Hum. Reprod. Advance Access originally published online on May 27, 2004
Human Reproduction 2004 19(8):1765-1769; doi:10.1093/humrep/deh337
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Interferon-
gene dinucleotide (CA) repeat and interleukin-4 promoter region (–590C/T) polymorphisms in Japanese patients with endometriosis
1 Department of Obstetrics and Gynecology and 2 Department of Endocrinology and Metabolism, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, 3 Department of Clinical Chemistry, Kobe Pharmaceutical University, Kobe and 4 Institute of Bio-Response Informatics, Kyoto, Japan
5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan. Email: kitawaki{at}koto.kpu-m.ac.jp
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Abstract |
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BACKGROUND: Endometriosis is a multifactorial disease with possible genetic predisposition and involvement of environmental factors in its pathogenesis. Cytokines may play important roles in the pathogenesis of endometriosis. The aim of this study was to investigate whether the interferon-
gene (IFNG) CA-repeat and interleukin-4 (IL-4) promoter region (–590C/T) polymorphisms may be responsible in part for genetic susceptibility to endometriosis. METHODS: IFNG CA-repeat and IL-4 –590C/T polymorphisms were determined for 185 patients with endometriosis and 176 healthy fertile women by quantitative genescan technology and PCR-restriction fragment length polymorphism analysis, respectively. Patients with endometriosis were analysed further according to their stage of disease, the presence or absence of chocolate cysts and whether or not their disease was associated with adenomyosis and/or lyomyomata. RESULTS: The global IFNG allele frequencies in the patients with endometriosis were significantly different from those in the control women (
2=12.964, 6 df, P=0.0436). The difference was due to an increase of the a13 (114 bp) allele in patients with endometriosis (2=10.222, P=0.0088, corrected P=0.0352, odds ratio=1.48, 95% confidence interval=1.10–1.98). There were no differences in IL-4 –590C/T genotypes and allele frequencies between control women and all patients with endometriosis or between control women and each subgroup of patients with endometriosis. CONCLUSION: The results suggest that the IFNG CA-repeat polymorphism is associated with susceptibility to endometriosis in a Japanese population.
Key words:
endometriosis/gene polymorphism/interferon- gene/interleukin-4 gene/risk factor
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Introduction |
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Endometriosis is a common gynaecological disorder in women of reproductive age and is characterized by pelvic pain and infertility. Approximately 5–10% of women of reproductive age are estimated to have endometriosis. Although the exact aetiology and pathogenesis of endometriosis are unclear, environmental and genetic factors have been implicated in the disease. A role for genetic factors has been supported by various familial and twin studies (Simpson et al., 1980
; Moen and Magnus, 1993; Kennedy, 1999; Treloar et al., 1999). However, several lines of evidence also support a role for cell-mediated and humoral immunity in the pathogenesis of endometriosis and endometriosis-associated infertility (Berkkanoglu and Arici, 2003). Defects in the ability of cytotoxic T cells and natural killer (NK) cells to recognize and lyse endometrial cells are considered crucial for the pathogenesis of endometriosis (Oosterlynck et al., 1991; Vigano et al., 1991; Ho et al., 1995). NK cells recognize specifically HLA class I molecules that inhibit the killing functions of these cells. A previous study in our laboratory demonstrated that the HLA-B*0702 allele is associated with an increased risk of endometriosis (Kitawaki et al., 2002).
Interferon (IFN)-, a cytokine secreted mainly by T helper type 1 (Th1) cells and NK cells, promotes macrophage activation and Th1 differentiation, in addition to upregulation of the expression of major histocompatibility complex (MHC) class I and II molecules and adhesion molecules. It has been reported that the concentration of IFN- is decreased in peritoneal fluid in patients with endometriosis (Ho et al., 1996; Hsu et al., 1997; Wu et al., 1998), although some groups have found no difference (Khorram et al., 1993; Keenan et al., 1994). IFN- is encoded by a single gene mapped in humans to chromosome 12q24.1. A dinucleotide (CA) repeat polymorphism in the first intron of the IFN- gene (IFNG) has been reported (Ruiz-Linares, 1993). An association between the IFNG CA-repeat polymorphism and multiple immunological diseases has been suggested (Awata et al., 1994; Siegmund et al., 1998; Miyake et al., 2002).
Interleukin-4 (IL-4) is a key cytokine for the T helper type 2 (Th2) response, which induces activation and maturation of B cells as well as differentiation of immature Th cells into Th2 cells. IL-4 inhibits the differentiation and function of the Th1 phenotype. An increased level of IL-4 mRNA and protein in the peripheral blood cells and peritoneal fluid of patients with endometriosis has been reported (Hsu et al., 1997). The gene encoding IL-4 has been mapped to chromosome 5. Rosenwasser et al. (1995) found a polymorphism with a C to T exchange at position 590 upstream from the open reading frame of the IL-4 gene (–590C/T) that is associated with greater luciferase activity.
In the present study, we investigated whether the IFNG CA-repeat and IL-4 promoter region (–590C/T) polymorphisms may be responsible in part for genetic susceptibility to endometriosis in Japanese women.
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Materials and methods |
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Subjects
One hundred and eighty-five patients with endometriosis, diagnosed by both macroscopic and histological examination, and 176 healthy fertile women with no history of uterine disease were included in the study. All subjects were of reproductive age (22–46 years old) and had undergone laparotomy or laparoscopy at the Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. Prior to the operation, the patients had not received any endocrine therapy such as GnRH analogues, danazol or estrogen–progestin that may have masked the presence of disease. The patients were classified into three different categories and each category was divided into two groups: pure or complicated, with or without chocolate cysts, and mild or severe. The pure group consisted of patients who had only endometriosis but no other gynaecological disease (n=132), and the complicated group consisted of patients who had endometriosis associated with adenomyosis and/or leiomyomata (n=53). Of the 185 patients with endometriosis, 125 had chocolate cysts. The stage of endometriosis was assigned according to the revised American Society for Reproductive Medicine scoring system (American Society for Reproductive Medicine, 1997
): stages I and II were grouped as mild (n=52) while stages III and IV were grouped as severe (n=133). All subjects were Japanese, unrelated and residents of the Kyoto and Osaka areas. This study protocol was approved by the Kyoto Prefectural University of Medicine institutional review board, and informed consent was obtained from all subjects.
DNA preparation and genotyping analysis of IFNG and the IL-4 gene
Genomic DNA was extracted from peripheral blood with a DNA extractor WB kit (Wako pure Chemicals, Osaka, Japan) according to the manufacturer's instructions.
The genotype of the IFNG CA-repeat polymorphism was determined by quantitative genescan technology. A region containing the CA-repeat polymorphism within the first intron of IFNG was amplified by PCR using the primers described by Miyake et al. (2002): forward (5'-TGA TTT TAT TCT TAC AAC ACA-3') and reverse (5'-CTT CCT GTA GGG TAT TAT TAT-3'). The 5' end of the forward primer was labelled with a fluorescent dye (6-Fam). An aliquot containing 50 ng of genomic DNA was mixed with 0.5 µmol/l of each primer, 0.2 mmol/l dNTPs and 1.25 IU of Taq polymerase (Takara Premix Ex Taq; Takara Biochemicals, Shiga, Japan) in a total volume of 50 µl of PCR buffer (provided by the manufacturer). The PCR procedure was as follows: an initial denaturation step at 95°C for 6 min, and then 30 cycles of amplification at 94°C for 1 min, 62°C for 45 s and 72°C for 45 s, followed by a final extension step at 72°C for 5 min. The PCR products were denatured for 4 min at 94°C, mixed with formamide-containing stop buffer, electrophoresed on a 4% polyacrylamide gel with an internal lane standard labelled with Genescan-500 Rox dye, and analysed on an ABI 377 DNA sequencing system using Genescan Analysis software (PE Applied Biosystems, Foster City, CA).
Genotyping of the IL-4 polymorphism at position –590 in the promoter region was performed by the PCR-restriction fragment length polymorphism (PCR-RFLP) method, as previously described (Rosenwasser et al., 1995), with a slight modification. Briefly, an aliquot containing 100 ng of genomic DNA was mixed with 0.5 µmol/l of each primer (forward, 5'-TAA ACT TGG GAG AAC ATG GT-3' and reverse, 5'-TGG GGA AAG ATA GAG TAA TA-3'), 0.2 mmol/l dNTPs and 1.25 IU of Taq polymerase (Takara Premix Ex Taq) in a total volume of 50 µl of PCR buffer (provided by the manufacturer). The PCR procedure was as follows: an initial denaturation step at 94°C for 5 min, and then 36 cycles of amplification at 94°C for 1 min, 48°C for 1 min and 72°C for 1 min, followed by a final extension step at 72°C for 5 min. The PCR products (195 bp) were digested with the restriction enzyme AvaII (Takara Biochemicals). The digested products were separated by 3% agarose gel electrophoresis, and visualized by ethidium bromide staining. The IL-4 genotypes were named according to the presence or absence of the enzyme restriction sites, i.e. AvaII CC, CT and TT are homozygous for the presence of the site (175/20 bp), heterozygous (195/175/20 bp) and homozygous for absence of the site (195 bp), respectively.
Statistical analysis
The sample size required to detect a difference between two proportions for the IFNG CA-repeat and IL-4 –590C/T polymorphisms was calculated using a computer program for 80% power, 5% type I error and odds ratio (OR). The global distribution of alleles of the IFNG CA-repeat polymorphism between the control subjects and the patient groups was evaluated by 2 test with a 2x7 contingency table. The frequency of genotypes and alleles in the different groups was evaluated by 2 test with a 2x3 contingency table (for genotypes) or a 2x2 table (for alleles), and the OR and 95% confidence intervals (CIs) were calculated. The corrected P-values (Pc) were obtained by multiplying the uncorrected P-value by the number of comparisons, according to Bonferroni's method.
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Results |
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In the present study, seven different IFNG alleles were identified by size. These alleles contained a sequence ranging from 10 (108 bp) to 16 CA repeats (120 bp) and were designated a10–a16 according to the number of CA repeats. The global allele frequencies of the IFNG CA-repeat polymorphism in the healthy control women, patients with endometriosis and each clinical subgroup of patients with endometriosis are illustrated in Table I. The global IFNG allele frequencies in all patients with endometriosis was significantly different from that in the control women (
2=12.964, 6 df, P=0.0436). The difference was due to an increase in the a13 (114 bp) allele in the patients with endometriosis (2=10.222, P=0.0088, Pc=0.0352, OR=1.48, 95% CI=1.10–1.98) (Tables I and II). No significant differences in global allele frequencies were found between the control women and each clinical subgroup of patients with endometriosis. The distribution of the IFNG a13 genotypes was significantly different between patients with endometriosis and the control women (2=6.075, 2 df, P=0.048) (Table II). The difference was due to over-representation of the IFNG a13 homozygote among the patients with endometriosis (2=5.176, P=0.0229, Pc=0.0916). An increased frequency of the IFNG a13 allele was observed in each subgroup of patients with endometriosis, except for the group of patients with Re-AFS stage I+II; however, these values were not statistically significant after corrections for multiple comparisons. No differences were evident in the IFNG a13 genotype, the allele frequencies between the groups with and without adenomyosis and/or leiomyomas, the groups with and without chocolate cysts, or between the groups with Re-AFS stage I+II and Re-AFS stage III+IV (Table II).
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The distribution of the IL-4 promoter region (–590C/T) genotype and allele frequencies among control women, all patients with endometriosis and each subgroup of patients with endometriosis is shown in Table III. There were no differences in IL-4 –590C/T genotype and allele frequencies between control women and all patients with endometriosis or between control women and each subgroup of patients with endometriosis. No differences were evident in the IL-4 –590 genotype and allele frequencies between the groups with and without adenomyosis and/or leiomyomas, the groups with and without chocolate cysts, or between the groups with Re-AFS stage I+II and Re-AFS stage III+IV.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Endometriosis is increasingly recognized as a complex trait, the development of which is influenced by interactions between multiple genes and environmental factors. It has been suggested that immunological abnormalities are associated with the presence of endometriosis. However, whether immune abnormalities are a cause or a result of endometriosis has not been resolved. Our primary aim was to study the genetic association of patients with endometriosis with IFN-
, which is a multifunctional cytokine secreted mainly by Th1 and NK cells. To our knowledge, the present study is the first attempt to examine polymorphic microsatellites of IFNG in patients with endometriosis. In this case–control study, we have shown that the frequency of the a13 polymorphism in the first intron of IFNG was significantly increased in patients with endometriosis. In the endometriosis group, the IFNG a13 genotype and allele frequencies were similar, regardless of whether or not the patients experienced complications of adenomyosis and/or leiomyomata, chocolate cysts, or whether the clinical stage was mild or severe. It has been reported that IFN- production by phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) bearing the IFNG a13 allele was significantly less than by PBMCs lacking the IFNG a13 allele (Miyake et al., 2002). Decreased peritoneal NK cell activity has been proposed as an aetiological factor that could contribute to the implantation, survival and proliferation of regurgitated endometrial cells in the peritoneal cavity, although the mechanisms that suppress NK activity are unclear. IFN- production will be low if Th1 and/or NK cells are inactivated. It has been reported that IFN- inhibits proliferation of human endometrial epithelium (Tabibzadeh et al., 1988). Therefore, our findings suggest that the IFNG CA-repeat polymorphism is associated, in part, with the development and growth of endometriosis. Further studies with a larger number of patients and different ethnic groups are required for further evaluation and confirmation.
Increased IL-4 production in peritoneal fluid in patients with endometriosis and normalization after hormone therapy has been reported (Hsu et al., 1997; Szyllo et al., 2003). Therefore, we also investigated whether the IL-4 –590C/T polymorphism may be responsible in part for genetic susceptibility to endometriosis. We did not find a significant association between the IL-4 –590C/T polymorphism and endometriosis. A similar result has been reported in Taiwan Chinese women (Hsieh et al., 2002).
In conclusion, we have demonstrated that the frequency of the IFNG a13 allele is significantly higher in patients with endometriosis than in controls. The data therefore suggest that the IFNG CA-repeat polymorphism may be a possible genetic susceptibility factor for endometriosis. Although the association should be verified in various ethnic populations, our findings may provide an important clue for elucidating the pathogenesis of the disease.
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Submitted on February 2, 2004; accepted on May 5, 2004.
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