1 CRC Human Cancer Genetics Group, Department of Oncology,
2 EPIC and
3 CRC Genetic Epidemiology Group, University of Cambridge, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 8RN, UK
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Abstract |
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Abbreviations: B[a]P: benzo[a]pyrene; CEs: catecholestrogens; HWE:HardyWeinberg equilibrium; PAHs: polycyclic aromatic hydrocarbons; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin
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Introduction |
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Increased lifetime exposure to endogenous and exogenous oestrogens have been implicated as a risk factor for breast cancer. The effects of increased oestrogen exposure have usually been assumed to be mediated through the oestrogen receptor. There is now increasing evidence that by-products of oestrogen metabolism in the body may themselves be genotoxic. Oxidative metabolism initially converts oestrogens to catecholestrogens (CEs) and further biotransformation of CEs forms quinones which can, in turn, form either stable or depurinating DNA adducts, resulting in carcinogenesis (1). CYP1A1 can convert oestradiol to 2-hydroxyestradiol, the initial step in this pathway, and so polymorphic variation in CYP1A1 activity could also modify breast cancer susceptibility through this mechanism.
Three common polymorphisms in CYP1A1 that have been reported in Caucasian populations: a ct substitution in the 3' non coding region; a ca substitution in exon 7 which results in an amino acid change (Thr461Asn); and an ag substitution in exon 7 which also results in an amino acid change (Ile462Val). These polymorphisms may also be designated CYP1A1*2A, CYP1A1*4, and CYP1A1*2B/2C respectively according to one nomenclature (http://www.imm.ki.se/CYPalleles/cyp1a1.htm), but other authors have used a different system of numbering (2). It has been shown that CYP1A1 activity is more easily induced in lymphocytes that carry a Val allele (3), and a recent study has shown that both the Thr461Asp and the Ile462Val polymorphism alter the enzyme kinetics properties to produce both the diol metabolites from B[a]P and diol epoxide 2 from benzo[a]pyrene-7,8-dihydrodiol (4), although an earlier study suggested that Ile462Val does not appear to affect enzymatic activity (5).
There have been four published studies investigating CYP1A1 polymorphic variants and breast cancer susceptibility (69). All four studies investigated the role of the Ile462Val polymorphism in breast cancer susceptibility, but none found a statistically significant association (reviewed in Table 4). However, the power of these studies to detect moderate risks was limited by study size; the largest study included data on >1000 subjects in total. Three studies have investigated the MspI RFLP in the 3' non-coding region of the gene (79). Only one of these reported a significant increase in risk associated, which was limited to African-American women that were homozygous for the rare variant (9). One study has investigated the Thr461Asp polymorphism, and again, no significant association was reported (7).
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The aim of this study was to clarify the importance of CYP1A1 in breast cancer susceptibility in the British, East Anglian population, using a casecontrol study design. We restricted our analysis to the two polymorphisms that alter the amino-acid sequence of the protein since the functional relevance of these would be most amenable to direct testing.
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Materials and methods |
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Female controls were randomly selected from the UK part of the European Prospective Investigation of Cancer (EPIC), a prospective study of diet and cancer being carried out in the same population from which the cases have been drawn (11). The whole EPIC cohort comprises 25 000 individuals resident in Norfolk (East Anglia), aged 4574 years. The controls were slightly older than the cases (median age 57). The ethnic background of both cases and controls was similar (99.0% of cases and 99.4% of controls being white Anglo-Saxon) and we have found no evidence for population stratification in the controls (12).
Genotyping
All samples were genotyped using the ABI PRISM® 7700 Sequence Detection System (PE Biosystems, US). For the Thr461Asn genotyping, PCR amplification was carried out on 20 ng DNA using 1x TaqMan® universal PCR master mix, 300 nM forward (GTGATTATCTTTGGCATGGGC) and reverse (GCCAGGAACAGAAAGACCTCC) primers, 150 nM of the VIC labelled probe (detecting Asn) (CGGGCAATGGTCTCACCGATAC) and 50 nM of the FAM labelled probe (detecting Thr) (GGGCAATGTTCTCACCGATACA) in a 15 ml reaction. Amplification conditions on an MJ Tetrad thermal cycler (GRI, UK) were as follows: 1 cycle of 50°C for 2 min, followed by 1 cycle of 95°C for 10 min and finally 30 cycles of 95°C for 15 s and 60°C for 1 min.
For Ile462Val PCR was carried out using the same conditions but with 300 nM forward (GCATGGGCAAGCGGAA) and reverse (GCCAGGAAGAGAAAGACCTCC) primers, 150 nM of the FAM labelled probe (detecting Ile) (TCGGTGAGACCATTGCCCG) and 100 nM of the TET labelled probe (detecting Val) (CGGTGAGACCGTTGCCCGC) in a 25 ml reaction. The polymorphic base is shown underlined.
Genotypes were determined using the Allelic Discrimination Sequence Detection Software (PE Biosystems, US). Eight each of no-template controls, common homozygote and rare homozygote templates were included in each 96 well plate. The Thr- and Asp461 templates were generated by cloning a heterozygote using the TA cloning kit (Invitrogen, Netherlands) according to manufacturers instructions. The Ile and Val462 templates were made by annealing appropriate oligonucleotides (SGS DNA, Sweden). TaqMan® primers and probes were designed using the Primer Express® Oligo Design Software v1.0 (PE Biosystems).
After initial genotyping of the Ile462Val polymorphism, we noted that there appeared to be an excess of rare homozygotes and the genotype distribution in the controls deviated from HWE. We therefore checked the genotype of all the rare homozygotes by direct sequencing. It was found that the genotype of some individuals that were heterozygous Ile462Val had been mis-classified as Val462 homozygotes if they were also heterozygous for Thr461Asp, and so we also checked the Ile462Val genotypes for all the Thr461Asp heterozygotes. The error was presumably due to inhibition of the binding of the Ile462 probe to the Ile allele by the presence of the Asn461 allele. Conversely, presence of the Val462 allele did not seem to affect binding of the Thr461 probe in the Thr461Asp assay.
Sequencing
Sequencing was carried out using Big-Dye Terminators on the ABI Prism 377 (PE Biosystems) according to manufacturers instructions. Forward (GGAGCTCCACTCACTTGACA) and reverse (AGGCATGCTTCATGGTTAGC) primers were used for the initial amplification across codons 461 and 462.
Statistics
Allele and genotype frequencies in cases and controls were compared by 2 tests. The genotypic specific risks were estimated as odds ratios and associated 95% confidence limits by unconditional logistic regression.
Potential interactions between smoking, CYP1A1 genotype and breast cancer risk were assessed using a case-only study design (13,14). The case-only approach does not estimate main effects, but seeks to identify interactions between two `exposures' that are important in disease aetiology. Here one exposure is genotype, the other smoking. In the absence of interaction, the risks associated with each exposure on its own combine in a multiplicative manner. Departures from multiplicativity are measured by the interaction odds ratio which is obtained from the case only 2 x 2 cross tabulation of genotype (+/) and risk factor (+/). In the absence of interaction, the interaction odds ratio is expected to be unity. The case-only approach has two major advantages over the casecontrol study for identification of interactions: firstly statistical power is increased, and secondly differential ascertainment of risk factors between cases and controls, which may cause bias, is avoided (15). The smoking habit of the cases was categorized as never, >10 pack years, 1019 pack years, and 20 pack years or greater. The smokinggenotype interaction odds ratios and associated 95% CI for each smoking habit category using never smoker as the referent group was calculated from the appropriate 2 x 2 table (see Table III).
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Results |
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The genotype distribution of Thr461Asp in controls was very close to that expected under HardyWeinberg equilibrium (Table I). We detected no significant differences between breast cancer cases and controls in either allele frequencies (
2 =0.02, 1df, P = 0.90) or genotype distributions (
2 = 0.17, 2df, P = 0.68). Table I
also shows the genotype-specific relative risks of breast cancer as estimated by the odds ratio (OR). No significant risk was found for either the Thr/Asp heterozygote [OR = 1.1 (95% CI 0.81.4)] or the Asp/Asp homozygote [OR = 0.4 (0.026.1)]. Subgroup analyses revealed no effect of age or menopausal status on genotypic risks.
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Table III shows the CYP1A1 genotype frequencies in cases by smoking habit. No interaction odds ratio differed significantly from unity and thus we found no evidence for an interaction. Furthermore we found no interaction between genotype and alcohol consumption (results not shown).
We have pooled our Ile462Val data with those of the previous four studies in a meta-analysis (Table IV), but even with >5000 subjects, none of the genotype-associated risks achieved statistical significance, and there was no consistent pattern to the risks associated with increasing Val allele dosage [Ile/Val OR = 0.9 (0.71.1), Val/Val OR = 2.3 (0.412), and Val carrier OR = 1.0 (0.91.1)].
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Discussion |
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We found no conclusive evidence for an interaction between CYP1A1 genotype and smoking habit and susceptibility to breast cancer. A slight increase in risk, that was of borderline statistical significance, in moderate smokers (1019 pack years) who also carry the valine allele of the Ile462Val polymorphism was not apparent in heavy smokers (20+ pack years), in whom the valine allele was protective. This is similar to the effect reported by Ambrosome et al., but does not seem to be biologically plausible, and is inconsistent with the data reported by Ishibe et al.
In interpreting these results some of the possible limitations of the study need to be considered. Our controls were not individually matched for ethnic group, but given the high proportion of both cases and controls that were white Anglo-Saxon and the absence of evidence for stratification in the controls (12), it seems unlikely that population stratification would have any effect on the results. Indeed, population stratification would be expected to result in bias away from the null, whereas we have reported a null result. A second potential source of bias to be considered is survival bias which may affect genotype frequencies in the prevalent cases if genotype were associated with differential survival. However, we found no evidence for a difference in genotype frequency between the two case series, and again, the effect would be predicted to be a bias away from the null. Finally, the possibility that other polymorphic variants in CYP1A1, either within the gene or in the regulatory region, are important determinants of breast cancer susceptibility cannot be excluded, because the extent of linkage disequilibrium across the whole region is not known.
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Notes |
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Acknowledgments |
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References |
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