Abstract
Background/Aim: Flap endonuclease 1 (FEN1), a protein with multiple functions in genome stability maintenance, is important in cancer prevention. The two functional germline variants of FEN1, rs174538 and rs4246215, regarding cancer susceptibility have been reported in lung, breast, liver, esophageal, gastric, colorectal cancer, glioma and leukemia, but not endometriosis. In this study, we firstly aimed at evaluating the contribution of FEN1 genotypes to endometriosis risk in a representative Taiwan population. Materials and Methods: In total, 153 patients with endometriosis and 636 non-cancer healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Results: The genotypes of FEN1 rs174538, but not those of rs4246215, were differently distributed between the endometriosis and control groups. In detail, the AA of FEN1 rs174538 genotypes were significantly less frequently found among endometriosis patients than among controls (odds ratio [OR]=0.43, 95% confidence interval [CI]=0.24-0.78, p=0.0125). The A allele at FEN1 rs174538 was also significantly less frequent among cases than controls (OR=0.65, 95%CI=0.50-0.86, p=0.0021). As for age of first menarche, those with first menarche at the age >12.8 carrying the FEN1 rs174538 AA genotype conferred lower OR of 0.29 (95%CI=0.11-0.78, p=0.0381) for endometriosis. Regarding the full pregnancy status, those without having had a full-term pregnancy carrying the FEN1 rs174538 AA genotype were of lower risk (ORs=0.12, 95%CI=0.03-0.53, p=0.0050). Conclusion: The FEN1 rs174538 A allele is a novel protective biomarker for endometriosis and this genotype may have interactions with age- and hormone-related factors on the development of endometriosis.
All over the world, about 10% of females at reproductive ages suffer from endometriosis, which is a hormone-dependent inflammatory benign gynaecological disease where functional endometrial tissue is unusually present outside the uterus (1, 2). In clinical, the main manifestations are dysmenorrhea, chronic pelvic pain, pain during intercourse and infertility (3). Although the mechanisms underlying endometriosis are still unrevealed, mounting evidence has shown that endometriosis is a multi-factorial, multi-step disease affected by inflammation, hormonal imbalance, genetic and environmental interactions (4-6).
It is believed that alterations in DNA repair genes are closely-related to genomic instability and carcinogenesis. Among these DNA repair genes, Flap endonuclease 1 (FEN1) is a multiple-function endonuclease in charge of removal of DNA adducts caused by alkylating agents and UV irradiation (7). As for the base excision repair (BER) pathway, FEN1 could efficiently remove 5’flap as an endonuclease (8, 9). During the DNA replication process, FEN1 could promote the maturation of Okazaki fragments in the lagging strand (8, 9). Additionally, FEN1 could also act as a 5’exonuclease and a gap-dependent endonuclease, promoting the DNA fragmentation during programmed cell death (10, 11). In cells with functionally impaired yeast FEN1 (known as RAD27 in yeast), significantly elevated spontaneous mutation rates are observed (11-13). In mice models, the knockout of FEN1 can lead to increased genomic instability and carcinogenesis (14). In human cancer cells, FEN1 mutations can result in reduced nuclease activity (15). Therefore, dysregulated expression of FEN1 resulting from genetic variations may contribute to an increased risk for the development of cancer.
In literature, most investigations of FEN1 genomic variations focused on the two common polymorphisms with minor allelic frequencies larger than 5%, promoter -69G>A (rs174538) and 3’UTR 4150G>T (rs4246215), which were both effective on influencing the expression level of FEN1 and enzyme activity (16). The polymorphic genotypes of rs174538 and/or rs4246215 are associated with the risk of several types of cancer such as esophageal (17, 18), lung (16), gastrointestinal (18, 19), gallbladder (20), breast cancer (21-23), glioma (24) and leukemia (25). However, the contribution of FEN1 genomic variation to endometriosis has never been investigated. The purpose of the present study was, therefore, to analyze the genetic polymorphisms of FEN1 promoter -69G>A (rs174538) and 3’UTR 4150G>T (rs4246215) in a population consisting of 153 endometriosis and 636 non-endometriosis cases, in order to investigate the association between these two FEN1 genotypes and endometriosis.
Materials and Methods
Study population and sample collection. One hundred and fifty-three endometriosis patients were recruited at the outpatient clinics of general surgery during 2000-2010 at Chung Shan Medical University Hospital in Taiwan. These endometriosis patients were diagnosed by laparoscopy, classified according to the American Society for Reproductive Medicine and confirmed histologically. Patients with pathological confirmation or clinical suspicion of leiomyoma, adenomyosis or invasive carcinoma of the uterine cervix or ovary were excluded from this study. No patient had received hormone therapy during the preceding 12 months. The mean age of the endometriosis patients was 40.3±4.9 years and 55 of them (35.9%) did not have a child or full pregnancy. The basal follicle-stimulating hormone (FSH) level was 7.2±1.4 IU/l. The non-endometriosis statuses were confirmed after detail ultrasonography. All operations were performed by the experienced surgeon Dr. Yin and his colleagues. According to the revised American Fertility Society classification, 32 (20.9%) had minimal or mild endometriosis (stage I-II) and 121 (79.1%) had moderate or severe endometriosis (stage III-IV). All women provided 3 to 5 ml of peripheral blood for genotyping analyses with written informed consent. The experiment was approved by the Ethical Committee and Institutional Review Board of the Chung Shan Medical University Hospital. At the same period, 636 non-endometriosis healthy volunteers were selected as controls via a matching system for age and habits after initial random sampling from the Health Examination Cohort of China Medical University Hospital. The exclusion criteria of the control group included previous malignancy, metastasized cancer from another or of unknown origin or any familial disease. Both groups completed a questionnaire, which included the individual smoking and drinking habits. Smokers were defined as daily or almost daily smokers who had smoked at least five packs of cigarettes in their lifetime. Smokers were asked for the age of initiation, whether they were currently smoking or had already quit and, if so, when they had quit and, on average, how many cigarettes they smoked or had smoked daily. The non-drinkers included those with social drinking behavior of less than 200 ml per week and less than twice per month. The selected characteristics of the control and patient groups are summarized and compared in Table I.
Distributions of characteristics among endometriosis cases and control subjects
Genotyping methodology. Genomic DNA was prepared from peripheral blood leukocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan), stored at −80°C, diluted and aliquoted for genotyping as working stock at −20°C (26, 27). Genotyping for FEN1 rs174538 and rs4246215 of all subjects was carried-out by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, as previously reported (28, 29). The primers were designed by Terry Fox Cancer Research team and the PCR-RFLP conditions for FEN1 rs174538 and rs4246215 are summarized in Table II. The success rate of PCR-RFLP was 100%, and the genotypes of 5% of the participants randomly selected from controls and cases were analyzed by PCR direct sequencing (Genomics BioSci & Tech Co., Taipei, Taiwan). The consistency between direct sequencing and PCR-RFLP was 100%.
Summary of the rs numbers, designed primer sequences, restriction enzymes, amplicon lengths before and after enzyme digestion for FEN1 rs174538 and rs4246215.
Distribution of the FEN1 genotypic and allelic frequencies among 636 healthy controls and 153 endometriosis patients.
Statistical analysis. The descriptive statistics of patients and controls were presented as the mean±standard deviations (SDs) or as percentages. The Pearson's Chi-square test or Fisher's exact test (when the number in any cell was less than five, such as “2” homo-variants in case group of no full pregnancy) was used to compare the distribution of the FEN1 genotypes. Associations were evaluated by odds ratios (ORs) with 95% confidence intervals (95%CIs). Statistical tests were deemed significant when the p-value was less than 0.05.
Results
The frequency distributions for the age, age at menarche, full pregnancy, smoking and alcohol drinking status, and clinical stages of 153 endometriosis patients and 636 non-endometriosis healthy controls are shown in Table I. The characteristics of patients and controls were well-matched according to age, age at menarche, smoking and alcohol drinking status (p>0.05), while the percentages of full pregnancy were less among endometriosis patients (64.1%) than among non-endometriosis subjects (75.9%) (Table I).
The genotypic and allelic frequencies for the FEN1 rs174538 and rs4246215 among endometriosis patients and non-endometriosis healthy controls are shown in Table III. The genotypic frequency distributions for FEN1 rs174538 were significantly different between endometriosis and control groups (p for trend=0.0125), while those for FEN1 rs4246215 polymorphism were not significantly different (p for trend=0.8677) (Table III). In detail, those who carried AA, AG plus AA genotypes had a significantly reduced risk of endometriosis with ORs of 0.43 and 0.63 respectively compared to those with the wild-type GG genotype (95%CI=0.24-0.78 and 0.45-0.91, respectively). From the results of allelic frequency analysis, we can find that the A allele of FEN1 rs174538 seemed to be a protective factor for endometriosis (p=0.0021, OR=0.65, 95%CI=0.50-0.86), while the T allele of FEN1 rs4246215 was not (p=0.6315, OR=1.06, 95%CI=0.83-1.37). The conclusion that can be deduced from Table III is that the FEN1 AA genotypes (A allele) seemed to be a protective factor for endometriosis among Taiwanese women.
Distribution of the FEN1 promoter rs174538 and 3’ UTR rs4246215 genotypes stratified by age at menarche and full pregnancy.
Because age- and hormone-related factors are risk factors for developing endometriosis, the contribution of the FEN1 genotype to endometriosis stratified by age at menarche and full pregnancy status were further analyzed and presented in Table IV. Since the average age of first menarche for the 318th and 319th subjects in the control and patient groups was 12.8 years, we have further stratified the groups into ≤12.8 and >12.8year-old sub-groups. Noticeably, in the later-menarche (>12.8 years) group, subjects with homo-variant AA genotypes for FEN1 rs174538 had lower risks for developing endometriosis than those with the wild-type GG genotype (p for trend=0.0381, OR=0.29, 95%CI=0.11-0.78), while the combined AG with AA group did not have the similar results (OR=0.64, 95%CI=0.38-1.09). On the contrary, this was not the case for the earlier-menarche (>12.8 years) group (Table IV). As for the full pregnancy status, those women with no full pregnancy and of AA or AG plus AA genotypes for FEN1 rs174538 were less likely to develop endometriosis than those with the homozygous GG genotype (p for trend=0.0050, OR=0.12 and 0.46, 95%CI=0.03-0.53 and 0.25-0.86 for AA and AG plus AA, respectively), but this was not the case for those with full pregnancy (Table III). As for the FEN1 rs4246215, there was no difference between the comparisons of stratified menarche age or full pregnancy status groups. In summary, stratified analyses revealed an interaction between the age of first menarche and no full pregnancy among FEN1 rs174538 genotypes in the endometriosis susceptibility.
Discussion
In the current study, our group firstly examined the contribution of FEN1 genotypes to endometriosis susceptibility. According to that, FEN1 plays a multiple role in DNA repair, DNA replication, and cell apoptosis, and is very possible that hereditary genomic variations may determine susceptibility to the development of cancer and endometriosis. In literature, polymorphic FEN1 genotypes, such as rs174538 and rs4246215, may determine FEN1 enzymatic activity and the rate of tumorigenesis at cell and animal levels (14, 15), and may be linked to the risk of cancers and endometriosis. In the current study, we found that the AA genotype or A allele at FEN1 rs174538 was significantly associated with a lower susceptibility to endometriosis among Taiwanese women (Table III). These findings are consistent with researches that have identified the A allele to be a protective factor for many types of cancer, including lung (16), gastrointestinal (19), esophageal (18), breast cancer (21), glioma (24) and childhood leukemia (25). As far as we are concerned, the current study was the first to provide evidence that the FEN1 genotype, rs174538 but not rs4246215, is associated with endometriosis risk (Table III).
Since the age- and hormone-related factors are environmental risk factors that contribute to endometriosis, we have also analyzed the gene-environment interactions via analyzing the distributions of the FEN1 rs174538 genotype and endometriosis status among the investigated subjects according to their age at first menarche and their full pregnancy status. Interestingly, in the later-menarche (>12.8 years) group, women with the AA genotype for FEN1 rs174538 had lower risks for developing endometriosis than those with the wild-type GG genotype (Table IV). On the contrary, no such age difference was observed in analyses of the FEN1 rs4246215 genotype. The mechanisms of FEN1 protein involved in the etiopathology and progression of endometriosis has never been studied. Although no statistical significance was found in the full pregnancy group in Table IV, we could not exclude the possibility our findings may not be the same when thousands of patients will be analyzed. Also, as for the positive findings, only five women with a homo-variant AA genotype had a first menarche at an age older than 12.8 years, an extremely small number (Table IV). The enlarged sample size may provide us with a more realistic answer. In the present article, we can only conclude that the protective impact of the A allele at FEN1 rs174538 with respect to endometriosis risk (Table III), and very possibly, the genotypes may have a gene-environment interaction with age- and hormone-related factors (Table IV). While the complete underlying mechanisms have not been discovered, sexual hormones and steroids reportedly play an important part in controlling the proliferation of cancer cells. For instance, in supporting the idea of gender differences in susceptibility to cancer, 17-β estrogen was found to have a stronger inhibitory effect than testosterone on human monoblastic U937 cells (30). At the molecular level, further measurement and analysis of the DNA repair capacity, cell proliferation and apoptosis rates among samples from endometriosis patients with differential FEN1 genotypes and age- and hormone-related status may help us further understand the development and gene-environment interaction in endometriosis.
In conclusion, the present study documented evidence of a positive association between the genotypes of FEN1 and endometriosis and examined the age- and hormone-related interactions with the genotype, in order to determine endometriosis susceptibility. The presence of the A allele of rs174538 was not only a novel detectable and predictive biomarker for endometriosis but a possible anti-endometriosis therapeutic target.
Acknowledgements
This study was partially supported by research grants from Taoyuan Armed Forces General Hospital to Dr. Huang (AFTYGH-105-27), Ministry of Science and Technology of Taiwan to Dr. Shen (MOST105-2628-B-039-002-MY3, 105-2815-C-039-049-B), China Medical University to Dr. Shen (CMU103-N-08); China Medical University Hospital to Dr. Shen (DMR-105-082) and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW106-TDU-B-212-113004).
Footnotes
↵* These Authors contributed equally to this study.
This article is freely accessible online.
Conflicts of Interest
The Authors declare that there are no conflicts of interest with any person or company.
- Received August 2, 2017.
- Revision received September 5, 2017.
- Accepted October 6, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
