1 Department of Obstetrics and Gynecology and 2 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: adenomyosis/endometriosis/leiomyomata/oestrogen receptor-alpha gene/restriction fragment length polymorphism
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Introduction |
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Recent studies have postulated that ER gene polymorphisms may influence its action as a modulator of the ligand oestrogens. Oestrogens are known to be important for the preservation of bone mass in females during menopause. Several studies have shown that among ER genotypes assessed by PvuII restriction fragment length polymorphism (RFLP), the PP genotype has higher bone mineral density than the Pp and pp genotypes (Kobayashi et al., 1996
; Ongphiphadhanakul et al., 1998
; Willing et al., 1998
; Kurabayashi et al., 1999
), and that in adolescent boys PP genotype has a greater body height than the others (Lorentzon et al., 1999
). These findings may suggest that the local oestrogenic action is more potent in those with PP genotype than in those with the Pp or pp genotypes. This is also supported by the presence of an association between ER
gene polymorphisms and oestrogen-dependent disease, including endometriosis (Georgiou et al., 1999
), and the risk of pre-menopausal hysterectomy and onset of natural menopause (Weel et al., 1999
).
The purpose of the present study was to investigate whether the polymorphism in the ER gene was related to oestrogen-dependent benign uterine disease such as endometriosis, adenomyosis and leiomyomata.
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Materials and methods |
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Genomic DNA analysis
Peripheral blood was drawn from each patient and collected in a tube with EDTA added. Genomic DNA was extracted from peripheral blood with DNA extractor WB kit (Wako pure chemical, Osaka, Japan) according to the manufacturer's instructions (Obayashi et al., 1999). Genotyping of the PvuII polymorphism in intron 1, 0.4 kb upstream of exon 2 of the ER
gene was determined by polymerase chain reaction (PCR)-RFLP analysis, essentially as previously described (Yaich et al., 1992
). Briefly, an aliquot of 100 ng DNA was mixed with 0.5 µmol/l each of the primers (forward, 5'-CTGCCACCCTATCTGTATCTTTTCCTATTCTCC-3'; and reverse, 5'-TCTTTCTCTGCCACCCTGGCGTCGATTATCTGA-3'), 0.2 mmol/l dNTPs, and 1.25 units Taq polymerase (Takara Premix Ex Taq; Takara Biochemicals, Tokyo, 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 5 min, and then amplified for 30 cycles at 94°C for 1 min, at 62°C for 1 min, and at 72°C for 1 min, followed by a final extension step at 72°C for 6 min. The PCR products were digested with restriction enzyme PvuII (Takara Biochemicals), separated by 4% agarose gel electrophoresis, and identified by ethidium bromide staining. Genotypes were defined as PP, Pp, or pp. Upper-case letters represent the absence of, and lower-case letters represent the presence of, restriction sites.
Statistics
Differences in the items of baseline characteristics of patients were analysed with one-factor analysis of variance and multiple comparisons were performed using Scheffé's procedure. The distributions of the ER genotypes and allele frequencies were evaluated by
2-test with 2x3 table (for genotypes) or 2x2 table (for alleles). The odds ratio (OR) and 95% confidence interval (CI) were calculated using logistic regression models. P < 0.05 was considered significant.
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Results |
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Discussion |
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Similar results were reported (Georgiou et al., 1999) in 57 Greek patients with endometriosis, who had a significantly lower frequency of PP genotype in the ER
gene compared with that in the control group. In contrast, a number of studies (Hill et al., 1989
; Parl et al., 1989
; Yaich et al., 1992
; Andersen et al., 1994
; Southey et al., 1998
) failed to show an association between polymorphisms in the ER
gene and breast cancer.
It is unclear how the anonymous intronic polymorphism of the ER gene influences its protein function. However, recent studies have postulated that ER
gene polymorphisms may influence its action as a modulator of the ligand oestrogens. Oestrogens are known to be important for preservation of bone mass in females during menopause. Several studies have shown that the PP genotype has higher bone mineral density than the Pp and pp genotypes (Kobayashi et al., 1996
; Ongphiphadhanakul et al., 1998
; Willing et al., 1998
; Kurabayashi et al., 1999
), while studies of Korean (Han et al., 1999
) and Caucasian (Vandevyver et al., 1999
) women have observed no association between ER
gene polymorphisms and bone mineral density. The PP genotype has a greater body height in adolescent boys (Lorentzon et al., 1999
), whereas bone density was not reduced in patients with endometriosis (Lane et al., 1991
; Ulrich et al., 1998
). These findings suggest that the local oestrogenic action is more potent in women carrying the P allele than those carrying the p allele. This is also supported by the finding that the PP genotype has a higher risk of premenopausal hysterectomy and earlier onset of natural menopause due to menorrhagia and fibroids than the Pp and pp genotypes (Weel et al., 1999
). These findings, however, contradict those of the present and the Greek studies (Georgiou et al., 1999
). The present study shows that women carrying the PP genotype have a lower risk for oestrogen-dependent uterine disease. Indeed, endometriotic implants and adenomyotic tissues express a reduced amount of ER protein (Prentice et al., 1992
; Bergqvist and Ferno, 1993
; Bergqvist et al., 1993
; Nisolle et al., 1994
) and lose the cyclic change of ER expression (Prentice et al., 1992
). This suggests that aberrant expression of ER may be partly involved in the onset or growth of oestrogen-dependent benign uterine disease. Similarly, women carrying the PP genotype show poorer response to ovarian stimulation (Georgiou et al., 1997
; Sundarrajan et al., 1999
).
Previously the distinction between normal or disease-free cases and those with endometriosis, adenomyosis and leiomyomata had not been clearly defined. In our laboratory (Kitawaki et al., 1997, 1999
), we demonstrated that the mRNA and protein of aromatase cytochrome P450, responsible for oestrogen biosynthesis, are expressed in the eutopic endometria of patients with endometriosis, adenomyosis, and/or leiomyomata, whereas neither are expressed in endometrial specimens obtained from normally menstruating women with cervical carcinoma in situ but no other gynaecological disease. Similarly, we demonstrated that the enzyme activity of 17ß-hydroxysteroid dehydrogenase type 2, which inactivates oestradiol to oestrone, is induced during the secretory phase in the endometrium of patients with endometriosis, adenomyosis, and/or leiomyomata, whereas the induction does not occur in disease-free endometrium (Kitawaki et al., 2000
). These findings indicate that, although the endometria of patients with endometriosis, adenomyosis, or leiomyomata resemble those of women without gynaecological disease, the oestrogen metabolism of these tissues is remarkably different. We carried out the present study using these criteria, strictly distinguishing disease-free cases from disease cases by abdominal examination and histopathology. This grouping is important for analysing studies relating to oestrogen and the disease-free group is an ideal control. However, we could only collect a small number of disease-free patients, because establishing a disease-free state was difficult in women undergoing surgery. This would have resulted in less statistically significant data. Alternatively, we enrolled the reference population, which consisted of a common female population in Japan. This group might include some with oestrogen-dependent uterine disease. Although the group consisted mostly of postmenopausal women with different profiles in physical constitution, such difference might not be related to genetic background. Indeed, the distribution of the ER
gene genotypes was compatible to that in Caucasians (Willing et al., 1998
; Weel et al., 1999
) and Japanese (Kobayashi et al., 1996
; Kurabayashi et al., 1999
). Furthermore, the distribution of the genotypes in the reference population was in-between those of the disease groups comprising the less frequent PP genotype and the disease-free group comprising the less frequent pp genotype. This supports the finding that the distribution of the ER
gene genotypes is different between the disease and disease-free groups.
The present study included cases who had undergone surgery but not those at early stages without symptoms or those who were not infertile. The higher mean age in the adenomyosis/leiomyomata group compared to that in the disease-free and endometriosis groups also adds to the problem. However, in the endometriosis group, the distribution of the ER gene genotypes was not related to complications of other diseases or to the clinical stage. We combined adenomyosis and leiomyomata into one group, because of the comparative numbers of cases that complicated both diseases, and the difficulty of being able to discriminate between the two diseases. Similarly, the disease-free group was not necessarily strictly `normal', but was associated with cervical carcinoma in situ or infertility. We have used this criterion previously and showed the lack of aromatase expression (Kitawaki et al., 1997
, 1999
) and the lack of 17ß-hydroxysteroid dehydrogenase type 2 induction (Kitawaki et al., 2000
) in the disease-free endometrium. We therefore conclude that the present study does not contain serious selection biases.
In conclusion, the PvuII polymorphism of the ER gene is associated with the risk for endometriosis, adenomyosis, and leiomyomata. The mechanism by which the anonymous intronic polymorphism affects protein function needs to be clarified. This will provide better understanding of the pathogenesis and pathophysiology of oestrogen-dependent uterine diseases.
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Notes |
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References |
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Submitted on June 23, 2000; accepted on September 29, 2000.