MOLECULAR CARCINOGENESIS 54:E122–E128 (2015)
Impact of Metallothionein Gene Polymorphisms on the Risk of Lung Cancer in a Japanese Population Hideo Nakane,1, 3 Minoru Hirano,2 Hidemi Ito,3 Satoyo Hosono,3 Isao Oze,3 Fumihiko Matsuda,4 Hideo Tanaka,3 and Keitaro Matsuo5* 1
Social Systems Research Laboratory, Toyota Central R&D Labs., Inc., Nagakute, Aichi, Japan Environmental & Applied Biotechnology Laboratory, Toyota Central R&D Labs., Inc., Nagakute, Aichi, Japan 3 Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan 4 Center for Genomic Medicine, Kyoto University Graduate School of Medicine and Faculty of Medical Sciences, Sakyo-ku, Kyoto, Japan 5 Department of Preventive Medicine, Kyushu University Faculty of Medical Sciences, Higashi-ku, Fukuoka, Japan 2
Metallothioneins (MTs) are cysteine-rich proteins that act as antioxidants. A case-control study was conducted to assess the effects of gene polymorphisms in the MT region on the risk of lung cancer in Japanese subjects: 769 lung cancer cases and 939 non-cancer controls. Associations were evaluated using logistic regression models with adjustment for potential confounders (age, sex, and lifestyle factors including smoking, drinking, and green-yellow vegetable intake). We found five polymorphisms in the MT-1 gene region that showed statistically significant associations with lung cancer. Of these polymorphisms, rs7196890 showed the strongest association (odds ratio: 1.30, P ¼ 0.004, 95% confidence interval: 1.09– 1.55). The impact of the polymorphism decreased with the increase of smoking, and virtually no association with lung cancer was observed among heavy smokers whose pack-year values were 30 or more (odds ratio: 1.02, P ¼ 0.93, 95% confidence interval: 0.67–1.55). These results suggest that polymorphisms in the MT gene are moderately associated with the risk of lung cancer and that the associations are modified by lifestyle factors. © 2014 Wiley Periodicals, Inc. Key words: single nucleotide polymorphism; case-control study; gene-environment interaction INTRODUCTION Lung cancer is the most common cancer in many countries. Smoking is a well-known risk factor of lung cancer. One possible cause of carcinogenesis by smoking is exposure to the various carcinogenic aromatic hydrocarbons contained in tobacco smoke, such as benzo(a)pyrene (B(a)P). Metallothioneins (MTs) are low-molecular-weight proteins with a high cysteine content that were discovered in 1957 as a cadmium-binding protein from horse kidney [1]. Cysteine residues in MTs serve to scavenge oxygen radicals, which protect cells from harmful oxidation of nucleic acids, proteins, and lipids [2]. Thus MTs are expected to prevent various diseases caused by oxidative damage. Takaishi et al. reported that the incidence of lung tumor in mice exposed to B(a)P was higher in MT-null mice than in wild-type mice [3], which suggests some involvement for MTs with tobaccosmoke carcinogenesis in the lung. In humans, four isoforms of MT have been identified (MT-1, MT-2, MT-3, and MT-4) [4]. In addition, various subtypes are known for MT-1 and MT-2. Of the 17 MT genes, including all isoforms and subtypes, the expression of MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT1X, MT-2A, and MT-3 are observed in the tissues of non-small cell lung cancer patients [5,6]. In the above context, it is possible that gene polymorphisms of MT are related to the risk of lung cancer. In this study, we examined the associations between single nucleotide polymorphisms (SNPs) of ß 2014 WILEY PERIODICALS, INC.
the MT genes and the risk of lung cancer in humans with consideration of lifestyle factors such as smoking. To our knowledge, this is the first report to identify SNPs in the MT gene region that are related to the risk of lung cancer. MATERIALS AND METHODS Subjects All subjects were first-visit outpatients at the Aichi Cancer Center Hospital (ACCH) aged 18–79 who gave
Abbreviations: B(a)P, benzo(a)pyrene; MT, metallothionein; SNP, single nucleotide polymorphism; ACCH, Aichi Cancer Center Hospital; HERPACC, Hospital-based Epidemiological Research Program at Aichi Cancer Center; MAF, minor allele frequency; PY, pack-yrs; OR, odds ratio; CI, confidence interval; LD, linkage disequilibrium. Grant sponsor: National Cancer Center Research and Development Fund; Grant number: 23-A-4; Grant sponsor: Health and Labor Sciences Research Grants for Research on Applying Health Technology; Grant number: 201136002A; Grant sponsor: Ministry of Health, Labor and Welfare, and a Grant-in-Aid for Scientific Research on Priority Areas of Cancer; Grant number: 17015018; Grant sponsor: Innovative Areas; Grant number: 221S0001; Grant sponsor: Japanese Ministry of Education, Culture, Sports, Science and Technology *Correspondence to: Department of Preventive Medicine, Kyushu University Faculty of Medical Sciences 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Received 29 October 2013; Revised 28 May 2014; Accepted 2 June 2014 DOI 10.1002/mc.22198 Published online 30 August 2014 in Wiley Online Library (wileyonlinelibrary.com).
METALLOTHIONEIN GENE POLYMORPHISMS AND LUNG CANCER
written informed consent to the enrollment in the Hospital-based Epidemiological Research Program at Aichi Cancer Center (HERPACC) during 2001–2005. Information on lifestyle factors was collected using a self-administered questionnaire, which was checked by a trained interviewer. The outpatients were also asked to provide blood samples. Approximately 95% of eligible subjects completed the questionnaire and 60% provided blood samples. Details of this program have been described elsewhere [7,8]. The cases included 769 patients who were newly and histologically diagnosed as having lung cancer. Controls (n ¼ 939) were randomly selected from outpatients who completed the questionnaire, provided blood samples, and were confirmed cancer-free. This study protocol was approved by the Institutional Ethical Committee of Aichi Cancer Center. Genotyping Procedure DNA of each subject was extracted from the buffy coat fraction using a DNA Blood mini kit (Qiagen, Tokyo, Japan). All samples were genotyped on an Illumina Human 610-Quad BeadChip (Illumina, San Diego, CA) with 576,736 SNP markers, at the Center for Genomic Medicine of Kyoto University Graduate School of Medicine. After removing SNPs that failed the quality control criteria (Hardy–Weinberg equilibrium P 1 106 [excluded SNPs: n ¼ 277]; SNP call rate > 0.95 [n ¼ 2,921]; and minor allele frequency [MAF] 0.01 [n ¼ 82,414]), 491,738 markers were selected as a source for this analysis (some SNPs were excluded based on two or more criteria). Functional human MT genes are clustered at a single locus on chromosome 16 (16q13) [9]; therefore, of the 491,738 source markers, we selected 75 SNPs in the region of the MT genes and their adjacent genes (BBS2 and NUP93). The process of SNPs selection is shown in Supplemental Figure S1. Assessment of Smoking, Alcohol Consumption, and Fruit and Green-Yellow Vegetable Intake All exposures were assessed from the self-administered questionnaire, completed during the first visit to ACCH before the diagnostic procedure was conducted. Subjects were questioned specifically about their lifestyle before the onset of the symptoms that occasioned their visit to ACCH. Smoking status was divided into three categories: never, former, and current. Former smokers were defined as those who quit smoking at least 1 yr before the administration of the survey. Cumulative doses of smoking were evaluated as pack-years (PY), the product of the number of packets consumed in 1 d and the number of years of smoking. Drinking status was divided into three categories: never, former, and current. Former drinkers were defined as those who quit drinking at least 1 yr before the administration of the survey. The intake of fruits and green-yellow vegetables was determined using a food frequency questionnaire Molecular Carcinogenesis
E123
(FFQ), described in detail elsewhere [10,11]. Briefly, the FFQ enables estimation of the quantity of intake using eight categories of intake frequency information: never or seldom, 1–3 times/mo, 1–2 times/wk, 3– 4 times/wk, 5–6 times/wk, once/d, twice/d, and three or more times/d. The intake was adjusted for total energy intake, and was classified into tertiles. Statistical Analysis Differences in categorized demographic variables between cases and controls were tested by a x2 test. Mean values for age and PY were compared by the Wilcoxon rank-sum test. To verify that the allele distribution for each SNP was in Hardy-Weinberg equilibrium, we used a x2 test with 1 degree of freedom. To assess the association between each polymorphism and the risk of lung cancer, odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated using unconditional logistic regression models adjusted for potential confounders. Potential confounders considered in this analysis were age, sex, smoking (PY), drinking status, and the energyadjusted intake of fruits and green-yellow vegetables. We evaluated the detailed effects of a selected SNP by considering the major histological subtypes of lung cancer (adenocarcinoma, squamous-cell carcinoma, and small-cell carcinoma) and interaction with lifestyle factors (smoking and the intake of fruits and green-yellow vegetables). Effect modification by lifestyle factors was evaluated by a test of heterogeneity using stratified analyses by smoking (PY ¼ 0,
Impact of Metallothionein Gene Polymorphisms on the Risk of Lung Cancer in a Japanese Population Hideo Nakane,1, 3 Minoru Hirano,2 Hidemi Ito,3 Satoyo Hosono,3 Isao Oze,3 Fumihiko Matsuda,4 Hideo Tanaka,3 and Keitaro Matsuo5* 1
Social Systems Research Laboratory, Toyota Central R&D Labs., Inc., Nagakute, Aichi, Japan Environmental & Applied Biotechnology Laboratory, Toyota Central R&D Labs., Inc., Nagakute, Aichi, Japan 3 Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan 4 Center for Genomic Medicine, Kyoto University Graduate School of Medicine and Faculty of Medical Sciences, Sakyo-ku, Kyoto, Japan 5 Department of Preventive Medicine, Kyushu University Faculty of Medical Sciences, Higashi-ku, Fukuoka, Japan 2
Metallothioneins (MTs) are cysteine-rich proteins that act as antioxidants. A case-control study was conducted to assess the effects of gene polymorphisms in the MT region on the risk of lung cancer in Japanese subjects: 769 lung cancer cases and 939 non-cancer controls. Associations were evaluated using logistic regression models with adjustment for potential confounders (age, sex, and lifestyle factors including smoking, drinking, and green-yellow vegetable intake). We found five polymorphisms in the MT-1 gene region that showed statistically significant associations with lung cancer. Of these polymorphisms, rs7196890 showed the strongest association (odds ratio: 1.30, P ¼ 0.004, 95% confidence interval: 1.09– 1.55). The impact of the polymorphism decreased with the increase of smoking, and virtually no association with lung cancer was observed among heavy smokers whose pack-year values were 30 or more (odds ratio: 1.02, P ¼ 0.93, 95% confidence interval: 0.67–1.55). These results suggest that polymorphisms in the MT gene are moderately associated with the risk of lung cancer and that the associations are modified by lifestyle factors. © 2014 Wiley Periodicals, Inc. Key words: single nucleotide polymorphism; case-control study; gene-environment interaction INTRODUCTION Lung cancer is the most common cancer in many countries. Smoking is a well-known risk factor of lung cancer. One possible cause of carcinogenesis by smoking is exposure to the various carcinogenic aromatic hydrocarbons contained in tobacco smoke, such as benzo(a)pyrene (B(a)P). Metallothioneins (MTs) are low-molecular-weight proteins with a high cysteine content that were discovered in 1957 as a cadmium-binding protein from horse kidney [1]. Cysteine residues in MTs serve to scavenge oxygen radicals, which protect cells from harmful oxidation of nucleic acids, proteins, and lipids [2]. Thus MTs are expected to prevent various diseases caused by oxidative damage. Takaishi et al. reported that the incidence of lung tumor in mice exposed to B(a)P was higher in MT-null mice than in wild-type mice [3], which suggests some involvement for MTs with tobaccosmoke carcinogenesis in the lung. In humans, four isoforms of MT have been identified (MT-1, MT-2, MT-3, and MT-4) [4]. In addition, various subtypes are known for MT-1 and MT-2. Of the 17 MT genes, including all isoforms and subtypes, the expression of MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT1X, MT-2A, and MT-3 are observed in the tissues of non-small cell lung cancer patients [5,6]. In the above context, it is possible that gene polymorphisms of MT are related to the risk of lung cancer. In this study, we examined the associations between single nucleotide polymorphisms (SNPs) of ß 2014 WILEY PERIODICALS, INC.
the MT genes and the risk of lung cancer in humans with consideration of lifestyle factors such as smoking. To our knowledge, this is the first report to identify SNPs in the MT gene region that are related to the risk of lung cancer. MATERIALS AND METHODS Subjects All subjects were first-visit outpatients at the Aichi Cancer Center Hospital (ACCH) aged 18–79 who gave
Abbreviations: B(a)P, benzo(a)pyrene; MT, metallothionein; SNP, single nucleotide polymorphism; ACCH, Aichi Cancer Center Hospital; HERPACC, Hospital-based Epidemiological Research Program at Aichi Cancer Center; MAF, minor allele frequency; PY, pack-yrs; OR, odds ratio; CI, confidence interval; LD, linkage disequilibrium. Grant sponsor: National Cancer Center Research and Development Fund; Grant number: 23-A-4; Grant sponsor: Health and Labor Sciences Research Grants for Research on Applying Health Technology; Grant number: 201136002A; Grant sponsor: Ministry of Health, Labor and Welfare, and a Grant-in-Aid for Scientific Research on Priority Areas of Cancer; Grant number: 17015018; Grant sponsor: Innovative Areas; Grant number: 221S0001; Grant sponsor: Japanese Ministry of Education, Culture, Sports, Science and Technology *Correspondence to: Department of Preventive Medicine, Kyushu University Faculty of Medical Sciences 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Received 29 October 2013; Revised 28 May 2014; Accepted 2 June 2014 DOI 10.1002/mc.22198 Published online 30 August 2014 in Wiley Online Library (wileyonlinelibrary.com).
METALLOTHIONEIN GENE POLYMORPHISMS AND LUNG CANCER
written informed consent to the enrollment in the Hospital-based Epidemiological Research Program at Aichi Cancer Center (HERPACC) during 2001–2005. Information on lifestyle factors was collected using a self-administered questionnaire, which was checked by a trained interviewer. The outpatients were also asked to provide blood samples. Approximately 95% of eligible subjects completed the questionnaire and 60% provided blood samples. Details of this program have been described elsewhere [7,8]. The cases included 769 patients who were newly and histologically diagnosed as having lung cancer. Controls (n ¼ 939) were randomly selected from outpatients who completed the questionnaire, provided blood samples, and were confirmed cancer-free. This study protocol was approved by the Institutional Ethical Committee of Aichi Cancer Center. Genotyping Procedure DNA of each subject was extracted from the buffy coat fraction using a DNA Blood mini kit (Qiagen, Tokyo, Japan). All samples were genotyped on an Illumina Human 610-Quad BeadChip (Illumina, San Diego, CA) with 576,736 SNP markers, at the Center for Genomic Medicine of Kyoto University Graduate School of Medicine. After removing SNPs that failed the quality control criteria (Hardy–Weinberg equilibrium P 1 106 [excluded SNPs: n ¼ 277]; SNP call rate > 0.95 [n ¼ 2,921]; and minor allele frequency [MAF] 0.01 [n ¼ 82,414]), 491,738 markers were selected as a source for this analysis (some SNPs were excluded based on two or more criteria). Functional human MT genes are clustered at a single locus on chromosome 16 (16q13) [9]; therefore, of the 491,738 source markers, we selected 75 SNPs in the region of the MT genes and their adjacent genes (BBS2 and NUP93). The process of SNPs selection is shown in Supplemental Figure S1. Assessment of Smoking, Alcohol Consumption, and Fruit and Green-Yellow Vegetable Intake All exposures were assessed from the self-administered questionnaire, completed during the first visit to ACCH before the diagnostic procedure was conducted. Subjects were questioned specifically about their lifestyle before the onset of the symptoms that occasioned their visit to ACCH. Smoking status was divided into three categories: never, former, and current. Former smokers were defined as those who quit smoking at least 1 yr before the administration of the survey. Cumulative doses of smoking were evaluated as pack-years (PY), the product of the number of packets consumed in 1 d and the number of years of smoking. Drinking status was divided into three categories: never, former, and current. Former drinkers were defined as those who quit drinking at least 1 yr before the administration of the survey. The intake of fruits and green-yellow vegetables was determined using a food frequency questionnaire Molecular Carcinogenesis
E123
(FFQ), described in detail elsewhere [10,11]. Briefly, the FFQ enables estimation of the quantity of intake using eight categories of intake frequency information: never or seldom, 1–3 times/mo, 1–2 times/wk, 3– 4 times/wk, 5–6 times/wk, once/d, twice/d, and three or more times/d. The intake was adjusted for total energy intake, and was classified into tertiles. Statistical Analysis Differences in categorized demographic variables between cases and controls were tested by a x2 test. Mean values for age and PY were compared by the Wilcoxon rank-sum test. To verify that the allele distribution for each SNP was in Hardy-Weinberg equilibrium, we used a x2 test with 1 degree of freedom. To assess the association between each polymorphism and the risk of lung cancer, odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated using unconditional logistic regression models adjusted for potential confounders. Potential confounders considered in this analysis were age, sex, smoking (PY), drinking status, and the energyadjusted intake of fruits and green-yellow vegetables. We evaluated the detailed effects of a selected SNP by considering the major histological subtypes of lung cancer (adenocarcinoma, squamous-cell carcinoma, and small-cell carcinoma) and interaction with lifestyle factors (smoking and the intake of fruits and green-yellow vegetables). Effect modification by lifestyle factors was evaluated by a test of heterogeneity using stratified analyses by smoking (PY ¼ 0,
