Short Communication Association of Genetic Polymorphisms in UGT1A1 with Breast Cancer and Plasma Hormone Levels

UDP-glucuronosyltransferases (UGTs) catalyze the detoxification and the elimination of a large number of endogenous and exogenous compounds in the liver and extrahepatic tissues. One of the UGT1A family members, UGT1A1, is involved in estradiol metabolism and, therefore, represents a candidate gene in breast carcinogenesis. A common insertion/deletion polymorphism in the TATA-box of the promoter region of UGT1A1 results in decreased initiation of transcription. In a previous study, we found a positive association between the UGT1A1 low-transcriptional alleles and premenopausal breast cancer risk in an African-American population. In the present study, we sought to determine whether the low-transcription UGT1A1 promoter allele, UGT1A1*28 [A(TA)7TAA], was associated with increased breast cancer risk among primarily Caucasian women in a nested case-control study within the Nurses’ Health Study cohort. No significant association between the UGT1A1*28 [A(TA)7TAA] allele and breast cancer was observed. Compared with women homozygous for the UGT1A1*1 [A(TA)6TAA] allele, the relative risk was 0.80 (confidence interval, 0.49–1.29) for women homozygous for the UGT1A1*28 allele. The effect of the UGT1A1 genotype on plasma hormone levels in postmenopausal women not using hormone replacement was also evaluated, and overall, no significant differences in hormone levels by genotypes were observed. When restricted to women who had at least one UGT1A1*28 allele and a body mass index at blood draw of >27 kg/m, particularly in combination with the cytochrome p450c17a genotype, estrone and estradiol levels tended to vary by UGT1A1 genotypes. The results presented do not support a strong association between the UGT1A1 promoter polymorphism and the risk of breast cancer. Introduction UGT represents one of the most important classes of phase II detoxification enzymes that catalyze the transfer of glucuronic acid to a large number of substrates in the liver and extrahepatic tissues (1). Compounds inactivated by glucuronic acid conjugation include steroid hormones, such as estrogens and catechol estrogens (2–5). Thus UGT enzymes may assist in the maintenance of steady-state levels of steroids in target tissues (6, 7). Moreover, there is evidence that one of the UGT1A family members, UGT1A1, is involved in E2 inactivation (8, 9) and expressed in the mammary gland (8), and, therefore, represents a good candidate gene in breast carcinogenesis. Interindividual variation in UGT1A1 expression is explained by a polymorphic alteration in the atypical TATA-box region of the UGT1A1 gene (10–12). This polymorphic site is characterized by a variation in the number of TA repeats in the A(TA)nTAA sequence of the promoter. The most common allele (UGT1A1*1) contains six TA repeats, whereas the principal variant allele (UGT1A1*28) contains seven TA repeats. Two other UGT1A1 alleles, the UGT1A1*33 [A(TA)5TAA] allele and the UGT1A1*34 [A(TA)8TAA] allele, have been found almost exclusively in the African-American population (13, 14). In a previous study, we investigated the association between breast cancer and A(TA)7TAA and A(TA)8TAA UGT1A1 alleles, which have been shown to have lower transcriptional activity in vitro (8). In the CBCS study, the alleles A(TA)7TAA and A(TA)8TAA were associated with a 2-fold increase in the risk of developing breast cancer in premenopausal African-American women. In the present study, we evaluated, among primarily Caucasian women, the relationship between UGT1A1 alleles and breast cancer risk in a nested case-control study within the NHS cohort. Because decreased transcription of the UGT1A1 gene has been found to be associated with increased plasma levels of substrates of the UGT1A1 protein, e.g., bilirubin, we also evaluated the relationship between the UGT1A1*28 allele and circulating estrogen levels and the interaction between UGT1A1 and CYP17 polymorphisms. Received 11/17/00; revised 3/23/01; accepted 4/9/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Medical Research Council of Canada (to C. G.) and NIH Grants CA40356, CA49449, and CA65725. 2 To whom requests for reprints should be addressed, at Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL), Faculty of Pharmacy, Laval University, Quebec G1V 4G2, Canada. E-mail: [email protected]. 3 The abbreviations used are: UGT, UDP-glucuronosyltransferase enzyme; NHS1, Nurses’ Health Study 1; OR, odds ratio; CI, confidence interval; CYP17, cytochrome p450c17a; CBCS, North Carolina Breast Cancer Study; E2, estradiol; E1, estrone; BMI, body mass index; RR, relative risk. 711 Vol. 10, 711–714, June 2001 Cancer Epidemiology, Biomarkers & Prevention on April 11, 2017. © 2001 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from Materials and Methods Study Population. The NHS was initiated in 1976, when 121,700 United States registered nurses between the ages of 30 and 55 returned an initial questionnaire reporting medical histories and baseline health-related exposures. Between 1989 and 1990, blood samples were collected from 32,826 women. Incident breast cancers are identified by self-report and confirmed by medical record review. Eligible cases in this study consisted of women from the subcohort with pathologically confirmed incident breast cancer who gave a blood specimen. Controls were matched to cases on the basis of year of birth, menopausal status, and postmenopausal hormone use, as well as time of day, month, and fasting status at blood draw. Menopause was defined as described previously (5). The nested case-control study consists of 461 incident breast cancer cases and 615 matched controls. The study sample for the plasma hormone analysis was restricted to postmenopausal controls not using hormone replacement therapy within 3 months of blood draw. The protocol was approved by the Committee on Human Subjects, Brigham and Women’s Hospital, Boston, MA. Detailed information on exposure data and hormone assay have been described previously (15). Molecular Analysis. All analyses were conducted with laboratory personnel blinded to case status. DNA was extracted from buffy coat fractions using the QIAGEN QIAamp Blood Kit (QIAGEN, Inc., Chatsworth, CA). DNA samples from cases and controls were genotyped for the dinucleotide insertion/deletion present in the promoter region of the UGT1A1 gene using previously described methods (8). Statistical Methods. ORs and 95% CIs were calculated using conditional and unconditional logistic regression. In addition to the matching variables, we adjusted for the following breast cancer risk factors: (a) BMI (kg/m) at age 18 (continuous); (b) weight gain since age 18 (,5, 5–19.9, and $20 kg); (c) age of menarche (,12, 12, 13, and $13 years); (d) parity/age at first birth (nulliparous; one to two children/age at first birth #24 years; one to two children/age at first birth $24 years; three children/age at first birth #24 years; and three or more children/age at first birth $24 years); (e) first-degree family history of breast cancer (yes/no); (f) history of benign breast disease (yes/no); and (g) duration of postmenopausal hormone use (never; past, ,5 and $5 years; current, ,5 and $5 years). We also adjusted for age at menopause (continuous in years) in analyses limited to postmenopausal women. Indicator variables for all three genotypes were created using the UGT1A1*1/UGT1A1*1 hypothesized low-risk genotypes as the reference category in the multivariable models. Genotype was also evaluated using a dichotomous variable with the UGT1A1*1/ UGT1A1*28 and UGT1A1*28/UGT1A1*28 subjects combined, because a gene dosage effect on breast cancer risk was not apparent. Unconditional multivariable models controlling for the matching factors enabled all controls to be included in analyses, limiting the cases to specified histopathological characteristics. Interactions between genotypes and breast cancer risk factors were evaluated by including interaction terms in multivariate logistic regression models. The likelihood ratio test was used to assess the statistical significance of these interactions. Mixed regression models were used to evaluate the association between genotype and circulating hormone levels among controls, controlling for BMI at blood draw and the matching variables (16). Hormone fractions were measured in two to three different batches; the laboratory batch was treated as a random variable in all hormone analyses except for dehydroepiandrosterone sulfate among never-users of hormone replacement, where a batch effect was not observed. We calculated least-square mean plasma steroid hormone levels to evaluate the differences in hormone levels between the genotypes; this was limited to postmenopausal women. The UGT1A1*1/UGT1A1*1 group was used as the reference category. To examine the interactive effect of the UGT1A1 gene and the CYP17 gene on hormone levels, we used the SAS Proc Mixed procedure. The natural logarithm of the plasma hormone values was used in the analyses to reduce the skewness of the regression residuals. We used the SAS statistical package for all analyses (SAS Institute, Inc.).