No Association Between Glutathione Peroxidase Pro198Leu Polymorphism and Breast Cancer Risk

Selenium-dependent glutathione peroxidase (GPX1) is a cytosolic antioxidant enzyme that neutralizes H₂O₂ to water and oxygen (1). A GPX1 Pro→Leu polymorphism exists at codon 198, with the variant Leu allele being less responsive than the common Pro allele to the stimulation of enzyme activity during selenium supplementation (2). The Leu allele has been associated with increased risk of lung (3), bladder (4), and breast (2) cancer, although two other studies found a null association with breast cancer risk (5, 6). Because oxidative stress may play a role in breast carcinogenesis (7) and the GPX1 polymorphism may confer interindividual variability in the response to reactive oxygen species, we evaluated the association between the 198 GPX1 polymorphism (RS#1050450) and risk of breast cancer, and assessed potential modifying influences of diet and lifestyle factors, which may affect reactive oxygen species, and tumor characteristics on risk relationships in the Long Island Breast Cancer Study Project.

The Long Island Breast Cancer Study Project, a population-based case-control study of breast cancer, was described previously (8). In brief, the cases were English-speaking women >20 years of age with newly diagnosed breast cancer who resided in Nassau and Suffolk Counties in Long Island, NY. Population-based controls were identified from the same geographic area, and frequency-matched to the expected age distribution of cases by 5-year age groups.

Known and suspected risk factors for breast cancer were ascertained by an in-person interview (8). Usual dietary intake was assessed by a self-administered modified National Cancer Institute–Block food frequency questionnaire (9). Genotyping was done by BioServe Biotechnologies (Laurel, MD) using Sequenom's high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as previously described (10), using PCR primers (5′-ACGTTGGATGATCGAGCCTGACATCGAAGC-3′ and 5′-ACGTTGGATGATCCCGAGACAGCAGCA-3′).

There was excellent observer agreement in the 8% of randomly selected duplicates of genotyping results that were included for quality control purposes (κ statistic: 0.95), with <1% assay failure rate. Among those with DNA available (1,038 cases and 1,088 controls), 94% of cases and 93% of controls were Caucasian.

Unconditional logistic regression (11) was used to calculate odds ratios (OR) and corresponding 95% confidence intervals (CI) for breast cancer, in relation to genotype. The final multivariate models shown include matching factor (age) as well as those factors that changed the estimated effect by 10% or more (11). Factors found not to confound the associations of interest included: race, body mass index, age at first birth, smoking status, age at menarche, hormone replacement therapy use, menopausal status, benign breast disease, and lifetime alcohol intake. We examined potential interactions between GPX1 genotypes and diet (fruit and vegetable consumption, and vitamin supplement; ref. 9), lifestyle factors (cigarette smoking, parity status, age at first birth, and lactation; refs. 8, 12), and tumor characteristics [in situ versus invasive, and estrogen receptor (ER)/progesterone receptor (PR) status; ref. 12]. Gene-environment interactions were evaluated by joint categories of GPX1 genotype and diet and lifestyle factors. To test interactions on a multiplicative scale, a cross-product term of the ordinal score for each genotype and the specific risk factors was included in multivariate models. To test for potential heterogeneity by tumor characteristics, stratified analysis was done.

Genotype distribution of GPX1 followed Hardy-Weinberg equilibrium (P = 0.34) among controls. Genotype distribution and allele frequencies (Pro, 69%; Leu, 31%) were comparable with those observed in other published studies (2, 3, 5, 6). As shown in Table 1, having at least one leu allele (Pro/Leu and Leu/Leu genotypes) was not associated with breast cancer risk.

As shown in Table 2, there was little evidence for interaction between GPX1 genotypes, selected breast cancer risk factors, and breast cancer risk in any of the models. In addition, there was little or no heterogeneity of risk with hormone receptor status. However, nulliparous women with variant Pro/Leu and Leu/Leu genotypes had increased risk [OR (95% CI) = 1.48 (0.99-2.23) and 2.12 (1.01-4.48), respectively], compared with parous women with common Pro/Pro genotypes, although cell sizes were small and risk estimates were somewhat unstable (P for multiplicative interaction = 0.21).

Our data do not support the hypothesis that variant GPX1 genotype is associated with an increased risk of breast cancer, confirming two previous studies. Knight et al. (6) reported that 198 GPX1 polymorphism was not associated with breast cancer risk; however, a second GPX1 allele containing four alanine repeats was associated with increased risk in same population. In the Nurses' Health Study, Cox et al. (5) found that both 198 Pro→Leu polymorphism and −1,040 G→A polymorphism (RS#3448) were not associated with the risk of breast cancer; significant linkage disequilibrium existed between them (D′ = 1.00; r = 0.4; P < 0.001).

There are several possible explanations for the null association between GPX1 genotypes and breast cancer. One possibility is that the effects of GPX1 on risk may only be observed in individuals with very high intake of selenium or fruits and vegetables, due to the observation that in vitro GPX1 enzyme activity differed between Pro and Leu alleles at high selenium supplementation (2). This is unlikely, however, because in our study, associations between GPX1 genotype and breast cancer risk were null even among vitamin supplement users or higher fruit and vegetable consumers. Furthermore, mean consumption of total fruits and vegetables in Long Island Breast Cancer Study Project participants was higher than that of average women in the U.S. (26 svg/wk among National Health and Nutrition Examination Survey women versus 30 svg/wk among the Long Island Breast Cancer Study Project participants, excluding juice). Finally, our findings are based on a large population-based study, and we have adequate power (0.80) to be able to detect an OR of 1.28 or greater, with the sample size available.

We observed a somewhat suggestive interaction with parity status, although the exact mechanisms whereby GXP1 effects may be greatest for nulliparous women need to be further investigated. Although our findings could be due to chance, it is also possible that women whose breast cells have never fully differentiated during a full-term pregnancy may be more susceptible to reduced capabilities for removal of reactive oxygen species by low-activity GPX1 genotype, and thereby at increased risk of breast cancer.

In summary, we did not find evidence for associations between variant GPX1 genotypes and breast cancer risk, nor was the association modified by diet or tumor characteristics in the Long Island Breast Cancer Study Project. However, we did find that risk was somewhat elevated among nulliparous women with the variant GPX1 genotype, compared with parous women with the common GPX1 genotype.

For their valuable contributions to the Long Island Breast Cancer Study Project, the authors thank: members of the Long Island Breast Cancer Network; the 31 participating institutions in Long Island and in New York City, NY; our NIH collaborators, Gwen Collman, PhD, National Institutes of Environmental Health Sciences; G. Iris Obrams, MD, PhD formerly of the National Cancer Institute; members of the External Advisory Committee to the population-based case-control study: Leslie Bernstein, PhD (Committee Chair); Gerald Akland, MS; Barbara Balaban, MSW; Blake Cady, MD; Dale Sandler, PhD; Roy Shore, PhD; and Gerald Wogan, PhD; as well as other collaborators who assisted with various aspects of our data collection efforts including Gail Garbowski, MPH; Mary S. Wolff, PhD; Steven D. Stellman, PhD; Maureen Hatch, PhD; Geoffrey Kabat, PhD; Jan Beyea, PhD; Bruce Levin, PhD; H. Leon Bradlow, PhD; David Camann, BS; Martin Trent, BS; Ruby Senie, PhD; Carla Maffeo, PhD; Pat Montalvan; Gertrud Berkowitz, PhD; Margaret Kemeny, MD; Mark Citron, MD; Freya Schnabel, MD; Allen Schuss, MD; Steven Hajdu, MD; and Vincent Vinceguerra, MD.