Estrogen receptor dinucleotide repeat and cytochrome P450c17 gene polymorphisms are associated with susceptibility to endometriosis

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1 ENDOMETRIOSIS Estrogen receptor dinucleotide repeat and cytochrome P450c17 gene polymorphisms are associated with susceptibility to endometriosis Yao-Yuan Hsieh, M.D., a,d Chi-Chen Chang, M.D., a Fuu-Jen Tsai, M.D., Ph.D., b Cheng-Chieh Lin, M.D., c and Chang-Hai Tsai, M.D., Ph.D. b,e a Department of Obstetrics and Gynecology, b Department of Pediatrics and Medical Genetics, and c Department of Family Medicine, China Medical University Hospital, Taichung; d Department of Biological Science and Technology, National Chiao Tung University, Hsinchu; and e Taichung Health Care and Management University, Taichung, Taiwan Objective: To investigate the association of endometriosis with estrogen receptor alpha (ER ) and cytochrome P450c17 (CYP17) gene polymorphisms in light of the fact that estrogen plays a role in the pathogenesis of endometriosis and the CYP17 enzyme is involved with estrogen biosynthesis. Design: Prospective study. Setting: Genetics and gynecology units. Patient(s): All patients were divided into two groups: group 1, women with endometriosis (n 119); group 2, normal controls (n 108). Intervention(s): A dinucleotide (thymine-adenine [TA]) repeat polymorphism lying upstream of the ER gene and A1/A2 polymorphism of the CYP17 gene were amplified by polymerase chain reaction, enzyme restriction, and electrophoresis. Main Outcome Measure(s): The ER genotypes were classified into A through T (TA repeats, 10 29). The CYP17 genotypes included indigestible (A1 homozygote), heterozygote, and digestible (A2 homozygote). We compared these polymorphism distributions in both groups. Result(s): The percentage of genotypes D G (TA, 13 16) in both groups were 10.5%, 29.4%, 13.0%, and 11.3% in group 1 and 7.9%, 16.7%, 19.9%, and 17.6% in group 2. The genotype E (14 TA repeats) is associated with a higher risk of endometriosis. Proportions of A1 homozygote/heterozygote/a2 homozygote for CYP17 were 26.1%/46.2%/27.7% for group 1 and 14.8%/44.5%/40.7% for group 2, respectively. The A1 homozygote and allele were associated with a higher susceptibility of endometriosis. Conclusion(s): ER * 14 TA repeats and the CYP17* A1 allele are associated with an increased risk of endometriosis. Both polymorphisms are useful markers for predicting endometriosis susceptibility. (Fertil Steril 2005;83: by American Society for Reproductive Medicine.) Key Words: Cytochrome P450c17, CYP17, dinucleotide repeat polymorphism, endometriosis, estrogen receptor, single nucleotide polymorphism It has been suggested that endometriosis, a frequent estrogen-dependent disease, has a genetic basis because of its familial tendency (1). Endometriosis is a complex disease that is caused by an interaction between multiple genes and the environment (2). Although the pathogenesis of endometriosis remains unclear, ectopic endometrium expresses persistent estrogen receptor (ER) with hormonal independence during the luteal phase with a possibly altered biologic Received February 12, 2004; revised and accepted July 26, Reprint requests: Fuu-Jen Tsai, M.D., Ph.D., Department of Pediatrics and Medical Genetics, China Medical University Hospital, No.2 Yuh- Der Road, Taichung, Taiwan (FAX: ; d0704@cmuh.org.tw). activity as well (3). Polymorphisms involved in steroid hormone biosynthesis and signaling may be useful genetic biomarkers for hormone-related diseases (4). The associations between the ER polymorphism and breast carcinoma or osteoporosis have been demonstrated. Sano et al. (5) investigated the thymine-adenine (TA) repeat polymorphism and observed that the 12 repeats of the TA allele had a significantly lower score of bone marrow density. Weiderpass et al. (6) demonstrated the correlation of ER polymorphism with endometrial cancer. Using TA repeat polymorphisms for ER, Georgiou et al. (7) first demonstrated that the higher percentage of patients with endometriosis had 15 repeats of the TA multiallele polymorphism. However, there have been a small number of reports concerning the relationship between ER polymorphism and endometriosis /05/$30.00 Fertility and Sterility Vol. 83, No. 3, March 2005 doi: /j.fertnstert Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 567

2 Cytochrome P450c17 gene (CYP17), the gene coding for the cytochrome P450c17 enzyme, is involved in estrogen biosynthesis (8). CYP17 mediates both steroid 17 hydroxylase and 17,20-lyase activities and functions at key steps in the genesis of human sex steroid hormones (9). The CYP17 gene maps to chromosome 10 and contains eight exons and seven introns (10). The 5=-untranslated region of CYP17 contains a single-bp polymorphism 34 bp upstream from the transcription start site (8). A single (A1 to A2) nucleotide change appears in the 5= region of CYP17, which contains a recognition site for the MspAI restriction enzyme. CYP17 polymorphism may play a crucial role in the etiology of hormone-related diseases such as endometriosis. Genetic studies of multifactorial diseases such as endometriosis are difficult to fully understand because of the uncertainty of the polygenic trait. The identification of the related genes is essential for genetic diagnosis and gene therapy for genetic-associated diseases. In this study, we further investigate the association of ER TA repeats and endometriosis in Taiwanese women. Furthermore, using the MspA1I restriction enzyme polymorphism in 3=-UTR of CYP17, we tried to evaluate whether the CYP17 polymorphism is a useful marker for predicting the susceptibility to endometriosis. To our knowledge, this is among the first few reports of this kind. MATERIALS AND METHODS Premenopausal Taiwanese Chinese women with surgically and histologically diagnosed endometriosis were included in the study. All patients were divided into two groups: group 1, women with moderate/severe endometriosis (n 119); and group 2, women with no endometriosis (n 108). Nonendometriosis status was confirmed during cesarean section or diagnostic laparoscopy. All operations were performed by two surgeons (Y.Y.H., C.C.C.). All women accepted the peripheral blood sampling for genotype analyses. The experiment was approved by the Ethics Committee and Institutional Review Board of the China Medical University Hospital. Informed consent was signed by all the women who donated their blood. The single nucleotide polymorphism (SNP) information for the genes involved was obtained through the Internet ( LocusLink/). ER Dinucleotide Repeat Polymorphism Genomic DNA was isolated from peripheral blood using the Genomaker DNA extractor kit (Blossom Biotechnology, Taipei, Taiwan). The multiallele (microsatellite) polymorphism, (TA)n, was surveyed as described by Sano et al. (5). Polymerase chain reaction (PCR) was performed using oligonucleotide primers designed to amplify the polymorphic (TA)n repeat at 1,174 bp upstream of the human ER gene. DNA was extracted from peripheral blood and subjected to analysis by PCR and gel electrophoresis of the PCR products. The primers for PCR were designed so that the PCR products contained upstream and downstream sequences of the TA dinucleotide repeat. The reaction was carried out in a final volume of 10 L containing 100 ng of genomic DNA, 5 pmol of each primer (5=-GACGCATGATATACT- TCACC-3= and 5=-GCAGAATCAAATATCCAGATG-3=), 400 M of dgtp, datp, and dttp, 40 M of dctp and 0.16 L of [32P]-dCTP (37 MBq/100 L), 10 mm Tris- HCL (ph 8.3), 50 mm KCL, 1.5 mm MgCl 2, 0.001% gelatin, and 1U Taq DNA polymerase (Takara Shuzo, Kyoto, Japan). Thirty PCR cycles (each for 2 minutes at 94 C, 1 minute at 58 C, and 1 minute at 74 C) were performed. Electrophoresis was performed on 5% polyacrylamide gels containing 7 M urea with control DNAs having a known length followed by autoradiography. The length of TA repeat in each amplified product was determined in comparison with a sequence ladder of control DNAs. The size of the PCR products ranged from 160 bp (containing 10 TA repeats with the 140 bp of amplified flanking sequences) to 198 bp (29 TA repeats). According to the number of the TA repeats, the genotypes were classified into A through T from 10 to 29. Eighteen alleles observed in this population were classified into genotypes A through R according to the number of dinucleotide repeats they contained (Fig. 1). CYP17 About 50 ng of genomic DNA was mixed with 20 pmol of each PCR primer in a total volume of 25 L containing 10 mm Tris-HCL, ph 8.3, 50 mm KCL, 1.5 mm MgCl 2, 0.2 mm deoxyribonucleotide triphosphate, and 1 U of Amplitaq DNA polymerase (Perkin-Elmer, Foster City, CA). The 169-bp fragment encompassing the polymorphic site in the promoter region of CYP17 A1/A2 was amplified by PCR using primers: 5=-CCACAAGGCAAGAGATAACA-3= and 5=-AGGGTAAGCAGCAAGAGAGC-3=. PCR amplification was performed in a programmable thermal cycler GeneAmp PCR System 2400 (Perkin Elmer). PCR was carried out in a 25- L aliquot containing 50 ng of genomic DNA, 50 pmol of each primer, 125 M deoxynucleotide triphosphates,1 U oftaq polymerase (Ampli- Taq Gold DNA polymerase, PE Applied Biosystems, Foster City, CA), and 1 reaction buffer supplied by the manufacturer (PE Applied Biosystems). The cycling condition for CYP17 A1/A2 gene polymorphism was set as follows: one cycle at 94 C for 5 minutes, 35 cycles at 94 C for 30 seconds, 55 C for 30 seconds, 72 C for 90 seconds, and one final cycle of extension at 72 C for 7 minutes. The PCR products were digested overnight with 10 U of MspA1I (New England Biolabs, Beverly, MA). When the MspA1I site was present, the 169-bp PCR fragment was divided into 102 and 67 bp by the endonuclease digestion. The genotypes were designated as A1 when the restriction site was absent and as A2 when the restriction site was present, as defined in other studies (8). PCR products were 568 Hsieh et al. ER and CYP17 in endometriosis Vol. 83, No. 3, March 2005

3 FIGURE 1 Frequency distributions of the TA repeat polymorphism for ER in patients with and without endometriosis. The PCR products ranged in length from 160 bp (10 repeats, genotype A) to 198 bp (27 repeats, genotype T) (*difference existed in the genotype between both groups). Hsieh. ER and CYP17 in endometriosis. Fertil Steril analyzed by electrophoresis on 3% agarose gel. Each allele was recognized according to its size. The polymorphism was divided into indigestible (A1 homozygote), A1/A2 heterozygote, and digestible (A2 homozygote). Statistical Analyses Genotypes and allelic frequencies for the ER dinucleotide repeat and CYP17 A1/A2 polymorphism in both groups were compared. Allelic frequencies are expressed as a percentage of the total number of alleles. The frequency and distribution of ER dinucleotide repeat polymorphisms were evaluated. The SAS system (version 8.1, SAS Institute, Cary, NC) with the 2 test, logistic regression method, and Fisher s exact tests were used for statistical analyses. P.05 was considered statistically significant. RESULTS There was a statistically significant difference in the distribution of TA repeats in the population with or without endometriosis. Their differences existed in the genotypes E (14 TA repeats), F (15 TA repeats), H (17 TA repeats), I (18 TA repeats), and O (24 TA repeats). The percentage of genotypes E, F, H, I, and O in groups 1 and 2 were 29.4%, 13.0%, 2.1%, 6.7%, and 5.5% in group 1 and 16.7%, 19.9%, 7.4%, 0.9%, and 0.9% in group 2 (Fig. 1). The main numbers of TA repeats in women with and without endometriosis were 14 (genotype E) and 15 repeats (genotype F), respectively (Fig. 1). Women with genotypes E, I, and O (14, 18, 24 TA repeats) have a higher risk of developing endometriosis. In contrast, women with genotypes F and H (15, 17 TA repeats) have a lower risk of developing endometriosis. In regard to the high prevalence of genotypes E and F in women with and without endometriosis, individuals possessing one allele of genotype E (14 TA repeats) had a higher risk of developing endometriosis compared with individuals not possessing genotype E. Women with phenotype F (15 TA repeats) had a lower risk of combination endometriosis. The logistic regression method was used in the analyses of all genotypes. Fisher s exact test was used in the analyses of genotype B, C, O, P, Q, R, and T. The 2 test was used in the analyses of genotype D, E, F, G, H, I, J, K, L, M, and N. Genotypes A and S were uncalculated. Genotype proportions of different CYP17 polymorphisms in both groups were also significantly different (Table 1). The percentage of A1 homozygote in the women with endometriosis was significantly higher than that in women without endometriosis. Proportions of A1 homozygote/heterozygote/a2 homozygote for CYP17 in both groups were Fertility and Sterility 569

4 TABLE 1 Genotype and allele frequencies of the CYP17 polymorphism in women with and without endometriosis. Endometriosis (n 119) (%) No endometriosis (n 108) (%) P a Genotype.04 A1/A1 31 (26.1) 16 (14.8) A1/A2 55 (46.2) 48 (44.5) A2/A2 33 (27.7) 44 (40.7) Allele frequencies.009 A1 117 (49.2) 80 (37.1) A2 121 (50.8) 136 (62.9) Note: Allelic sizes (bp) after enzyme digestion were as follows: A1 allele, 169 bp, uncuttable; A2 allele, bp, cuttable. a P values were calculated by the 2 test. Hsieh. ER and CYP17 in endometriosis. Fertil Steril %/46.2%/27.7% in group 1 and 14.8%/44.5%/40.7% in group 2, respectively (Table 1). Furthermore, the A1 allele was related with a significantly higher susceptibility to endometriosis (Table 1). A1 and A2 allele frequencies in both groups were 49.2%/50.8% in group 1 and 37.1%/62.9% in group 2 (Table 1). DISCUSSION Numerous chronic disorders, such as osteoporosis, hypertension, diabetes, and asthma, have been attributed to genetic susceptibility. Susceptibility-causing genes are thought to interact with other genes and the environment to produce the corresponding disorder (5). Unlike mutations, polymorphisms are not directly linked to a certain disease, but they are useful tools in the study of multifactorial disorders (11). Polymorphism is related with endometriosis development, including ER gene polymorphisms (7), glutathione S- transferase M1 gene polymorphism (12), and N314D polymorphism of galactose-1-phosphate uridyl transferase (13). In our previous reports, we have observed the correlation between endometriosis and a series of gene polymorphisms, including estrogen and androgen receptors and IL-1, IL-4, TNF, p53, and p21 polymorphisms (IL promoter, IL-1 exon 5) (14 17). The ER gene presents itself in endometrial tissue and the pelvic organs, which are the targets of endometriotic implants (3). Estrogen receptor polymorphism is associated with numerous chronic diseases, including endometriosis (7), breast carcinoma (18), recurrent abortion (19), ovarian dysfunction (20), osteoporosis (5), and arthritis (21). Georgiou et al. (7) first demonstrated that the higher percentage of patients with endometriosis had 15 repeats of the TA multiallele polymorphism. They also observed that the distributions of (TA)n repeats were bimodal, with two peaks at 15 repeats and 23 repeats, respectively. In contrast, our data revealed that the women who had 14 repeats displayed a more advanced stage of endometriosis. Genotype F (15 TA repeat) appeared prominent (19.9%) in women without endometriosis. The higher prevalence of genotype E (14 TA repeat) suggested its influence upon endometriosis formation. This reflected the genetic contribution to endometriosis. The genotype E at the microsatellite locus may be associated with some variation of the ER gene that causes endometriosis formation. We also observed a monopeak distribution instead of a bimodal distribution. The different distributions may be due to the racial difference between Asians and Caucasians. CYP17 is a key enzyme in sex steroid synthesis (22). The enzyme has both 17 -hydroxylase and 17,20-lyase activities, which are involved in the production of estrogen (10). CYP17 gene polymorphism may be related with numerous tumors, including breast cancer (23) and prostate cancer (9), among others. Some investigators have demonstrated that the A1 allele has a more androgenic effect on men. The A1 allele of the CYP17 polymorphism is associated with an increased risk of prostate cancer and benign prostatic hyperplasia (24). In contrast, the A2 allele has an estrogenic effect on women. The A2 allele is associated with an increased risk of advanced breast cancer (25), polycystic ovary syndrome (26), and higher levels of serum E 2 (27). However, some investigators have indicated that CYP17 polymorphism is not associated with certain individual diseases, including ovarian cancer (28), breast cancer (29, 30), polycystic ovaries (31), prostate cancer (32, 33), and steroid hormone levels (29, 34). Nedelcheva Kristensen et al. (29) demonstrated that the age at onset, tumor grade, metastases, and ER for breast cancer were not associated with the CYP17 genotype. Haiman et al. (35) demonstrated that the A2 allele of CYP17 gene is not a strong risk factor for breast cancer. Furthermore, Haiman et al. (27) demonstrated that 570 Hsieh et al. ER and CYP17 in endometriosis Vol. 83, No. 3, March 2005

5 the A2 allele of CYP17 was associated with a decreased risk of endometrial cancer. These controversies may be due to the multiple enzymatic processes and interactions, different illness classification, and racial, environmental, and disease variations. Although the exact reason for these contradictory results remains unclear, the identical CYP17 genotype may play either a protective or a promoting role in endometriosis given different environmental and/or genetic backgrounds. In this study, we observed a higher percentage of the A1 homozygote and allele in the women with endometriosis compared with the women without endometriosis. The presence of the A2 allele was associated with a decreased risk for endometriosis. The A1 and A2 alleles may be serving as markers of a functional variant in a nearby gene. Presumably, the distinct biological condition caused by the CYP17 genotype will be among various genetic, dietary, and environmental factors regulating hormonal and nonhormonal conditions in the development of endometriosis. This polymorphism may also be in linkage disequilibrium with an unidentified functional polymorphism in CYP17 that influences endometriosis risk. In conclusion, it is likely that the ER gene polymorphism contributes to the pathogenesis of endometriosis. The 14 TA repeats are associated with endometriosis formation. This study could be extended to understand whether the genotype E also affects ER function and endometriosis formation. Furthermore, the A1 allele frequency may be useful in predicting endometriosis susceptibility. These findings provide a database for the further survey of the ER and CYP17 polymorphisms. Although the real roles of ER and CYP17 gene polymorphisms have not yet been clarified, these polymorphisms deserve more attention to examine their roles in endometriosis development. After the clarification of its role in endometriosis, these gene polymorphisms may become useful markers to predict the future development of endometriosis and to permit early therapeutic intervention in women at high risk of endometriosis. Furthermore, the influence of other hormone gene polymorphisms on endometriosis development also merits further investigation. REFERENCES 1. Hadfield RM, Mardon HJ, Barlow DH, Kennedy S. Endometriosis in monozygotic twins. Fertil Steril 1997;68: Kennedy S. The genetics of endometriosis. J Reprod Med 1998;43: Nisolle M, Casanas-Roux F, Donnez J. Immunohistochemical analysis of proliferative activity and steroid receptor expression in peritoneal and ovarian endometriosis. Fertil Steril 1997;68: Dunning AM, Healey CS, Pharoah PD, Teare MD, Ponder BA, Easton DF. 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