ORIGINAL STUDY
Apurinic/Apyrimidinic Endonuclease 1 Polymorphisms Are Associated With Ovarian Cancer Susceptibility in a Chinese Population Xiaohong Zhang, MD, Xiaoyan Xin, PhD, Jianfang Zhang, PhD, Jia Li, PhD, Biliang Chen, PhD, and Wei Zou, PhD
Objective: Apurinic/apyrimidinic endonuclease 1 (APE1) plays an essential role in the base excision repair pathway. Recent studies have shown that APE1 polymorphisms are associated with an increased risk for many types of cancers. This study investigated the association between APE1 polymorphisms and the susceptibility of ovarian cancer. Methods: A case-control study was performed on 124 patients with ovarian cancer and 141 controls. We genotyped the rs1760944 and rs1130409 polymorphisms and assessed their associations with the risk for ovarian cancer. Results: The rs1130409 polymorphism was significantly associated with a risk for ovarian cancer. The TG/GG genotype and the G allele were associated with a decreased risk for ovarian cancer (adjusted odds ratio [aOR], 0.495; 95% confidence interval [CI], 0.267Y0.920 for TG vs TT; aOR, 0.263; 95% CI, 0.132Y0.521 for GG vs TT; aOR, 0.486; 95% CI, 0.344Y0.0.688 for the G allele vs the T allele). In the stratified analyses, we found that when comparing the TG/GG genotype versus the TT genotype, the lower risk was more evident in subgroups of patients 50 years or older (aOR, 0.753; 95% CI, 0.604Y0.938), patients with menarche age of 15 years or older (aOR, 0.722; 95% CI, 0.573Y0.910), patients with gravidity of 3 or more times (aOR, 0.732; 95% CI, 0.587Y0.912), and postmenopausal women (aOR, 0.763; 95% CI, 0.615Y0.947). Meanwhile, the rs1760944 polymorphism was not found to be associated with a risk for ovarian cancer. However, by haplotype analysis, we found that the T-G and G-G haplotypes were associated with a decreased risk for ovarian cancer. Conclusions: Our results suggest that in a Han Chinese population, the APE1 rs1130409 polymorphism may correlate with ovarian cancer susceptibility. Key Words: APE1, Single nucleotide polymorphism, Ovarian cancer Received March 15, 2013, and in revised form June 11, 2013. Accepted for publication July 1, 2013. (Int J Gynecol Cancer 2013;23: 1393Y1399)
Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, Xi’an, China. Address correspondence and reprint requests to Xiaoyan Xin, PhD, Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, 17 Changle West Rd, Xi’an, 710032, China. E-mail: [email protected]. This research was supported by the National Science Foundation of China (NSFC 81172460/M162). The authors declare no conflicts of interest. Copyright * 2013 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0b013e3182a33f07 International Journal of Gynecological Cancer
cancer is the most lethal malignancy among gyO varian necological carcinomas. In China, the annual number of
new cases is more than 192,000, and approximately 114,000 die of ovarian cancer every year.1 Owing to a lack of symptoms in the early stages and effective measures for early diagnosis, more than 70% of patients are diagnosed at an advanced stage. Although the pathogenesis of ovarian cancer is still not fully known, the patient characteristics found to be associated with ovarian cancer incidence include age older than 55 years, late age at menopause, family history of breast or ovarian cancer, infertility, and use of hormone replacement
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therapy. It has also been suggested in a previous study that genomic alterations such as mutations in BRCA1, BRCA2, P53, and the PI3K genes and that some common genetic variants play an important role in modulating susceptibility to ovarian cancer.2 Single nucleotide polymorphisms (SNPs) are the most common and simplest DNA sequence variation, which occurs when a single base mutates, but can affect coding sequences, splicing, and transcription regulation.1 Single nucleotide polymorphisms are thought to be key enablers in determining how humans develop diseases, respond to drugs, and may be valuable in realizing the concept of personalized medicine. Moreover, many SNPs have been used as high-resolution markers related to carcinomas, including ovarian cancer. Therefore, identification of SNPs in predisposing genes is very likely a good way to determine highrisk groups and achieve early diagnosis for ovarian cancer. Genetic variations in DNA repair genes can modulate the DNA repair capability, which may cause permanent mutations in the genome and thus contribute to carcinogenesis.3 Among all the DNA repair systems, the base excision repair (BER) pathway is the most ubiquitous approach for correcting DNA alterations, which arise spontaneously or from alterations caused by endogenous reactive chemicals, and is the preferential mechanism for repairing a large number of base lesions caused by oxidative damage, alkylation, or methylation.4,5 Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential enzyme, which plays a central role in the BER pathway for the repair of damaged bases and single-stranded DNA breaks caused by oxidation or alkylation, and also protects cells from cytotoxicity induced by endogenous or exogenous apurinic/apyrimidinic (AP) sites.6 In addition, APE1 also can regulate transcription by activating numerous transcription factors, which are involved in some important cell functions, such as apoptosis, cell cycle regulation, cell growth, proliferation, and differentiation. Apurinic/apyrimidinic endonuclease 1 has been demonstrated to play a pivotal role in carcinogenesis and progression of several cancers, including cervical cancer, colon cancer, glioma, lung cancer, and so on.7 In addition, APE1 has been found to be correlated with tumorigenesis, angiogenesis, progression of ovarian cancer, and drug resistance to platinum in ovarian cancer.8,9 Thus, genetic variations of APE1 may alter its functions and DNA repair capacity, ultimately leading to an increased risk for cancers. Until now, many epidemiological studies suggested that SNPs in APE1 may confer an individual’s susceptibility to cancer. A total of 10 SNPs in APE1 have been identified in CHB (HapMap database and dpSNP database), and rs1760944 (j656 T 9 G in the promoter region) and rs1130409 (1349 T 9 G in the fifth exon) have been the SNPs within APE1, which have been most frequently investigated; however, the results of the previous studies remain inconclusive. Furthermore, the minor allele frequency of these 2 SNPs were more than 0.05, and their linkage disequilibrium analysis in CHB suggested that they were not correlated (D¶ = 0.409, r2= 0.153, HapMap database). Based on the previous studies, we speculate that polymorphisms in APE1 may be associated with an individual’s susceptibility to ovarian cancer. To test this hypothesis, we investigated the relationship in a hospital-based, case-control study in a Han Chinese population.
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MATERIALS AND METHODS Subjects and Specimen Collection This study involved 124 patients with ovarian cancer and 141 cancer-free controls, who were all Han Chinese. Cases were first diagnosed and treated at Xijing Hospital (Xi’an, China) from October 2010 to November 2010 and from October 2011 to January 2013. All patients had never received chemotherapy, surgery, or other treatments. In addition, all the patients had no history of other malignancies. There were no restrictions on recruitment in terms of age, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor grade, or histological type of disease. As controls, 141 women having no blood relationship to the cases were recruited from the same hospital for regular gynecological examinations; to be eligible, these individuals were required to be healthy and not have a history of cancers. Whole blood (4 mL) was extracted from all the subjects who agreed to participate in the study and preserved in tubes coated with K2-EDTA. All blood samples were stored in a j80-C freezer at the gynecological laboratory of Xijing Hospital. The ethics committees of the Fourth Military Medical University approved this research, and all the cases and controls signed consent forms.
DNA Extraction and Genotyping Genomic DNA was extracted from blood samples with the Blood Genomic DNA Isolation Kit (Sangon, Shanghai, China), according to the manufacturer’s instructions. An UV spectrophotometer (Jenway, England) was used to measure the DNA concentration and purity of each sample. The target fragments were amplified by polymerase chain reaction (PCR), and genotyping was performed by direct sequencing (ABI 3730xl DNA Analyzer). The following primers were used: 5¶-TCTGCCCCACCTCTTGATT-3¶ (forward) and 5¶-TGCC ACATTGAGGTCTCCA-3¶ (reverse) for rs1130409, and 5¶-CCTAACCCGAGCACAAAGAA-3¶ (forward), and 5¶-CGAGC CAACTCTGTGCCTTA-3¶ (reverse) for rs1760944. The PCR reaction mix included 7.6 HL of template DNA, 1.2 HL of forward primer, 1.2 HL of reverse primer, 20 HL of 2Taq PCR MasterMix, and 20 HL of ddH2O. Samples were subjected to denaturation at 95-C for 3 minutes, followed by 35 cycles of heating at 94-C for 30 seconds, annealing at 58-C (rs1760944) or 63-C (rs1130409) for 30 seconds, then extension at 72-C for 40 seconds, and a final incubation at 72-C for 5 minutes. After verification by 2% agarose gel electrophoresis, the PCR products were sent to Sangon Biotech Company and directly sequenced using the ABI 3730xl DNA Analyzer. The results of the sequencing were analyzed by Chromas2 software and compared with the sequence of APE1 in GenBank using DNAMAN V6 software by 2 persons independently in a blind fashion. If the sequence of one sample could not be read accurately, DNA was again extracted from the sample, and direct sequencing was repeated. Ten percent of the samples were randomly selected for repeat sequences, and the results were fully concordant.
Statistical Analysis
For each SNP in the control subjects, the W2 test was used to test for Hardy-Weinberg equilibrium. The Student t test and * 2013 IGCS and ESGO
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TABLE 1. Distribution of selected variables among cases and controls and test of the hardy-weinberg equilibrium for the SNPs of APE1 Variables
Case (124)
Age, mean (SD), y 49.30 (14.67) Menarche age, mean (SD), y 14.97 (1.96) Gravidity, n (%) G3 54 (43.55) Q3 70 (56.45) Parity, n (%) G3 93 (75.00) Q33 31 (25.00) Menopausal status, n (%) Yes 70 (57.38) No 52 (42.62)
Control (141)
P
48.05 (13.15)
0.718*
14.70 (1.84)
0.522*
56 (39.72) 85 (60.28)
0.528†
98 (69.50) 43 (30.50)
0.320†
77 (54.61) 64 (45.39)
0.652†
*Two-sided Student t test for the difference between the cases and controls. †Two-sided W2 test for the distribution between the cases and controls.
W2 test were used to compare demographic characteristics between cancer cases and controls. Multiple logistic regression analysis was performed to evaluate the distribution analyses of rs1130409 and rs1760944 genotypes between the cases and controls and to evaluate the stratified analysis between the genotypes and characteristic variables. P values, odds ratios (ORs) and 95% confidence intervals (CIs) were adjusted for age, age at menarche, gravidity, parity, and menopausal status. The association between genotype distributions and clinicopathological characteristics of the cases was analyzed by W2 test. The haplotypes and their frequencies were estimated by PHASE 2.1, and Pearson W2 test was used to compare the frequency of all haplotypes between the case and control participants. A P G 0.05 was considered significant in all statistical tests, and all P values were based on 2-way tests. All statistical tests described previously were conducted in SPSS 16.0 (SPSS Inc, Chicago, Ill).
RESULTS Parametric distributions of the study subjects are shown in Table 1. No significant differences were found for the age,
menarche age, gravidity, parity, and menopausal status between the cases and controls. Of the 124 ovarian cancer cases, 106 (85.48%) were epithelial ovarian cancers, which were classified as serous (76), mucinous (10), endometrioid (4), clear cell (3), malignant Brenner (1), and mixed/other roughly reported carcinomas (12). Eleven (8.87%) were germ cell tumors, including 7 immature teratomas and 4 endodermal sinus tumors, and 7 (5.65%) were sex gonad stromal tumors, which were all granulosa cell tumors. In addition, 42 patients with ovarian cancer were classified as FIGO stages I to II and 82 as FIGO stages III to IV. Fifty-five patients were detected as having lymphatic metastasis, which was not present in the remaining patients. Genotype frequencies of the 2 SNPs in the case and control groups conformed to Hardy-Weinberg equilibrium (W2rs1760944 = 2.052, Prs1760944 = 0.152 and W2rs1130409 = 1.122, Prs1130409 = 0.289 for the case group; W2rs1760944 = 0.614, Prs1760944 = 0.433 and W2rs1130409 = 1.313, Prs1130409 = 0.252 for the control group). The sequencing map view for all genotypes of rs1760944 and rs1130409 is shown in Figures 1 to 6. The genotype and allele frequencies of the rs1760944 and rs1130409 polymorphisms among the case and control groups and the association with the risk for ovarian cancer are shown in Table 2. The distribution of rs1130409 was significantly different between the ovarian cancer cases and controls. Compared with the control group, the cases exhibited a lower frequency of the G allele (P G 0.001). For the rs1130409 polymorphism, genotypic tests and allelic tests revealed significant associations between the TG/GG genotype and the G allele and a lower risk for developing ovarian cancer (adjusted OR [aOR], 0.495; 95% CI, 0.267Y0.920 for TG vs TT; aOR, 0.263; 95% CI, 0.132Y0.521 for GG vs TT; aOR, 0.486; 95% CI, 0.344Y0.0.688 for the G allele vs the T allele). However, the rs1760944 polymorphism in the APE1 gene was not found to be associated with the risk for ovarian cancer. Further stratified analyses showed that when the TG/GG genotype was compared with the TT genotype, the lower risk was more evident in subgroups of patients with age 50 years or older (aOR, 0.753; 95% CI, 0.604Y0.938), patients with menarche age of 15 years or older (aOR, 0.722; 95% CI, 0.573Y0.910), patients with gravidity of 3 or more times (aOR, 0.732; 95% CI, 0.587Y0.912), and postmenopausal women (aOR, 0.763; 95% CI, 0.615Y0.947), as shown in Table 3. This protective effect was observed in women whether parity is less than 3 or more than 3. However, our analysis did not show an association between rs1130409 polymorphisms and clinicopathological characteristics of
FIGURE 1. Sequencing map view of TT genotype for rs1760944 * 2013 IGCS and ESGO
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FIGURE 2. Sequencing map view of GG genotype for rs1760944
FIGURE 3. Sequencing map view of TG genotype for rs1760944
ovarian cancer risk stratified according to tumor histology, clinical stage, or lymph node status (Table 4). In addition, our study found that rs1760944 polymorphisms had no association with the risk for ovarian cancer in stratified analyses comparing characteristic variables and clinicopathological characteristics (data not shown). To further analyze the combined effects of polymorphisms of rs1760944 and rs1130409, we performed haplotype analysis. As shown in Table 5, when 4 haplotypes were constructed, the distribution of the haplotype frequency was different between the cases and controls (P = 0.001), with haplotype T-G and G-G showing a decreased risk for ovarian
cancer compared with haplotype T-T (OR, 0.646; 95% CI, 0.500Y0.836 for T-G vs T-T; OR, 0.627, 95% CI, 0.472Y0.823 for G-G vs T-T).
DISCUSSION In this study, we observed that the rs1130409 G allele and the TG/GG genotypes were significantly associated with a decreased risk for ovarian cancer susceptibility; however, the rs1760944 polymorphism was not associated with a risk for ovarian cancer (Table 2). In addition, the correlation analysis of APE1 variants and clinical features revealed that
FIGURE 4. Sequencing map view of TT genotype for rs1130409
FIGURE 5. Sequencing map view of GG genotype for rs1130409
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FIGURE 6. Sequencing map view of TG genotype for rs1130409
the protective effect of the TG/GG genotype compared with the rs1130409 TT genotype was more pronounced among subgroups of patients aged 50 years or older, patients with menarche age of 15 years or older, patients with gravidity of 3 or more times, and postmenopausal women (Table 3). Moreover, we found that the protective effect was observed in women whose parity was both less than 3 and more than 3. However, we did not find any associations between rs1130409 polymorphism variants and clinicopathological variables of ovarian cancer (Table 4). From the haplotype analysis, the distribution of the 4 haplotypes between the case and control groups was significantly different, with the T-G and G-G genotypes associated with a lower risk for ovarian cancer. Apurinic/apyrimidinic endonuclease 1 is the rate-limiting enzyme in the BER pathway and produces normal 3¶-hydroxyl nucleotide termini, which are important for DNA repair synthesis and ligation at single- or double-strand breaks by hydrolyzing 3¶-blocking fragments from oxidized DNA.10,11 Recently, many studies have investigated the relationship between APE1 polymorphisms and cancer susceptibility; however, little is known about the role of APE1 polymorphisms and ovarian cancer risk. The rs1130409 (T 9 G) polymorphism, which results in a single amino acid substitution (Asp 9 Glu), is the most extensively studied, and the results concerning
the rs1130409 polymorphism and cancer susceptibility remain inclusive. In the previous study, it was found that the GG genotype was associated with a significantly increased risk for colon cancer and gastric cancer and a decreased risk for bladder cancer; moreover, no associations were found between rs1130409 polymorphisms and susceptibilities to lung cancer, breast cancer, and prostate cancer.12Y15 Our results showed that the G allele and the GG genotype were associated with a lower risk for ovarian cancer. The discrepancy between different studies might be caused by rs1130409 polymorphisms playing a different role at different cancer sites. However, even for the sam type of cancer, the results could be different. For example, Gu et al13 and Canbay et al16 found that the frequency of the rs1130409 GG genotype in patients with gastric cancer was different from the control subjects, but Palli et al17 did not detect association of the polymorphism with gastric cancer. These different findings might be due to sample size differences or ethnic diversity. Moreover, it was reported that the rs1130409 GG genotype prolonged cell cycle G2 delays relative to the TT and TG genotypes; however, it had no impact on the endonuclease and DNA binding activities of APE1. Even so, the rs1130409 polymorphism might affect susceptibility to some cancers because these alterations might contribute to interactions of the polymorphism with other genes. For example, it was reported
TABLE 2. Genotype and allele frequencies of APE1 and the association with risk for ovarian cancer SNP and Genotype
Cases, n (%)
rs1760944 (j656T 9 G) TT 48 (38.71%) TG 52 (41.94%) GG 24 (19.35%) T allele 148 (59.68%) G allele 100 (40.32%) rs1130409 (1349T 9 G,Asp148Glu) TT 47 (37.90%) TG 54 (43.55%) GG 23 (18.55%) T allele 148 (59.68%) G allele 100 (40.32%)
Controls, n (%)
aOR (95% CI)*
P
46 65 30 157 125
(32.62%) (46.10%) (21.28%) (55.67%) (44.33%)
1.00 1.065 0.727 0.849
(reference) (0.547Y2.074) (0.365Y1.446) (0.601Y1.199)
0.852 0.363 0.352
28 62 51 118 164
(19.86%) (43.97%) (36.17%) (41.84%) (58.16%)
1.00 0.495 0.263 0.486
(reference) (0.267Y0.920) (0.132Y0.521) (0.344Y0.688)
0.026 G0.001 G0.001
*Adjusted for age, menarche age, gravidity, parity, and menopausal status in logistic regression.
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TABLE 3. Stratified analyses for rs1130409 polymorphisms in ovarian cancer cases and controls Cases/ Controls, n Variables
TT
Age, y G50 22/17 Q50 25/11 Menarche age, y G15 19/16 Q15 27/12 Gravidity G3 19/12 Q3 28/16 Parity G3 35/22 Q3 12/6 Menopausal status Yes 27/15 No 19/13
TT vs TG/GG
TG/GG
P
aOR (95% CI)
36/61 41/52
0.040 0.010
0.794 (0.629Y1.002) 0.453 (0.604Y0.938)
37/59 39/54
0.107 0.004
0.840 (0.673Y1.048) 0.722 (0.573Y0.910)
36/44 41/69
0.124 0.003
0.833 (0.658Y1.055) 0.732 (0.587Y0.912)
58/76 19/37
0.022 0.014
0.804 (0.665Y0.973) 0.712 (0.525Y0.966)
43/62 33/51
0.010 0.052
0.763 (0.615Y0.947) 0.796 (0.626Y1.013)
*Adjusted for age, menarche age, gravidity, parity, and menopausal status in logistic regression.
that the rs1130409 TT genotype and the combination of the TT genotype and hOGG1-Cys variants increased the risk for p53 mutation for nonYsmall cell lung cancer.18 In our study, the rs1130409 G allele and GG genotype were associated with a lower risk for ovarian cancer. Thus, investigation of the interactions between APE1 polymorphisms and other DNA repair genes, oncogenes, tumor suppressor genes, and genes related to steroid hormone mechanisms would be beneficial to exploring the pathogenesis of ovarian cancer. In addition, it is well-known that the prevalence of ovarian cancer is increased in nulliparous, infertile, early menstruating, and late-onset menopause women. In our
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study, we found that when the rs1130409 TG/GG genotype was compared with the TT genotype, the lower risk was more evident in the subgroup of patients with menarche age of 15 years or older, patients with gravidity of 3 or more times, and postmenopausal women, which was consistent with the previous study. However, we also found that this protective effect occurred in a subgroup of patients aged 50 years or older but not in younger subjects. It was reported that ovarian cancer tends to occur in women whose median age is approximately 50 years. The lower risk for ovarian cancer in older individuals with the GG genotype may be due to direct genetic effects rather than the aging effect. In the stratified analyses concerning and parity, the protective effect of the TG/GG genotype was also shown, which suggested that the correlation between nulliparity and a high risk for ovarian cancer may also be related to genetic effects rather than parity. Moreover, our analysis did not find associations between rs1130409 polymorphisms and clinicopathological characteristics of ovarian cancer, and future studies are needed to expand the sample size to confirm the results. For rs1760944, we did not find any association between this polymorphism and ovarian cancer susceptibility; we also did not find any association in the stratified analyses. Although in a previous study, the G allele of rs1760944 was reported to significantly influence transcriptional activity when compared with the T allele, the results were not consistent.19,20 Regarding the relationship between rs1760944 polymorphisms and cancer susceptibility, the results differed in multiple studies.19,21Y25 Given the disparity between the mechanistic studies and the genetic studies, the rs1760944 polymorphism requires further investigation. In the haplotype analysis, we found that a decreased ovarian cancer risk was associated with the G allele of the rs1130409 polymorphism regardless of the rs1760944 polymorphism. Despite the T-G and G-G haplotypes having a lower risk for ovarian cancer compared with the T-T haplotype, exploring the associations between the haplotypes concerning the rs1130409 and rs1760944 polymorphisms and the function of APE1 is still necessary. In conclusion, our study demonstrates that the APE1 rs1130409 polymorphism, but not the rs1760944 polymorphism, may contribute to ovarian cancer susceptibility in northwestern Han Chinese. Furthermore, a larger
TABLE 4. Relationship between rs1130409 polymorphism and selected clinicopathological variables of ovarian cancer Tumor Histology SNP and Genotype
Epithelial, n (%)
TT TG GG T allele G allele
41 45 20 127 85
(38.68) (42.45) (18.87) (59.91) (40.09)
Others, n (%) 6 9 3 21 15
(33.33) (50.00) (16.67) (58.33) (41.67)
Clinical Stage P
I-II, n (%)
III-IV, n (%)
0.836 15 (35.71) 32 (39.02) 18 (42.86) 36 (43.90) 9 (21.43) 14 (17.07) 0.859 48 (57.14) 100 (60.98) 36 (42.86) 64 (39.02)
Lymph Node Status P 0.831
0.560
Positive, n (%)
Negative, n (%)
22 (40.00) 21 (38.18) 12 (21.82) 65 (59.09) 45 (40.91)
25 33 11 83 55
(36.23) (47.82) (15.95) (60.14) (39.86)
P 0.521
0.867
Two-sided W2 test for distributions of rs1130409 genotypes among clinicopathological variables in the cases.
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TABLE 5. Frequencies of APE1 haplotype between cases and controls and the results of association analysis Haplotype
Cases
T-T T-G G-T G-G
95 53 54 46
(38.24%) (21.43%) (21.84%) (18.49%)
Controls 70 87 49 76
(24.73%) (30.91%) (17.37%) (26.99%)
OR (95% CI)
P
1.00 (reference) 0.646 (0.500Y0.836) 0.880 (0.650Y1.191) 0.627 (0.472Y0.832)
0.001
Haplotype frequencies were composed according to PHASE software and analyzed by Pearson W2 test.
number of patients and well-matched controls with detailed individual data are required to validate our results.
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Apurinic/Apyrimidinic Endonuclease 1 Polymorphisms Are Associated With Ovarian Cancer Susceptibility in a Chinese Population Xiaohong Zhang, MD, Xiaoyan Xin, PhD, Jianfang Zhang, PhD, Jia Li, PhD, Biliang Chen, PhD, and Wei Zou, PhD
Objective: Apurinic/apyrimidinic endonuclease 1 (APE1) plays an essential role in the base excision repair pathway. Recent studies have shown that APE1 polymorphisms are associated with an increased risk for many types of cancers. This study investigated the association between APE1 polymorphisms and the susceptibility of ovarian cancer. Methods: A case-control study was performed on 124 patients with ovarian cancer and 141 controls. We genotyped the rs1760944 and rs1130409 polymorphisms and assessed their associations with the risk for ovarian cancer. Results: The rs1130409 polymorphism was significantly associated with a risk for ovarian cancer. The TG/GG genotype and the G allele were associated with a decreased risk for ovarian cancer (adjusted odds ratio [aOR], 0.495; 95% confidence interval [CI], 0.267Y0.920 for TG vs TT; aOR, 0.263; 95% CI, 0.132Y0.521 for GG vs TT; aOR, 0.486; 95% CI, 0.344Y0.0.688 for the G allele vs the T allele). In the stratified analyses, we found that when comparing the TG/GG genotype versus the TT genotype, the lower risk was more evident in subgroups of patients 50 years or older (aOR, 0.753; 95% CI, 0.604Y0.938), patients with menarche age of 15 years or older (aOR, 0.722; 95% CI, 0.573Y0.910), patients with gravidity of 3 or more times (aOR, 0.732; 95% CI, 0.587Y0.912), and postmenopausal women (aOR, 0.763; 95% CI, 0.615Y0.947). Meanwhile, the rs1760944 polymorphism was not found to be associated with a risk for ovarian cancer. However, by haplotype analysis, we found that the T-G and G-G haplotypes were associated with a decreased risk for ovarian cancer. Conclusions: Our results suggest that in a Han Chinese population, the APE1 rs1130409 polymorphism may correlate with ovarian cancer susceptibility. Key Words: APE1, Single nucleotide polymorphism, Ovarian cancer Received March 15, 2013, and in revised form June 11, 2013. Accepted for publication July 1, 2013. (Int J Gynecol Cancer 2013;23: 1393Y1399)
Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, Xi’an, China. Address correspondence and reprint requests to Xiaoyan Xin, PhD, Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, 17 Changle West Rd, Xi’an, 710032, China. E-mail: [email protected]. This research was supported by the National Science Foundation of China (NSFC 81172460/M162). The authors declare no conflicts of interest. Copyright * 2013 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0b013e3182a33f07 International Journal of Gynecological Cancer
cancer is the most lethal malignancy among gyO varian necological carcinomas. In China, the annual number of
new cases is more than 192,000, and approximately 114,000 die of ovarian cancer every year.1 Owing to a lack of symptoms in the early stages and effective measures for early diagnosis, more than 70% of patients are diagnosed at an advanced stage. Although the pathogenesis of ovarian cancer is still not fully known, the patient characteristics found to be associated with ovarian cancer incidence include age older than 55 years, late age at menopause, family history of breast or ovarian cancer, infertility, and use of hormone replacement
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therapy. It has also been suggested in a previous study that genomic alterations such as mutations in BRCA1, BRCA2, P53, and the PI3K genes and that some common genetic variants play an important role in modulating susceptibility to ovarian cancer.2 Single nucleotide polymorphisms (SNPs) are the most common and simplest DNA sequence variation, which occurs when a single base mutates, but can affect coding sequences, splicing, and transcription regulation.1 Single nucleotide polymorphisms are thought to be key enablers in determining how humans develop diseases, respond to drugs, and may be valuable in realizing the concept of personalized medicine. Moreover, many SNPs have been used as high-resolution markers related to carcinomas, including ovarian cancer. Therefore, identification of SNPs in predisposing genes is very likely a good way to determine highrisk groups and achieve early diagnosis for ovarian cancer. Genetic variations in DNA repair genes can modulate the DNA repair capability, which may cause permanent mutations in the genome and thus contribute to carcinogenesis.3 Among all the DNA repair systems, the base excision repair (BER) pathway is the most ubiquitous approach for correcting DNA alterations, which arise spontaneously or from alterations caused by endogenous reactive chemicals, and is the preferential mechanism for repairing a large number of base lesions caused by oxidative damage, alkylation, or methylation.4,5 Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential enzyme, which plays a central role in the BER pathway for the repair of damaged bases and single-stranded DNA breaks caused by oxidation or alkylation, and also protects cells from cytotoxicity induced by endogenous or exogenous apurinic/apyrimidinic (AP) sites.6 In addition, APE1 also can regulate transcription by activating numerous transcription factors, which are involved in some important cell functions, such as apoptosis, cell cycle regulation, cell growth, proliferation, and differentiation. Apurinic/apyrimidinic endonuclease 1 has been demonstrated to play a pivotal role in carcinogenesis and progression of several cancers, including cervical cancer, colon cancer, glioma, lung cancer, and so on.7 In addition, APE1 has been found to be correlated with tumorigenesis, angiogenesis, progression of ovarian cancer, and drug resistance to platinum in ovarian cancer.8,9 Thus, genetic variations of APE1 may alter its functions and DNA repair capacity, ultimately leading to an increased risk for cancers. Until now, many epidemiological studies suggested that SNPs in APE1 may confer an individual’s susceptibility to cancer. A total of 10 SNPs in APE1 have been identified in CHB (HapMap database and dpSNP database), and rs1760944 (j656 T 9 G in the promoter region) and rs1130409 (1349 T 9 G in the fifth exon) have been the SNPs within APE1, which have been most frequently investigated; however, the results of the previous studies remain inconclusive. Furthermore, the minor allele frequency of these 2 SNPs were more than 0.05, and their linkage disequilibrium analysis in CHB suggested that they were not correlated (D¶ = 0.409, r2= 0.153, HapMap database). Based on the previous studies, we speculate that polymorphisms in APE1 may be associated with an individual’s susceptibility to ovarian cancer. To test this hypothesis, we investigated the relationship in a hospital-based, case-control study in a Han Chinese population.
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MATERIALS AND METHODS Subjects and Specimen Collection This study involved 124 patients with ovarian cancer and 141 cancer-free controls, who were all Han Chinese. Cases were first diagnosed and treated at Xijing Hospital (Xi’an, China) from October 2010 to November 2010 and from October 2011 to January 2013. All patients had never received chemotherapy, surgery, or other treatments. In addition, all the patients had no history of other malignancies. There were no restrictions on recruitment in terms of age, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor grade, or histological type of disease. As controls, 141 women having no blood relationship to the cases were recruited from the same hospital for regular gynecological examinations; to be eligible, these individuals were required to be healthy and not have a history of cancers. Whole blood (4 mL) was extracted from all the subjects who agreed to participate in the study and preserved in tubes coated with K2-EDTA. All blood samples were stored in a j80-C freezer at the gynecological laboratory of Xijing Hospital. The ethics committees of the Fourth Military Medical University approved this research, and all the cases and controls signed consent forms.
DNA Extraction and Genotyping Genomic DNA was extracted from blood samples with the Blood Genomic DNA Isolation Kit (Sangon, Shanghai, China), according to the manufacturer’s instructions. An UV spectrophotometer (Jenway, England) was used to measure the DNA concentration and purity of each sample. The target fragments were amplified by polymerase chain reaction (PCR), and genotyping was performed by direct sequencing (ABI 3730xl DNA Analyzer). The following primers were used: 5¶-TCTGCCCCACCTCTTGATT-3¶ (forward) and 5¶-TGCC ACATTGAGGTCTCCA-3¶ (reverse) for rs1130409, and 5¶-CCTAACCCGAGCACAAAGAA-3¶ (forward), and 5¶-CGAGC CAACTCTGTGCCTTA-3¶ (reverse) for rs1760944. The PCR reaction mix included 7.6 HL of template DNA, 1.2 HL of forward primer, 1.2 HL of reverse primer, 20 HL of 2Taq PCR MasterMix, and 20 HL of ddH2O. Samples were subjected to denaturation at 95-C for 3 minutes, followed by 35 cycles of heating at 94-C for 30 seconds, annealing at 58-C (rs1760944) or 63-C (rs1130409) for 30 seconds, then extension at 72-C for 40 seconds, and a final incubation at 72-C for 5 minutes. After verification by 2% agarose gel electrophoresis, the PCR products were sent to Sangon Biotech Company and directly sequenced using the ABI 3730xl DNA Analyzer. The results of the sequencing were analyzed by Chromas2 software and compared with the sequence of APE1 in GenBank using DNAMAN V6 software by 2 persons independently in a blind fashion. If the sequence of one sample could not be read accurately, DNA was again extracted from the sample, and direct sequencing was repeated. Ten percent of the samples were randomly selected for repeat sequences, and the results were fully concordant.
Statistical Analysis
For each SNP in the control subjects, the W2 test was used to test for Hardy-Weinberg equilibrium. The Student t test and * 2013 IGCS and ESGO
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TABLE 1. Distribution of selected variables among cases and controls and test of the hardy-weinberg equilibrium for the SNPs of APE1 Variables
Case (124)
Age, mean (SD), y 49.30 (14.67) Menarche age, mean (SD), y 14.97 (1.96) Gravidity, n (%) G3 54 (43.55) Q3 70 (56.45) Parity, n (%) G3 93 (75.00) Q33 31 (25.00) Menopausal status, n (%) Yes 70 (57.38) No 52 (42.62)
Control (141)
P
48.05 (13.15)
0.718*
14.70 (1.84)
0.522*
56 (39.72) 85 (60.28)
0.528†
98 (69.50) 43 (30.50)
0.320†
77 (54.61) 64 (45.39)
0.652†
*Two-sided Student t test for the difference between the cases and controls. †Two-sided W2 test for the distribution between the cases and controls.
W2 test were used to compare demographic characteristics between cancer cases and controls. Multiple logistic regression analysis was performed to evaluate the distribution analyses of rs1130409 and rs1760944 genotypes between the cases and controls and to evaluate the stratified analysis between the genotypes and characteristic variables. P values, odds ratios (ORs) and 95% confidence intervals (CIs) were adjusted for age, age at menarche, gravidity, parity, and menopausal status. The association between genotype distributions and clinicopathological characteristics of the cases was analyzed by W2 test. The haplotypes and their frequencies were estimated by PHASE 2.1, and Pearson W2 test was used to compare the frequency of all haplotypes between the case and control participants. A P G 0.05 was considered significant in all statistical tests, and all P values were based on 2-way tests. All statistical tests described previously were conducted in SPSS 16.0 (SPSS Inc, Chicago, Ill).
RESULTS Parametric distributions of the study subjects are shown in Table 1. No significant differences were found for the age,
menarche age, gravidity, parity, and menopausal status between the cases and controls. Of the 124 ovarian cancer cases, 106 (85.48%) were epithelial ovarian cancers, which were classified as serous (76), mucinous (10), endometrioid (4), clear cell (3), malignant Brenner (1), and mixed/other roughly reported carcinomas (12). Eleven (8.87%) were germ cell tumors, including 7 immature teratomas and 4 endodermal sinus tumors, and 7 (5.65%) were sex gonad stromal tumors, which were all granulosa cell tumors. In addition, 42 patients with ovarian cancer were classified as FIGO stages I to II and 82 as FIGO stages III to IV. Fifty-five patients were detected as having lymphatic metastasis, which was not present in the remaining patients. Genotype frequencies of the 2 SNPs in the case and control groups conformed to Hardy-Weinberg equilibrium (W2rs1760944 = 2.052, Prs1760944 = 0.152 and W2rs1130409 = 1.122, Prs1130409 = 0.289 for the case group; W2rs1760944 = 0.614, Prs1760944 = 0.433 and W2rs1130409 = 1.313, Prs1130409 = 0.252 for the control group). The sequencing map view for all genotypes of rs1760944 and rs1130409 is shown in Figures 1 to 6. The genotype and allele frequencies of the rs1760944 and rs1130409 polymorphisms among the case and control groups and the association with the risk for ovarian cancer are shown in Table 2. The distribution of rs1130409 was significantly different between the ovarian cancer cases and controls. Compared with the control group, the cases exhibited a lower frequency of the G allele (P G 0.001). For the rs1130409 polymorphism, genotypic tests and allelic tests revealed significant associations between the TG/GG genotype and the G allele and a lower risk for developing ovarian cancer (adjusted OR [aOR], 0.495; 95% CI, 0.267Y0.920 for TG vs TT; aOR, 0.263; 95% CI, 0.132Y0.521 for GG vs TT; aOR, 0.486; 95% CI, 0.344Y0.0.688 for the G allele vs the T allele). However, the rs1760944 polymorphism in the APE1 gene was not found to be associated with the risk for ovarian cancer. Further stratified analyses showed that when the TG/GG genotype was compared with the TT genotype, the lower risk was more evident in subgroups of patients with age 50 years or older (aOR, 0.753; 95% CI, 0.604Y0.938), patients with menarche age of 15 years or older (aOR, 0.722; 95% CI, 0.573Y0.910), patients with gravidity of 3 or more times (aOR, 0.732; 95% CI, 0.587Y0.912), and postmenopausal women (aOR, 0.763; 95% CI, 0.615Y0.947), as shown in Table 3. This protective effect was observed in women whether parity is less than 3 or more than 3. However, our analysis did not show an association between rs1130409 polymorphisms and clinicopathological characteristics of
FIGURE 1. Sequencing map view of TT genotype for rs1760944 * 2013 IGCS and ESGO
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FIGURE 2. Sequencing map view of GG genotype for rs1760944
FIGURE 3. Sequencing map view of TG genotype for rs1760944
ovarian cancer risk stratified according to tumor histology, clinical stage, or lymph node status (Table 4). In addition, our study found that rs1760944 polymorphisms had no association with the risk for ovarian cancer in stratified analyses comparing characteristic variables and clinicopathological characteristics (data not shown). To further analyze the combined effects of polymorphisms of rs1760944 and rs1130409, we performed haplotype analysis. As shown in Table 5, when 4 haplotypes were constructed, the distribution of the haplotype frequency was different between the cases and controls (P = 0.001), with haplotype T-G and G-G showing a decreased risk for ovarian
cancer compared with haplotype T-T (OR, 0.646; 95% CI, 0.500Y0.836 for T-G vs T-T; OR, 0.627, 95% CI, 0.472Y0.823 for G-G vs T-T).
DISCUSSION In this study, we observed that the rs1130409 G allele and the TG/GG genotypes were significantly associated with a decreased risk for ovarian cancer susceptibility; however, the rs1760944 polymorphism was not associated with a risk for ovarian cancer (Table 2). In addition, the correlation analysis of APE1 variants and clinical features revealed that
FIGURE 4. Sequencing map view of TT genotype for rs1130409
FIGURE 5. Sequencing map view of GG genotype for rs1130409
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FIGURE 6. Sequencing map view of TG genotype for rs1130409
the protective effect of the TG/GG genotype compared with the rs1130409 TT genotype was more pronounced among subgroups of patients aged 50 years or older, patients with menarche age of 15 years or older, patients with gravidity of 3 or more times, and postmenopausal women (Table 3). Moreover, we found that the protective effect was observed in women whose parity was both less than 3 and more than 3. However, we did not find any associations between rs1130409 polymorphism variants and clinicopathological variables of ovarian cancer (Table 4). From the haplotype analysis, the distribution of the 4 haplotypes between the case and control groups was significantly different, with the T-G and G-G genotypes associated with a lower risk for ovarian cancer. Apurinic/apyrimidinic endonuclease 1 is the rate-limiting enzyme in the BER pathway and produces normal 3¶-hydroxyl nucleotide termini, which are important for DNA repair synthesis and ligation at single- or double-strand breaks by hydrolyzing 3¶-blocking fragments from oxidized DNA.10,11 Recently, many studies have investigated the relationship between APE1 polymorphisms and cancer susceptibility; however, little is known about the role of APE1 polymorphisms and ovarian cancer risk. The rs1130409 (T 9 G) polymorphism, which results in a single amino acid substitution (Asp 9 Glu), is the most extensively studied, and the results concerning
the rs1130409 polymorphism and cancer susceptibility remain inclusive. In the previous study, it was found that the GG genotype was associated with a significantly increased risk for colon cancer and gastric cancer and a decreased risk for bladder cancer; moreover, no associations were found between rs1130409 polymorphisms and susceptibilities to lung cancer, breast cancer, and prostate cancer.12Y15 Our results showed that the G allele and the GG genotype were associated with a lower risk for ovarian cancer. The discrepancy between different studies might be caused by rs1130409 polymorphisms playing a different role at different cancer sites. However, even for the sam type of cancer, the results could be different. For example, Gu et al13 and Canbay et al16 found that the frequency of the rs1130409 GG genotype in patients with gastric cancer was different from the control subjects, but Palli et al17 did not detect association of the polymorphism with gastric cancer. These different findings might be due to sample size differences or ethnic diversity. Moreover, it was reported that the rs1130409 GG genotype prolonged cell cycle G2 delays relative to the TT and TG genotypes; however, it had no impact on the endonuclease and DNA binding activities of APE1. Even so, the rs1130409 polymorphism might affect susceptibility to some cancers because these alterations might contribute to interactions of the polymorphism with other genes. For example, it was reported
TABLE 2. Genotype and allele frequencies of APE1 and the association with risk for ovarian cancer SNP and Genotype
Cases, n (%)
rs1760944 (j656T 9 G) TT 48 (38.71%) TG 52 (41.94%) GG 24 (19.35%) T allele 148 (59.68%) G allele 100 (40.32%) rs1130409 (1349T 9 G,Asp148Glu) TT 47 (37.90%) TG 54 (43.55%) GG 23 (18.55%) T allele 148 (59.68%) G allele 100 (40.32%)
Controls, n (%)
aOR (95% CI)*
P
46 65 30 157 125
(32.62%) (46.10%) (21.28%) (55.67%) (44.33%)
1.00 1.065 0.727 0.849
(reference) (0.547Y2.074) (0.365Y1.446) (0.601Y1.199)
0.852 0.363 0.352
28 62 51 118 164
(19.86%) (43.97%) (36.17%) (41.84%) (58.16%)
1.00 0.495 0.263 0.486
(reference) (0.267Y0.920) (0.132Y0.521) (0.344Y0.688)
0.026 G0.001 G0.001
*Adjusted for age, menarche age, gravidity, parity, and menopausal status in logistic regression.
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TABLE 3. Stratified analyses for rs1130409 polymorphisms in ovarian cancer cases and controls Cases/ Controls, n Variables
TT
Age, y G50 22/17 Q50 25/11 Menarche age, y G15 19/16 Q15 27/12 Gravidity G3 19/12 Q3 28/16 Parity G3 35/22 Q3 12/6 Menopausal status Yes 27/15 No 19/13
TT vs TG/GG
TG/GG
P
aOR (95% CI)
36/61 41/52
0.040 0.010
0.794 (0.629Y1.002) 0.453 (0.604Y0.938)
37/59 39/54
0.107 0.004
0.840 (0.673Y1.048) 0.722 (0.573Y0.910)
36/44 41/69
0.124 0.003
0.833 (0.658Y1.055) 0.732 (0.587Y0.912)
58/76 19/37
0.022 0.014
0.804 (0.665Y0.973) 0.712 (0.525Y0.966)
43/62 33/51
0.010 0.052
0.763 (0.615Y0.947) 0.796 (0.626Y1.013)
*Adjusted for age, menarche age, gravidity, parity, and menopausal status in logistic regression.
that the rs1130409 TT genotype and the combination of the TT genotype and hOGG1-Cys variants increased the risk for p53 mutation for nonYsmall cell lung cancer.18 In our study, the rs1130409 G allele and GG genotype were associated with a lower risk for ovarian cancer. Thus, investigation of the interactions between APE1 polymorphisms and other DNA repair genes, oncogenes, tumor suppressor genes, and genes related to steroid hormone mechanisms would be beneficial to exploring the pathogenesis of ovarian cancer. In addition, it is well-known that the prevalence of ovarian cancer is increased in nulliparous, infertile, early menstruating, and late-onset menopause women. In our
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study, we found that when the rs1130409 TG/GG genotype was compared with the TT genotype, the lower risk was more evident in the subgroup of patients with menarche age of 15 years or older, patients with gravidity of 3 or more times, and postmenopausal women, which was consistent with the previous study. However, we also found that this protective effect occurred in a subgroup of patients aged 50 years or older but not in younger subjects. It was reported that ovarian cancer tends to occur in women whose median age is approximately 50 years. The lower risk for ovarian cancer in older individuals with the GG genotype may be due to direct genetic effects rather than the aging effect. In the stratified analyses concerning and parity, the protective effect of the TG/GG genotype was also shown, which suggested that the correlation between nulliparity and a high risk for ovarian cancer may also be related to genetic effects rather than parity. Moreover, our analysis did not find associations between rs1130409 polymorphisms and clinicopathological characteristics of ovarian cancer, and future studies are needed to expand the sample size to confirm the results. For rs1760944, we did not find any association between this polymorphism and ovarian cancer susceptibility; we also did not find any association in the stratified analyses. Although in a previous study, the G allele of rs1760944 was reported to significantly influence transcriptional activity when compared with the T allele, the results were not consistent.19,20 Regarding the relationship between rs1760944 polymorphisms and cancer susceptibility, the results differed in multiple studies.19,21Y25 Given the disparity between the mechanistic studies and the genetic studies, the rs1760944 polymorphism requires further investigation. In the haplotype analysis, we found that a decreased ovarian cancer risk was associated with the G allele of the rs1130409 polymorphism regardless of the rs1760944 polymorphism. Despite the T-G and G-G haplotypes having a lower risk for ovarian cancer compared with the T-T haplotype, exploring the associations between the haplotypes concerning the rs1130409 and rs1760944 polymorphisms and the function of APE1 is still necessary. In conclusion, our study demonstrates that the APE1 rs1130409 polymorphism, but not the rs1760944 polymorphism, may contribute to ovarian cancer susceptibility in northwestern Han Chinese. Furthermore, a larger
TABLE 4. Relationship between rs1130409 polymorphism and selected clinicopathological variables of ovarian cancer Tumor Histology SNP and Genotype
Epithelial, n (%)
TT TG GG T allele G allele
41 45 20 127 85
(38.68) (42.45) (18.87) (59.91) (40.09)
Others, n (%) 6 9 3 21 15
(33.33) (50.00) (16.67) (58.33) (41.67)
Clinical Stage P
I-II, n (%)
III-IV, n (%)
0.836 15 (35.71) 32 (39.02) 18 (42.86) 36 (43.90) 9 (21.43) 14 (17.07) 0.859 48 (57.14) 100 (60.98) 36 (42.86) 64 (39.02)
Lymph Node Status P 0.831
0.560
Positive, n (%)
Negative, n (%)
22 (40.00) 21 (38.18) 12 (21.82) 65 (59.09) 45 (40.91)
25 33 11 83 55
(36.23) (47.82) (15.95) (60.14) (39.86)
P 0.521
0.867
Two-sided W2 test for distributions of rs1130409 genotypes among clinicopathological variables in the cases.
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TABLE 5. Frequencies of APE1 haplotype between cases and controls and the results of association analysis Haplotype
Cases
T-T T-G G-T G-G
95 53 54 46
(38.24%) (21.43%) (21.84%) (18.49%)
Controls 70 87 49 76
(24.73%) (30.91%) (17.37%) (26.99%)
OR (95% CI)
P
1.00 (reference) 0.646 (0.500Y0.836) 0.880 (0.650Y1.191) 0.627 (0.472Y0.832)
0.001
Haplotype frequencies were composed according to PHASE software and analyzed by Pearson W2 test.
number of patients and well-matched controls with detailed individual data are required to validate our results.
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