[CANCER RESEARCH 58. 2107-2110. May 15. IWX] Genetic Polymorphisms in Catechol-0-Methyltransferase, Menopausal Status, and Breast Cancer Risk1 Patricia A. Thompson, Peter G. Shields, Jo I.. Freudenheim, Angie Stone, John E. Vena, James R. Marshall, Saxon Graham, Rosemary Laughlin, Takuma Nemoto, Fred F. Kadlubar, and Christine B. Ambrosone2 Division of Molecular fyiidetniolof>y. National Center for Toxicological Research. Jefferson, Arkansas 72205 {P.A. T., A. S., F. F. K., C.B.AJ; Ltiboratory irf Human Carcinogenesis, National Cancer Institute. Betliesda. Maryland 20X92 ¡P.G. S. /; Department of Social & Preventive Medicine. State University of New York at Buffalo. Muffalo, New York 14214 ¡J.L F.. J. E. V., S. G., K. L. T. N.J: and Ari-unti Cancer Center. Tucson. Ari-ona 85724 ¡J.R. MJ ABSTRACT Polymorphic catechol-0-methyltransferase (COMT) catalyzes the O- methylation of estrogen catechols. In a case-control study, we evaluated the association of the low-activity alÃ-ele(COM7"M") with breast cancer risk. Compared to women with CO/V/7"VlilAal, COMj*t"/M" was associated with an increased risk among premenopausal women [odds ratio (OR), 2.1; confidence interval (CI), 1.4-4.3] but was inversely associated with postmenopausal risk (OR, 0.4; CI, 0.2-0.7). The association of risk with at least one low-activity COMT*1" alÃ-elewas strongest among the heaviest premenopausal women (OR, 5.7; CI, 1.1-30.1) and among the leanest postmenopausal women (OR, 0.3; CI, 0.1-0.7), suggesting that COMT, mediated by body mass index, may be playing differential roles in human breast carcinogenesis, dependent upon menopausa! status. INTRODUCTION It is widely believed that estrogen exposure is an important etio- logical agent in breast carcinogenesis. However, studies of excreted estrogens or estrogen metabolites have demonstrated only weak as sociations between high levels of plasma or urinary estrogens and breast cancer risk (1-3). The catechol estrogens (i.e., 2-hydroxy estrogens) are the major metabolites of estrogens in humans and animals (4). The 2- and 4-catechol estrogens have been reported to demonstrate both cancer-promoting and -inhibiting activities through interactions with the estrogen receptor or with macromolecules (i.e., cellular proteins and DNA; Refs. 3-9). Interindividual differences in steroid metabolism have been noted and attributed to both genetic polymorphisms in and differential expression of metabolizing en zymes that hydroxylate and conjugate the steroid hormones (4, 10- 12). COMT3 is one of several phase II enzymes involved in the conjugation and inactivation of the catechol estrogens (13). COMT is found in various mammalian tissues, with high levels in liver and kidney and significant amounts in RBCs. endometrium, and breast ( 14). An amino acid change (valine to methionine) at position 158/108 in the membrane-bound/cytosolic form of the protein has been linked to decreased methylation activity of the enzyme (15). This amino acid change is believed to be closely associated with the observed trimodal distribution of COMT enzyme activity in the human population as sociated with high COMrVill/Val. intermediate COMTVMMa. and low COMT COMTMct/M" activity toward certain combination therapeu- Received 11/24/97; accepted 3/19/98. 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. ' This work was a collaborative effort by the Division of Molecular 1-pidemiology. National Center for Toxicological Research, the Department of Social and Preventive Medicine, State University of New York at Buffalo, and Ihe Laboratory of Human Carcinogenesis. National Cancer Institute. This work was supported, in part, by Grants CAI 1535. CA/ES62995. and CA01633 from the National Cancer Institute and the National Institute for Environmental Health Sciences and USAMRMC#CAMC17-°4-J- 4108. This work is solely Ihe responsibility of the authors and does not necessarily represent the views of the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Division of Molecular Epidemiology. National Center for Toxicological Research. 39(X) NCTR Road. Jefferson. AR 7207". K-mail:camhrosone<!*nctr.fda.gov. 'The abbreviations used are: COMT. catechol (J-methyltransferasc; BMI. body mass index; OR, odds ratio; CI. confidence interval; HRT. hormone replacement therapy. tics used in Parkinson's disease (16. 17). Because the genetic poly morphism in COMT correlates with decreased enzyme activity and because COMT is a major conjugation pathway for the catechol estrogens, we sought to determine whether polymorphisms in Ihe COMT gene may be associated with increased risk of breast cancer and whether the association between genotypes and risk may be modified by menopausa! status and body mass index. MATERIALS AND METHODS Study Population. These research dala were collected from an earlier case-control study (1986-1991) of 617 premenopausal and 933 postmeno pausal Caucasian women in Western New York; the detailed methods have been reported (18. 19). The protocol for the study was reviewed by the Institutional Review Board of the State University of New York at Buffalo and of all of the participating hospitals. Informed consent was received from all participants for interview and medical record review. Women diagnosed with incident, primary, histologically confirmed breast cancer were frequency- matched by age and county of residence with controls randomly selected from the New York State Motor Vehicle lists (<65 years) and the Health Care Finance Administration rolls (>65 years). Interview data included medical, reproductive, and lifestyle histories. Approximately 45% of premenopausal and 63% of postmenopausal women provided blood samples. DNA was extracted from blood clots, as reported previously, and analy/ed tor COMT genotype in case and control specimens having adequate DNA. Laboratory Analysis. To determine the polymorphic COMT genotype. DNA was subjected to PCR as described (20). Briefly. Ihe reaction conditions included buffer [ 10 mM Tris-HCI (pH 8.3). 50 mM KC1], 2 IHMMgC12. 0.2 mM 2'-deoxynucleoside-3'-lriphosphate (Boehringer Mannheim. Indianapolis In diana)]. 2.5 units ol Taq DNA polymerase (Promega Corp.. Madison. Wl). and primers specific for COMT (10 pmol each; 5'-ACTGTGGCTACTCAGCT- GTG-3' and 5'-CCTTTTTCCAGGTCTGACAA-.V) in a total reaction volume of 100 fi\ using 100 ng of sample DNA. PCR producÃ-s( 169 bp) were digested with Hsp92II (Promega) and analyzed by gel electrophoresis (2.5% Metaphor agarosc; FMC BioProducts. Rockland. ME). Digestion of the COMT product with Hsp92II gives rise to fragment sizes of 114. 23. and 32 bp for the high-activity alÃ-eleand 96. 23. 32. and 18 bp for Ihe low-activity alÃ-ele.This assay was validated hy confirming inheritance patterns in eight family lines encompassing three generations (National Institute General Medical Scientist Human Genetic Mutant Cell Repository; Coriell Institute. Camdcn. NJ). All assays were conducted and interpreted by two reviewers (P. T. and A. S.) blinded to case-control status. Statistical Analysis. Student's i tests were performed to assess mean differences in reproductive and lifestyle factors by COMT genotypes within case and control groups. ORs and 95% CIs were calculated using unconditional logistic regression to evaluate associations between COMT genotypes and breast cancer risk separately for premenopausal and postmenopausal women. ORs were adjusted for age. education, age at menarche. age at first pregnancy, reported family history of breast cancer, body mass index, and age at meno pause for postmenopausal women. Possible modification of risk by body muss was evaluated by calculating ORs for genotype and breast cancer risk within fertiles of body mass index, determined by the distribution among controls. RESULTS Genotype data for COMT were available for 281 women with breast cancer and 289 community controls. For the most part, asso- 2107 Research. on February 29, 2016. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from