Association of postmenopausal endogenous sex hormones with global methylation level of leukocyte DNA among Japanese women

Subjects were postmenopausal women who participated as controls in a multicenter, hospital-based case–control study of breast cancer conducted from May 2001 to September 2005 at four hospitals in Nagano Prefecture, Japan. Details of this study have been described previously [14, 15]. The study protocol was approved by the institutional review board of the National Cancer Center, Tokyo, Japan.

Briefly, the case subjects were a consecutive series of women aged 20–74 years with newly diagnosed, histologically confirmed invasive breast cancer who were admitted to one of the four hospitals during the survey period. Of the 412 eligible patients, 405 (98%) agreed to participate. Healthy controls were selected from medical checkup examinees at two of the hospitals and confirmed to not have any cancer. They were matched with one case subject each by age (within three years) and residential area during the study period. Among potential control women, one declined to participate and two refused to provide blood samples. Consequently, written informed consent was obtained from 405 matched pairs.

Of 405 control women, we selected postmenopausal women aged over 55 years who provided blood samples and reported an energy intake between 500 and 4000 kcal. The present study included a total of 185 women.

Participants were asked to complete a self-administered questionnaire which included questions on demographic characteristics, anthropometric factors, smoking habit, family history of cancer, physical activity, medical history, and menstrual and reproductive history. Dietary habits were investigated using a 136-item semi-quantitative food-frequency questionnaire (FFQ) which was developed and validated in a Japanese population [16, 17]. In the FFQ, participants were questioned on how often they consumed individual food items (frequency of consumption), as well as relative sizes compared to standard portions. Daily food intake was calculated by multiplying frequency by standard portion and relative size for each food item in the FFQ. Daily intakes of nutrients were calculated using the fifth revised and enlarged edition of the Standard Tables of Food Composition in Japan [18]. The FFQ included questions on supplement use, but nutrient intake from supplements was not included in the analyses because no comprehensive database for supplements was available. Any influence is assumed to be minimal, however, because none of the present subjects used folate supplementation and only eight subjects (4.3%) used vitamin B supplementation. We previously documented that the questionnaire’s assessments of folate intake, and vitamin B2, B6, and B12 intake were reasonably valid [14, 17].

Participants provided blood samples at the time they returned their self-administered questionnaire. Whole blood in 7-mL EDTA-2Na vacutainers and serum samples were stored at −80°C until analysis.

Details of hormone assays have been described previously [19]. Briefly, we measured estradiol, estrone, androstenedione, dehydroepiandrosterone sulfate (DHEAS), testosterone, and free testosterone in serum by radioimmunoassay. Bioavailable estradiol (free and albumin-bound estradiol) was measured by the ammonium sulfate precipitation method. Sex-hormone binding globulin (SHBG) was measured by immunoradiometric assay. Lower detection limits (LODs) were 5 pg/mL for estradiol, 15 pg/mL for estrone, 6.25 nmol/L for SHBG, 0.1 ng/mL for androstenedione, 5 ug/dL for DHEAS, 0.05 ng/mL for testosterone, and 0.4 pg/mL for free testosterone. Measurement values below the LOD were assigned half the value of the LOD if measurable values below the LOD were not available. Intra-assay coefficients of variation (CV) were 6.5% for estradiol at a mean concentration of 24.9 pg/mL (n = 12), 10.6% for bioavailable estradiol at a mean level of 48.1% (n = 10), 5.6% for estrone at a mean concentration of 101.7 pg/mL (n = 10), 4.7% for SHBG at a mean concentration of 104.6 nmol/L (n = 10), 9.4% for androstenedione at a mean concentration of 1.33 ng/mL (n = 10), 5.2% for DHEAS at mean concentration of 75 ug/dL (n = 10), 4.5% for testosterone at a mean concentration of 0.83 ng/mL (n = 10), and 11.6% for free testosterone at a mean concentration of 5.4 pg/mL (n = 10). A commercial laboratory performed all hormone assays and provided information on intra-assay CVs (Mitsubishi Kagaku Bio-Clinical Laboratories, Tokyo, Japan).

Genomic DNA was extracted from the peripheral blood using a Qiagen FlexiGene DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.

Global DNA methylation was quantified by LUMA (LUminometric Methylation Assay) [20, 21]. Three hundred nanograms of Genomic DNA were cleaved with HapII + EcoRI or MspI + EcoRI in two separate 20-μl reactions containing 2 μl of 10 × T buffer (330 mM Tris-acetate, 100 mM Mg-acetate, 660 mM K-acetate, 5 mM dithiothreitol), 2 μl of 0.1% BSA, and 5 units of each of the restriction enzymes. The reactions were set up in a PSQ 96 Plate Low (Qiagen) and incubated at 37°C for 1 h. Then, 20 μl of annealing buffer that contained 200 mM Tris-acetate and 50 mM Mg-acetate pH 7.6 was added to the cleavage reactions, and samples were assayed using PSQ96 MA system (Biotage AB, Uppsala, Sweden). The instrument was programmed to add dNTPs in six steps, including Step 1, dATPαS; Step 2, mixture of dGTP + dCTP; Step 3, dTTP; Step 4, mixture of dGTP + dCTP; Step 5, water; and Step 6, dATP. Peak heights were calculated using the PSQ96 MA software. HapII/EcoRI and MspI/EcoRI ratios were calculated as (dGTP + dCTP)/dATP for each reaction. The HapII/ MspI ratio was then calculated as (HapII/EcoRI)/(MspI/EcoRI), which corresponds to the proportion of unmethylated CCGG. Restriction enzymes (HapII, MspI, and EcoRI) were purchased from Takara Bio (1053A, 1150A and 1040A, respectively; Shiga, Japan). PyroMark Gold Q96 Reagents for pyrosequencing were purchased from Qiagen (972804). DNA quantification was performed using the Quan-iT PicoGreen dsDNA Reagent and kit (P7581, Invitrogen, CA, USA). Intra-assay CV was 6.4% at a mean level of 74% (n = 20).

Five polymorphisms in methylenetetrahydrofolate reductase (MTHFR) (rs1801133 and rs1801131), methionine synthase (MTR) (rs1805087), and methionine synthase reductase (MTRR) (rs10380 and rs162049) were genotyped by TaqMan SNP Genotyping Assays developed by Applied Biosystems (Foster City, CA). Genotype frequencies were tested for deviation from the Hardy–Weinberg equilibrium with the χ2-test as quality control for genotyping (all P values > 0.05).

Hormone levels were divided into median or quartile categories. Adjusted mean global methylation levels of leukocyte DNA according to endogenous sex hormone levels were calculated using a multivariable liner regression model. To test linear trends for mean hormone levels, regression coefficients (β) were calculated in the multivariable linear regression model using median or quartile categories of individual hormone levels as ordinal variables. We performed the initial analyses with age-adjustment. In multivariate model, the following variables were used for adjustment: age, family history of breast cancer, history of benign breast disease, age at menarche, age at menopause, number of births, age at first birth, height, body mass index (BMI), smoking status, alcohol drinking habit, physical activity in the past five years, and energy-adjusted folate intake, most of which are known as potential breast cancer risk factors [11, 12]. Dietary folate intake was adjusted for total energy intake with the use of the residual method [22, 23]. To investigate potential effect modification, subgroup analyses were performed by dietary and genetic factors related to one-carbon metabolism and tests for interaction were carried out. All P values reported are two-sided, and significance level was set at P < 0.05. All statistical analyses were performed with SAS software version 9.1 (SAS Institute, Inc., Cary, NC).