Association between single nucleotide polymorphisms (SNPs) of XRCC2 and XRCC3 homologous recombination repair genes and triple-negative breast cancer in Polish women

XRCC2 and XRCC3 genes involved in homologous recombination repair (HRR) of DNA and in the maintenance of the genome integrity play a crucial role in protecting against mutations that lead to cancer. The aim of the present work was to evaluate associations between the risk of triple-negative breast cancer (TNBC) and polymorphisms in the genes, encoding for two key proteins of HRR: XRCC2 Arg188His (c. 563 G>A; rs3218536, Genbank Accession Number NT 007914) and XRCC3 Thr241Met (c. 722 C>T; rs861539, Genbank Accession Number NT 026437). The polymorphisms of the XRCC2 and XRCC3 were investigated by PCR–RFLP in 70 patients with TNBC and 70 age- and sex-matched non-cancer controls. In the present work, a relationship was identified between XRCC2 Arg188His polymorphism and the incidence of triple-negative breast cancer. The 188His allele and 188His/His homozygous variant increased cancer risk. An association was confirmed between XRCC2 Arg188His and XRCC3 Thr241Met polymorphisms and TNBC progression, assessed by the degree of lymph node metastases and histological grades. In conclusion, XRCC2 Arg188His and XRCC3 Thr241Met polymorphisms may be regarded as predictive factors of triple-negative breast cancer in female population.

Molecular profiling indicated that triple-negative breast cancer represents heterogeneous subgroup of breast cancer. Triple-negative breast cancer shares histological and genetic abnormalities with basal-like subtype of breast cancer, however, this overlap is incomplete. Triple-negative breast cancer do not benefit from hormonal therapies or treatments targeted against HER2 [1][2][3][4][5]. Many of targeted therapeutic agents show promise in early stage studies, but their clinical performance has yet to be definitively proven.
Molecular epidemiological studies have provided the evidence that an individual's susceptibility to precancerous lesions and cancer is modulated by both genetic and environmental factors [6,7]. Genomic rearrangements (translocations, deletions, and duplications) are extremely frequent in breast cancer cells [8][9][10][11]. These rearrangements are believed to result from an aberrant repair of DNA double-strand breaks (DSBs).
Double-strand DNA breaks are the most dangerous DNA damage. If not repaired leads to down-regulation of transcription and various cancers development [12,13]. DSB are repaired by two mechanisms: recombination (HR) and non-homologous end joining (NHEJ) [14,15].
A recent study on the Caucasian population has provided the first epidemiological evidence, supporting the association between DSBs repair gene variants and breast cancer development [16].
Polymorphisms in DNA repair genes may alter the activity of the proteins and thus modulate cancer susceptibility [17].
RAD51 is involved in homologous recombination and repair of double-strand breaks in DNA and DNA crosslinks and for the maintenance of chromosome stability. RAD51 gene is highly polymorphic in nature. In the literature, many reports confirm the significance RAD51 gene G135C polymorphism (c. 98 G[C; rs1801320; Genbank Accession Number NT 010194), regarding the risk of breast carcinoma [21][22][23][24].
XRCC2 Arg188His polymorphism (c. 563 G[A; rs3218536, Genbank Accession Number NT 007914) may limit effect on gene activity, although it can modify the breast cancer risk in female patient with low levels of plasma a-carotene or plasma folate [16,25].
The C722T substitution is the most thoroughly analyzed polymorphism in the XRCC3 gene (c. 722 C[T; rs861539, Genbank Accession Number NT 026437). Although the functional relevance of XRCC3 Thr241Met variation is unknown, some studies have reported that the 722T/T genotype is associated with increased risk of breast cancer [26][27][28].
In the present study, the association between the Arg188His polymorphism of XRCC2 gene and Thr241Met polymorphism of XRCC3 gene and triplenegative breast cancer in the population of Polish women was investigated.

Patients
In the reported study, paraffin-embedded tumor tissue was collected from 70 women with triple-negative breast carcinoma, treated at the Department of Oncology, Institute of Polish Mother's Memorial Hospital, Lodz, Poland. The age of the patients ranged from 36 to 68 years (the mean age 46.2 ± 10.12). No distant metastases were found in any of the patients at the time of treatment onset. The median follow-up of patients at the time of analysis was 38 months (the range 2-70 months). The average tumor size was 20 mm (the range 17-32 mm). All the tumors were graded by a method, based on the criteria of Scarff-Bloom-Richardson. The demographic data and the pathologic features of the patients are summarized in Table 1 The breast tissue samples (cancerous and non-cancerous) were fixed routinely in formaldehyde, embedded in paraffin, cut into thin slices, and stained with hematoxylin/ eosin for pathological examination. DNA for analysis was obtained from an archival pathological paraffin-embedded tumor and non-cancerous breast samples which were deparaffinized in xylene and rehydrated in ethanol and distilled water. In order to ensure that the chosen histological material is representative for cancerous and noncancerous tissue, every tissue sample qualified for DNA extraction was initially checked by a pathologist. DNA was extracted from material using commercially available QIAmp Kit (Qiagen GmbH, Hilden, Germany) DNA purification kit according to manufacturer's instruction.

Genotyping
Polymorphism of XRCC2 and XRCC3 gene was determined by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism), using the appropriate primers ( Table 2).

Determination of XRCC2 genotype
The 25 lL PCR mixture contained 100 ng of DNA, 12.5 pmol of each primer, 0.2 mmol/l of dNTPs, 2 mmol/l of MgCl

Results
The genotype frequency of the XRCC2 Arg188His polymorphism in the TNBC patients and controls is summarized in Table 3. It can be seen from the Table that there are significant differences in the frequency of genotypes (p \ 0.05) between the two investigated groups. A weak association was observed between triple-negative breast carcinoma occurrence and the presence of at least one 188His allele. A stronger association was observed for 188His/His than for 188Arg/His heterozygous variant. In case of the Arg188His polymorphism of XRCC2 gene, the distribution of the genotypes in the patients differed significantly from one expected from the Hardy-Weinberg equilibrium (p \ 0.05).
No statistically significant differences were observed in genotype frequencies of XRCC3 Thr241Met polymorphism between the control group and the TNBC patients (see Table 4). Among the patients, all genotype distributions did not differ significantly (p [ 0.05) from those expected by the Hardy-Weinberg equilibrium.
Histological grading was related to XRCC2 Arg188His and the XRCC3 Thr241Met polymorphisms. Histological stages were evaluated in all the cases (n = 70). There were as follows: stage I-20 cases, stage II-45 cases, and stage III-5 cases. Stages II and III were accounted together for statistical analysis (see Table 5). Some correlation was observed between the XRCC2-Arg188His and XRCC3-Thr241Met polymorphisms and triple-negative breast cancer invasiveness. An increase was observed, regarding 188Arg/His heterozygotes frequency (OR 2.45; 95 % CI  That increase was, however, not statistically significant. Table 6 shows the distribution of genotypes and the frequency of alleles in patients with (N?) and without (N-) lymph node metastases. A tendency for a decreased risk of breast cancer was observed with the occurrence of 188His/His genotype and 188His allele of XRCC2 and 241Met/Met genotype and 241Met allele of XRCC3 polymorphism. That decrease was, however, not statistically significant (p [ 0.05) (see Table 6). There were no differences either in the distribution of genotypes or the frequency of alleles in the group of patients with different tumor size (Table 6).

Discussion
According to our data, it is the first time that polymorphisms in XRCC2 and XRCC3 genes involved in the DNA repair pathway were analyzed in the population of Polish women with TNBC. The combined effect of XRCC2 and XRCC3 polymorphisms on TNBC occurrence was not investigated before. The study was performed on an ethnically homogenous population, which may improve our knowledge, regarding to what an extent the genotypephenotype relationship variations are population-related.
The polymorphisms, chosen for the study, had previously been shown to have functional significance and to be responsible factors for low DNA repair capacity phenotype, characteristic for patients with cancer including those with breast carcinoma [20].
The genes involved in DNA repair and in the maintenance of genome integrity play a crucial role in providing protection against mutations that may lead to cancer [29].
XRCC2 and XRCC3 proteins are structurally and functionally related to RAD51, which plays an important role in the homologous recombination, the process being frequently involved in cancer transformation [30].
RAD51, XRCC2, and XRCC3 gene are highly polymorphic. A single nucleotide polymorphism, 135G/C, has been identified in the 5 0 untranslated region of the RAD51 gene and has been shown to influence gene transcription activity [31]. As it was mentioned in the Introduction above, the reports on the relationship between RAD51 G135C polymorphism and breast cancer incidence are suggest that the RAD51 135C variant allele was associated  with an increased risk of female breast cancer [22,23,32,33]. By contrast, Brooks et al. [34] showed that RAD51 gene variants were found to be not associated with breast cancer risk.
Other studies have shown that the RAD51 135C variant allele was associated with an increased risk of female breast cancer [35][36][37].
135C/C genotype may be associated with an elevated tumor risk among the European populations, regarding sporadic breast cancer [36]. Similar results were obtained in the Polish population [38].
In our earlier study, RAD51 135C allele variant was associated with an elevated risk of triple-negative breast cancer in the Polish women [39].
It is possible that the presence of C allele remains in a linkage disequilibrium with another, so far unknown, mutation located outside the coding region in the RAD51 gene, which may be important, regarding RAD51 concentrations in plasma.
In the presented study, XRCC2 Arg188His genotype was associated with an elevated risk of triple-negative breast cancer in the Polish population. There was a 6.25-fold increased risk of TNBC for the individuals, carrying XRCC2-188His/His genotype, compared with subjects carrying XRCC2-188Arg/Arg, 188Arg/His genotype, respectively. XRCC2 Arg188His polymorphism was not related, either to tumor size or cancer type or grade.
In the reported study, the Arg188His polymorphism of XRCC2 gene and Thr241Met of XRCC3 were correlated with breast carcinoma progression. Arg188His and Thr241Met heterozygote were associated with an increased risk of stage I breast cancer. However, other literature data were also found [40][41][42]. No significant associations were observed between the Thr241Met and breast cancer in Iowa and Cypriot women (40,43).
In the Polish population, Thr241Met genotype of XRCC3 polymorphism increased the risk of breast cancer development [41,42,44].
Similar to our observation, the recent reports demonstrate that XRCC3 Thr241Met allele seems associated with an elevated breast cancer risk in non-Chinese subjects (28).
The role of position 188 in the aminoacid chain for XRCC2 protein functionality is still unknown. The several data suggest that XRCC2 Arg188His polymorphism is not directly associated with breast cancer risk [45,46].
In conclusion, the reported study is another evidence for the significance of Thr241Met and Arg188His genotype in breast carcinoma staging.
The obtained data show that Arg188His and Thr241Met polymorphisms of XRCC2/3 genes may be associated with the risk of triple-negative breast carcinoma occurrence. On the other hand, a protective effect was observed of all the polymorphisms in the patients without (N-) lymph node metastasis. The obtained data suggest that the reported study may be the first observation of the polymorphisms in XRCC2 and XRCC3 genes, involved in the DNA repair pathway, to be associated with triple-negative breast carcinoma risk in the population of Polish women.
Finally, it is postulated that these polymorphisms may be used as predictive factors for TNBC in the Polish female population. Further studies, conducted on a larger group, are suggested to clarify this point.