1 Department of Obstetrics and Gynecology and 2 The First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 and 3 Department of Clinical Research, Kyoto Microbiological Institute, Kyoto 607-8482, Japan
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Abstract |
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Key words: gene/CYP19 gene/endometriosis/insertion-deletion polymorphism/restriction fragment length polymorphism
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Introduction |
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Endometriosis grows and regresses in an estrogen-dependent manner. Indeed, ectopic endometriotic implants contain estrogen, progesterone and androgen receptors (Lessey et al., 1989; Prentice et al., 1992
; Bergqvist and Fernö, 1993
) as well as aromatase, an enzyme that catalyses the conversion of androgens to estrogens. Local estrogen production in conjunction with circulating estrogen stimulates the growth, which is mediated by the estrogen receptor (Kitawaki et al., 1997
). Adenomyosis and leiomyomas are separate entities from endometriosis. They share a common pathophysiology in that their growth is estrogen-dependent with the expression of both estrogen receptors and aromatase (Kitawaki et al., 1997
), and they have a complicated pattern of occurrence. An anonymous intronic polymorphism of the estrogen receptor
gene assessed by PvuII restriction fragment length polymorphism (RFLP) is associated with endometriosis as well as adenomyosis and leiomyomas (Georgiou et al., 1999
; Kitawaki et al., 2001
).
The CYP17 gene, which is located on chromosome 10 and contains eight exons, encodes the cytochrome P450c17 enzyme (Picado-Leonard and Miller, 1987
). This enzyme mediates both steroid 17
-hydroxylase and 17,20-lyase activities and plays a key role in androgen biosynthesis. The 5' untranslated region of CYP17 contains a single nucleotide polymorphism that creates an SP-1 type (CCACC box) promoter site 34 bp upstream from an initiation of translation and 27 bp downstream from the transcription start site (Carey et al., 1994
). This polymorphism can be assessed by MspA1 RFLP, giving two alleles; A1 that does not contain the restriction site and A2 that contains the restriction site. Serum levels of androgens and estrogens have been shown to be elevated in women who carry the A2 allele (Feigelson et al., 1998
; Haiman et al., 1999
). These findings suggest that the A2 allele may have greater promoter activity, resulting in an increased transcription of the P450c17
enzyme. This may lead to an increased production of precursor androgens and to subsequent conversion of estrogens.
The CYP19 gene located on chromosome 15q21.2 (Chen et al., 1988) contains 10 exons and encodes aromatase cytochrome P450, the major component of the enzyme aromatase (Mendelson et al., 1990
). Multiple untranslated exons I control the tissue-specific gene expression (Harada et al., 1993
; Mahendroo et al., 1993
). Endometriotic implants use both the adipose-type promoter I.4 and gonadal-type promoter II for P450arom expression (Noble et al., 1996
). A tetranucleotide (TTTA) simple tandem repeat polymorphism, which is located in intron 4 of the CYP19 gene ~80 nucleotides downstream from exon 4 (Polymeropoulos et al., 1991
), has been reported to be associated with the risk of breast cancer, which is known to grow in an estrogen-dependent manner in Caucasian (Kristensen et al., 1998
; Siegelmann-Danieli and Buetow, 1999
; Haiman et al., 2000
) and Japanese women (Miyoshi et al., 2000
). Another polymorphism, a 3 bp insertion (I)/deletion (D) polymorphism, is located at 50 bp upstream of the TTTA repeat region of the CYP19 gene (Kurosaki et al., 1997
). However, several studies have failed to show the association between the 3 bp I/D polymorphism and the risk of breast cancer (Probst-Hensch et al., 1999
; Healey et al., 2000
; Young et al., 2000
).
The purpose of the present study was to investigate whether the MspA1 polymorphism of the CYP17 gene, the TTTA repeat polymorphism and the 3 bp I/D polymorphism of the CYP19 gene are related to estrogen-dependent benign uterine diseases such as endometriosis, adenomyosis and leiomyomas.
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Materials and methods |
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The patients were classified into two groups defined as the endometriosis (n = 140) or adenomyosis/leiomyomas group (n = 67) (Kitawaki et al., 1997, 1999
, 2001
). The mean (± SD) ages were 36.3 ± 8.1 and 44.4 ± 7.7 years, and the mean body mass index (BMI) was 20.9 ± 3.2 and 22.4 ± 3.2 kg/m2 respectively. The endometriosis group was subclassified into two different categories: with or without chocolate cysts, and light or severe. The stage of endometriosis was assigned according to the revised American Fertility Society scoring system (American Fertility Society, 1985
); stages I and II were classified as light while stages III and IV were classified as severe. The group of adenomyosis/leiomyomas consisted of patients with adenomyosis and/or leiomyomas but without endometriosis. Controls consisted of 177 Japanese healthy fertile women with no history of gynaecological disease who had undergone an annual health examination. The mean age (63.8 ± 6.1 years) was higher than the groups with endometriosis (P < 0.001) and adenomyosis/leiomyomas (P < 0.001), but the mean BMI (22.3 ± 3.2 kg/m2) was not different from these two groups.
Genotyping analysis of the CYP17 gene and the CYP19 gene
Peripheral blood was drawn from each patient and collected in an EDTA-containing tube. Genomic DNA was extracted from peripheral blood with DNA extractor WB kit (Wako Pure Chemical, Osaka, Japan) (Obayashi et al., 1999) according to the manufacturer's instructions.
Genotyping of the MspA1 polymorphism in the 5' untranslated region of the CYP17 gene was determined by PCRRFLP analysis, as previously described (Carey et al., 1994) with slight modifications. Briefly, an aliquot of 100 ng DNA was mixed with 1.0 µmol/l each of the primers (forward, 5'-CATTCGCACTCTGGAGTC-3'; reverse, 5'-AGGCTCTTGGGGTACTTG-3'), and 1.25 units Taq polymerase (Takara Premix Taq; Takara Biochemicals, Tokyo, Japan) with 0.4 µmol/l dNTPs 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 5 min, and then amplified for 30 cycles at 94°C for 1 min, 60°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 were digested with restriction enzyme MspA1 (Promega Corp., Madison, WI, USA), separated by 3% agarose gel electrophoresis and identified with ethidium bromide staining.
For the detection of the tetranucleotide (TTTA) repeat polymorphism in the intron 4 of the CYP19 gene including the 3 bp I/D polymorphism region at the 50 bp upstream of the (TTTA)n tract, a set of the primers as previously described (Kurosaki et al., 1997) (forward, 5'-GCAGGTACTTAGTTAGCTAC-3'; reverse, 5'-TTACAGTGAGCCAAGGTCGT-3') was used. The forward primer was 5'-labelled with a fluorescent dye (FAM, 5-carboxy-fluorescein) for automated fragment analysis. An aliquot of 50 ng of DNA was mixed with 1.25 µmol/l each of the primers, 1.25 U Taq polymerase (Takara Biochemicals) with 0.4 µmol/l dNTPs. The PCR amplification was carried out for 30 cycles (denaturation at 94°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 1 min). In order to determine the absolute allele sizes in bp, amplified products were subjected to rapid fragment detection using the ABI Prism 377 DNA sequencing system (PE Applied Biosystems, Foster City, CA, USA) and Genescan Analysis software (PE Applied Biosystems) with a 4% polyacrylamide gel. Fragment sizes were determined by comparison with internal lane size standards, Genescan-500 Rox dye (PE Applied Biosystems). Aliquots of the DNA products were sequenced by the dye terminator method using a model 100 DNA analyser (PE Applied Biosystems) and the sequences were confirmed to be equal to previously published descriptions (Kurosaki et al., 1997
).
Statistics
The sample size required to detect a difference between two proportions for CYP19 I/D polymorphism was calculated using the computer program JavaStat, based on fixed 80% power, 5% type I error and odds ratio (OR). Differences in the items of baseline characteristics of patients were analysed with one factor analysis of varience and multiple comparisons were performed using Scheffé's post-hoc procedure. The distribution of the CYP17 genotypes and allele frequencies were evaluated by 2-test with 2x3 table (for genotypes) or 2x2 table (for alleles). The distribution of the CYP19 genotypes and allele frequencies were evaluated by
2-test or Fisher's exact test for small sample sizes with 2x5 table [for genotypes, rare alleles (TTTA)13, (TTTA)10, (TTTA)9 and (TTTA)8 were combined] or 2x2 table (for alleles of 3 bp I/D polymorphism of the CYP19 gene). The corrected P-values (Pc) were obtained by multiplying the uncorrected P-values (Pu) with the number of comparisons, according to Bonferroni's method. A Pc < 0.05 was considered significant. The OR with 95% confidence interval (CI) was calculated using logistic regression models.
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Results |
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Discussion |
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The CYP19 gene, encoding aromatase, plays the most direct role in estrogen production. In this study we examined two sites of polymorphisms in intron 4 of the CYP19 gene: the TTTA repeat and 3 bp I/D polymorphisms. We found no significant difference between the TTTA repeat polymorphism and estrogen-dependent benign uterine disease including endometriosis, adenomyosis or leiomyomas. In breast cancer, relatively longer alleles, such as (TTTA)10, (TTTA)11 and (TTTA)12, were reported to be associated with a risk of breast cancer (Kristensen et al., 1998; Haiman et al., 2000
; Miyoshi et al., 2000
), while several studies failed to show an association with the TTTA repeat polymorphism and a risk of breast cancer (Probst-Hensch et al., 1999
; Healey et al., 2000
; Young et al., 2000
).
On the other hand, our results showed a weak but significant association between the 3 bp I/D polymorphism and endometriosis. The patients carrying the D/D genotype were more frequent in the endometriosis group than those in the control group. This evidence was confirmed in the endometriosis subgroups with chocolate cysts and at severe stages. This strengthens the association, as according to the revised American Fertility Society scoring system (American Fertility Society, 1985) many cases associated with chocolate cysts are scored high, which leads to classification as the severe stage. In contrast, we found no significant association between the group with adenomyosis/leiomyomas and controls. In the present study and in others (Kurosaki et al., 1997
; Healey et al., 2000
; Miyoshi et al., 2000
; Young et al., 2000
), the D allele has been found only in the (TTTA)7 allele, except in three of 349 (0.86%) cases found in the (TTTA)8 allele in the AfricanAmerican population (Probst-Hensch et al., 1999
). Several reports failed to show the association between the 3 bp I/D polymorphism and a risk of breast cancer (Probst-Hensch et al., 1999
; Healey et al., 2000
; Young et al., 2000
). An increased frequency of the (TTTA)7 allele with 3 bp I in patients with breast cancer compared with controls has been shown (Siegelmann-Danieli and Buetow, 1999
). However, the function of these variants has not been established. One may speculate that either variant has an elevated aromatase activity that is localized in breast cancer and endometriosis. Unexpectedly, the present results showing increased D/D genotype in endometriosis are completely opposed to the finding showing increased (TTTA)7 allele with 3 bp I in breast cancer (Siegelmann-Danieli and Buetow, 1999
). Although endometriotic implants express aromatase, which is not expressed in the normal endometrium or myometrium (Kitawaki et al., 1999
), we speculate that the onset or growth of endometriosis may be associated with an aberrant transcription of CYP19. For this reason, among PvuII genotypes of the estrogen receptor
gene, the PP genotype has higher bone mineral density than the Pp and pp genotypes, suggesting more potent local estrogen action in those with the PP genotype (Kitawaki et al., 2001
). In contrast, the PP genotype was decreased in estrogen-dependent benign uterine disease, suggesting that the aberrant expression of estrogen receptors may be involved in the disease (Kitawaki et al., 2001
). Altered estrogen metabolism or action in the local environment may contribute to estrogen-dependent disease. Our results suggest that the local expression of aromatase in the endometriotic lesion contributes to some extent to the growth of the disease.
Although age-matched, disease-free women would have made an ideal control group, we had only a small number of such patients, because surgery is required to establish definitively the disease-free state. This small number would have resulted in less statistically significant data. To increase numbers in the control group, we enrolled healthy fertile women with no history of gynaecological disease. Although the group might include <5% with asymptomatic estrogen-dependent benign uterine disease, there was no statistical difference in the distribution of genotypes examined. In addition, although the group consisted mostly of post-menopausal women, such differences might not be related to genetic background. Moreover, we have combined adenomyosis and leiomyomas into one group because of the comparative number of cases that combined both diseases, and the difficulty in being able to strictly discriminate between the two. A minor possibility that might have caused selection biases was that the present study included only cases that had undergone surgery, but not those at early stages without symptoms or those who were not infertile.
In conclusion, our current study in a Japanese population suggests that the 3 bp I/D polymorphism of the CYP19 gene is weakly associated with the risk of endometriosis. However, there is no association between the MspA1 polymorphism of the CYP17 gene or the TTTA repeat polymorphism of the CYP19 gene and the risk of endometriosis, adenomyosis or leiomyomas. The function of the polymorphisms needs to be determined in order to provide a better understanding of the pathogenesis and pathophysiology of endometriosis.
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Notes |
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References |
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Submitted on July 9, 2001; resubmitted on September 13, 2001; accepted on December 11, 2001.