Abstract
X-ray repair cross-complementing group 2 (XRCC2) gene is important for the repair of double-strand DNA breaks (DSB) by homologous recombination (HR). XRCC2 polymorphisms may be associated with the development of certain types of cancers, but little is known about their association with ovarian carcinoma. XRCC2 −41657C/T (rs718282) polymorphisms were genotyped by the PCR–RFLP (restriction fragment length polymorphism) method in 608 patients with ovarian cancer and in 400 cancer-free women, who served as controls. In the present work, a relationship was identified between XRCC2 −41657C/T polymorphism and the incidence of ovarian cancer. An association was observed between ovarian carcinoma occurrence and the presence of T/T genotype [OR = 3.50 (2.46–4.97), p < 0.0001]. A tendency for an increased risk of ovarian cancer was detected with the occurrence of T allele of XRCC2 polymorphism. There were no significant differences between the distribution of XRCC2 −41657C/T genotypes in the subgroups assigned to histological grades. We suggest that the −41657C/T polymorphism of the XRCC2 gene may be risk factors for ovarian cancer development.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
The ovarian cancer is diagnosed in Poland in more than three thousand women a year, out of whom, two-thirds die during five subsequent years [1, 2]. For the time being, there are no reliable diagnostic methods, allowing for an early identification of this neoplasm. The ovarian cancer is diagnosed late, in advanced stages of the disease, as the first, non-characteristic symptoms are ignored by patients. Despite the growing knowledge on the ovarian cancer, no effective screening test has yet been designed. It is then necessary to identify new risk factors.
In the recent years, there has been a considerable progress in the understanding of DNA repair mechanisms. Our knowledge of these processes, their control and various factors which affect their performance and effectiveness may, in future, provide more effective prophylactics against a number of diseases, associated with insufficient or incomplete repair of DNA defects, leading, in consequence, to mutations, genomic instability and neoplastic diseases.
Out of all the DNA damages, double-strand breaks (DSB) are especially dangerous for the cell. Because of the multiplicity of DSB-causing exo- and endogenous factors, the effectiveness of their repair is of key significance for proper cell functionality, preventing DNA fragmentation and the translocation and deletion of chromosomes [3]. The DSB repair system functions via two mechanisms: homologous recombination (HR) and non-homologous DNA end joining (NHEJ) [3, 4].
Mutations in DNA of double-strand breaks repair genes are involved in the pathogenesis of cancers; however, it is still unclear whether any defects in this pathway may play any role in the aetiology of ovarian cancer.
XRCC2 (X-ray repair cross-complementing group 2) with RAD51 (RecA homolog, E. coli; S. cerevisiae), XRCC3 (X-ray repair cross-complementing group 3), BRCA1 (breast cancer-1), BRCA2 (breast cancer-2) and other DNA repair proteins are involved in the homologous recombination and repair of double-strand breaks in DNA and DNA cross-links, as well as in the maintenance of chromosome stability [3].
XRCC2 gene, located in 7q36.1, is an essential part of the homologous recombination repair pathway and a functional candidate for involvement in cancer progression [5].
It is known that polymorphisms in XRCC2 may modify individual susceptibility to various cancer diseases [6–13].
There are also some reports of associated XRCC2 gene variants, particularly a coding single-nucleotide polymorphism (SNP) in exon 3 (Arg188His, R188H, rs3218536), and the development of ovarian cancer [14–16].
Indeed, recently, several studies have shown that the XRCC2 −41657C/T genotype was related to increased esophageal squamous cell carcinoma (ESCC), gastric cardia adenocarcinoma (GCA) and in smoking-/drinking-related laryngeal cancer risk [11, 17].
Unfortunately, it is difficult to find in the literature reports directly binding −41657C/T (rs718282) single-nucleotide polymorphism in DNA repair gene XRCC2 with ovarian cancer occurrence. These data stimulated us to search for an association between ovarian cancer development and the −41657C/T (rs718282) polymorphisms in XRCC2 gene.
Materials and methods
Patients
Six hundred and eight patients with histologically proven diagnosis of ovarian cancer were included in the reported study (Table 1). Paraffin-embedded tumour tissues were obtained from women with ovarian carcinoma (age range 37–84, mean age 52.23 ± 11.12) treated at Department of Menopausal Diseases, Institute of Polish Mother’s Memorial Hospital (Lodz, Poland), between 2000 and 2014. All the diagnosed tumours were graded according to the criteria of the International Federation of Gynaecology and Obstetrics (FIGO) [18]. DNA from normal ovarian tissue obtained from non-cancer women (n = 400) served as control. They were non-related women who have never been diagnosed with ovarian tumours, other tumours or chronic disease and were randomly selected and frequency matched to the cases on age (age range 34–83, mean age 51.27 ± 11.18). Informed consent was obtained from patients and controls, and the Ethical Committee approved the study.
The ovarian tissue samples (cancerous and non-cancerous) were fixed routinely in formaldehyde, embedded in paraffin, cut into thin slices and stained with hematoxyline/eosin for pathological examination. DNA for analysis was obtained from archival pathological paraffin-embedded tumour and healthy ovarian samples which were deparaffinised in xylene and rehydratated in ethanol and distilled water. The pathological evaluation report was obtained for each patient (Department of Pathology, Institute of Polish Mother’s Memorial Hospital, Lodz, Poland). Genomic DNA was prepared from the material, using a commercially available QIAmp DNA purification kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instruction.
Genotype determination
The PCR- restriction fragment length polymorphism method (PCR–RFLP) was used to detect the genotypes of the −41657C/T polymorphism as described previously [17].
Polymorphism −41657C/T of the XRCC2 gene was determined by PCR–RFLP, using primers (forward 5′-GGAGGCCGCAATGAGCTGAGATG-3′ and reverse 5′-TCGGGAAGCTGAGGTGGGAGGA-3′). The PCR was carried out in a PTC-100 TM (MJ Research, INC, Waltham, MA, USA) thermal cycler. PCR amplification was performed in the final volume of 25 μl of reaction mixture, which contained 100 ng of genomic DNA, 0.2 μmol of each primer (ARK Scientific GmbH Biosystems, Darmstadt, Germany), 2.5 mM of MgCl2, 1 mM of dNTPs and 1 unit of Taq Polymerase (Qiagen GmbH, Hilden, Germany). PCR cycle conditions were the following: 95 °C for 45 s, 72 °C for 45 s and 72 °C for 60 s, repeated in 35 cycles. PCR products were electrophoresed in a 2 % agarose gel and visualised by ethidium bromide staining. The cleavage with MvaI (New England BioLabs, Frankfurt am Main, Germany) produced fragments of 315/59/42, 357/315/59/42 and 357/59 bp corresponding to the C/C, C/T and T/T genotypes of the XRCC2 gene, respectively.
Statistical analysis
For each polymorphism, departure of the genotype distribution from that expected from Hardy–Weinberg equilibrium was assessed using the standard χ 2 test. Genotype frequencies in the study cases and the controls were compared by χ 2 test. Genotype specific risks were estimated as odds ratios (ORs) with associated 95 % intervals (CIs) by unconditional logistic regression. p values <0.05 were considered significant. All the statistical analyses were performed, using the STATISTICA 6.0 software (Statsoft, Tulsa, OK, USA).
Results
All the recruited samples both ovarian cancer (n = 608) and control samples (n = 400) were successfully genotyped for XRCC2 polymorphisms. Table 2 shows genotype distribution −41657C/T XRCC2 polymorphism between endometrial cancer patients and controls. It can be seen from the table that there are significant differences (p < 0.05) between the two investigated groups. A weak association was observed between ovarian carcinoma occurrence and the presence of T/T and C/T genotypes. A stronger association was observed for T/T than for C/T heterozygous variant. Variant T allele of XRCC2 increased cancer risk (p < 0.05). The distribution of the genotypes in the patients differed significantly from one expected from the Hardy–Weinberg equilibrium (p < 0.05).
Because we were interested in the association between the distribution of genotypes and frequencies of alleles of investigated genetic variability on the tumour grade evaluated according to FIGO criteria, these data were also analyzed.
Histological grading was evaluated in all the cases (n = 608): grade 1 (G1)—332 cases, grade 2 (G2)—256 cases and grade 3 (G3)—20 cases. G2 and G3 were accounted together for statistical analysis (see Table 3). We did not find any association of the XRCC2 polymorphisms in the patients group with cancer progression assessed by ovarian cancer grading (p > 0.05).
Clinical FIGO staging was also related to XRCC2 polymorphisms (Table 4). Staging was evaluated in all the cases (n = 608). We did not find any association of the XRCC2 polymorphisms in the patients group with cancer progression assessed by ovarian cancer staging (p > 0.05).
Our data did not demonstrate any statistically significant correlation between XRCC2 −41657C/T polymorphism and the risk factors for ovarian cancer, such as number of pregnancy, size of tumour, age, menarche, ascites, tumour wall infiltration and women with ovarian cancer, here again erase the remark “(data not shown)”.
Discussion
DNA changes, imposed by damaging factors and replication errors, may bring about serious consequences for the cell. Its survival is then determined by the availability of DNA repair systems, eliminating damages and reducing the frequency of DNA mutations. The systems present with high substrate specificity and specialisation. Changes in the encoding genes may lead to an increased general incidence of mutations, development of neoplasms and other serious conditions, including hereditary diseases.
In the process of neoplastic transformation, beside mutations in protooncogenes and suppressor genes, genetic polymorphisms may also play an important role, including the single-nucleotide polymorphisms (SNPs). Our research addressed the role of single-nucleotide polymorphism −41657C/T of DNA repair gene XRCC2 as a risk factor for ovarian cancer.
Homologous recombination repair plays a critical role in repairing DNA damage [4, 19]. The cellular reaction to DNA damaging agents can modulate the susceptibility towards tumour development [20]. This reaction is mainly determined by the efficacy of DNA repair, which may, in turn, be influenced by the variability of DNA repair genes, expressed by their polymorphisms [21–24].
Defects of genes, involved in double-strand breaks repair, often lead to better cancer development [20, 25]. XRCC2 and XRCC3 gene are structurally and functionally related to RAD51, which plays an important role in the homologous recombination, the process being frequently involved in cancer transformation [5]. RAD51, XRCC2 and XRCC3 gene are highly polymorphic.
A large number of molecular epidemiologic studies have been performed on various neoplasms, such as cancer of breast, bladder, lung, head and neck and skin, to evaluate the role of RAD51 polymorphisms [26–30].
Our earlier study of RAD51 G135C polymorphism in Polish population identified a haplotype associated with ovarian cancer [31, 32]. The RAD51 135C allele was associated with a significantly increased risk of ovarian cancer in Poland [31, 32].
The XRCC2 polymorphisms were found to be correlated with various cancer diseases [7, 14, 16, 33, 34], but little data are available on the association or its lack in ovarian cancer [14–16, 31, 32].
As mentioned in Introduction, XRCC2 −41657C/T genotype has been associated with an increased risk of esophageal squamous cell carcinoma (ESCC), gastric cardia adenocarcinoma (GCA) and in smoking-/drinking-related laryngeal cancer [11, 15].
The role of XRCC2 −41657C/T polymorphisms and ovarian cancer development is still unknown. To date, none studies have addressed the association between alterations in this region of the XRCC2 gene and ovarian cancer. Because a proper functioning of the XRCC2 gene is important for the genomic stability, its alternations may be associated with higher cancer susceptibility.
Therefore, we analysed the role of −41657C/T genetic variations in the homologous recombination repair gene and for the risk of developing ovarian cancer.
We succeeded to demonstrate—a series of 608 samples from patients with ovarian cancer—that T/T genotype of −41657C/T polymorphism of XRCC2 gene was associated with an increased risk of ovarian cancer in studied population, almost four times (3.50) higher than in case of the other genotypes, while having no effect on the degree of disease progression, tumour size, number of pregnancy, age or menarche.
It is possible that the presence of T allele remains in some linkage disequilibrium with another, so far unknown, mutation, located outside of the coding region in the XRCC2 gene, which may be of importance for the XRCC2 concentration in plasma and more severe cancer development.
In conclusion, the reported study is another evidence for the significance of -41657C/T genotypes in ovarian cancer occurrence. Thus, we conclude that our observations may be an important signal, prompting to appreciate the role of XRCC2 in ovarian cancer development and likely triggering further studies on this interesting subject.
References
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer Statistics, 2009. CA Cancer J Clin. 2009;59:225–49.
Wojciechowska U, Didkowska J, Zatoński W. Corpus uteri cancer. In: Zatoński W, editor. Cancer in Poland in 2006. Warszawa: Department of Epidemiology and Cancer Prevention; 2008. p. 30–2.
Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis. 2002;23:687–96.
Helleday T. Pathways for mitotic homologous recombination in mammalian cells. Mutat Res. 2003;532:103–15.
Kuschel B, Auranen A, McBride S. Variants in DNA double-strand break repair genes and breast cancer susceptibility. Hum Mol Genet. 2002;11:1399–407.
Smolarz B, Makowska M, Samulak D, Michalska MM, Mojs E, Wilczak M, Romanowicz H. Association between single nucleotide polymorphisms (SNPs) of XRCC2 and XRCC3 homologous recombination repair genes and triple-negative breast cancer in Polish women. Clin Exp Med. 2014. doi:10.1007/s10238-014-0284-7.
Romanowicz-Makowska H, Smolarz B, Zadrozny M, Westfa B, Baszczyński J, Kokołaszwili G, Burzyfiski M, Połać I, Sporny S. The association between polymorphisms of the RAD51-G135C, XRCC2-Arg188His and XRCC3-Thr241 Met genes and clinico-pathologic features in breast cancer in Poland. Eur J Gynaecol Oncol. 2012;33:145–50.
Romanowicz-Makowska H, Smolarz B, Zadrozny M, Baszczynski J, Polac I, Sporny S. Single nucleotide polymorphisms in the homologous recombination repair genes and breast cancer risk in Polish women. Tohoku J Exp Med. 2011;224:201–8.
Silva SN, Tomar M, Paulo C, Gomes BC, Azevedo AP, Teixeira V, Pina JE, Rueff J, Gaspar JF. Breast cancer risk and common single nucleotide polymorphisms in homologous recombination DNA repair pathway genes XRCC2, XRCC3, NBS1 and RAD51. Cancer Epidemiol. 2010;34:85–92.
Lin WY, Camp NJ, Cannon-Albright LA, Allen-Brady K, Balasubramanian S, Reed MW, et al. A role for XRCC2 gene polymorphisms in breast cancer risk and survival. J Med Genet. 2011;48:477–84.
Romanowicz-Makowska H, Smolarz B, Gajęcka M, Kiwerska K, Rydzanicz M, Kaczmarczyk D, Olszewski J, Szyfter K, Błasiak J, Morawiec-Sztandera A. Polymorphism of the DNA repair genes RAD51 and XRCC2 in smoking- and drinking-related laryngeal cancer in a Polish population. Arch Med Sci. 2012;8:1065–75.
Mohamed FZ, Hussien YM, AlBakry MM, Mohamed RH, Said NM. Role of DNA repair and cell cycle control genes in ovarian cancer susceptibility. Mol Biol Rep. 2013;40:3757–68.
Krupa R, Sliwinski T, Wisniewska-Jarosinska M, Chojnacki J, Wasylecka M, Dziki L, Morawiec J, Blasiak J. Polymorphisms in RAD51, XRCC2 and XRCC3 genes of the homologous recombination repair in colorectal cancer–a case control study. Mol Biol Rep. 2011;38:2849–54.
He Y, Zhang Y, Jin C, Deng X, Wei M, Wu Q, Yang T, Zhou Y, Wang Z. Impact of XRCC2 Arg188His polymorphism on cancer susceptibility: a meta-analysis. PLoS ONE. 2014;9:e91202.
Shi S, Qin L, Tian M, Xie M, Li X, Qi C, Yi X. The effect of RAD51 135 G > C and XRCC2 G > A (rs3218536) polymorphisms on ovarian cancer risk among Caucasians: a meta-analysis. Tumour Biol. 2014;35:5797–804.
Zhang Y, Wang H, Peng Y, Liu Y, Xiong T, Xue P, Du L. The Arg188His polymorphism in the XRCC2 gene and the risk of cancer. Tumour Biol. 2014;35:3541–9.
Wang N, Dong X-J, Zhou R-M, Guo W, Zhang X-J, Li Y. An investigation on the polymorphisms of two DNA repair genes and susceptibility to ESCC and GCA of high-incidence region in northern China. Mol Biol Rep. 2009;36:357–64.
Report Meeting. The new FIGO staging system for cancers of the vulva, cervix, endometrium and sarcomas. Gynecol Oncol. 2009;115:325–8.
O’Driscoll M, Jeggo PA. The role of double-strand break repair-insights from human genetics. Nat Rev Genet. 2006;7:45–54.
Smilenov LB. Tumor development: haploinsufficiency and local network assembly. Cancer Lett. 2006;240:17–28.
Akisik E, Yazici H, Dalay N. ARLTS1, MDM2 and RAD51 gene variations are associated with familial breast cancer. Mol Biol Rep. 2011;38:343–8.
Li C, Jiang Z, Liu X. XPD Lys (751) Gln and Asp (312) Asn polymorphisms and bladder cancer risk: a meta-analysis. Mol Biol Rep. 2010;37:301–9.
Stanczyk M, Sliwinski T, Cuchra M, Zubowska M, Bielecka-Kowalska A, Kowalski M, Szemraj J, Mlynarski W, Majsterek I. The association of polymorphisms in DNA base excision repair genes XRCC1, OGG1 and MUTYH with the risk of childhood acute lymphoblastic leukemia. Mol Biol Rep. 2011;38:445–51.
Wu J, Wang D, Song L, Li S, Ding J, Chen S, Li J, Ma G, Zhang X. A new familial gastric cancer-related gene polymorphism: T1151A in the mismatch repair gene hMLH1. Mol Biol Rep. 2011;38:3181–7.
Johnson RD, Liu N, Jasin M. Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination. Nature. 1999;401:397–9.
Webb PM, Hopper JL, Newman B, Chen X, Kelemen L, Giles GG, Southey MC, Chenevix-Trench G, Spurdle AB. Double-strand break repair gene polymorphisms and risk of breast or ovarian cancer. Cancer Epidemiol Biomark Prev. 2005;14:319–23.
Matullo G, Guarrera S, Sacerdote C, Polidoro S, Davico L, Gamberini S, Karagas M, Casetta G, Rolle L, Piazza A, Vineis P. Polymorphisms/haplotypes in DNA repair genes and smoking: a bladder cancer case–control study. Cancer Epidemiol Biomark Prev. 2005;14:2569–78.
Garcia-Closas M, Egan KM, Brinton LA, Titus-Ernstoff L, Chanock S, Welch R, Lissowska J, Peplonska B, Szeszenia-Dabrowska N, Zatonski W, Bardin-Mikolajczak A, Struewing JP. Polymorphisms in DNA double-strand break repair genes and risk of breast cancer: two population-based studies in USA and Poland, and metaanalyses. Hum Genet. 2006;119:376–88.
Zienolddiny S, Campa D, Lind H, Ryberg D, Skaug V, Stangeland L, Phillips DH, Canzian F, Haugen A. Polymorphisms of DNA repair genes and risk of non-small cell lung cancer. Carcinogenesis. 2006;27:560–7.
Kietthubthew S, Sriplung H, Au WW, Ishida T. Polymorphism in DNA repair genes and oral squamous cell carcinoma in Thailand. Int J Hyg Environ Health. 2006;209:21–9.
Smolarz B, Makowska M, Samulak D, Michalska MM, Mojs E, Romanowicz H, Wilczak M. Association between polymorphisms of the DNA repair gene RAD51 and ovarian cancer. Pol J Pathol. 2013;64:290–5.
Romanowicz-Makowska H, Smolarz B, Samulak D, Michalska M, Lewy J, Burzyński M, Kokołaszwili G. A single nucleotide polymorphism in the 5′ untranslated region of RAD51 and ovarian cancer risk in Polish women. Eur J Gynaecol Oncol. 2012;33:406–10.
Pérez LO, Crivaro A, Barbisan G, Poleri L, Golijow CD. XRCC2 R188H (rs3218536), XRCC3 T241M (rs861539) and R243H (rs77381814) single nucleotide polymorphisms in cervical cancer risk. Pathol Oncol Res. 2013;19:553–8.
García-Quispes WA, Pérez-Machado G, Akdi A, Pastor S, Galofré P, Biarnés F, Castell J, Velázquez A, Marcos R. Association studies of OGG1, XRCC1, XRCC2 and XRCC3 polymorphisms with differentiated thyroid cancer. Mutat Res. 2011;709–710:67–72.
Conflict of interest
The authors declare that there are no conflicts of interest.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Michalska, M.M., Samulak, D. & Smolarz, B. An association between the −41657 C/T polymorphism of X-ray repair cross-complementing 2 (XRCC2) gene and ovarian cancer. Med Oncol 31, 300 (2014). https://doi.org/10.1007/s12032-014-0300-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s12032-014-0300-5