Glutathione S-Transferases M 1 , T 1 , and P 1 and Breast Cancer 1

We examined associations for glutathioneS-transferases M1 (GSTM1), T1 (GSTT1), and P1 (GSTP1) genotypes and breast cancer in the Carolina Breast Cancer Study, a population-based, case-control study in North Carolina. Odds ratios were close to the null value for eachGST locus among African-American women (278 cases and 271 controls) and white women (410 cases and 392 controls), as well as preand postmenopausal women. For women with a history of breast cancer in one or more first-degree relatives, odds ratios were 2.1 (95% confidence interval, 1.0–4.2) forGSTM1 null and 1.9 (0.8–4.6) forGSTT1null genotypes. Among women with a family history, age at diagnosis was significantly earlier for those with the GSTM1 null genotype. We did not observe strong evidence for modification of odds ratios for smoking according to GST genotypes. There was no evidence for combined effects of GSTM1, GSTT1,and GSTP1genotypes, and there were no combined effects for GST genotypes and the catechol O-methyltransferase genotype. We conclude thatGSTM1, GSTT1,and GSTP1 genotypes do not play a strong role in susceptibility to breast cancer. However, the role ofGST genotypes in age at onset and risk of breast cancer among women with a family history merits further investigation. Introduction GSTs are a family of enzymes involved in detoxication of benzo(a)pyrene and other carcinogens found in tobacco smoke, cytotoxic drugs, and chemical solvents (1, 2). Deletions in two GST genes, GSTM1andGSTT1, occur at frequencies of 15% or greater in human populations (3). Individuals who are deletion homozygotes, classified as GSTM1null or GSTT1null, exhibit absence of enzymatic activity and are hypothesized to be at increased risk for the carcinogenic effects of a wide range of environmental exposures. Associations between GSTM1and GSTT1null genotypes and cancer of the lung, bladder, and colon have been reported, but results are inconsistent across studies (2–5). An amino acid substitution variant in a third GST gene,GSTP1codon 105Ile3Val, has been identified recently that encodes an enzyme with reduced catalytic activity (6). The GSTP1 Valallele is common in human populations but has not been extensively examined in association with cancer. Several previous studies investigated GSTM1andGSTT1 genotypes and breast cancer risk (reviewed in Refs. 4, 7, and 8), and one study examined the role of GSTP1genotype (9). We examined the relation of GSTM1,GSTT1,and GSTP1genotypes and breast cancer risk in the CBCS, a large, populationbased, case-control study of African-American and white women residents of North Carolina. To address issues raised by previous studies, we estimated main effects for each GSTlocus; conducted analyses stratified on smoking, family history, and other factors; and determined age at onset according to GST genotype and family history. We investigated joint effects for combinations of GSTgenotypes, as well as joint effects for GST genotypes and the COMT genotype, a gene involved in detoxication of catechol estrogens (10). Materials and Methods Study Population. The CBCS is a population-based, casecontrol study of breast cancer conducted in 24 counties of North Carolina (11). Incident cases of invasive breast cancer among women of ages 20–74 were identified in cooperation with the North Carolina Central Cancer Registry, and controls were identified using Division of Motor Vehicles and Medicare beneficiary lists. Randomized recruitment (12) was used to oversample younger women and African-American women. Between 1993 and 1996, 889 cases of primary invasive breast cancer and 841 population-based controls were enrolled. Overall response rates (number of completed interviews/number of eligible women) were 74% for cases and 53% for controls (13). In-person interviews were conducted in participants’ homes. Over 98% of women who were interviewed agreed to provide a blood sample. Genotyping forGSTswas conducted for the first 688 cases and 663 controls enrolled in the CBCS (278 African-American cases and 271 African-American controls; 410 white cases and 392 white controls). There were no appreciable differences in risk factors for breast cancer or response rates between participants genotyped for GSTsand the remaining participants in the CBCS (data not shown). Laboratory Analysis. DNA was extracted from peripheral blood lymphocytes using standard methods (14). Genotyping for GSTM1, GSTT1, and GSTP1was conducted using PCRRFLP methods, as described in Helzlsouer et al.(9), with slight Received 11/2/99; revised 3/8/00; accepted 4/11/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Specialized Program of Research Excellence in Breast Cancer, NIH/NCI P50-CA58223; Pesticides and Breast Cancer in North Carolina, NIH/ NIEHS R01-ES07128; and Environment and Breast Cancer Program, R21CA66201. 2 To whom requests for reprints should be addressed, at Department of Epidemiology, CB #7400, School of Public Health, University of North Carolina, Chapel Hill, NC 27599-7400. 3 The abbreviations used are: GST, glutathione S-transferase; CBCS, Carolina Breast Cancer Study; BMI, body mass index; CI, confidence interval; COMT, catecholO-methyltransferase; df, degrees of freedom; ETS, environmental tobacco smoke; OR, odds ratio. 567 Vol. 9, 567–573, June 2000 Cancer Epidemiology, Biomarkers & Prevention on August 5, 2021. © 2000 American Association for Cancer Research. cebp.aacrjournals. org Downloaded from modifications forGSTT1; the forward primer sequence was 59-gcc ctg gct agt tgc tga ag, the reverse primer was 5 9-gca tct gat ttg ggg acc aca, and the annealing temperature was 59°C. The genotyping assays for GSTM1and GSTT1classify individuals with one or two copies of the relevant gene as “present” and individuals with homozygous deletions as “null. ” The assay forGSTP1classifies individuals according to the allelesIle andVal. Assays that were unreadable for each locus are reported as “missing. ” Approximately half of the samples listed as missing did not amplify for the relevant locus, and the remaining amplified too poorly to assign genotypes. The proportions of missing values were similar in cases and controls (5% of cases and 4% of controls were missing for GSTM1;6% of cases and 4% of controls for GSTT1;11% of cases and 9% of controls forGSTP1). Missing values for genotypes were not related to smoking or other covariates (data not shown). Sensitivity analyses were conducted by replacing missing values with different genotype combinations, and ORs remained within the confidence limits presented. Positive and negative control samples were included with each batch of samples (one batch, 94 samples). Gels were scored by two different readers, and discordant samples were repeated. Reliability was assessed by selecting a random sample of 10% of samples from each batch. Batches with ,95% agreement were rerun. Methods for genotyping of COMT and associations with breast cancer in the CBCS have been reported previously (15). Statistical Analysis. Genotype frequencies and 95% CI for GSTM1,GSTT1,andGSTP1were calculated as the proportion of individuals with a given genotype divided by the total number of participants. For GSTP1, allele frequencies and 95% CI were calculated as the number of alleles divided by the number of chromosomes, and tests for Hardy-Weinberg equilibrium were conducted by comparing observed and expected genotype frequencies using a x test (16). Adjusted OR for breast cancer and 95% CI were calculated from unconditional logistic regression models using SAS (version 6.11; SAS Institute, Cary NC). PROC GENMOD was used to incorporate offset terms derived from the sampling probabilities used to identify eligible participants (12) and to adjust for race (as two-level categorical variable) and age (as an 11-level ordinal variable reflecting 5-year age categories). Race was defined according to self-report. Table 1 Genotype frequencies and ORs for breast cancer for GSTM1, GSTP1, andGSTT1among African-American and white participants Genotype Cases Controls OR (95% CI) n Frequency (95% CI) n Frequency (95% CI) A. African-Americans N 5 278 N 5 271