Introduction

Breast cancer is the most invasive malignant tumor disease in women worldwide [1]. The morbidity and mortality of breast cancer have grown rapidly in both urban and rural areas of China. Breast cancer has become the leading cause of malignancy in Chinese females [2]. 2012 GLOBOCAN data show that in China, the incidence of breast cancer has reached 22.1/100 thousand, and the mortality rate is 5.4/100 thousand [1]. The pathogenesis of breast cancer is complicated, suggesting that genetic factors play a significant role [3, 4]. Recent genome-wide association studies (GWAS) have discovered numerous risk-associated variants more than 90 common genetic susceptibility genes/loci for breast cancer, as reported in the National Human Genome Research Institute catalog (NHGRI GWAS Catalog, available at:www.genome.gov/gwastudies). However, fewer than 30 loci have been validated in the Chinese Han population [510]. Most GWAS have been conducted among women of European ancestry. However, these novel loci in women of European descent tend to be weakly associated with breast cancer in women of Chinese descent [5] or have not been determined in Chinese Han populations. Because of the genetic architecture difference between two ethnic populations, additional validation studies in Chinese Han population are of great interest [11].

We have previously performed three validation studies in Chinese Han women [5, 12, 13] and, in total, have evaluated 84 SNPs that were discovered before 2013 in 47 genes/loci and confirmed nine reported breast cancer susceptibility loci, including SIAH2, INHBB-GLI2,TGFBR2, ADAM29, FTO, DUSP4-MIR3148, ESR1, FGFR2, and TOX3. For the novel susceptibility loci/genes in Europeans after 2013, in this study, we evaluated these SNPs in Chinese Han women [1423].

Materials and methods

Subjects

A total of 3036 patients suffering from breast cancer and 3036 healthy controls (female only) were recruited through collaborations with the No. 1 Hospital and No. 2 Hospital of Anhui Medical University in the province of Anhui. The basic breast cancer characteristics are shown in (Table 1). The diagnosis of each case was confirmed by at least two oncologists. All of the enrolled patients were newly diagnosed. All of the Chinese controls were clinically confirmed to be free of breast cancer, other neoplastic diseases, systemic disorders, and a family history of neoplastic diseases (including first-, second-, and third-degree relatives). Uniform criteria were used to recruit patients and controls. The same questionnaire was used to collect the clinical and demographic information of each participant. After written informed consent was obtained, a peripheral blood sample was collected from each participant. This study was approved by the Institutional Ethical Committee of each hospital and was conducted in accordance with the Declaration of Helsinki.

Table 1 The basic breast cancer characteristics

SNP selection

For this study, we choose 32 candidate SNPs that were discovered from other populations after 2013. Most of these studies were conducted on women of European descent. These 32 SNPs represent 22 independent loci that are present in either genes or intergenic regions (Table 2) [1423].

Table 2 The 32 candidate SNPs from other population

Genotyping and quality controls

Genotyping analyses were conducted using the Sequenom Mass Array system at the State Key Laboratory Incubation Base of Dermatology, Ministry of National Science and Technology, Hefei, Anhui, China. Genomic DNA was extracted from whole-blood or buffy-coat samples using FlexiGene® DNA kits (QIAGEN, Hilden, Germany). The DNA quality of all of the samples was analyzed using a Nanodrop Spectrophotometer ND-2000 (Thermo Scientific, Wilmington, USA), and agarose gel electrophoresis was performed to ensure the genomic integrity of the samples. Approximately 15 ng of genomic DNA was used to genotype each sample. Locus-specific PCR and detection primers were designed using MassARRAY Assay Design 3.0 software (Sequenom, San Diego, USA). Following the manufacturer’s instructions, the DNA samples were amplified by multiplex PCR reactions, and the PCR products were then used for locus-specific single-base extension reactions. The resulting products were desalted and transferred to a 384-element SpectroCHIP array. Allele detection was performed using matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS). The mass spectrograms were analyzed by MassARRAY Typer software (Sequenom). The exclusion criteria for the genotyped SNPs included a call rate of <95 % and deviation from Hardy–Weinberg equilibrium (HWE, P < 0.05/32) in the controls. In total, 23 SNPs passed the quality control test and were subjected to statistical analysis.

Statistical analysis

The association between the SNPs and breast cancer susceptibility was assessed using logistic regression, adjusting for age. The strength of the association was estimated by calculating the odds ratio (OR) with a 95 % confidence interval (CI). The Hardy–Weinberg equilibrium was assessed using the Chi-square test. All of the statistics were analyzed using the SPSS 13.0 and Plink 1.07 software packages. In total, 23 SNPs were subjected to statistical analysis. Conservatively accounting for multiple comparisons by Bonferroni correction, the threshold for statistical significance was P < 2.17 × 10−3 (0.05/23).

Results

To identify additional susceptibility loci associated with breast cancer in the Han Chinese population, we selected 32 SNPs from studies of breast cancer susceptibility genes/loci that were published after 2013 for a validation study (Table 2). Of the 32 SNPs, 9 were excluded from further analyses because they did not pass the quality control tests. Of the remaining 23 SNPs (Table 3), 10 SNPs were significantly associated with breast cancer in Chinese women. For 5q11.2, eight reported SNPs were replicated (rs16886181, P = 5.29 × 10−6, OR = 1.19; rs1017226, P = 5.24 × 10−4, OR = 1.22; rs16886034, P = 2.00 × 10−3, OR = 1.21; rs16886113, P = 1.24 × 10−3, OR = 1.20; rs16886364, P = 9.20 × 10−4, OR = 1.21; rs16886397, P = 1.17 × 10−3, OR = 1.20; rs16886448, P = 1.62 × 10−3, OR = 1.20; and rs2229882, P = 5.14 × 10−4, OR = 1.31). For 5q14.3, one reported SNP was replicated (rs421379, P = 2.83 × 10−13, OR = 1.83). For 10q26.1, one reported SNP was replicated (rs35054928, P = 7.73 × 10−6, OR = 1.18). 5q11.2 and 5q14.3 are first confirmed here as susceptibility loci for breast cancer in Chinese Han women. 10q26.1 has been confirmed in our previous studies as a susceptibility locus for breast cancer in Chinese Han women [5].

Table 3 Association of breast cancer susceptibility in the Chinese population with 23 SNPs previously identified by GWAS

Discussion

We could not confirm all of the SNPs that we selected, which were confirmed in other populations. Such SNPs were in linkage disequilibrium (LD), with the functional variant potentially being located somewhere in particular chromosomal regions. Because of differences in LD patterns according to genetic ancestry, SNPs identified in studies including individuals of other population may not be in high LD with the functional variant in Chinese Han women [24, 25]. Genetic interactions with other SNPs that differ in frequency between populations could also manifest as effect heterogeneity [11].

In the present study, we confirmed for the first time two breast cancer susceptibility loci in Chinese Han women: 5q11.2 (rs16886181, rs1017226, rs16886034, rs16886113, rs16886364, rs16886397, rs16886448, and rs2229882) and 5q14.3 (rs421379).

At 5q11.2, eight SNPs were confirmed. These SNPs were respectively located in the intronic region of the mitogen-activated protein kinase kinase kinase 1(MAP3K1) gene. MAP3K1 is of particular interest for breast carcinogenesis among the genes located in the 5q11.2 region. The MAP3K1 gene is involved in the MAPK signaling pathway and plays a pivotal role in regulating the transcription of important cancer genes by encoding MAP3K1, a serine/threonine kinase protein [26]. MAPKs include MAP3K1, MAP2K, and MAPK, which regulate diverse cellular functions by modulating transcription factor activity to affect gene expression. Among these, MAP3K1 regulates immune system development and function, injury repair, vasculature remodeling, and tumor progression [27, 28]. Recently, several studies have demonstrated that MAP3K1 is a genetic susceptibility marker for some tumors, such as breast cancer and gastric cancer [2931]. Breast cancer risk alleles, such as rs62355900 [iCHAV1], rs16886397 [iCHAV2a], and rs17432750 [iCHAV3], increase MAP3K1 expression in vivo and might promote breast cancer cell survival [29]. A recent study investigated the impact of MAP3K1-targeting miRNA on the growth and invasive behavior of breast cancer in vitro and in vivo by delivering using a miRNA-expressing lentivirus system an artificial miRNA (Map3k1 amiRNA) that targets MAP3K1 to 4T1 breast cancer cells; this finding suggests that MAP3K1-targeting artificial miRNA may have promising therapeutic effects for the treatment of breast cancer [30]. Another study noted that the MAP3K1 protein expression level in breast cancer cells was higher than that in normal mammary gland cells. MAP3K1 siRNA transfection can significantly reduce the expression level of MAP3K1 and enhance paclitaxel-induced cell proliferation inhibition and cell cycle arrest in breast cancer cells. Targeting MAP3K1 expression through small RNA interference can promote the therapeutic effects of paclitaxel on breast cancer [27].

The SNP rs421379 lies upstream of the arrestin domain containing 3 (ARRDC3) gene on chromosome 5q14.3. The associated SNP rs421379 is located in the 5′ region of the ARRDC3 gene and might affect a transcription binding site and ARRDC3 gene expression, permitting the development of a more aggressive and invasive tumor [23]. The ARRDC3 gene belongs to the arrestin gene family and functions in a novel regulatory pathway that controls the cell surface adhesion molecule, b-4 integrin (ITGb4), a protein associated with aggressive tumor behavior [23, 32]. The upregulation of the ARRDC3 gene in a breast cancer cell line has been shown to repress cell proliferation, migration, invasion, and in vivo tumorigenesis [33]. The data indicate that ARRDC3 binds directly to a phosphorylated form of ITGb4, leading to its internalization, ubiquitination, and ultimate degradation, which identifies the ARRDC3-ITGb4 pathway as a new therapeutic target in breast cancer and demonstrates the importance of connecting genetic arrays with mechanistic studies searching for new treatments [33]. A gene cluster at 5q11-q23 that includes ARRDC3 was deleted in 17 % of breast cancer tumor tissue [34].

Conclusions

In summary, we performed a large-scale case–control study and for the first time confirmed two reported breast cancer susceptibility loci (5q11.2 and 5q14.3) in a Chinese Han population; these loci have already been confirmed in other populations. We once again confirmed 10q26.1, a susceptibility locus for breast cancer in Chinese Han women. These data, along with data on other reported susceptibility loci, collectively demonstrate the complexity of the heritable contribution to the pathogenesis of breast cancer and highlight the contribution of multiple variants of modest effect. Moreover, further investigation and functional studies will be required if we are to advance our understanding of how the loci confirmed in this study influence the etiology of breast cancer.