Hum. Reprod. Advance Access published online on February 12, 2008
Human Reproduction, doi:10.1093/humrep/den016
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Association of tumor necrosis factor-
gene polymorphisms with advanced stage endometriosis
1 Department of Obstetrics and Gynecology, Seoul Medical Center, Seoul, South Korea 2 Department of Obstetrics and Gynecology, The Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University College of Medicine, Seoul, South Korea 3 Department of Obstetrics and Gynecology, College of Medicine, University of Ulsan, Asan Medical Center, Seoul, South Korea 4 Department of Obstetrics and Gynecology, Chonnam National University College of Medicine, Gwangju, South Korea 5 Department of Obstetrics and Gynecology, St Vincent’s Hospital, College of Medicine, Catholic University of Korea, Seoul, South Korea
6 Correspondence address. Tel: +82-2-2072-2385; Fax: +82-2-762-3599; E-mail: ymchoi{at}snu.ac.kr
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Abstract |
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BACKGROUND: This study was performed to investigate whether specific haplotypes and several single nucleotide polymorphisms in the promoter region of the tumor necrosis factor (TNF)-
gene are associated with the risk of advanced stage endometriosis in a Korean population.
METHODS: This study comprised women with (n = 246) or without (n = 248) endometriosis. The TNF:g.[-1031T > C], TNF:g.[-863C > A] and TNF:g.[-857C > T] polymorphism in the TNF- gene were assessed by PCR-restriction fragment length polymorphism analysis, which utilized digestion by BbsI, HypCH4IV and HypCH4IV restriction enzymes, respectively. In silico haplotypes were deduced by using the Haploview version 3.32.
RESULTS: The genotype distribution of TNF:g.[-1031T > C] was significantly different between total endometriosis patients and the controls (T/T of 56.9 versus 60.1%, T/C of 35.4 versus 37.5% and C/C of 7.7 versus 2.4%, respectively, P = 0.027). This difference at the TNF:g.[-1031T > C] tends to increase in Stage IV endometriosis (P = 0.01). However, there was no difference in the TNF:g.[-863C > A] and TNF:g.[-857C > T] site between the two groups. Even when the endometriosis cases were subdivided into American Society for Reproductive Medicine Stages III and IV, genotype differences were not found. The CC homozygote at TNF:g.-863 was more frequently found in the controls than Non-CC group (P = 0.04; odds ratio = 0.67; 95% confidence interval = 0.45–0.98). All haplotypes and diplotypes, deduced by in silico analysis, showed no association with subgroups or controls.
CONCLUSIONS: Our results suggest that the genotype frequencies at the TNF:g.[-1031T > C] and the TNF:g.[-863C > A] sites may be associated with advanced stage endometriosis in the Korean population.
Key words: endometriosis/tumor necrosis factor/polymorphism
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Introduction |
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Since the human genomic project was completed (Celera Genomics, 2001
; Consortium of the IHGS, 2001), several complex trait diseases have been the main targets of association and linkage studies using single nucleotide polymorphisms (SNPs; biallele sequence variants found with a frequency of over 1% in the human population) or microsatellite markers.
Endometriosis (MIM 131200
[OMIM]
) is a complex trait gynecologic disease characterized by the growth of endometrial tissue implants outside the uterus. It is known to cause diverse clinical manifestations such as infertility, pelvic pain and dysmenorrhea in about 10% of women of reproductive age (Wheeler, 1989).
Among the large-scale studies, the report of the International Endogene Study groups is worth noting as specific, shared sites in affected siblings were found (Treolar et al., 2005). Several candidate genes in detoxification enzymes, and the estrogen and progesterone receptors have been investigated to examine the biological plausibility of their involvement with endometriosis (Georgiou et al., 1999; Bischoff et al., 2002; Wieser et al., 2002).
Although there are many studies reporting multiple distinct pathways that could be involved in its pathogenesis, endometriosis is still among the most enigmatic diseases. Since up to 90% of women exhibit retrograde menstruation during the mense (Halme et al., 1984), a defective immunosurveillance is thought to contribute to the first and the most important step in the implantation and growth of ectopic endometrial tissue. Of the various inflammatory proteins, it is well known that the concentration of tumor necrosis factor (TNF) in peritoneal fluid is high in patients suffering from endometriosis and primary infertility compared to women with normal pelvic anatomy (Eisermann et al., 1988).
Of the three TNFs, TNF-, TNF-β (also known as lymphotoxin , LT-) and LT-β (Eigler et al., 1997), the active form of early endometriosis contains a higher amount of TNF- depending on the size and the number of red lesions (Harada et al., 1999). Because of this, TNF- is considered to play an important role in the first pathogenesis event of endometriosis that involves angiogenic activity.
The human TNF- gene, so called TNF, is located at 6p21.31 and is one of the 200 genes in the HLA system. However, TNF, which is located in the HLA Class III region, is known to be unrelated to HLA Class I and Class II genes, either structurally or functionally (Klein and Sato, 2000). Specific SNPs, such as those at positions –1031, –863 and –857 relative to the transcription start site of TNF, are known to have direct effects on TNF regulation and transcriptional efficiency (Wilson et al., 1997; van Heel et al., 2002; Soga et al., 2003). On the basis of these findings, several researchers have investigated these polymorphisms for possible effects on TNF regulation that could be associated with susceptibility to several autoimmune diseases (Negoro et al., 1999; Park et al., 2006).
Recently, a study of Japanese subjects with endometriosis showed that specific haplotypes [T; C; C/T; C; C] tended to increase significantly in patients compared to controls [P = 0.043, P-corrected = 0.344, odds ratio (OR) = 1.62] as analysed using a PCR-preferential homoduplex formation assay (Teramoto et al., 2004).
Considering that there has been no further supporting evidence and that the implicated polymorphic sites and allele frequencies appear to vary substantially among ethnic groups, we performed this study to evaluate whether TNF:g.[-1031T > C], TNF:g.[-863C > A] and TNF:g.[-857T > C] are independently associated with endometriosis stage and to confirm the in silico haplotypes associated with endometriosis in a Korean population.
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Materials and Methods |
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Subjects
Peripheral blood was obtained from a total of 494 patients. All subjects were of Korean origin, which is made up of a single ethnic trait. A total of 246 patients had surgical and histological evidence of advanced endometriosis, whereas another 248 patients without the disease served as controls. In the endometriosis group, the extent of disease was staged according to the guidelines of the American Society for Reproductive Medicine (ASRM) (American Society for Reproductive Medicine, 1997). Fifty-six patients were diagnosed as having Stage III, and 190 patients had Stage IV endometriosis. None of the subjects had received hormone therapy during the previous 12 months. Endometriosis status was confirmed by diagnostic laparoscopy, pelviscopic surgery, exploratory laparotomy or transabdominal hysterectomy in both groups. Approval for this study was obtained from the Institutional Review Board at the Seoul National University Hospital, and informed consent was obtained from each woman.
Ages ranged from 19 to 53 years (33.9 ± 7.2, mean ± SD) in the endometriosis group, and from 21 to 55 years (44.2 ± 9.8) in the control group (P < 0.05).
Genomic DNA analysis
Genomic DNA was extracted from the peripheral blood with the Wizard DNA purification kit (Promega, Madison, WI, USA). Genotyping of TNF:g.[-1031T > C], TNF:g.[-863C > A] and TNF:g.[-857T > C] was performed using the PCR-restriction fragment length polymorphism method. This analysis was made using Ashgar’s protocol with the specifically designed reverse primers (Asghar et al., 2004). Briefly, genomic DNA was amplified using specific primers (Table I). It is noteworthy that the reverse primer for the TNF:g.[-863C > A] polymorphism overlaps with the TNF:g.[-857T > C] polymorphism and contains the wild-type C nucleotide at the –857 position. The forward primer was same for both the –863 and –857 SNP sites.
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For the PCR, 0.1 µg of genomic DNA was added to a PCR mixture containing 0.2 mM of dNTPs, 0.4 mM of each primer and 1.25 U of Taq polymerase (Takara, Shiga, Japan). The concentration of MgCl2 varied between the PCR and was 1.0 mM for the TNF:g.[-1031T > C], 1.25 mM for the TNF:g.[-863C > A] and 2.0 mM for the TNF:g.[-857T > C]. The PCR cycling conditions were as follows: an initial denaturation step at 94°C for 12 min, amplification for 35 cycles at 94°C for 30 s, 59°C for 60 s and 72°C for 120 s, followed by a final extension step at 72°C for 2 min (MJ Research, Waltham, MA, USA). The PCR products were digested overnight using 2 IU of BbsI for TNF:g.[-1031T > C] and HypCH4IV (New England Biolabs, Beverly) for both TNF:g.[-863C > A] and TNF:g.[-857T > C] at 37°C, separated by 2% agarose gel electrophoresis and visualized using ethidium bromide staining.
Statistical analysis
The ages of each group were normally distributed as tested by Kolmogorov–Smirnov test and thus were compared using a Student’s t-test.
Genotype distributions were examined for significant departure from Hardy–Weinberg equilibrium (HWE) by a goodness-of-fit 
2-test with one degree of freedom. The 2-test was used to examine difference in the proportions of genotypes between endometriosis cases and controls. When the assumption of the 2-test was violated because >20% of the cells had an expected count of <5, a Binomial test was used. P < 0.05 was considered statistically significant.
ORs and 95% confidence intervals (CIs) were used to compare categorical variables. Cases were divided into subgroups containing women with Stage III and IV diseases, and the distribution of the three polymorphisms in these subgroups was analysed separately.
The haplotype combination at TNF:g.-1031, TNF:g.-863 and TNF:g.-857 and the linkage disequilibrium (LD) value between two specific loci (TNF:g.-1031 and TNF:g.-863, TNF:g.-1031 & TNF:g.-857, TNF:g.-863 & TNF:g.-857) were deduced using Haploview version 3.32 based on an accelerated EM algorithm (Wall et al., 2003; Barrett et al., 2005). The differences in the distribution of the haplotype and diplotype distributions between the two groups were assessed with a 2-test. P < 0.05 was considered statistically significant.
Additional statistical analyses using Bonferroni correction were applied to adjust for multiple comparisons.
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TNF genotype and allele frequencies
The genotype of the three promoter polymorphisms in the TNF gene of endometriosis patients and controls is shown in Table II. The genotype frequencies of both cases and controls are compatible with HWE.
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There is a significant association between total endometriosis groups and controls at the genotype level for TNF:g.[-1031T > C] (P = 0.027). In addition, the patients suffering from a more severe case of endometriosis appeared to be more genetically influenced based on the finding that the major difference occurs between Stage IV endometriosis and controls (P = 0.01). The more severe the endometriosis, the higher the frequency. Specifically, the TNF:g.-1031CC homozygote is high in the total endometriosis group (7.7%) compared to the control group (2.4%), meaning endometriosis cases carry the C-allele in the homozygous state (CC group) more so than the TC heterozygous and TT homozygous state (Non-CC group) (P = 0.01; OR = 3.38; 95% CI = 1.32–8.6). This pattern is more pronounced in Stage IV endometriosis subgroup (P = 0.002; OR = 3.96; 95% CI = 1.53–10.25) (Table II).
In the TNF:g.[-863C > A] SNP site, the CC genotype at the TNF:g.-863 appears to have a protective effect on endometriosis compared to the Non-CC group (P = 0.04; OR = 0.67; 95% CI = 0.45–0.98) (Table III). However, there is no significant difference between subgroups in the frequencies of the TNF:g.[-863C > A] polymorphism.
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The genotype difference in the TNF:g.[-857C > T] is not found between endometriosis cases and controls.
TNF promoter haplotype and diplotype
In silico analysis, using the Haploview, shows LD at the three specific SNP sites of the TNF gene in a Korean population. TNF:g.-1031T was strongly linked with TNF:g.-863C (D’ = 0.721) and TNF:g.-857C (D’ = 0.845). TNF:g.-863C is also strongly linked with TNF:g.-857C (D’ = 0.921).
Among the eight (23) expected haplotypes, Haploview showed that TNF:g.-1031T- TNF:g.-863C-TNF:g.-857C [T; C; C] (58.6%) constituted the dominant upstream promoter haplotypes in controls, and the frequencies of [T; C; T], [C; A; C], [C; C; C] and [T; A; C] were 16.7, 13.8, 7.3 and 3.5%, respectively (Table IV). As the default option of Haploview is to report haplotype frequency above 1.0%, another three haplotypes are omitted.
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Although the frequency of TCC haplotype was significantly different between endometriosis cases and controls in Teramoto’s data (Teramoto et al., 2004
), all haplotypes in our data were not associated with either total cases or subgroups based on ASRM stage (Table IV). For the control groups, the [T; C; C] haplotype in our study was significantly less frequent than reported by Teramoto (Table IV) (P = 0.04). The most common diplotype for the SNPs was TCC/TCC, for which the frequencies for control and total endometriosis were 34.7 and 28.9%, respectively. However, the frequency of diplotypes with a total frequency above 5.0% was not significantly associated with the risk of endometriosis in this study (Table V).
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Discussion |
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TNF-
plays a critical role not only in protection from microbial infection, but also in the pathogenesis of a wide range of inflammatory or autoimmune diseases, and specific polymorphisms have been reported to play a role in the implantation and the growth of ectopic endometrial implants (Kroeger et al., 1997; Higuchi et al., 1998). In this study, we observed three polymorphic sites at the genotype, haplotype and diplotype level and found that there are significant associations in the TNF:g.[-1031T > C] (P = 0.027) and the TNF:g.[-863C > A]. This association is more pronounced when Stage IV endometriosis patients are compared with the controls in terms of CC versus Non-CC group. All the haplotype and diplotype frequencies were not significantly associated either with total cases or any subgroup.
These findings are in opposition with those reported by Asghar’s group, who showed that the TNF:g.-1031C alleles may have some protective functions against severe endometriosis (Asghar et al., 2004). Contrary to the expectation, we observed the opposite result when the case (n = 246) and control (n = 248) numbers were over 200. Women who were TNF:g.-1031CC homozygotes were more frequently found in the total endometriosis group and tended to have more severe endometriosis. This contrast can be explained by two hypotheses. One possibility to account for this discrepancy is a false positive error, which may be the case in Asghar’s group due to the small number of cases and controls (n = 130 and 183, respectively). The other possibility revolves around the differences in ethnic makeup. Subtle genetic differences between ethnicities may underlie the pathogenesis of endometriosis, even though the Korean and Japanese populations are closely linked. Some reports showing that TNF:g.-238A and TNF:g.-308A are not associated with immune-mediated diseases resulted in our exclusion of the above SNP sites from the study lists (Lee et al., 2002; Asghar et al., 2004).
With reference to haplotype activity at the site of TNF:g.-1031, TNF:g.-863 and TNF:g.-857, some specific haplotypes [T; C; T & C; A; C] are known to be associated with T helper 1 (Th1)-dominant diseases, such as rheumatoid arthritis, insulin dependent diabetes mellitus and Crohn’s disease, due to high promoter activity. In contrast, the [T; C; C] haplotype is associated with low promoter activity and is consequently related to Th2-dominant diseases such as atopic dermatitis and ulcerative colitis (Matsushita et al., 1999). In our study, we found no difference in the haplotype and diplotype frequencies between groups. We also calculated the LD, which represents a non-random pattern of alleles at different loci found together more or less often than expected based on their frequencies (Freeman et al., 2006). The Lewontin’s D’ between all SNP markers were all above 0.7, but unlike the high level of D’, R2 behaved very differently. The maximal value R2, measured to be between TNF:g.[-1031T > C] and TNF:g.[-863C > A], was 0.408.
Our results are also further supported and validated by the fact that all loci were in HWE, not deviating from random mating.
The mean age of the control group in the present study was higher than that of the endometriosis group. However, this distribution of age has some advantage in revealing the OR of SNPs between the two groups, as described by Hadfield et al. (2001). Since all the controls underwent surgery, this type of control group may not be representative of the general population. Consequently, this control group, completely free of endometriosis and of an older age, can give us a clue about the genetic contribution to the pathogenesis of endometriosis.
In conclusion, this study demonstrated that the TNF:g.-1031CC homozygote may be associated with advanced stage endometriosis and that the TNF:g.-863CC homozygote has a protective role in the pathogenesis of endometriosis. Despite these findings, the in silico analysis did not show any additive power in the development of endometriosis. Further studies should be executed on a larger population to further clarify the real value of the three polymorphic markers in the TNF promoter region in determining the genetic susceptibility to endometriosis.
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This study was supported by a grant (01-PJ10-PG6-01GN13-0002) from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea.
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Submitted on September 30, 2007; resubmitted on December 21, 2007; accepted on January 11, 2008.
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