research ethics committee, and the Anglia and Oxford Multi-Centre research ethics committee, and written informed consent was obtained from all subjects.

We used the National Center for Biotechnology Information dbSNP database (http://www.ncbi.nlm.nih.gov) and literature searches to identify nonsynonymous coding SNPs in the CASP8, CASP10, and DR4 genes. For CASP8 and CASP10, we identified only one nonsynonymous SNP with rare allele frequency greater than 0.05 (D302H in exon 12 of CASP8 and I522L in exon10 of CASP10, respectively). For DR4, the least conservative variant from two nonsynonymous SNPs in tight linkage disequilibrium was chosen for genotyping (R209T in exon 4 of DR4) (12).

Genotyping was carried out by 5' nuclease polymerase chain reaction using the Taqman allelic discrimination system (Applied Biosystems, Warrington, U.K.), using primers and probes described in Supplementary Table A (http://jncicancerspectrum.oupjournals.org/jnci/content/vol96/issue24). The SNP assays were validated by regenotyping 10%–20% of samples, and concordance rates of more than 99.5% were attained for all SNPs. As a further check for possible genotype error, we compared genotype frequencies in the control subjects with those expected under Hardy–Weinberg equilibrium (HWE). CASP8 G50358A was inconsistent with HWE in the Sheffield sample and was not analyzed further (P = .01; Table 1); T21914C was consistent with HWE in the Sheffield sample but not in the East Anglian sample (P = .39 and .05, respectively; Table 1). Because the T21914C SNP is rare, this slight deviation from HWE will not have any major effect on the frequencies of the common haplotypes.


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Table 1. Genotype frequencies for apoptosis gene SNPs in case patients and control subjects*

 
Genotype frequencies for individual markers were compared in case patients and control subjects using Pearson’s chi-square test (2 degrees of freedom [df]). Unconditional logistic regression was used to determine odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for study when the study populations were combined. Tests for trend of odds were based on likelihood ratio tests assuming a multiplicative model. Adjustment for potential confounding variables, including age at diagnosis, parity, age at first full-term pregnancy, menopausal status, age at menarche, and age at menopause, was carried out within the logistic regression framework, with variables coded as described in the legend to Supplementary Table B (http://jncicancerspectrum.oupjournals.org/jnci/content/vol96/issue24). The hapipf command within Stata (version 8.0; Stata Corporation, College Station, TX) was used to estimate haplotype frequencies and test for association with breast cancer, stratifying for study (13). The hapipf command uses the expectation-maximization algorithm to resolve phase combined with a log-linear model to estimate expected haplotype frequencies. Haplotype odds ratios and standard errors (and hence confidence intervals) were calculated from the estimated haplotype frequencies obtained from hapipf. This method underestimates the true standard errors because it does not take into account that the haplotype frequency estimates were based on unphased data. All statistical tests were two-sided.

The Sheffield study sample of 999 case patients and 996 control subjects was genotyped for CASP10 I522L, CASP8 D302H, and DR4 R209T. No statistically significant difference in CASP10 or DR4 genotypes between case patients and control subjects was observed (P = .13 and .33, respectively; Table 1). However, the CASP8 DH and HH genotype frequencies were lower in case patients than in control subjects, and this association was statistically significant (P = .03; Table 1).

To confirm the observed association of CASP8 D302H with breast cancer, we undertook a replication study using 2262 control subjects and 2192 case patients from East Anglia. We also genotyped additional SNPs in CASP8. Three SNPs were chosen that, together with D302H, had previously been identified from a total of 13 CASP8 SNPs as capturing 95% of the haplotype diversity of the CASP8 gene in a European population (14). These haplotype-tagging SNPs were T21914C (exon 3), G50121A (intron 12), and G50358A (exon 13). T21914C showed no association in either sample (P = .22 and .81 for the Sheffield and East Anglian studies, respectively; Table 1). G50121A showed some evidence of association with breast cancer in the Sheffield sample but not in the East Anglian sample (P = .04 and .60, respectively; Table 1). G50358A showed no association in the Sheffield sample (P = .12; Table 1) and was not investigated further because the Sheffield control genotype frequencies were inconsistent with HWE (P = .01; Table 1). In contrast with the other CASP8 SNPs, a statistically significant association of D302H with breast cancer was evident in the East Anglian sample (P = .03; Table 1). When the data from Sheffield and East Anglia were combined, the CASP8 D302H association with breast cancer remained statistically significant and was dose dependent (for DH, OR = 0.83, 95% CI = 0.74 to 0.94; for HH, OR = 0.58, 95% CI = 0.39 to 0.88; Ptrend = .0002; Table 2). There was no evidence of heterogeneity in odds ratios between the two studies (P = .47 and .98, respectively, for DH and HH; Table 2). D302H genotype frequencies were similar between case patients above and below the median age at diagnosis (P = .64 and .80 [2 df] for Sheffield and East Anglia, respectively). Furthermore, there was no evidence of confounding of the D302H association in the combined studies by known breast cancer risk factors such as age, parity, and endogenous hormone-related variables (Supplementary Table B at http://jncicancerspectrum.oupjournals.org/jnci/content/vol96/issue24).


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Table 2. Odds ratios for CASP8 SNPs in the Sheffield and East Anglian populations*

 
Haplotype analysis indicated that the haplotype carrying the H allele at D302H was associated with a reduced risk of breast cancer, with an odds ratio similar to that of the D302H SNP alone (haplotype OR = 0.88, 95% CI = 0.79 to 0.98). The odds ratios for the association of all of the other haplotypes with breast cancer were close to 1.0 (Supplementary Table C at http://jncicancerspectrum.oupjournals.org/jnci/content/vol96/issue24).

Taken together, these data are consistent with a model in which D302H is either itself a causative variant or is in strong linkage disequilibrium with an unknown causative variant. The functional effect, if any, of the aspartate-to-histidine change at residue 302 in caspase-8 is as yet unknown. Aspartate-302 is conserved between mouse and human caspase-8 and lies on the surface of the protein. Thus, the D302H change could affect autoprocessing of procaspase-8 molecules or caspase-8 interactions with the antiapoptotic molecule CASP8 and FADD-like apoptosis regulator (CFLAR). Functional studies are required to test these possibilities. With genetic association studies of this type, the possibility of false-positive results must also be considered, and Wacholder et al. have recently suggested the false-positive report probability (FPRP) as an indicator (15). The FPRP depends on the power of the study, the observed P value, and the prior probability that the SNP or gene under investigation is involved in the disease (15). The authors suggest prior probabilities in the .01 to .001 range for functional SNPs or strong candidate genes, or .0001 to .00001 for anonymous noncoding SNPs. Using a prior probability of .001, and the observed Ptrend of CASP8 D302H for both populations combined of .0002, the FPRP is .27. This finding indicates that the result presented here has a less than 30% probability of being a false positive using these criteria. Further, large case–control studies of D302H will ultimately resolve this issue.

Few published studies have reported associations between apoptosis gene SNPs and cancer, although mutations in CASP8, CASP10, and DR4 have been reported in hematologic and digestive tract cancers (1619). The DR4 T209R SNP has been found to be associated with head and neck cancer and lung cancer in a small study (12). In a separate study, the rare allele was associated with a reduced risk of bladder cancer (20). We found no statistically significant association of either the DR4 T209R SNP or the CASP10 I522L SNP with breast cancer risk. However, the use of the haplotype-tagging approach for these genes would provide more confidence in these negative results.

In summary, CASP8 D302H was associated with a reduced risk of breast cancer in a dose-dependent manner in two large case–control series. Individuals with two copies of the H allele were at approximately 40% lower risk of breast cancer compared with those homozygous for the D allele. To our knowledge, this is the first report of an association between an apoptosis gene and breast cancer.


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Supported by the Breast Cancer Campaign, Yorkshire Cancer Research and Cancer Research UK (CRUK). PDPP is a Senior Research Fellow of CRUK, and BAJP is a Gibb Fellow of CRUK.

We thank Helen Cramp, Jane McDaid, and Sue Higham for assistance with recruitment and Dan Connley for data management of the Sheffield study; the ABC Study team and general practitioners who helped recruit the East Anglian case patients and the EPIC team for access to, preparation, and selection of DNA from East Anglian control subjects; and all the people who took part in the study.


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Manuscript received April 14, 2004; revised October 2, 2004; accepted October 14, 2004.


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