This case-control study enrolled 266 pathologically confirmed BC patients who were referred to the Ali Ebneh Abitaleb hospital (Iran) and 225 age- and population-matched healthy women who participated in a screening project for metabolic syndrome; they were unrelated to the patients and had no history of any type of cancer. The clinicopathologic characteristics of the patients are summarized in Table 1. Ethical approvals for recruitment were obtained from the local Ethics Committee of Zahedan University of Medical Sciences, and informed consent was obtained from all patients and healthy individuals. Blood samples from patients and healthy controls were collected in EDTA-containing tubes and DNA was extracted using the salting out method, as described previously 27 . The quality of the isolated DNA was verified using electrophoresis on 1% agarose gel, quantitated spectrophotometrically and stored at -20°C until further use.

Polymerase chain reaction (PCR) was used to genotype the MNS16A variable number of tandem repeat polymorphisms with the primer set, as previously reported 22 . The forward and reverse primer sequences were 5′-AGGATTCTGATCTCTGAAGGGTG-3′ and 5′-TCTGCCTGAGGAAGGACGTATG-3′, respectively. PCR was performed using 2X Prime Taq Premix (Genet Bio, Korea). The amplification procedure consisted of an initial denaturing step for 5 min at 95°C followed by 30 cycles for 30 s at 95°C, 20 s at 67.5°C, and 17 s at 72°C, as well as a final extension step for 10 min at 72°C. The PCR products were visualized on 3% agarose gel containing 0.5 μg/ml of ethidium bromide (Figure 1a) and genotypes were assigned as previously reported 22 : the 243 bp and 272 bp bands were classified as the short (S*) allele, and the 333 bp and 302 bp bands were classified as the long (L*) allele, thus defining the MNS16A genotypes as L*/L*, L*/S* and S*/S*.

hTERT 2736098 genotyping was accomplished using RFLP. The forward and reverse primers were 5′AGGACGCGTGGACCGAGTGA-3′ and 5′- GGAACCCAGAAAGATGGTCTC-3′, respectively. In each 0.20 ml reaction, 1 μl of genomic DNA (~100 ng/ml), 1 μl of each primer and 10 μl of 2X Prime Taq Premix (Genet Bio, Korea) and 7 μl ddH₂O were added. The PCR conditions were set as follows: 95°C for 5 min, 30 cycles of 95°C for 30 s, 67°C for 30 s, and 72°C for 28 s and a final extension step of 72°C for 10 min. The PCR product (10 μl) was digested using Bsp120I restriction enzyme. The G allele was digested and produced 137 bp and 187 bp fragments while the A allele was undigested and produced a 324 bp fragment (Figure 1b).

hTERT 177 bp insertion/deletion genotyping was performed using PCR with forward (5′-GACCATCCTGGACTGATGGC-3′) and reverse (5′-AGGGGTGAACAATGGCGAAT-3′) primers, which can produce 366 bp and 189 bp insertion and deletion alleles, respectively. The PCR cycling conditions were 95°C for 5 min, 30 cycles of 95°C for 30 s, 66°C for 30 s, and 72°C for 26 s and a final extension step of 72°C for 10 min. The PCR products were verified on 2% agarose gels containing 0.5 μg/ml ethidium bromide, and observed under UV light (Figure 1c).

The hTERT rs2735940 polymorphism was genotyped using the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) method. The primer sequences were as follows: generic primer, 5′-CGGCAAACACTGAAATGCTA-3′; T allele, 5′-GGGATTTCTAGAAGAGCGACAT-3′; and C allele, 5′-GGGATTTCTAGAAGAGCGACAC-3′. The product size for the allele was 197 bp. Beta-2 microglobulin (B2MF: 5′-TGTAAACACTTGGTGCCTGATATAGCTTGA-3′, B2MR: 5′-CATCAGTATCTCAGCAGGTGCCACTAATCT-3′), which produces 574 bp fragments, was used as an internal control.

In each 0.20 ml reaction solution, 1.3 μl of genomic DNA (~100 ng/ml), 0.5 μl of each primer and 10 μl of 2X Prime Taq Premix (Genet Bio, Korea) and 6.7 μl ddH₂O were added. The PCR cycling condition was an initial denaturation at 95°C for 5 min followed by 30 cycles of 30 s at 95°C, annealing temperature for 23 s at 60°C, and 30 s at 72°C, with a final extension of 72°C for 10 min. The PCR products were verified on 2% agarose gels containing 0.5 μg/ml ethidium bromide, and observed under UV light (Figure 1d). Product sizes were 252 bp for either of the Ins or Del alleles, and 574 bp for the internal control. The presence of a 252 bp allele-specific band, in conjunction with a 574 bp control band, was considered to be positive evidence for each particular allele. The absence of an allele-specific band and the presence of a control band were considered to be evidence for the absence of an allele (Figure 1d).

Statistical analysis was performed using the statistical software package SPSS 18. The association between genotypes and BC were assessed by computing the odds ratio (OR) and 95% confidence intervals (95% CI) from logistic regression analyses. Haplotype analysis was performed using SNPStats software 28 29 . The Hardy–Weinberg equilibrium was tested for polymorphisms. A p-value of <0.05 was considered statistically significant.

The study groups included 266 BC patients with a mean age of 48.9 ± 11.1 years and 225 healthy women with a mean age of 50.0 ± 12.9 years. The patient group demographic information is summarized in Table 1. No significant difference in age was found between the groups (p = 0.306). The frequency distribution of the MNS16A genotypes in BC patients was: LL, 43.2%; LS, 51.1%; and SS, 5.7%, and the distribution in controls was: LL, 29.5%; LS, 68.3%; and SS, 2.2% (Table 2). Our finding showed that the L/S and L/S + S/S decreased the risk of BC (OR = 0.51, 95% CI = 0.35-0.75, p < 0.001 and OR = 0.55, 95% CI = 0.38-0.81, p = 0.002, respectively) compared with the L/L genotype.

The hTERT rs2736098 A/G variant was associated with BC risk (Table 2). Our results indicated that AG as well as GG and AG + GG increased the risk of BC (OR = 1.80, 95% CI = 1.12-2.88, p = 0.017; OR = 1.80, 95%CI = 1.06-3.06, p = 0.033 and OR = 1.87, 95% CI = 1.19-2.94, p = 0.006, respectively) compared with the AA genotype. The rs2736098 G allele increased the risk of BC compared with the A allele (OR = 1.38, 95% CI = 1.06-1.78, p = 0.016). Our results also demonstrated that the hTERT rs2735940 polymorphism was not associated with BC risk/protection, while the rs2735940 T allele increased the risk of BC compared with the C allele (OR = 1.35, 95% CI = 1.05-1.75, p = 0.020). The 177 bp ins/del polymorphism was not polymorphic in our population so that all patients and controls had the insertion allele for the hTERT 177 bp ins/del polymorphism.

Haplotype analysis is shown in Table 3. Haplotypes LCA and STA decreased the risk of BC compared with LTG (MNS16A T/rs2735940 T/rs2736098 G). No significant association was observed among the hTERT polymorphisms and clinicopathologic parameters, including tumor stage, tumor grade, estrogen and progesterone receptors (ER, PgR), tumor size, and human growth factor receptor 2 (HER2) (Table 4).

Telomeres are involved in maintaining genomic stability 30 . In the current study, we investigated the impact of hTERT variants on BC risk in a sample of the Iranian population in southeast Iran. Our data demonstrated that MNS16A LS and L/S + S/S decreased the risk of BC. AG as well as GG and AG + GG increased the risk of BC for the rs2736098 A/G polymorphism. The rs2736098 G allele was associated with an increased risk of BC. Although the hTERT rs2735940 C/T polymorphism was not associated with BC risk/protection, the rs2735940 T allele was significantly associated with BC risk. The 177 bp ins/del polymorphism was not polymorphic in our population (all individuals were the ins/ins genotype).

Earlier studies showed that hTERT mRNA expression is regulated by MNS16A in lung cancer 31 , while studies in BC patients showed that MNS16A and BC risk association are strongly related to the geographic area of the study and the selection of the patient population 25 32 . Glioblastoma multiforme studies have also confirmed that the MNS16A association with the risk of cancer incidence is highly dependent on the population’s ethnicity 23 33 34 . Overall, it can be concluded that there is much controversy regarding the association of MNS16A with different cancers, which highlights the importance of cancer origin and ethnicity in the results.

Studies on the hTERT rs2736098 variant also showed a significant controversy in association of the variant and BC risk 35 36 37 . Haiman et al. 37 observed a positive association between the 5p15 locus and the increased risk of BC while Savage et al. 35 suggested a protective effect of three correlated SNPs in this region, including rs2736098, among Polish women with a positive family history. For familial cancers, association was also observed, although not statistically significant, after Bonferroni adjustment. In the present study, we found that the rs2736098 polymorphism increased the risk of BC in our population, and it can be concluded that an association between BC risk and hTERT rs2736098 variant is generally related to ethnicity of the study population and the geographical location of the sample.

A functional variant located in the promoter of the hTERT gene, -1327C > T (rs2735940), is associated with telomere length 38 . There are few reports about the correlation between the hTERT rs2735940 variant and BC. Recently, Pellatt et al. found no association between the hTERT rs2735940 polymorphism and BC risk. They found that this variant was associated with estrogen receptor negative/progesterone receptor positive (ER-/PR-) tumors (OR = 0.73, 95% CI = 0.59-0.91) 39 .