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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Key words:
endometriosis/gene polymorphism/interferon- gene/interleukin-4 gene/risk factor
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Introduction |
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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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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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Discussion |
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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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References |
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Submitted on February 2, 2004; accepted on May 5, 2004.
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