Tumor Biol. DOI 10.1007/s13277-013-1348-0
RESEARCH ARTICLE
Association between polymorphisms within the susceptibility region 8q24 and breast cancer in a Chinese population Yu Zhang & Pengfei Yi & Wei Chen & Jie Ming & Beibei Zhu & Zhi Li & Na Shen & Wei Shi & Juntao Ke & Qunzi Zhao & Xuzai Lu & Xueqiong Xun & Li Liu & Ranran Song & Hui Guo & Rong Zhong & Liming Liang & Tao Huang & Xiaoping Miao
Received: 29 August 2013 / Accepted: 22 October 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Recent publications have found associations between single-nucleotide polymorphisms (SNPs) in 8q24 and the risk of breast cancer (BC) in some populations, but the conclusions are inconsistent. In order to further investigate the association between variants in this region and BC risk in Chinese population, we conducted an independent hospitalbased case–control study to discern the effects of these SNPs on BC risk. We genotyped three 8q24 SNPs (rs13281615, Yu Zhang, Pengfei Yi, and Wei Chen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13277-013-1348-0) contains supplementary material, which is available to authorized users. Y. Zhang : W. Chen : B. Zhu : N. Shen : J. Ke : X. Lu : R. Song : R. Zhong : X. Miao State Key Laboratory of Environment Health (Incubation), MOE (Ministry of Education) Key Laboratory of Environment and Health, Ministry of Environmental Protection Key Laboratory of Environment and Health, Wuhan, China X. Miao (*) Department of Epidemiology and Biostatistics, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China e-mail: [email protected] P. Yi : J. Ming : Z. Li : W. Shi : Q. Zhao : X. Xun : H. Guo : T. Huang (*) Department of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China e-mail: [email protected] L. Liu Guangdong Key Lab of Molecular Epidemiology and Department of Epidemiology and Biostatistics, School of Public Health, Guangdong Pharmaceutical University, Guangzhou, China L. Liang Department of Biostatistics, Harvard School of Public Health, Boston, MA 02115, USA
rs6983267, and rs9642880) in 485 cases and 530 cancer-free controls. The results indicated that the rs13281615 G allele significantly increased BC risk, with an odds ratio (OR) of 1.23 (95 % confidence interval (CI)=1.03–1.46) under the allelic model. Besides, stratification analysis reported that the significant association remained in the estrogen receptor (ER)+/progesterone receptor (PR)+ subgroup with a P value of 0.007 under the allelic model (OR=1.33, 95 % CI=1.08–1.63). For the rs9642880 variant, only a feeble association was observed for the GT genotype compared with the GG genotype (OR=1.33, 95 % CI=1.01–1.74). In addition, there was a negligible association between rs6983267 and BC risk in the ER−/PR− subgroup. However, no significant finding was observed in the overall participants. The findings suggested that polymorphisms in 8q24 may contribute to susceptibility to BC risk. However, functional studies are warranted to further elucidate the mechanisms of the association. Keywords Breast cancer 8q24 . Case–control study Abbreviations SNP single-nucleotide polymorphism BC breast cancer GWAS genome-wide association studies OR odds ratio CI confidence interval
Introduction Breast cancer (BC) is the most common malignancy affecting women worldwide with rapidly increasing prevalence in many countries including China [1, 2]. Although the precise mechanism underlying the development of BC has not been elucidated, substantial evidence indicates that genetic factors
Tumor Biol.
play a crucial role in the pathogenesis of BC. The most important advance in understanding genetic susceptibility to BC was the detection of two major predisposing genes BRCA1 and BRCA2, which were identified through the genetic linkage mapping and the positional cloning in the last decade of the twentieth century [3]. However, germ line mutations in these highly penetrant susceptibility genes account for only 5–10 % of BC cases [4, 5]. Therefore, the remaining inheritance is likely to be a consequence of a combination of low-penetrance alleles. In the past few years, genome-wide association studies (GWAS) have identified multiple single-nucleotide polymorphisms (SNPs) on chromosome 8q24, not only to BC but also to prostate, colorectal, and bladder cancer susceptibility [6–11]. Although these risk alleles are located in a “gene desert,” accumulating evidence supports the notion that these 8q24 genetic variants may affect MYC oncogene expression by altering its regulation or amplification status. The protooncogene MYC functions as a transcriptional activator. It is involved in a complex regulatory network modulating cell growth, differentiation, apoptosis, and other cellular responses [12]. It has been demonstrated that breast tumors frequently carried amplifications of the 8q24 region, spanning a large region covering the MYC [13]. Easton et al. [7] firstly identified a significant association between rs13281615 (8q24.21) and BC risk through a three-staged GWA study in a European population. However, other studies reported inconsistent results in different populations [14–22]. In addition, it is worth noting that multiple low-penetrance, commonly inherited SNPs in the 8q24 region have been demonstrated to be associated with more than one type of cancer. For example, rs6983267 appears to confer susceptibility to both prostate and colorectal cancer [9, 23]. rs13254738 has been implicated to be associated with both BC and prostate cancer [6, 14]. Moreover, the involvement of several BC predisposition loci, such as P53, FGFR, and BRCA2 , in bladder and other cancers, indicates that different types of cancers may share common predisposition mechanisms which include commonly inherited SNPs. Therefore, we genotyped three 8q24 SNPs identified from GWAS to examine their relationships with BC risk among a Chinese population in Hubei province of the central region of China. In these three 8q24 SNPs, rs13281615, rs9642880, and rd6983267 were previously identified as a risk allele for BC, urinary bladder cancer, and both colorectal and prostate cancer, respectively.
Materials and methods Study population In this hospital-based case–control study, a total of 485 breast cancer patients and 530 cancer-free controls were recruited
between June 2009 and December 2011 in the Union Hospital of Huazhong University of Science and Technology, Wuhan, China. All subjects were unrelated ethnic Han Chinese females. Cases were histopathologically confirmed with BC and without any treatment (radiotherapy or chemotherapy) prior to blood sample collection. Cancer-free controls, frequently matched to the cases on age (±5 years), were randomly selected from individuals who participated in medical examination at the same hospital during the same period. The subjects with breast-related diseases were excluded from the control group. At recruitment, a 2-ml peripheral venous blood sample was drawn from each participant after written informed consent has been received. In addition, the characteristics of each participant were collected by interview or from medical records. However, some characteristics, such as age of menarche and family history, were not completely collected due to the poor memory of some subjects. This study was approved by the institutional review board of Union Hospital of Huazhong University of Science and Technology and Tongji Medical College, Huazhong University of Science and Technology. Genotyping Genomic DNA was extracted from 2 ml of peripheral blood sample using the RelaxGene Blood System DP319-02 (Tiangen, Beijing, China). The genotypes of three SNPs at 8q24 (rs13281615, rs6983267, and rs9642880) were determined by the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The call rate of the three SNPs was 95.2, 98.5, and 99.4 % for rs9642880, rs6983267, and rs13281615, respectively. Additionally, genotyping was performed without the knowledge of subjects' case–control status. To ensure quality control, 5 % duplicated samples were randomly selected to check for consistency, with a concordance rate of 100 %. Statistical analyses Pearson χ 2 test and t test were employed to analyze differences in demographic characteristics and distributions of genotypes between cases and controls, where appropriate. The Hardy–Weinberg equilibrium (HWE) for each SNP was examined among control subjects with the use of goodnessof-fit χ 2 test. In unconditional multivariate logistic regression model, the genotypic odds ratios (ORs) and 95 % confidence interval (CI) were calculated after adjusting for age and menopause status, with the common homozygote as reference. All the above statistical analyses were carried out with the SPSS 12.0 software. Linkage disequilibrium (LD)
Tumor Biol.
of the four SNPs was estimated with Haploview [24]. Haplotypes were reconstructed and analyzed using Phase 2.1 [25].
Results Population characteristics The characteristics of cases and controls are listed in Table 1. The mean age (±SD) was 48.23 (±9.73) years for cases and 48.28 (±11.76) years for controls. No significant differences were observed for age (P =0.943) and menopause status (P =0.103) between cases and controls. The association of variants in 8q24 and BC risk First, the LD analysis suggested that the three SNPs were in very weak linkage disequilibrium with each other (the r 2 was 0.023, 0.001, and 0.004 for rs13281615 and rs6983267, rs6983267 and rs9642880, and rs13281615 and rs9642880, respectively). Table 2 summarized the results from the current case–control study. The genotype frequencies of the three SNPs were in accordance with HWE in the control group (P >0.05). After adjustment for age and menopause, two SNPs of rs13281615 and rs9642880 were found to be associated with the risk of BC at a significance level of P
RESEARCH ARTICLE
Association between polymorphisms within the susceptibility region 8q24 and breast cancer in a Chinese population Yu Zhang & Pengfei Yi & Wei Chen & Jie Ming & Beibei Zhu & Zhi Li & Na Shen & Wei Shi & Juntao Ke & Qunzi Zhao & Xuzai Lu & Xueqiong Xun & Li Liu & Ranran Song & Hui Guo & Rong Zhong & Liming Liang & Tao Huang & Xiaoping Miao
Received: 29 August 2013 / Accepted: 22 October 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Recent publications have found associations between single-nucleotide polymorphisms (SNPs) in 8q24 and the risk of breast cancer (BC) in some populations, but the conclusions are inconsistent. In order to further investigate the association between variants in this region and BC risk in Chinese population, we conducted an independent hospitalbased case–control study to discern the effects of these SNPs on BC risk. We genotyped three 8q24 SNPs (rs13281615, Yu Zhang, Pengfei Yi, and Wei Chen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13277-013-1348-0) contains supplementary material, which is available to authorized users. Y. Zhang : W. Chen : B. Zhu : N. Shen : J. Ke : X. Lu : R. Song : R. Zhong : X. Miao State Key Laboratory of Environment Health (Incubation), MOE (Ministry of Education) Key Laboratory of Environment and Health, Ministry of Environmental Protection Key Laboratory of Environment and Health, Wuhan, China X. Miao (*) Department of Epidemiology and Biostatistics, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China e-mail: [email protected] P. Yi : J. Ming : Z. Li : W. Shi : Q. Zhao : X. Xun : H. Guo : T. Huang (*) Department of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China e-mail: [email protected] L. Liu Guangdong Key Lab of Molecular Epidemiology and Department of Epidemiology and Biostatistics, School of Public Health, Guangdong Pharmaceutical University, Guangzhou, China L. Liang Department of Biostatistics, Harvard School of Public Health, Boston, MA 02115, USA
rs6983267, and rs9642880) in 485 cases and 530 cancer-free controls. The results indicated that the rs13281615 G allele significantly increased BC risk, with an odds ratio (OR) of 1.23 (95 % confidence interval (CI)=1.03–1.46) under the allelic model. Besides, stratification analysis reported that the significant association remained in the estrogen receptor (ER)+/progesterone receptor (PR)+ subgroup with a P value of 0.007 under the allelic model (OR=1.33, 95 % CI=1.08–1.63). For the rs9642880 variant, only a feeble association was observed for the GT genotype compared with the GG genotype (OR=1.33, 95 % CI=1.01–1.74). In addition, there was a negligible association between rs6983267 and BC risk in the ER−/PR− subgroup. However, no significant finding was observed in the overall participants. The findings suggested that polymorphisms in 8q24 may contribute to susceptibility to BC risk. However, functional studies are warranted to further elucidate the mechanisms of the association. Keywords Breast cancer 8q24 . Case–control study Abbreviations SNP single-nucleotide polymorphism BC breast cancer GWAS genome-wide association studies OR odds ratio CI confidence interval
Introduction Breast cancer (BC) is the most common malignancy affecting women worldwide with rapidly increasing prevalence in many countries including China [1, 2]. Although the precise mechanism underlying the development of BC has not been elucidated, substantial evidence indicates that genetic factors
Tumor Biol.
play a crucial role in the pathogenesis of BC. The most important advance in understanding genetic susceptibility to BC was the detection of two major predisposing genes BRCA1 and BRCA2, which were identified through the genetic linkage mapping and the positional cloning in the last decade of the twentieth century [3]. However, germ line mutations in these highly penetrant susceptibility genes account for only 5–10 % of BC cases [4, 5]. Therefore, the remaining inheritance is likely to be a consequence of a combination of low-penetrance alleles. In the past few years, genome-wide association studies (GWAS) have identified multiple single-nucleotide polymorphisms (SNPs) on chromosome 8q24, not only to BC but also to prostate, colorectal, and bladder cancer susceptibility [6–11]. Although these risk alleles are located in a “gene desert,” accumulating evidence supports the notion that these 8q24 genetic variants may affect MYC oncogene expression by altering its regulation or amplification status. The protooncogene MYC functions as a transcriptional activator. It is involved in a complex regulatory network modulating cell growth, differentiation, apoptosis, and other cellular responses [12]. It has been demonstrated that breast tumors frequently carried amplifications of the 8q24 region, spanning a large region covering the MYC [13]. Easton et al. [7] firstly identified a significant association between rs13281615 (8q24.21) and BC risk through a three-staged GWA study in a European population. However, other studies reported inconsistent results in different populations [14–22]. In addition, it is worth noting that multiple low-penetrance, commonly inherited SNPs in the 8q24 region have been demonstrated to be associated with more than one type of cancer. For example, rs6983267 appears to confer susceptibility to both prostate and colorectal cancer [9, 23]. rs13254738 has been implicated to be associated with both BC and prostate cancer [6, 14]. Moreover, the involvement of several BC predisposition loci, such as P53, FGFR, and BRCA2 , in bladder and other cancers, indicates that different types of cancers may share common predisposition mechanisms which include commonly inherited SNPs. Therefore, we genotyped three 8q24 SNPs identified from GWAS to examine their relationships with BC risk among a Chinese population in Hubei province of the central region of China. In these three 8q24 SNPs, rs13281615, rs9642880, and rd6983267 were previously identified as a risk allele for BC, urinary bladder cancer, and both colorectal and prostate cancer, respectively.
Materials and methods Study population In this hospital-based case–control study, a total of 485 breast cancer patients and 530 cancer-free controls were recruited
between June 2009 and December 2011 in the Union Hospital of Huazhong University of Science and Technology, Wuhan, China. All subjects were unrelated ethnic Han Chinese females. Cases were histopathologically confirmed with BC and without any treatment (radiotherapy or chemotherapy) prior to blood sample collection. Cancer-free controls, frequently matched to the cases on age (±5 years), were randomly selected from individuals who participated in medical examination at the same hospital during the same period. The subjects with breast-related diseases were excluded from the control group. At recruitment, a 2-ml peripheral venous blood sample was drawn from each participant after written informed consent has been received. In addition, the characteristics of each participant were collected by interview or from medical records. However, some characteristics, such as age of menarche and family history, were not completely collected due to the poor memory of some subjects. This study was approved by the institutional review board of Union Hospital of Huazhong University of Science and Technology and Tongji Medical College, Huazhong University of Science and Technology. Genotyping Genomic DNA was extracted from 2 ml of peripheral blood sample using the RelaxGene Blood System DP319-02 (Tiangen, Beijing, China). The genotypes of three SNPs at 8q24 (rs13281615, rs6983267, and rs9642880) were determined by the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The call rate of the three SNPs was 95.2, 98.5, and 99.4 % for rs9642880, rs6983267, and rs13281615, respectively. Additionally, genotyping was performed without the knowledge of subjects' case–control status. To ensure quality control, 5 % duplicated samples were randomly selected to check for consistency, with a concordance rate of 100 %. Statistical analyses Pearson χ 2 test and t test were employed to analyze differences in demographic characteristics and distributions of genotypes between cases and controls, where appropriate. The Hardy–Weinberg equilibrium (HWE) for each SNP was examined among control subjects with the use of goodnessof-fit χ 2 test. In unconditional multivariate logistic regression model, the genotypic odds ratios (ORs) and 95 % confidence interval (CI) were calculated after adjusting for age and menopause status, with the common homozygote as reference. All the above statistical analyses were carried out with the SPSS 12.0 software. Linkage disequilibrium (LD)
Tumor Biol.
of the four SNPs was estimated with Haploview [24]. Haplotypes were reconstructed and analyzed using Phase 2.1 [25].
Results Population characteristics The characteristics of cases and controls are listed in Table 1. The mean age (±SD) was 48.23 (±9.73) years for cases and 48.28 (±11.76) years for controls. No significant differences were observed for age (P =0.943) and menopause status (P =0.103) between cases and controls. The association of variants in 8q24 and BC risk First, the LD analysis suggested that the three SNPs were in very weak linkage disequilibrium with each other (the r 2 was 0.023, 0.001, and 0.004 for rs13281615 and rs6983267, rs6983267 and rs9642880, and rs13281615 and rs9642880, respectively). Table 2 summarized the results from the current case–control study. The genotype frequencies of the three SNPs were in accordance with HWE in the control group (P >0.05). After adjustment for age and menopause, two SNPs of rs13281615 and rs9642880 were found to be associated with the risk of BC at a significance level of P
