Introduction

Breast cancer is the most commonly diagnosed cancer in women worldwide. It is responsible for 16 % of all and 22.9 % of invasive female cancers (http://www.who.int/cancer/detection/breastcancer/en/index1.html). Despite lots of investigation on the molecular pathogenesis of breast cancer, the predisposing biological processes are still not well characterized [1]. In most cases, genetic alterations of the pathways controlling growth, differentiation, apoptosis, or genomic repair lead to the malignancy [2]. Most of these changes are mediated through transcriptional-regulatory elements [3].

Data are accumulating regarding the protective role of vitamin D in various types of cancers [4]. In vitro studies revealed that vitamin D enhanced the differentiation and apoptosis of cancer cells in culture [5] including mammary glands [6]. The effects of vitamin D are mediated via the vitamin D receptor (VDR) which is expressed in most cell types, including breast tissues [7]. VDR belongs to nuclear receptor superfamily with a well-known role of transcriptional-regulatory factor [8]. It has been shown that breast tumors which express more VDR have better prognosis [9]. Animal model study exhibits enhanced growth and branching of breast tissue in VDR knockout mice [10].

The gene encoding the VDR is mapped on the long arm of chromosome 12 (12q12–14) composed of 9 exons, with an alternatively spliced promoter region [11, 12]. A series of polymorphisms in the VDR gene were reported to be linked to various biological processes [13]. FokI restriction enzyme can identify a variable site (rs2228570) in exon 2 of the gene [14]. The alteration made by a C/T transition (ACG to ATG) at the translation initiation sites and results in three amino acids longer proteins [15]. BsmI polymorphism which is located at the 3′ end of the gene apparently does not change the translated protein and has no known function on VDR [13]. This G → A polymorphism is located on intron 8 and is linked in a haplotype with variable-length polyA sequence within the 3′-untranslated region that has an impact on VDR mRNA stability [16].

Polymorphisms of the VDR gene are also associated with some pathological situations including osteoporosis [17, 18]. Although the impact of these allelic variation on breast cancer is still controversial [15, 19–21]. Recently, it has been shown that the ATG genotype at FokI site enhanced aggressive breast cancer via a poor response to vitamin D treatment [22]. In the present study, we focused on the distribution of VDR FokI and BsmI polymorphisms in Iranian breast cancer patients compared to healthy population.

Subjects and methods

A case–control study was conducted to investigate association of the two VDR gene polymorphisms with the risk of breast cancer. The breast cancer patients (n = 140) were randomly selected from patients who referred to three different breast cancer clinics. Diagnosis criteria and managements of the centers were similar and based on the standard international guidelines. All patients were diagnosed with ductal carcinoma. Histopathology reports were reviewed, and personal information such as age and menopausal status was obtained from their hospital records. The age of the patients at the time of breast cancer diagnosis was assigned. Also, 156 volunteer women participated in the study as normal controls. They were healthy women without family history of breast cancer who have referred to state-run health care services for routine mother and baby examinations. Each patient contributed to the study signed a written consent approved by the ethics committee of the Iran National Science Foundation (INSF).

We received cancer group samples either as whole blood or frozen peripheral blood mononuclear cells (PBMCs). Extraction of DNA was carried out using DNA genomic kit (QIAamp Mini kit, Qiagen). Peripheral blood sample (2 mL) was obtained from each subject in the control group. DNA extraction performed according to the standard salting out protocol. The concentration and quality of the DNA was measured using NanoDrop® ND-1000 spectrophotometer at 260 and 280 nm. DNA samples with the A260/A280 ratios of more than 1.7 were selected for analysis. DNA Sample aliquots were stored at −20 °C, and fresh working solutions (10–40 ng/μL) were prepared immediately before each experiment. Specific PCR primers were designed and verified using single nucleotide polymorphisms (SNPs) database (dbSNP 129; http://www.ncbi.nlm.nih.gov/projects/SNP/) and BLAST website (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Table 1).

Table 1 The sequence of the primers and their PCR–RFLP product characteristics
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Genomic DNA was amplified by PCR according to the following program: an initial denaturation step for 5 min at 94 °C then 30 amplification cycles of : denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s, and extension at 72 °C for 30 s. Final extension was allowed to proceed for 5 min at 72 °C.

For BsmI and FokI genotyping, restriction fragment length polymorphism (RFLP) analysis was performed. Each PCR product was digested with the appropriate restriction endonuclease as recommended by the manufacturer’s instruction (Fermentas, Cinaclon, Iran). The digested products were separated by electrophoresis on 2 % agarose gels and were visualized by ethidium bromide staining under short-wave UV light. In the case of cutting by the enzymes, alleles were indicated by b and f, whereas undigested alleles were assigned as B and F, respectively. Randomly selected PCR products were subjected to sequencing to verify the RFLP results.

Statistical methods

The collected data were loaded on statistical analysis software SPSS V.16. The data were analyzed using conditional logistic regression along with 95 % confidence intervals (CIs) in a 2 × 2 table. Chi-square test was selected to weigh up the null hypothesis (H0) among the groups. The p values less than 0.05 were considered to be statistically significant.

Results

Subject’s data

Patients mean age was 45.44 ± 10.61, and Controls mean age was 42.64 ± 9.67 years. The age ranges were 30–72 and 30–67 years for the case and control groups, respectively. Controls were matched also by menopausal status, parity, and breast feeding experience.

Histopathology reports of the patients’ tumors showed 78.5 % strong and 12.6 % week estrogen receptor expression (ER+). Progesterone receptor expression was strong in 54 % and weak in 17 % of PR+ patients. The Her2 overexpression was detected in 38 % of the breast tumors.

Genotyping and statistics

The desired fragments of BsmI and FokI containing amplicons were revealed after restriction enzyme digestion (Fig. 1). The allele frequencies of the two SNPs are summarized in Table 2. There was no major deviation from the expected Hardy–Weinberg equilibrium in control samples.

Fig. 1
figure 1

Electrophoresis results of BsmI restriction enzyme digestion on 2 % agarose. Lanes 3 and 8 BB genotype, lane 11 bb genotype, and the other lanes Bb heterozygotes

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Table 2 VDR BsmI and FokI allele frequency in patients and controls
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BsmI

The b allele of BsmI polymorphism was detected in 62.5 % of the patients versus 54 % of the controls. The relative frequency of BsmI minor allele was 0.37 which is comparable to the reports from other populations in whom VDR BsmI polymorphism showed an ethnicity-related variation with a highest rate in Caucasian (0.41) and the lowest rate in Japanese American (0.14) [23].

To analyze the correlation between BsmI SNP and breast cancer risk, the BB genotype was assigned as reference genotype according to the previous studies [15, 24, 25]. When the other two genotypes were compared with the reference genotype, a significantly (p value = 0.01) increased risk of breast cancer was observed with both BsmI bb and Bb genotypes (Table 3). The corresponding odds ratio was 2.39 (CI 1.17–4.85) for BsmI bb and 2.28 (CI 1.16–4.47) for BsmI Bb genotype. This data suggests that the b allele may contribute in susceptibility to breast cancer, either in heterozygote or homozygote state. There were no association between BsmI SNP and tumor characteristics including ER, PR, and HER2 status.

Table 3 Odds ratios for the association of breast cancer with VDR gene BsmI and FokI polymorphisms
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FokI

The relative frequency of FokI minor allele was 0.27 and 0.28 in patients and controls, respectively (Table 2). These values were lower than most of the previously published data. The reported minor allele frequency of FokI exhibits very slight variation within populations, with a few exceptions [23]. In our study, statistical analysis for cancer predisposing effect of FokI revealed no significantly increased risk in patients with FokI ff genotype compared to the control group (OR 0.83, CI 0.36–1.92). Also, the analysis of FokI heterozygous genotype demonstrates no association between this genetic variation and breast cancer in Iranian patients (OR 1.09, CI 0.67–1.77). There was no correlation between VDR FokI and patients clinical findings or tumor specification.

Discussion

The susceptibility and modifier genes play a major role in developing malignancies in concert with environmental factors. In this respect, impact of sunlight exposure, diet vitamin D, and VDR polymorphisms in breast cancer pathogenesis have attracted much interest worldwide. The effects of Vitamin D on proliferation and differentiation of several cancer cell lines have been demonstrated [26, 27]. Because this vitamin acts via VDR, the study of VDR gene SNPs can elucidate important factors in environmental exposure and breast cancer.

VDR FokI cause a longer protein with a reduced activity [28]. In vitro study has shown that FF(CC) genotype promotes gene transcription by interaction with transcription factor IIB [29]. Using MCF-7 breast cancer cells, a recent study revealed that FF genotype promotes vitamin D anti cancer function by different approaches such as regulation of estrogen receptor-α expression [22].

The distribution of two VDR gene SNPs, FokI and BsmI, has been extensively investigated in breast cancer patients. However, the reported results regarding both polymorphisms are controversial. Two comprehensive case–control study provided evidence that ff(TT) genotype of FokI was associated with a higher breast cancer risk [30, 31]. Nevertheless, there are other reports that have not supported this correlation, and in contrast, some researches even showed lower risk of breast malignancy in the subjects with ff genotype [19, 32–34]. Meta-analyses assessments have demonstrated that FokI ff genotype is linked to susceptibility to breast cancer [35, 36].

In our study, 140 Iranian breast cancer patients were analyzed for VDR gene BsmI and FokI polymorphisms. No statistically significant association was observed between FokI ff genotype and the predisposition to malignancy development. Although, in our study, the distribution of FokI minor allele mildly differs from other studies, but BsmI minor allele distribution showed the same pattern as most of the previous reports.

BsmI SNP is an intronic polymorphism, which is linked to polyA variants in a haplotype. Epidemiological studies assessing correlation of this SNP to breast cancer have reported controversial results [15, 24, 30, 37–40]. Since BsmI has various geographical distributions [23], the selected subjects and controls have a great impact on the results by population stratification. Most of the studies which were performed on Caucasian women reported increased risk of breast cancer with the BsmI bb(GG) genotype [15, 23, 37, 38]. Whereas, BsmI BB(AA) genotype was correlated to breast cancer among Hispanic [19] and Taiwanese population [20]. Both reports demonstrated diverse allele frequencies compared to the Caucasian population. We observed approximately twofold increased risk of breast cancer in carriers of BsmI b allele in either homozygote or heterozygote state. The BsmI exhibits strong linkage disequilibrium with other polymorphisms, located at the 3′ untranslated region (3′ UTR) of the VDR gene. This haplotype may affect mRNA stability and processing as well as regulation of VDR transcription and translation [41, 42].

In summary, the present study demonstrated that Iranian women with the BsmI Bb or bb genotype were at twice higher risk for breast cancer. To our knowledge, this is the first report regarding Iranian patients and may be applied for prevention and early intervention strategies in this population.