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Carcinogenesis Advance Access originally published online on January 8, 2007
Carcinogenesis 2007 28(5):1079-1086; doi:10.1093/carcin/bgl256
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Breast cancer risk associated with genotypic polymorphism of the mitotic checkpoint genes: a multigenic study on cancer susceptibility

Yen-Li Lo1,3, Jyh-Cherng Yu4, Shou-Tung Chen6, Giu-Cheng Hsu5, Yi-Chien Mau1, Show-Lin Yang1, Pei-Ei Wu1 and Chen-Yang Shen1,2,7,*

1 Institute of Biomedical Sciences
2 Life Science Library, Academia Sinica, Taipei 11529, Taiwan
3 Division of Biostatistics and Bioinformatics, National Health Research Institute, Chu-Nan 350, Taiwan
4 Department of Surgery
5 Department of Radiology, Tri-Service General Hospital, Taipei 114, Taiwan
6 Department of Surgery, Changhua Christian Hospital, Changhua 500, Taiwan
7 Graduate Institute of Environmental Science, China Medical University, Taichung 407, Taiwan

* To whom correspondence should be addressed. Tel: +886 2 2789 9036; Fax: +886 2 2782 3047; Email: bmcys{at}ibms.sinica.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aneuploidy occurs early during tumorigenesis and may contribute to tumor formation. Tumor cells become aneuploid as a result of aberrant mitotic divisions, suggesting a tumorigenic contribution of the mechanisms in maintaining chromosomal number stability. We therefore speculated that the genes TTK, MAD2L1, BUB1, BUB1B and PTTG1 (Securin), jointly implicated in the regulation of mitotic checkpoint, might be associated with breast tumorigenesis. To test this hypothesis, this case–control study of 698 primary breast cancer patients and 1492 healthy controls examined single-nucleotide polymorphisms (SNPs) in these mitotic checkpoint genes to define their tumorigenic contribution. Because estrogen is known to promote breast cancer development via its mitogenic effect leading to malignant proliferation of breast epithelium and the mitotic checkpoint genes are involved in regulating mitosis, we were also interested in knowing whether any association between genotypes and breast cancer risk was modified by reproductive risk factors. Support for these hypotheses came from the observations that (i) two SNPs in TTK and PTTG1 were associated with breast cancer risk; (ii) haplotype and haplotype combination analyses in TTK, BUB1B and PTTG1 revealed a strong association with breast cancer risk; (iii) a trend to an increased risk of breast cancer was found in women harboring a greater number of putative high-risk genotypes/haplotypes of mitotic checkpoint genes and (iv) a significant interaction between high-risk genotypes/haplotypes and reproductive risk factors in determining breast cancer risk was defined. This study provides new support for the mutator role of mitotic checkpoint genes in breast cancer development, suggesting that breast cancer could be driven by genomic instability associated with variant mitotic checkpoint genes, the tumorigenic contribution of which could be enhanced as a result of increased mitosis due to estrogen exposure.

Abbreviations: aOR, adjusted odds ratio; BMI, body mass index; CI, confidence interval; CIN, chromosomal instability; FFTP, first full-term pregnancy; FTP, full-term pregnancy; LD, linkage disequilibrium; OR, odds ratio; SNP, single-nucleotide polymorphism


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Aneuploidy, or an abnormal chromosome content, the most common characteristic of human solid tumors, occurs early during tumorigenesis and may contribute to tumor formation (1,2). Tumor cells become aneuploid as a result of aberrant mitotic divisions (3,4) providing support for the idea of a tumorigenic contribution of the mechanisms responsible for maintaining chromosomal number stability. However, if the genes responsible for maintaining the fidelity of the mitotic machinery are as important a cause of chromosome instability leading to cancer formation as suggested, it is puzzling that, except in the case of a rare aneuploidy-cancer predisposition syndrome (5), there is no genetic evidence linking common cancers and mutated mitotic checkpoint genes (5), thus preventing any conclusion to be made about the role of these genes in human cancer development. Furthermore, in contrast to the high frequency of aneuploidy seen in cancers, the frequency of somatic mutations in mitotic checkpoint genes, including MAD1 and BUB1 (6,7), is rare. We recently proposed a hypothesis (the ‘hide-then-hit’ hypothesis) (8) that may explain these paradoxes and suggested that, since mitotic checkpoint genes are crucial for cells to maintain chromosomal stability, any severe defects (e.g. mutation) in these genes would result in genomic instability and trigger cell death by cell-cycle surveillance (9–11). Thus, for these high-penetrant mitotic checkpoint genes, only subtle defects arising from low-penetrance (risk) alleles (e.g. hypomorphic mutants or polymorphic variants) would escape cell-cycle surveillance and lead to the accumulation of the unrepaired DNA damage required for tumor formation (8). Our recent finding that polymorphic variants of Aurora-A/STK15/BTAK, a gene implicated in the regulation of centrosome duplication, are associated with breast tumorigenesis provides support for this hypothesis (12). Since single-nucleotide polymorphism (SNP) is the most frequent and most subtle genetic variation in the human genome and has great potential for application to association studies of complex diseases (13), the first aim of the present study was to comprehensively examine the association between SNPs in the mitotic checkpoint genes, TTK, MAD2L1, BUB1, BUB1B and PTTG1 (Securin), and the risk of breast cancer development. The functions of these genes in regulating individual steps of mitosis, including the correct attachment of chromosomes to the spindle (MAD2L1, BUB1, BUB1B and TTK) and sister chromatid separation (PTTG1), are well defined (1–4).

Since estrogen is known to promote breast cancer development via its mitogenic effect, leading to malignant proliferation of the breast epithelium (14,15), it is tempting to speculate that there might be a joint effect on cancer risk of the genotype of mitotic checkpoint genes and reproductive risk factors (prolonged exposure to estrogen or susceptibility to estrogen). The second aim of the present study was therefore to examine this hypothesis, as the definition of a joint effect could yield valuable clues to the association between breast tumorigenesis and estrogen.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Study subjects
This case–control study is part of an ongoing cooperative study aimed at understanding the causes of breast cancer in Taiwan that is characterized by a low incidence (16), early tumor onset (17), hormone dependency (18) and novel genomic alterations (19,20). Because of the low incidence, which suggests an overall lower effect of common risk factors (21), and because of its homogenous genetic background (22), the Taiwanese population has certain advantages for studying the effects of subtle genetic variations, such as SNPs. Furthermore, the use of this genetically homogenous population reduces the chance of false positives due to population stratification (12). The present study included 698 female breast cancer patients and 1492 healthy female controls. This study was approved by the Institutional Review Board, and all subjects gave their informed consent.

All breast cancer patients had pathologically confirmed incident primary infiltrating ductal carcinoma of the breast (cases of lobular carcinomas were not included) and 6% had a family history of breast cancer (mother or sister). All were diagnosed and treated at the Tri-Service General Hospital and the Changhua Christian Hospital between March 2002 and July 2004; these patients accounted for almost all (>90%) women with breast cancer attending our breast cancer clinics during the study period, the remaining patients being excluded because of a lack of suitable blood specimens. No significant differences in breast cancer risk factors were found between the included and excluded women. More importantly, because the clinics in which this study was performed are two of the major breast cancer clinics in northern Taiwan, our patients accounted for a significant proportion (~20%) of all breast cancer cases diagnosed during the study period in this region.

Control subjects were cancer-free individuals randomly selected from women attending the health examination clinic of the same hospital during the same study period. Women with a prior history of cancer or breast disease were excluded. These women underwent a 1-day comprehensive health examination (including routine breast screening using X-ray mammography and ultrasonic examination of the breasts), and any showing any evidence of breast cancer, suspicious precancerous lesions of the breast or other cancers were excluded. These controls accounted for ~40% of all women attending the clinics, and no significant differences were found in terms of socio-economic status between those included and those not included. The response rates were 85% for the controls and 92% for the cases.

Considerations regarding methodological issues in the present study (such as study design, sampling scheme and potential bias) have been described in detail previously (8,12,18,23), and the validity of our study approach was addressed and confirmed in these studies.

Questionnaire
Two experienced research nurses were assigned to administer a structured questionnaire to both cases and controls. The validity of this questionnaire was addressed and confirmed in our previous studies (8,12,18,23). The information collected included demographic characteristics (ethnic background, residence area, family income and educational level), reproductive risk factors [age at menarche and/or menopause, age at first full-term pregnancy (FFTP), number of full-term pregnancies (FTPs), parity, history of breast-feeding and menopausal status], medical history [age at diagnosis of breast cancer, family history of breast cancer (first-degree relatives), history of breast biopsy and history of breast screening] and exogenous hormone exposure (use of oral contraceptives and hormone replacement therapy). The body mass index (BMI), diet and history of alcohol consumption or cigarette smoking were also recorded. Women younger than 55 years who had undergone hysterectomy, but not bilateral oophorectomy, were classified as unknown in terms of menopausal status.

Genotyping
Genomic DNA was extracted from peripheral blood samples using a QIAamp DNA extraction kit (Qiagen) following the manufacturer's protocol. The SNP information was obtained from both public SNP databases (e.g. the database maintained by the National Institute of Health, USA, http://www.ncbi.nlm.nih.gov/SNP/index.html) and the recently published results by HapMap (24).

All SNPs were genotyped using a high-throughput genotyping platform based on the 5' nuclease allelic discrimination TaqMan assay (25) in a 96-well format on an ABI Prism 7000HT Sequence Detection System (Applied Biosystems). The PCR primers and probes for individual SNPs were designed using the Assays-by-Design Service (Applied Biosystems). To ensure that the observed polymorphisms were specific and not the result of experimental variation, 10% of the samples were randomly selected and run in duplicates with complete concordance.

Data analysis
We followed our previously established sequential steps (8,12,18,23) for the statistical analysis of an association study:

(i) Univariate and multivariate analyses were used to determine risk factors and to establish background risk profiles for breast cancer in this series of study subjects. Important reproductive risk factors were used as indices to estimate the level of estrogen exposure or susceptibility to estrogen exposure in the later analysis.
(ii) To ensure that the controls used were representative of women in the general population and to exclude the possibility of genotyping error, deviation of the genotype frequencies of each SNP in the control subjects from those expected under Hardy–Weinberg equilibrium was assessed using the goodness-of-fit test (26). The degrees of linkage disequilibrium (LD) between markers were indicated using Lewontin's D' value (27).
(iii) Differences in genotypic frequencies of individual SNPs between cases and controls were tested using chi-square and Fisher's exact tests and multiple logistic regression models with simultaneous consideration of known risk factors of breast cancer and adjusted odds ratios (aORs) for the association were estimated.
(iv) The global haplotype frequencies were estimated employing the expectation–maximization algorithm (28) under Hardy–Weinberg equilibrium. A global test was first performed for differences in haplotype frequencies between cases and controls (i.e. overall haplotype effect) using a model-free permutation test, which was conducted using the program FASTEHPLUS with 10 000 permutations (29). A chi-square test was applied using procedure HAPLOTYPE in SAS/GENETICS and the exact P value was calculated by permuting randomly the disease status with 10 000 permutations (30). We also estimated the odds ratio (OR) of breast cancer associated with harboring individual specific haplotypes, each haplotype being compared with a shared reference haplotype. Consequently, we applied the program PHASE (version 2.0.2) to reconstruct haplotypes and to estimate the diplotype (haplotype pair) probabilities for each subject (31). The PHASE algorithm is a Bayesian approach to haplotype inference. The haplotype risks were estimated as ORs and 95% confidence intervals (CIs) by haplotype-based logistic regression by using the procedure HAPLOTYPE in SAS/GENETICS (32). The effect of haplotype combinations (diplotype) was also analyzed. We defined the at-risk haplotype as that having an increased effect on breast cancer risk, the other haplotypes being combined as the reference. Diplotype data were treated as categorical variable and incorporated as dummy variables in the logistic regression models.
(v) The relationship between the mitotic checkpoint genes and breast cancer risk in women with different levels of estrogen exposure or with different degrees of estrogen susceptibility was examined using joint and stratified methods (33). We calculated the risk of breast cancer associated with the combination of the putative high-risk genotype of the mitotic checkpoint genes and a reproductive risk factor. Using ß estimates from the logistic regression model (34), in which we used a set of dummy variables representing different combinations of gene (i.e. whether harboring the putative high-risk genotypes) and risk factor, we assessed the relative excess risk from harboring putative high-risk genotype within reproductive risk factor strata (joint method). Furthermore, estrogen promotes breast tumorigenesis by triggering the breast epithelium to proliferate (14), and, thus, the relationship between breast cancer risk and polymorphisms of the mitotic checkpoint genes might be modified by estrogen exposure; this was evaluated by calculating the risk (aOR) of breast cancer associated with polymorphism of the mitotic checkpoint genes in different groups of women stratified by their estrogen-related reproductive risk factor status (stratified method).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The distributions of selected demographic characteristics and major risk factors for breast cancer were similar to those reported in our previous studies (8,12,18,23) based on similar study subjects. The cases and controls had a similar average age (cases, 50.3 years and controls, 47.2 years). In terms of reproductive risk factors associated with estrogen exposure, 64.1% of the cases experienced menarche before the age of 14 years compared with 68.1% of the controls, whereas only 39.2% of the cases had undergone the menopause compared with 49.8% of the controls. Using multiple logistic regression analysis, an increased risk (multivariate aOR, 1.54; 95% CI, 1.13–2.09) was found to be conferred by a family history of breast cancer in female first-degree relatives. Although not all differences were statistically significant, reproductive risk factors of breast cancer occurred more frequently among the cases than among the controls. Of these, pregnancy-related risk factors were consistently found to be highly associated with an increased risk. Compared with controls, cases had a lower frequency of a history of FTPs (no history versus at least one FTP; aOR, 1.80; 95% CI, 1.23–2.65) and were older at FFTP (>29 years versus ≤23 years; aOR, 1.66; 95% CI, 1.22–2.26). The significant protection against the development of breast cancer conferred by pregnancy has been suggested to be due to its causing permanent differentiation of the vulnerable breast stem cells, thus reducing susceptibility to estrogen exposure (35,36). This was also confirmed by our finding that a history of breast-feeding was significantly associated with a decreased risk of breast cancer (history of breast-feeding versus no history; aOR, 0.79; 95% CI, 0.64–0.97). No association was found between cancer risk and smoking status, radiation exposure, hormone replacement therapy, hormonal contraceptive use or dietary intake of specific kinds of foods, vegetables or micronutrients, but obese women (BMI > 24 kg/m2) showed a significantly higher risk (aOR, 1.33; 95% CI, 1.09–1.63).

To define the tumorigenic contribution of mitotic checkpoint genes, we examined whether the frequency distribution of SNPs in these genes differed between cases and controls. Thirty SNPs of the spindle checkpoint genes (MAD2L1, BUB1, BUB1B and TTK) and the checkpoint gene of sister chromatid separation (PTTG1) described in SNP databases were genotyped in an initial screening of 188 cases and 188 controls. Of these, 15 were not observed and 2 were infrequent (minor allele frequency < 1%), so these 17 were not genotyped in the rest of the samples. The remaining 13 SNPs (three for TTK, three for MAD2L1, one for BUB1, three for BUB1B and three for PTTG1) were genotyped in all cases and controls. The genotyping results for these 13 SNPs in the controls were compared with the expected distribution of genotypes estimated on the basis of the observed allelic frequency for each SNP, and no significant difference was found (i.e. none of the loci showed a significant P value in the chi-square test). This suggests that the SNPs examined were in agreement with Hardy–Weinberg equilibrium (26).

To explore a possible association between breast cancer and individual SNPs of the mitotic checkpoint genes, the genotype distributions of the 13 SNPs were compared between cases and controls and the effects of breast cancer risk factors were simultaneously adjusted in the multiple logistic regression model (Table I). Two SNPs, one in TTK (G21650C) and the other in PTTG1 (C1892G), were found to show statistically significant differences (Table I).


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  		Table I. Genotype frequencies of variants in the genes involved in mitotic checkpoint and their association with risk of breast cancer

 
Since only two SNPs in two genes showed a significant association with breast cancer risk (Table I), we considered that single SNP might fail to capture all of the contribution of a locus to a particular trait. To better describe the contribution of the mitotic checkpoint genes to breast cancer risk, we studied genetic variants in the context of their haplotypes, using inferred haplotypes assigned to cases and controls (28–30). For the five genes in this study, four genes (but not BUB1) had more than one SNP genotypes in breast cancer cases and control subjects. To this end, we first estimated the degree of LD between each pair of the SNP loci, and all SNPs in the same gene were found to be in LD (data not shown). Notably, haplotype analyses revealed a stronger association with breast cancer risk than genotype analysis at each locus alone (Table II), the difference in frequency distribution of the haplotypes of three genes, i.e. TTK, BUB1B and PTTG1, between cases and controls being significant in the global test (P < 0.0001) (29), showing a significant effect of haplotype on breast cancer risk. The effect of haplotype combinations was also analyzed. We defined the at-risk haplotype as that having an increased effect on breast cancer risk (i.e. OR > 1 in Table II), the other haplotypes (i.e. OR ≤ 1 in Table II) being combined as the reference. Interestingly, a significantly increased trend of risk to develop breast cancer was found in women harboring a higher number of at-risk haplotypes of TTK, BUB1B and PTTG1 (Table III), suggesting a dose effect of the at-risk haplotypes of these mitotic checkpoint genes.


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  		Table II. Inferred haplotype frequencies of selected mitotic checkpoint genes and their association with risk of breast cancer

 


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  		Table III. Diplotypes (combined haplotypes) of mitotic checkpoint genes and breast cancer risk

 
Given that individual genes in the mitotic checkpoint participate cooperatively in maintaining chromosomal stability and given that an increased risk of cancer due to a combined effect of genes belonging to a common tumorigenic pathway has been demonstrated in a mouse model (37), cell line-based functional studies (38) and epidemiological studies (8,23), we examined whether a joint effect of these mitotic checkpoint genes might be associated with breast cancer development. To this end, we summed the number of putative high-risk genotypes for all five mitotic checkpoint genes and examined the breast cancer risk associated with the number of these putative high-risk genotypes, since the genes belonging to a common tumorigenic pathway may act combinatorially in a dose-dependent manner to determine cancer predisposition (39). The SNP for each individual gene showing the highest statistical significance (Table I) was chosen for analysis, and the heterozygous and homozygous variant genotypes were grouped together and compared with the homozygous wild-type genotype. A greater combined effect was seen with a higher number of putative high-risk genotypes (aOR of 1.50 for having at least one putative high-risk genotype; 95% CI, 1.16–1.93) (P value of test for trend = 0.007), and a >6-fold increase in risk (aOR, 6.47; 95% CI, 1.88–22.26) was found in women harboring all putative at-risk genotypes of these five genes. However, it was not totally unlikely that there might be a false combined effect due to a non-homogenous effect of individual genes. To exclude this possibility, we employed a more conservative definition of the joint effect, only considering the contribution of genotypic polymorphisms of TTK and PTTG1, the two genes that showed a significant association with breast cancer risk in the single gene analysis, and found that the results obtained using this conservative definition (Table IV) were consistent with those based on all five genes. The harboring of a higher number of putative high-risk genotypes of TTK or PTTG1 was significantly associated with an increased risk, and women harboring high-risk genotypes of both genes showed a 3.14-fold increase in risk (95% CI, 1.62–6.11) (Table IV). The increase in breast cancer risk associated with this gene–gene interaction was more than multiplicative, because, when the log-likelihood scores of the logistic models with and without a cross-product term containing TTK and PTTG1 were compared, a significant P value (P = 0.035) was obtained.


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  		Table IV. Combined effect of TTK and PTTG1 on breast cancer risk

 
This significant association between a joint effect of individual genes and breast cancer prompted us to explore interactions between mitotic checkpoint genotypes and an established significant risk factor for breast cancer, namely older at FTP, as this risk factor is known to be an indicator of increased susceptibility to estrogen exposure (35,36) and was significant in determining breast cancer risk in the present study. To carry out this analysis, we first classified our women into two groups: those with >1 and those with ≤1 putative high-risk genotype in the five mitotic checkpoint genes; as such a definition would give sufficient statistical power to address relevant questions. We also examined the effect of estrogen exposure during the critical period between menarche and FFTP. The reference group consisted of women harboring ≤1 putative high-risk genotype and having been exposed to estrogen for a shorter period (<10 years) before FFTP. In the absence of long exposure to estrogen before FFTP, the harboring of a higher number (>1) of putative high-risk genotypes of the mitotic genes was associated with an increased, but not significant, risk (aOR, 1.08; 95% CI, 0.74–1.56). However, in the presence of the reproductive risk factor, the harboring of more than one putative high-risk genotype of the mitotic checkpoint genes was associated with a much greater combined risk of breast cancer (aOR, 1.62; 95% CI, 1.12–2.35). Similarly, if we employed the more conservative definition, only considering the effect of TTK and PTTG1, a similar result was obtained (Table V). Furthermore, to confirm this gene–reproductive risk factor interaction, we investigated the potential importance of a protective effect of the number of FTPs in conjunction with the putative high-risk genotypes of TTK and PTTG1. The aORs associated with additional putative high-risk genotypes within strata with different number of FTPs were estimated. Modification of the risk was supported by our findings, shown in Table VI, that a significant increase in cancer risk associated with the number of putative high-risk genotypes was only seen in women with a lower number of FTPs, who supposedly are more susceptible to estrogen exposure (35,36). The possibility of a difference in statistical power in the detection of cancer risk due to different sample sizes in the subsets of women can be excluded, since the sample sizes in these two strata were similar. In addition, statistical examination of the interaction between the number of high-risk genotypes and the number of FTPs in relation to increased breast cancer risk yielded a significant P value (P = 0.03). These results indicate the presence of an interaction between the mitotic checkpoint mechanisms and susceptibility to estrogen exposure during breast tumorigenesis.


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  		Table V. Combined effect of TTK and PTTG1 and reproductive risk factor (estrogen exposure before FFTP, i.e. interval between menarche and FFTP) on breast cancer risk

 


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  		Table VI. Combined effect of TTK and PTTG1 on breast cancer risk, stratified by number of FTPsa

 
Finally, we re-examined the breast cancer risk associated with a joint effect of TTK and PTTG1 and with an interaction between genes and reproductive risk factors in the context of haplotypes. A higher number of at-risk haplotypes was significantly associated with an increased risk of breast cancer (P for trend = 0.0001), and the significance levels of this increasing trend were modified by reproductive risk factors, and were more significant in the strata of women with a long period of estrogen exposure before FFTP or with a lower number of FTPs (Figure 1). These findings are consistent with our hypothesis of a combined effect in determining breast cancer risk of more than one gene in the mitotic checkpoint pathway and of genes and reproductive risk factors.


Figure 1
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  		Fig. 1. Breast cancer risk (aOR) associated with the number of at-risk haplotypes [i.e. the haplotypes having an increased effect on breast cancer risk (i.e. OR > 1) in Table II] of TTK and PTTG1 in all women, in all women stratified by the years of estrogen exposure before FFTP (i.e. the interval between menarche and FFTP) or in parous women stratified by the number of FTP.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
On the basis of a multigenic model, the present study comprehensively examined the tumorigenic contribution to breast cancer development of critical genes participating in mitotic checkpoint, a mechanism essential for maintaining genomic stability and defects which have been linked to cancer development (1–4). The SNPs, haplotypes and haplotype combinations of some of the mitotic checkpoint genes, including TTK and PTTG1, were found to be associated with increased breast cancer risk, supporting our hypothesis that certain high-penetrance genes, originally thought to confer an extremely high risk, could play an etiological role via the effect of low-penetrance alleles. Because maintenance of chromosomal integrity is crucial for prevention of a wide variety of adverse cellular outcomes, it is not surprising that homozygous disruption of checkpoint genes causes widespread apoptosis and cell death rather than chromosomal instability (CIN) (9–11). The generation of CIN can occur through the deletion of not only mitotic checkpoint but also TP53 (11) that emphasizes that escape from cell-cycle surveillance by TP53 is essential for cells harboring defective mitotic checkpoint genes to acquire CIN without triggering cell death (40). Thus, in tumor formation, which is distinct from other genetic diseases in that an extended period of time is needed to accumulate the genetic changes required for its development, cells harboring subtle defects arising from low-penetrance variants would have the chance to grow without activating cell-cycle surveillance and serious pathological outcomes by TP53. However, because of their suboptimal capacity to maintain genomic stability, cells harboring variant alleles of mitotic checkpoint genes would be subject to a higher rate of genomic change caused by improper mitosis. This suggestion is consistent with our recently proposed hide-then-hit model (8) to explain the tumorigenic contribution of SNPs of the high-penetrance DNA repair genes. On the other hand, although CIN is a very common feature in breast cancer (19,20), whether this is a cause or a consequence of tumor development remains unclear. The findings of the present study therefore provide important support for the idea that variant mitotic checkpoint genes may serve as the mutators predisposing to a higher risk of developing CIN, essential to drive breast tumorigenesis.

In considering whether our finding represents a true association between the SNPs of the mitotic checkpoint genes and breast cancer, the most important issue is the interpretation of the identified association between the SNPs and the trait. The present study used a candidate gene approach, based on SNPs locating in the genes of the mitotic checkpoint pathway. Since the SNPs analyzed are in introns and do not affect amino acid coding and therefore probably may not affect protein function, the observed associations between breast cancer risk and SNPs might be interpreted as the presence of LD between these SNPs and other SNPs in exons, resulting in functional polymorphism, or in regulating regions, affecting the expression of these genes. We used more than one SNP in these genes to assign the haplotypes and to examine haplotype effects on cancer risk, and the information generated by haplotype analysis supported our suggestion that genotypic polymorphisms of mitotic checkpoint genes are associated with susceptibility to develop breast cancer. On the other hand, it is less probable that the observed association reflects the effects of other adjacent genes, because the newly published haplotype map of the human genome (24) shows that no well-defined cancer-associated genes are located in the same haplotype blocks of these mitotic checkpoint genes (e.g. TTK and PTTG1). Furthermore, genetic heterogeneity is less of a concern in the Taiwanese population than in the USA (22), and, as a result, bias due to population stratification is less probable to be seen in our study, and the probability that the functional variants targeted by the same SNPs of individual mitotic checkpoint genes are different in cases and controls due to differences in genetic background is small. However, we recognize that the sequencing of the entire gene and promoter region is the definitive approach to identifying all of the important sequence variants and that a large-scale evaluation of these variants and functional assessments are needed to address this question.

The contribution of estrogen to the development of breast cancer has been documented in epidemiological and molecular and cell biology studies. Hypotheses that estrogen is involved in tumorigenesis are based on the general concept that cell division plays a crucial role in cancer development and that reproductive factors that increase mitotic activity in the breast epithelium also increase cancer risk (15). Estrogen triggers cell growth and tumor promotion by binding to the estrogen receptor (ESR1) in the nucleus, leading to the expression of many target genes involved in cell-cycle progression, either directly or indirectly by activating the signaling pathway resulting in proliferation (41). Based on this well-known mechanism, some epidemiological studies have evaluated the association between genetic polymorphisms in the ESR1 gene and breast cancer risk (42). The present study tested the breast tumorigenic contribution of mitogenic mechanisms involving estrogen using a different approach, in which we examined whether breast tumorigenesis was linked to the key genes involved in ensuring the fidelity of mitosis, and the results supported the idea that increased exposure to estrogen confers a higher risk of breast cancer by promoting mitosis and cell proliferation. Interestingly, our finding suggesting an interaction between mitotic checkpoint genes and reproductive risk factors reflecting susceptibility to estrogen exposure is consistent with recent results from a mouse model, in which exposure to steroid hormones is sufficient to generate a high frequency of aneuploidy in mammary cells by increasing the rate of chromosome missegregation due to alterations in the levels of mitotic checkpoint proteins (43). Furthermore, the combined effect of mitotic checkpoint genes and reproductive risk factors leading to an increased risk of breast cancer can explain the tissue specificity by suggesting that estrogen may impose a selective microenvironment beneficial for breast cells that have lost mitotic checkpoint. Because mitotic checkpoint genes are essential, suboptimal function due to variant alleles of these genes would predispose the cells to acquire an abnormal chromosome content, leading to a severe decrease in proliferation and reducing the likelihood that additional mutations will occur, allowing tumor formation. In contrast, in the breast epithelium, the presence of tissue-specific survival factors resulting from estrogen stimulation may have a protective effect on cells harboring variant mitotic checkpoint genes, allowing them to survive and proliferate for the prolonged period of time essential for tumor formation (44).

Given that tumorigenesis is usually a multistep, multigenic process and that it is unlikely that any single genetic polymorphism would have a dramatic effect on cancer risk, a multigenic approach, such as that used in the present study, should permit a more precise evaluation of the risks associated with individual susceptibility genes. In addition, we were especially interested in knowing whether the association between the joint effect of mitotic checkpoint genes and breast cancer was modified by reproductive risk factors reflecting susceptibility to estrogen exposure. However, it has not yet been determined how the cancer risk associated with either a joint effect of genes in the same functional pathway or with a combined effect of genes and risk factors can best be measured. We examined these gene–gene/gene–risk factor interactions using three methods. Firstly, using the joint method, we calculated the risk of breast cancer associated with the combination of the number of putative high-risk genotypes of mitotic checkpoint genes and a reproductive risk factor. Using ß estimates from the logistic regression model, in which we used a set of dummy variables representing different combinations of genes (i.e. the number of putative high-risk genotypes) and risk factors, we assessed the risk associated with harboring different numbers of putative high-risk genotypes within risk factor strata. Secondly, using the stratified method, possible modification of risk associated with mitotic checkpoint genes by estrogen exposure was evaluated by calculating the aOR of breast cancer in relation to the number of high-risk genotypes/haplotypes within different levels (categories) of estrogen-related risk factors. Finally, if applicable, we also performed a log-likelihood test comparing a ‘full’ model that contained both the main effects of genotypes and risk factors plus a (gene x risk factor) interaction term and a ‘reduced’ model that did not contain the cross-product variable. The rationales underlying the individual methods are straightforward, which yield results that can be easily interpreted biologically, and all are frequently used, but each has specific limitations (33). Because of this, we explored the interactions using multiple methods and found that these yielded consistent results, supporting the validity of our suggestion that the mitotic checkpoint genes, in particular TTK and PTTG1, act jointly and in combination with reproductive risk factors reflecting susceptibility to estrogen exposure to determine breast cancer risk. However, it has recently been recognized that other sophistical modeling methods and statistical programs (e.g. MDR and CART) based on complicated assumptions may be helpful in exploring these joint effects and interactions.

Using a candidate gene approach, this present study provided evidence supporting the breast tumorigenic contribution of mitotic checkpoint genes, of which TTK and PTTG1 were the most important. Additional functional analyses of these genes and polymorphisms are worthy to explore the mechanisms by which TTK and PTTG1 affect breast cancer risk. Interestingly, a physical and functional interaction between TTK and CHEK2 was demonstrated in our recent study and was shown to participate in the regulation of the DNA damage checkpoint response (45). Furthermore, PTTG1 can interact with TP53 and modulate TP53-mediated transcriptional activity and apoptosis (46). Because CHEK2 and TP53 are two well-documented breast cancer susceptibility genes, these links provide essential support for the tumorigenic contribution of TTK and PTTG1 to breast cancer development.


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 27, 2006; revised November 26, 2006; accepted December 20, 2006.


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