Departments of 1 Obstetrics and Gynecology, 2 Cancer Research Institute and 3 Human Genome Research Institute, College of Medicine, Seoul National University, Seoul, Korea
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 28 Yongun-Dong, Jongno-Gu, Seoul, 110-744, Korea. Email: kjwksh{at}snu.ac.kr
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Abstract |
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Key words: DNA repair gene XRCC1/leiomyoma/neoplasm/polymorphism/uterus
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Introduction |
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Several predisposing factors of this tumour have been identified, including age (late reproductive years), African-American ethnicity, nulliparity and obesity. Estrogen and progesterone are recognized as promoters of tumour growth. Several growth factors are elevated in uterine leiomyomas and may be the effectors of estrogen and progesterone promotion (Flake et al., 2003). Although the initiator(s) of leiomyomas remains unknown, considering their extremely high incidence, initiating conditions must be common to most or all women (Flake et al., 2003
). If in fact this is the case, one could hypothesize that leiomyoma development is related to individual susceptibilities to a common initiator(s). In addition, the fact that leiomyomas are monoclonal tumours derived from a single myometrial cell suggests that DNA repair failure may be an early event in myoma development (Townsend et al., 1970
).
In this study we have focused on the XRCC1 gene, the product of which plays an important role in DNA repair. Indeed, although lacking any known enzymatic activity itself, XRCC1 interacts with enzymatic components of each stage of DNA strand break repair, including PARP-1, AP endonuclease-1, polynucleotide kinase, DNA polymerase- and DNA ligase III
(Caldecott, 2003
). Thus, polymorphisms in XRCC1 that cause amino acid substitutions may impair the interaction of XRCC1 with the other enzymatic proteins and consequently alter DNA strand break repair.
Hence, we undertook this hospital-based case-control study to determine the association between the Arg399Gln polymorphism of XRCC1 and susceptibility to uterine leiomyoma in Korean women.
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Materials and methods |
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Genotyping
DNA was extracted from peripheral blood samples. XRCC1 genotypes were determined by PCR restriction fragment length polymorphism (PCR-RFLP). The codon 399 region was amplified using the following primers: forward, 5'-CCCCAAGTACAGCCAGGTC-3' and reverse, 5'-TGTCCCGCTCCTCTCAGTAG-3'. The PCR was started with a mixture containing the following reagents: 0.1 µg of DNA, 5 mmol/l of dNTPs, 5 pmol/l of primer and 1 U of Taq polymerase, which was added into PCR buffer containing 10 mmol/l of TrisHCl, 1.5 mmol/l MgCl2 and 50 mmol/l KCl. PCR conditions were 95°C for 3 min, followed by 40 cycles of 94°C for 30 s, 57°C for 45 s, 72°C for 45 s and a final elongation step at 72°C for 5 min. Following PCR, 20 µl aliquots were removed and subjected to restriction digestion with MspI (New England Biolabs Inc., Beverly, MA, USA). The digested products were resolved on 1.5% agarose gels and stained with 0.5 µg/ml ethidium bromide. To test the reliability of the assay, 50 randomly selected samples were re-tested with identical results being obtained. Genotyping failures resulted from the DNA extraction error led to the exclusion of seven (2.1%) and nine (4.4%) subjects from the case and control groups, respectively, leaving 327 patients and 197 controls available for analysis.
Statistical analysis
Statistical analyses were carried out using the SPSS version 11.0 for Windows (SPSS Inc., Chicago, IL, USA). The 2-test and the t-test were used to compare variables. A logistic regression model was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs) and corresponding P-values for each genotype controlling age, parity, age at menarche and body mass index (BMI) as covariates. All the variables listed were forced into the equation when running logistic regression. P-values of <0.05 were considered statistically significant.
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Results |
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Discussion |
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Genetic aspects of leiomyomas have been suggested by studies that viewed the issue from different perspectives, i.e. ethnic predisposition, twin studies, familial aggregation and cytogenetic changes (Kurbanova et al., 1989; Marshall et al., 1997
; Ligon and Morton, 2000
; Luoto et al., 2000
; Sato et al., 2002
; Van Voorhis et al., 2002
; Dal Cin et al., 2003
). In terms of the association between genetic polymorphisms and susceptibility to uterine leiomyoma, the estrogen receptor gene polymorphism has been investigated by several researchers (Kitawaki et al., 2001
; Massart et al., 2001
; Hsieh et al., 2003
; Massart et al., 2003
); however, these studies have produced inconsistent findings.
The results of the present study, on the other hand, corresponded well with those of other studies on the XRCC1 polymorphism and proliferative disease: carcinoma. Various types of carcinoma were investigated, namely esophageal cancer, prostate cancer, colorectal cancer, lung cancer and hepatocellular carcinoma (Park et al., 2002; Yu et al., 2003
; Zhou et al., 2003
; Krupa and Blasiak, 2004
; Rybicki et al., 2004
; Yu et al., 2004
). All of these studies found that the 399Gln allele is associated with a higher risk of cancer than the 399Arg allele. In addition, these conclusions corresponded well with the finding that subjects with the 399Gln allele have higher numbers of chromosomal breaks per cell than those with other genotypes (Wang et al., 2003
), and that these subjects are more susceptible to chemically induced genetic damage (Li et al., 2003
).
However, an almost equally impressive number of studies have reported that the XRCC1 399Gln allele has no or an inverse relationship with the risk of neoplasm development (Olshan et al., 2002; Mort et al., 2003
; Shen et al., 2003
; Smith et al., 2003
; Kelsey et al., 2004
; Matsuo et al., 2004
; Mertens et al., 2004
; Sanyal et al., 2004
; Wu et al., 2004
). A credible explanation for this discrepancy is that the variant protein has an altered repair efficiency; the resultant increased levels of damage might give rise to enhanced apoptosis at the time of cell division, which would finally manifest as reduced risk for exposure-induced cancer (Nelson et al., 2002
) and the possibility of XRCC1-independent single-strand break repair (Caldecott, 2003
). However, the exact mechanism by which the XRCC1 399Gln allele can have apparently opposite effects on the risk of neoplasm development is not known, and this should be the subject of further investigation.
In this study, age and parity were similar between the two groups, although we expected low parity in the case group because the nulliparity is a known predisposing factor for leiomyomas. A possible explanation for this unexpected result is that nulliparous women in Korea generally are unwilling to have an operation on their uterus and clinicians also have a similar attitude. Therefore, our inclusion criteriapatients who had an operation and histopathological confirmation of leiomyomamight not have led to the enrollment of a sufficient number of nulliparous women in this study.
We included menarche age in the analysis on the grounds that early menarche leads to increased duration of estrogen exposure and could result in high susceptibility to leiomyomas. Contrary to our expectations, menarche age was 0.7 years later in the case group. Although menarche age was significantly different between cases and controls, we think that it has minimal, if any, biological significance because of the size of the study, the fact that the difference was small, only 0.7 years, and it would have been extremely affected by recall bias. BMI showed a statistical difference between the two groups and this difference was conserved even after logistic regression (data not shown). This corresponds well with the long-standing knowledge that obesity is a predisposing factor of leiomyomas, although our patients were not particularly obese.
The present study is limited by its hospital-based, casecontrol nature. Moreover, patients and controls were selected at a single institution (Seoul National University Hospital) and thus may be unrepresentative of the general population. However, in the control group, the observed agreement between XRCC1 genotype frequencies and HardyWeinberg equilibrium expectation suggested no selection bias. In addition, the present study produced significant results after adjusting for several possible confounders: age, menarche age and BMI.
In conclusion, our study is the first to determine the significance of the XRCC1 polymorphism in uterine leiomyoma, and suggests that the XRCC1 399Gln allele is associated with an increased risk of uterine leiomyoma in Korean women. Although further studies are needed, we hope that this work increases knowledge about the pathogenesis of uterine leiomyomas and may give clues for the primary and secondary prevention of the disease.
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Acknowledgements |
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References |
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Submitted on December 27, 2004; resubmitted on February 6, 2005; accepted on February 10, 2005.
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