1 Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, chuo-ku, Kobe, 650-0017, Japan, 2 Nuffield Department of Obstetrics and Gynecology, University of Oxford, OX3 9DU, UK and 3 Department of Gastroenterology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-Cho, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
4 To whom correspondence should be addressed. Email: maruo{at}kobe-u.ac.jp
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Abstract |
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Key words:
endometriosis/gene polymorphism/PCR/restriction fragment length polymorphism/TNF-
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Introduction |
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Tumor necrosis factor alpha (TNF-) gene is one of many genes previously implicated, based on the finding that elevated TNF-
levels in peritoneal fluid have been associated with up-regulated TNF-
production in peritoneal macrophages and peripheral monocytes of women with endometriosis (Keenan et al., 1995
; Braun et al., 1996
). It has been suggested that a peritoneal immuno-surveillance network involving different cell types may sub-serve this role, with the consequence that the immune system appears critical for the development of this disease (Braun and Dmowski, 1998
; Libvoic et al., 2001
). Although the functional role of TNF-
in endometrial tissue is unknown, it is conceivable that TNF-
may be associated with the susceptibility to endometriosis.
TNF- is a multifunctional proinflammatory cytokine that plays an important role in the initiation and regulation of immune responses. It activates inflammatory leukocytes and stimulates macrophages to produce other cytokines, such as interleukin (IL)-1, IL-6 and TNF-
itself (Lee et al., 2002
). Upon interaction of TNF-
with one of its receptors, TNF receptor 1 or TNF receptor 2 (Beutler et al., 1994), a variety of responses are elicited which affect the regulation of a large number of genes (Beutler, 1995
).
TNF- is also a significant source of genetic variability. Sequence polymorphisms, especially in the promoter region, have been identified that could play a part in the transcriptional regulation of the TNF-
gene. A G to A substitution at position 238 (D'Alfonso et al., 1994), a G to A substitution at position 308 (Wilson et al., 1992
), a C to T substitution at position 857 (Hermann et al., 1998
), a C to A substitution at position 863 (Uglialoro et al., 1998
) and a T to C substitution at position 1031 (Higuchi et al., 1998
) have been described in the proximal promoter of the TNF-
gene.
Two studies (Lee et al., 2002; Wieser et al., 2002
) have previously examined the association between endometriosis and TNF-
gene promoter polymorphisms at 238G/A and 308G/A in Caucasian and Korean populations, but no evidence of association was found. However, there has been no published study examining the association between endometriosis and the 857, 863 and 1031 polymorphisms in the promoter region of TNF-
gene. In this study, we investigated whether these five common polymorphisms in the promoter region of the TNF-
gene are associated with endometriosis in a Japanese population.
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Materials and methods |
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Genotyping
Genomic DNA was extracted from EDTA anti-coagulated whole blood using the Wizard DNA purification kit (Promega, Madison, WI). The 238, 308, 857, 863 and 1031 polymorphisms in the promoter region of TNF- gene were determined by using PCRRFLP analysis, as previously described (Skoog et al., 1999
; Van Heel et al., 2002
). Genotyping for the 238G/A polymorphism was performed using a PCR fragment amplified by the forward primer 5'-AAACAGACCACAGACCTGGTC and the reverse primer 5'-CTCACACTCCCCATCCTCCCGGATC. The reverse primer contained two nucleotide mismatches, which made it possible to use the restriction enzyme BamHI for the detection of the 238G/A polymorphism. The 308G/A polymorphism was analyzed using the forward primer 5'-GAGGCAATAGGTTTTGAGGGCCAT and the reverse primer 5'-GGGACACACAAGCATCAAG. The forward primer contained one nucleotide mismatch, which made it possible to use the restriction enzyme NcoI for the detection of the 308G/A polymorphism. Genotyping for the 857C/T and 863C/A polymorphisms was performed using the forward primer 5'-GGCTCTGAGGAATGGGTTAC and the reverse primers 5'-CCTCTACATGGCCCTGTCTAC and 5'-CTACATGGCCCTGTCTTCGTTACG, respectively. The two reverse primers each contain a nucleotide mismatch, which made it possible to detect both polymorphisms using the restriction enzyme HypCH4IV. It is noteworthy that the reverse primer for the 863C/A polymorphism overlaps with the 857C/T polymorphism and contains the wild-type C nucleotide at the 857 position. The 1031T/C polymorphism was evaluated using the forward primer 5'-TATGTGATGGACTCACCAGGT and the reverse primer 5'-CCTCTACATGGCCCTGTCTT, followed by digestion with the restriction enzyme BbsI.
The conditions for the genotyping were: PCR in a 20 µl reaction mixture containing 20 ng of genomic DNA, 10 pmol of each primer, 250 µM of each dNTP and 1.0 units Taq gold DNA polymerase. The concentration of MgCl2 varied between the PCR reactions for the different polymorphisms and was 1.5 mM for the 238 locus, 1.0 mM for 308, 2.0 mM for 857, 1.25 mM for 863 and 1.0 mM for 1031. The PCR was conducted with ABI 9700 thermocycler (PE Applied Biosystem, Foster City, CA) using the following thermal profile: an initial denaturing cycle of 96°C for 12 min; 35 cycles of denaturing at 94°C for 30 sec, annealing at 59°C for 1 min, and extension at 72°C for 2 min; and a final cycle of 72°C for 2 min. Digestions with the appropriate restriction enzymes were performed as described by the manufacturer (New England Biolabs, Beverly, MA) at 37°C for 16 h. DNA fragments were subjected to electrophoresis in a 4% agarose gel in the case of 238, 308, 857, 863 and a 2% agarose gel in the case of 1031. Gel was stained with ethidium bromide (0.1 µg/ml) and visualized by ultraviolet illumination.
Statistical methods
Genotypic distribuions were examined for significant departure from the HardyWeinberg equilibrium by a goodness of fit 2 test.
2 test was used to examine differences in the proportions of genotypes of the five polymorphisms between endometriosis cases and controls. The Fisher correction was applied when appropriate. Odds ratios (OR) and 95% confidence intervals (CI) were used to compare categorical variables. Cases were also divided into a subgroup containing women with stage IV disease only, and the distribution of the five polymorphisms in this subgroup was analyzed separately.
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Results |
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Discussion |
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A review of the literature highlighted the importance of several polymorphisms in the TNF- gene region (Wilson et al., 1992
). The polymorphisms at positions 238, 308, 857, 863 and 1031 have been associated with increased transcriptional activity and production of TNF-
in several studies (Wilson et al., 1997
; Higuchi et al., 1998
; Ahmad et al., 2003
). The polymorphism at the 238 G/A has been associated with insulin resistance syndrome and obesity (Rasmussen et al., 2000
). The 308 G/A polymorphism has been associated with various inflammatory and autoimmune diseases (Verjans et al., 1994
; Cabrera et al., 1995
; Mizuki et al., 1995
; Wilson et al., 1995
; Fong et al., 1996
; Nadel et al., 1996
). The 857, 863 and 1031 polymorphisms are associated with both increased luciferase activity and increased concanavalin-A stimulated TNF-
production from peripheral blood mononuclear cells (Higuchi et al., 1998
). Associations between the three polymorphisms 857, 863, 1031 and immune-mediated diseases such as rheumatoid arthritis and Crohn's disease have been reported (Wilson et al., 1992
; Negoro et al., 1999
). To date, two studies have investigated the TNF-
gene polymorphisms at 238G/A and 308G/A in patients with endometriosis in the Austrian and Korean populations, but failed to find any evidence of association. The frequencies of the 238A and 308A alleles in TNF-
varies significantly between ethnic groups, and it is rare in the Japanese population (<3%) (Sakao et al., 2001
). Therefore, large sample populations are necessary to evaluate associations between the 238G/A and 308G/A polymorphisms and diseases in the Japanese population. At present, there is limited evidence showing that these allelic variants, at positions 238 and 308, are involved in the regulation of cytokine production (Wilson et al., 1997
; Kaluza et al., 2000
; Koch et al., 2000
).
The 1031T/C polymorphism has recently been reported to be associated with Behcet's disease (Ahmad et al., 2003), extra intestinal manifestations of Crohn's disease including uveitis, erythema nodosum and large joint arthropathy (Orchard et al., 2002
) and Crohn's disease itself (Negoro et al., 1999
). Negoro et al., (1999)
and Ahmad et al., (2003)
both reported an increased frequency of the 1031C allele in patients with Crohn's disease and in patients with Behcet's disease compared to controls. In contrast, a lower frequency of 1031C allele is reported in patients with hyperandrogenism and ulcerative collitis than in controls (Negoro et al., 1999
; Escobar-Morreale et al., 2001
). Our data are in agreement with the results of Escobar-Morreale et al., (2001)
and Negoro et al., (1999)
who showed a decreased frequency of the 1031C allele in these related diseases compared to controls, while the functional correlate of decreased 1031C in various diseases remain unclear. Further investigation is necessary to determine the functional significance of the TNF-
1031C allele and how it interacts with the inflammatory dysregulation associated with endometriosis. In our study, a decreased frequency of 1031C allele is associated with severe endometriosis but not with less severe disease. The results imply that the C allele could provide protection from severe forms of endometriosis. It is striking that TNF-
promoter polymorphisms have been associated with the most severe manifestations of a number of other diseases (McGuire et al., 1994
; Nadel et al., 1996
; Knight et al., 1999
).
The TNF gene is located on the short arm of chromosome 6, very close to the HLA class II locus. This region remains a major challenge because of the extensive linkage disequilibrium across this highly polymorphic region. Higuchi et al., (1998) investigated the transcriptional promoter activity of the 857T or 1031C/863A allele in response to concanavalin A stimulation, and reported that these activities were 1.7- or 2.0-fold higher than those of the dominant allele, respectively. They also reported that the 1031C allele is in linkage disequilibrium with HLA-DRB1*0901. Ishii et al., (2002)
have examined the possible association between endometriosis and HLA-DR. They showed that the prevalence of the HLA-DRB1 *1403 allele was significantly higher in endometriosis patients than controls, suggesting that HLA might be involved in the etiology of endometriosis. However, they did not find an association between endometriosis and HLA-DRB1*0901. Taken together, it is possible that the low frequency of 1031C allele in endometriosis is not caused by linkage disequilibrium with HLA-DR, but the allele itself may independently protect women from the disease. Interestingly, Ahmed et al., (2003)
reported that the association with TNF-1031C appeared to be independent of linkage disequilibrium with other polymorphic genes in the HLA region in Behcet's disease. It remains incompletely determined whether the 1031T/C polymorphism of the TNF-
promoter is directly associated with susceptibility to endometriosis, or if it only represents a marker for some other closely linked susceptibility gene.
A relationship between the immune system and endometriosis was recognized almost 15 years ago, with the first description of TNF- in peritoneal fluid of women with endometriosis (Eisermann et al., 1988
). It has become clear since then that several molecular entities produced by cells in the immune system play a determining role in the establishment and maintenance of endometriosis (Bischof and Simpson, 2000
; Harada et al., 2001
; Nothnick, 2001
; Van den Linder, 2001; Braun et al., 2002
). The most widely studied cytokines in that regard are interleukin-1 (IL-1), IL-6 and TNF-
. TNF-
is therefore a good candidate for studies related to the development and progression of endometriosis.
A genome-wide screen with micro-satellite markers testing for linkage in sister pairs affected with endometriosis is already under way (Kennedy et al., 2001), but doubts remain about the power of this approach alone to identify genes implicated in the disease processhence the need for studies of functional candidates. The hypothesis driven investigation of functional candidate genes (i.e. those chosen on the basis of biological plausibility or known aberrant mechanisms in a disease) is a complementary approach to linkage analysis, which is hypothesis free, in the identification of susceptibility genes.
In conclusion, our data demonstrate a lower frequency of the 1031C polymorphism in the promoter region of the TNF- gene in the most severe cases of endometriosis in a Japanese population, suggesting that the 1031C polymorphism may play some protective role from the progression of endometriosis. Although this model is biologically plausible, we recognize that our conclusions are based on relatively small numbers and will require verification from additional independent studies.
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Acknowledgements |
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References |
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Submitted on September 19, 2003; accepted on November 20, 2003.
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