Association between endometriosis and genetic polymorphisms of the estradiol-synthesizing enzyme genes HSD17B1 and CYP19

M. Tsuchiya1,2, H. Nakao1, T. Katoh1,6, H. Sasaki3, M. Hiroshima3, T. Tanaka3, T. Matsunaga4, T. Hanaoka5, S. Tsugane5 and T. Ikenoue2

Departments of 1 Public Health and 2 Obstetrics and Gynecology, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, 3 Department of Obstetrics and Gynecology, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 4 Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo and 5 Epidemiology and Prevention Division, Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo, Japan

6 To whom correspondence should be addressed. Email: katoht{at}med.miyazaki-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Endometriosis, an estrogen-dependent disease, is believed to be influenced by multiple genetic and environmental factors. Here, we evaluated whether the risk and severity of endometriosis are associated with polymorphisms in estradiol-synthesizing enzyme genes: the Ser312Gly polymorphism in 17-beta-hydroxysteroid dehydrogenase type 1 (HSD17B1) and the Arg264Cys polymorphism in cytochrome P450, subfamily XIX (CYP19). METHODS: All participants underwent diagnostic laparoscopy, and the stage of endometriosis was determined according to the Revised American Fertility Society classification. Of the 138 women enrolled, 59 had no endometriosis, 21 had stage I, 10 had stage II, 23 had stage III and 25 had stage IV. SNPs were discriminated by allele-specific oligonucleotide hybridization. RESULTS: Individuals having at least one A-allele (A/G or A/A genotype) of HSD17B1 showed a significantly increased risk of endometriosis (A/G genotype: adjusted OR, 3.06; 95%CI 1.21–7.74; A/A genotype: adjusted OR, 3.02; 95%CI 1.08–8.43). There was a significant trend associating A/G + A/A genotypes with severity of endometriosis (P for trend <0.01). No statistically significant association was found for the CYP19 polymorphism. CONCLUSIONS: Evidence for association between the Ser312Gly polymorphism in HSD17B1 and endometriosis was found in a Japanese population. The A-allele of HSD17B1 appears to confer higher risk for endometriosis.

Key words: CYP19/endometriosis/estrogen synthesis/genetic polymorphism/HSD17B1


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Endometriosis, one of the most common causes of female infertility and chronic pelvic pain, is defined as the presence of endometrial tissue outside the uterus. Three predominant theories have been proposed for the etiology of this disease: Mullerian remnants, metaplasia and direct implantation of endometrial cells (El-Mahgoub and Yaseen, 1980Go; Murphy et al., 1986Go; Fujii et al., 1991Go). Although the exact prevalence is still not known, endometriosis affects up to 5–10% of women of reproductive age (Wheeler et al., 1989Go). The prevalence of endometriosis is as high as 20–50% in infertile women (Strathy et al., 1982Go; Rawson et al., 1991Go).

Endometriosis is regarded as a complex trait, in which genetic and environmental factors contribute to the disease phenotype (Kennedy et al., 1998Go). A variety of factors affect the development of endometriosis, including hormonal status and genetic factors. For example, women with shorter intervals between menstruation and longer duration of menses are at higher risk for endometriosis (Vercellini et al., 1997Go). The risk of endometriosis is seven times higher if a first-degree relative has been affected by endometriosis (Simpson et al., 1980Go). However, the interaction between genetic susceptibility and environmental factors is not yet adequately understood.

The development of endometriosis is estrogen-dependent. Endometrial implants contain estrogen and progesterone receptors (Lessey et al., 1989Go) and respond to ovarian hormonal changes, causing local bleeding, inflammation and formation of adhesions. The three main estrogens are estradiol, estrone and estriol. Estradiol, the most active form, is produced either from estrone via 17-{beta}-hydroxysteroid dehydrogenase type 1 (HSD17B1) or from testosterone via cytochrome P450, subfamily XIX (CYP19, aromatase) (Mitrunen and Hirvonen, 2003Go).

Current evidence indicates that polymorphisms in genes of drug-metabolizing enzymes can affect phenotypic metabolic variations. The HSD17B1 gene is located in chromosome 17q12 and has a polymorphism consisting of an A to G substitution in exon 6, resulting in an amino acid change of Ser312Gly (Puranen et al., 1994Go). The CYP19 gene, located in chromosome 15q21, has a polymorphism consisting of C to T substitution in exon 7, resulting in an amino acid change of Arg264Cys (Toda et al., 1990Go).

Genetic polymorphisms involved in estrogen synthesis and metabolism may play an important role in the variation of endometriosis among individuals by altering local estrogen production or circulating levels of estrogen. Here, we evaluate whether the Ser312Gly polymorphism in HSD17B1 and Arg264Cys polymorphism in CYP19 are associated with the risk and severity of endometriosis. A case-control study was conducted on these two polymorphisms in patients with different stages of endometriosis and controls.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The protocol for the study was approved by the Institutional Review Board of University of Miyazaki, The Jikei University School of Medicine and National Cancer Center. All subjects gave their written informed consent before the laparoscopic examination.

Study participants
This study was a part of a case–control study of endometriosis. During the years 1999–2000, 139 women were recruited at the Department of Obstetrics and Gynecology, The Jikei University School of Medicine Hospital. Participants were patients between the ages of 20 and 45 who had complained of infertility and attended the hospital. The study protocol excluded all women from the study who had ever given birth or lactated. Of the 139 women recruited, only one was excluded from subsequent analysis because a DNA sample was not available.

All participants underwent diagnostic laparoscopy, and the stage of endometriosis was determined according to the Revised American Fertility Society classification (r-AFS) (American Fertility Society, 1985Go). Of the 138 women enrolled, 59 women had no endometriosis, 21 had stage I, 10 had stage II, 23 had stage III and 25 had stage IV.

Cases and controls were similar in several confounding factors. Risk factors for endometriosis include age, shorter menstrual cycles and longer duration of menstrual flow (Vessey et al., 1993Go; Eskenazi and Warner, 1997Go). The mean age of the cases was 32.4 years and 33.1 in the controls (P=0.35). No significant difference was observed in the duration of menstruation. However, there was a significant trend towards cases having shorter menstrual cycles compared to the controls (28.8 days for cases and 30.7 for controls, P=0.03).

Genotyping
Blood samples were obtained before the laparoscopic examination. Genomic DNA samples were extracted from peripheral white blood cells by using a DNA Extractor WB Kit (Wako, Osaka, Japan).

A 67 bp fragment in HSD17B1, including an SNP site located at 27 bases from the 5' end, was amplified using sense (5'-CTGGGGCAGAGGACGAGG) and biotin-labeled antisense (5'-GCGGCCGGAGGATCG) primers. A 56 bp fragment of CYP19, including an SNP site located at 31 bases from the 5' end, was amplified using biotin-labeled sense (5'-GCCATAGAAGTTCTGATAGCAG) and antisense (5'-AGTTTCTCTTCTGTGGAAATCCT) primers. PCR amplifications were performed using a TPC-200 thermal cycler (MJ Research Inc., Watertown, MA) in a total reaction volume of 25 µl containing 20 ng of DNA sample, 0.6 U AmpliTaq DNA polymerase (Applied Biosystems), 0.25 mM dNTPs, 0.2 µM primers and PCR buffer [1 x GC buffer II (Takara Bio Inc., Otsu, Japan) for HSD17B1 and 1 x GeneAmp PCR buffer (Applied Biosystems) for CYP19]. The amplification protocol comprised initial denaturation at 95 °C for 5 min, then 35 cycles of denaturation at 95 °C for 15 s and annealing at 55 °C for 30 s.

SNP discriminations were conducted in a manner similar to that described previously (Maruyama et al., 2004Go) with details as follows, based on allele-specific oligonucleotide hybridization using bio-nano magnetite particles (Takeyama et al., 2000Go; Matsunaga et al., 2001Go). Cy3- and Cy5-labeled detection probes were designed for each SNP as follows: Cy3-labeled HSD17B1 A-allele detection probe (5'-CCGGGCGCAGTGCGGTG), Cy5-labeled HSD17B1 G-allele detection probe (5'-CCGGGCGCGGTGCGGTG), Cy3-labeled CYP19 T-allele detection probe (5'-AATCCTGCATCTTTTTT) and Cy5-labeled CYP19 C-allele detection probe (5'-AAATCCTGCGTCTTTTTT). All the following experiments were performed using the semi-automated SNP detection processor (Tanaka et al., 2003Go).

Biotinylated PCR product (12.5 µl) and streptavidin-immobilized bio-nano magnetic particles (25 µg/12.5 µl), which were prepared according to the method described by Yoshino et al. (2002), were mixed and incubated for 15 min at room temperature for capturing the PCR products on the magnetic particles. PCR products were denatured into single strands by alkali treatment (10 mM NaOH). After neutralization of the mixture by neutralization buffer (100 mM Tris–HCl pH 7.5, 3 mM EDTA, 0.1% BSA), 25 µl of hybridization buffer (1 M tetramethylammonium chloride, 37.5 mM Tris–HCl pH7.5 and 3 mM EDTA) containing 12.5 pmoles of Cy3-labeled and Cy5-labeled detection probes was added to the PCR products captured on magnetic particles.

The mixture was rapidly heated up to 70 °C, and then cooled slowly to 25 °C over 10 min to allow hybridization of the detection probes and biotinylated PCR products. The optimum temperature for dissociating single-base mismatched probes was determined by analysis of dissociation curves. The mixture was heated to the optimum temperature: 56 °C for HSD17B1 or 54 °C for CYP19, and the detection probes dissociated were removed at this temperature using an automated SNP detection processor (Maruyama et al., 2004Go). Finally, the mixture was heated to 80 °C to liberate the hybridized detection probe into the supernatant.

The fluorescence intensity of the liberated detection probe was measured at Ex: 540 nm, Em: 570 nm for Cy3 and Ex: 645 nm, Em: 675 nm for Cy5 by a microplate reader (BMG Labtech, Offenburg, Germany), respectively. The samples were classified into three distinct categories according to the signal ratio of Cy5/Cy3: (i) those with values >2, representing samples with the homozygous G/G genotype in HSD17B1 or the homozygous C/C genotype in CYP19; (ii) values <0.5, representing samples with the homozygous A/A genotype in HSD17B1 or the homozygous T/T genotype in CYP19; (iii) intermediate values, representing samples with the heterozygous genotype.

Statistical analysis
To estimate the risk of endometriosis, crude odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for subgroups with the different genotypes with respect to the Ser312Gly polymorphism in HSD17B1 and the Arg264Cys polymorphism in CYP19. ORs were adjusted for three endometriosis risk factors: age, menstrual cycle and duration of menstruation, using multiple logistic regression analysis by SPSS for Windows software (version 11.0.1J, SPSS Japan, Tokyo, Japan).

The Wilcoxon rank-sum test for trend was also used to estimate the associations between the two polymorphisms and stage of endometriosis (Goodman et al., 1954Go). All statistical tests were based on two-tailed probability. P<0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Of the 138 DNA samples, 10 could not be reliably genotyped and were excluded from subsequent statistical analysis (six subjects for HSD17B1, four subjects for CYP19). The observed genotype distributions were in Hardy–Weinberg equilibrium in the control subjects.

Test for association with risk of endometriosis
Table I shows the genotypic and allelic distributions of HSD17B1 and CYP19 polymorphisms. Individuals with at least one HSD17B1 A-allele (A/G or A/A genotype) showed a significantly increased risk of endometriosis (A/G genotype: adjusted OR, 3.06; 95%CI 1.21–7.74; A/A genotype: adjusted OR, 3.02; 95%CI 1.08–8.43). A similar result was obtained in comparison of the combined A/A + A/G genotypes with the G/G genotype (adjusted OR, 3.05; 95%CI 1.30–7.14). No significant association was observed between the Arg264Cys polymorphism in CYP19 and risk of endometriosis (C/T genotype: adjusted OR, 1.35; 95%CI 0.62–2.95; T/T genotype: adjusted OR, 0.41; 95%CI 0.09–1.85).


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Table I. Genotypic and allelic distribution of HSD17B1 and CYP19 polymorphisms

 
Test for association with severity of endometriosis
To evaluate whether the Ser312Gly polymorphism in HSD17B1 and the Arg264Cys polymorphism in CYP19 are associated with the severity of endometriosis, participants were categorized into three groups according to r-AFS classification: controls, stage I–II and stage III–IV (Table II). There was a significant trend between the combined A/G + A/A genotypes and stage of endometriosis (P for trend <0.01). No statistically significant association was found between the Arg264Cys polymorphism in CYP19 and stage of endometriosis (data not shown).


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Table II. Severity of endometriosis associated with HSD17B1 polymorphism

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, we evaluated whether two polymorphisms in estradiol-synthesizing enzyme genes are associated with the risk and severity of endometriosis. The results of this study suggested that the Ser312Gly polymorphism in HSD17B1 is associated with both risk and severity of endometriosis, while no associations were found for the Arg264Cys polymorphism in CYP19.

In this study, we applied strict clinical criteria for the definition of cases and controls. One third of women with endometriosis are asymptomatic, and laparoscopy or laparotomy is indispensable for the diagnosis of endometriosis. All participants in the present study underwent diagnostic laparoscopy and were diagnosed according to the r-AFS classification. In addition, bias from confounding variables was minimized by adjusting ORs for the risk and stage of endometriosis for variables known to affect endometriosis: age, menstrual cycle and duration of menstrual flow. Detailed questionnaires were designed to determine patients' menstrual cycle and duration of menstruation. The questionnaires were administered by a trained interviewer before the laparoscopic examination to minimize recall bias.

Retrograde menstruation is thought to be one of the main causes of endometriosis. However, retrograde menstruation is a common phenomenon (Kruitwagen et al., 1991Go), and not all the women of reproductive age are affected by endometriosis. In short, women with endometriosis can be considered to have defects in the regulation of endometrial proliferation: (i) a tendency to proliferate endometrial tissue, and (ii) impaired clearance of abnormal endometrial tissue (Vinatier et al., 2001Go).

Development of endometriosis is estrogen-dependent, and several features of this disease can be explained on the basis of overproduction of estrogen. Current therapy consists of hormone treatments aimed at lowering circulating estrogen. Genetic polymorphisms in the estrogen-synthesis or estrogen-metabolizing enzymes may cause inter-individual variation of levels and activity of circulating estrogen.

Estradiol, the most physiologically active form of estrogen, stimulates the proliferation of the endometrium during the ovulatory phase of the menstrual cycle. The HSD17B1 genotypes found to be associated with endometriosis may confer increased activity or expression of HSD17B1 enzyme, causing increased exposure to estradiol. Our results suggest that possessing at least one A-allele of the Ser312Gly polymorphism in HSD17B1 increases the risk of endometriosis. Furthermore, AG + AA genotypes showed a significantly increased risk for severe endometriosis (P for trend <0.01). These findings support the idea that the Ser312Gly polymorphism in HSD17B1 is associated with the risk and progression of endometriosis, especially in terms of the inter-individual variation of estradiol synthesis.

Breast and endometrial cancer, like endometriosis, are considered to be estrogen-dependent diseases. The results of this study are consistent with those of a previous study that examined the Ser312Gly polymorphism in HSD17B1 and breast cancer risk. When the HSD17B1 A-allele and CYP17 A2-allele were considered as the high-risk alleles, the risk of advanced breast cancer among women carrying four high-risk alleles (HSD17B1 AA and CYP17 A2A2) was 2.21 compared with that of women who carried none (Feigelson et al., 2001Go).

The CYP19 gene, encoding aromatase, plays a crucial role in estradiol synthesis. However, we could not find any association between the Arg264Cys polymorphism in CYP19 and endometriosis. A possible explanation for this negative result might be a lack of a functional effect for this polymorphism. A previous study reported that aromatase activity was not affected by the Arg264Cys polymorphism (Watanabe et al., 1997Go).

Three major limitations must be considered when evaluating the results of this study. First is the relatively small number of subjects. Although we found statistically significant differences, the 95% confidence intervals were relatively wide, reflecting the small number of cases. The sample size was sufficient to detect odds ratios of three or larger with 80% power at the 5% level of significance. The lack of a significant association with the CYP19 Arg264Cys polymorphism means only that we failed to detect a difference. It remains unclear whether this polymorphism affects endometriosis.

Secondly, the distribution of HSD17B1 alleles in the control group deviated considerably from the expected Hardy–Weinberg equilibrium, a difference that was not significant (P=0.06), but was on the edge of being so. The discrepancy may result from the small number of subjects or from the characteristics of the control group. The setting of this study is a hospital, and the control group is women complaining of infertility. Because of the requirement for a surgical diagnosis, the selection of a control group in case–control studies of endometriosis has been particularly difficult (Zondervan et al., 2002Go), and it is difficult to exclude a selection bias completely. Development of non-invasive methods for diagnosis or a prospective randomized trial will minimize any sampling bias.

Lastly, endogenous estrogen levels were not measured in this study. HSD17B1 is not expressed in normal endometrium or endometrial hyperplasia (Utsunomiya et al., 2001Go). One possible explanation of the apparent influence of HSD17B1 is that the HSD17B1 A-allele increases the level of circulating estradiol. Although an in vitro study failed to demonstrate any change in HSD17B1 catalytic activity produced by the Ser312Gly polymorphism (Puranen et al., 1994Go), a recent molecular epidemiological study showed that HSD17B1 A/A genotype was associated with higher estradiol levels in lean women (Setiawan et al., 2004Go). The functional effects of the Ser312Gly polymorphism in HSD17B1 and the Arg264Cys polymorphism in CYP19 have not yet been clearly established. Further information on functional changes and more epidemiologic studies will help clarify the association between these polymorphisms and endometriosis.

Many alleles have been reported to vary in frequency among different ethnic or geographic populations. In the present study, allelic frequencies of HSD17B1 Ser312Gly polymorphism in control individuals were similar to previously reported distributions in Chinese and American populations: A 0.43 and G 0.57 in Japanese and Chinese populations, and A 0.51 and G 0.49 in an American population (Wu et al., 2003Go; Setiawan et al., 2004Go). On the other hand, large variations were found between Japanese and Caucasians in the CYP19 Arg264Cys polymorphism: C 0.70 and T 0.30 in a Japanese population, versus C 0.96 and T 0.04 in a Caucasian population (Hefler et al., 2004Go).

In summary, we demonstrated that the Ser312Gly polymorphism in HSD17B1 was associated with the risk and severity of endometriosis in a Japanese population. The A-allele of the HSD17B1 gene is considered to be a high-risk allele for endometriosis. However, no association was found between the Arg264Cys polymorphism in CYP19 and endometriosis. The results of this study are not conclusive and further investigation is warranted. Endometriosis is a complex trait. The many factors contributing to the disease phenotype make its pathophysiology very difficult to understand. Progress in the genetics of the disease, including understanding of genetic polymorphisms, will facilitate research on endometriosis.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Drs Haruko Takeyama and Kouhei Maruyama, Department of Biotechnology, Tokyo University of Agriculture and Technology, for genetic analysis. This work was supported by a Grant-in-Aid for Risk Analysis Research on Food and Pharmaceuticals and Cancer Research from the Ministry of Health, Labour and Welfare of Japan.


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Submitted on November 17, 2004; accepted on December 8, 2004.





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