- Email: [email protected]
Association of estrogen receptor α and β3-adrenergic receptor polymorphisms with endometrial cancer
Association of Estrogen Receptor ␣ and 3-Adrenergic Receptor Polymorphisms With Endometrial Cancer Ichiro Iwamoto, MD, Toshinori Fujino, MD, Tsutomu Douchi, MD, and Yukihiro Nagata, MD OBJECTIVE: To investigate the association of estrogen receptor ␣ and 3-adrenergic receptor polymorphisms with endometrial cancer risk in Kagoshima, Japan. METHODS: Ninety-two patients with endometrial cancer and 65 healthy women were enrolled in this study. Blood samples were collected, and deoxyribonucleic acid (DNA) was extracted. Estrogen receptor ␣ and 3-adrenergic receptor gene variants were analyzed by restriction fragment length polymorphisms using the restriction enzymes, Pvu II, Xba I for estrogen receptor ␣, and Mva I for 3-adrenergic receptor. Multivariable logistic regression analysis was performed, and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. RESULTS: The Pvu II PP genotype was associated with a decreased risk of endometrial cancer (multivariable OR 0.23; 95% CI 0.07, 0.82) compared with the pp genotype. The Xba I XX genotype was associated with a decreased risk for endometrial cancer (multivariable OR 0.26; 95% CI 0.09, 0.79) compared with the xx genotype. The Mva I variants were not associated with endometrial cancer risk (multivariable OR 0.55; 95% CI 0.20, 1.51). CONCLUSION: Estrogen receptor ␣ polymorphisms, but not 3-adrenergic receptor gene, may be associated with a risk of endometrial cancer. (Obstet Gynecol 2003;102:506 –11. © 2003 by The American College of Obstetricians and Gynecologists.)
Endometrial cancer is one of the most common gynecologic cancers, and its incidence is increasing in Japan, similarly to colorectal and breast cancers. In general, cancers develop as a result of the combination of several genetic causes and environmental stimuli. There have been many reports regarding the genetic changes of somatic cells in endometrial cancer; mutational activation of oncogene1 and inactivation of tumor suppression genes2,3 are examples. Apart from genetic changes in somatic cells, germline mutation or polymorphism has become another target From the Department of Obstetrics and Gynecology and School of Health Sciences, Faculty of Medicine, Kagoshima University, Kagoshima, Japan.
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for investigating the causes of cancer. Endometrial cancer is an estrogen-dependent neoplasm, and estrogen exerts its effects by binding to its receptor. The estrogen receptor ␣ gene is located on chromosome 6, and it consists of eight exons and seven introns. An estrogen receptor ␣ polymorphism was found in intron 1. The polymorphism results from a point mutation (T3 C). There are two restriction sites, Pvu II and Xba I; the Pvu II restriction fragment length polymorphism (RFLP) site is located in intron, 1400 base pair (bp) upstream of exon 2, and the Xba I RFLP site is approximately 50 bp apart from the Pvu II site.4 Recently, Weiderpass et al5 reported that variants of the estrogen receptor ␣ gene might be associated with an altered risk of endometrial cancer in the Swedish population. They calculated odds ratios (ORs) using multivariable logistic regression analysis; the presence of endometrial cancer was the dependent variable, with age, parity, age at last birth, smoking, diabetes mellitus, hypertension, body mass index (BMI), use of contraceptives, menopausal status, age at menopause, and estrogen receptor ␣ variants being independent variables. The 3-adrenergic receptor gene is mainly expressed in brown adipose tissues,6 and its major role is thought to be regulation of the resting metabolic rate. A knockout mouse for the 3-adrenergic receptor gene showed increased body weight and fat stores.7 Thus, the 3-adrenergic receptor gene is thought to be one of the key genes for obesity and insulin resistance. Walston et al identified a missense mutation in the 3-adrenergic receptor gene that resulted in the replacement of tryptophan at position 64 by arginine (Trp64Arg),8 and reported an association of genetic variation in the 3-adrenergic receptor with the onset of noninsulin-dependent diabetes mellitus in Pima Indians.8 In the same issue of the journal, the association of the 3-adrenergic receptor gene variation with obesity9 and insulin-resistance syndrome10 was reported. Thereafter, many reports regarding the relationships between the 3-adrenergic receptor gene mutation (Trp64Arg) and obesity, diabetes mellitus, and body fat
VOL. 102, NO. 3, SEPTEMBER 2003 © 2003 by The American College of Obstetricians and Gynecologists. Published by Elsevier.
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distribution have been reported.11–14 Obesity and diabetes mellitus are well-known predisposing factors for endometrial cancer.15 In the present study, we investigated the association of estrogen receptor ␣ and 3-adrenergic receptor polymorphisms with endometrial cancer risk in Japanese women along with the endometrial cancer-associated factors, such as obesity, diabetes mellitus, hypertension, nulliparity, menopausal status, and serum lipids levels. MATERIALS AND METHODS Ninety-two patients with endometrial cancer (78 menopausal women), who had been referred to the Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan, between 1997 and 2000, were consecutively recruited in this study. All patients underwent an operation, and the diagnosis of endometrial cancer was confirmed by the histopathology of the extirpated uterus in Kagoshima University. They all agreed to participate in this study. Control subjects who were at their peri-or postmenopausal state were recruited in this study during the same period. They came to the Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, for periodic gynecologic checkup. They fulfilled the following recruitment criteria: no abnormal genital bleeding and no abdominal thickening of the endometrium observed by transvaginal ultrasonography. Control subjects were not randomly selected, but were recruited consecutively. A total of 65 control subjects (38 postmenopausal women) were asked to participate in this study, and they all agreed. None of them had ever received hormone replacement therapy and none of them had smoking. Age, height, and weight were measured. Also, BMI was calculated by the equation: weight (kg)/(height [m])2. Fasting blood was drawn, and serum total cholesterol level, triglyceride level, and high-density lipoprotein (HDL) cholesterol level were measured. Diabetes mellitus and hypertension met the diagnostic criteria of the World Health Organization. This study was conducted following our institutional guidelines. Oral or written informed consent was obtained from each subject before study entry. Genomic deoxyribonucleic acid (DNA) was extracted from blood, and DNA analysis was carried out to identify RFLPs. Polymerase chain reaction (PCR) amplification of estrogen receptor ␣ using specific primers4 was conducted as follows: denaturation at 94C for 30 seconds, annealing at 62C for 20 seconds, and polymerase extension at 72C for 90 seconds. The PCR amplification was per-
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formed through 30 cycles. After amplification, the PCR products were digested with restriction enzyme Pvu II or Xba I. After digestion, fragments obtained were separated on a 1.5% agarose gel. Genotypes for Pvu II polymorphism were defined as follows: no restriction site for the P allele and fragments of 850 and 450 bp for the p allele. Genotyping for Xba I was as follows: no restriction site for the X allele and fragments of 950 and 400 bp for the x allele. The specific primers of 3-adrenergic receptor16 were used as described in previous studies. The PCR amplification consisted of the following steps: denaturation at 94C for 30 seconds, annealing at 61C for 30 seconds, and polymerase extension at 72C for 30 seconds. The PCR was performed through 30 cycles. The PCR products were digested with the restriction enzyme Mva I. After digestion, fragments obtained were separated on a 3% agarose gel. Genotypes were defined as follows: fragments of 99, 62, 30, 12, and 7 bp for the Trp allele, and eliminating one of the above fragments, yielding a novel 161-bp fragment for the Arg allele. Differences in the means of age, height, weight, BMI, serum total cholesterol level, triglyceride level, and HDL cholesterol level between patients with endometrial cancer and control women were evaluated by two-tailed unpaired t test. The prevalence of hypertension, diabetes mellitus, and nulliparity was compared between groups by 2 test. We calculated the ORs and 95% confidence intervals (CIs) using logistic regression analysis. Genotype frequencies for the Pvu II, Xba I, and Mva I polymorphisms were compared using subjects homozygous for p, x, and Trp, respectively, which had previously been reported as the most common allele as the reference. As the number of Arg/Arg genotype was very small in the present study, we examined the association of Mva I polymorphism with endometrial cancer risk, combining Arg/Arg and Trp/Arg into one group. We first estimated unadjusted (univariable) ORs for estrogen receptor ␣ and 3-adrenergic receptor genotypes along with clinical characteristics. We subsequently included the variables that were found to be associated with endometrial cancer in the present study into the logistic regression models, and calculated the multivariable ORs of Pvu II, Xba I, and Mva I polymorphisms for endometrial cancer risk. All calculations were performed with a Macintosh microcomputer using StatView-j 5 (SAS Institute Inc., Cary, NC). Significance was recognized at P ⬍ .05. RESULTS Table 1 shows the clinical characteristics of the endometrial cancer cases and controls. There were no significant
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Table 1. Clinical Characteristics of the Subjects
Age (y) Height (cm) Weight (kg) BMI (kg/m2) Age at menopause (y) T-CHO (mg/dL) TG (mg/dL) HDL-C (mg/dL) Hypertension (%) Diabetes mellitus (%) Nulliparity (%)
Cases (n ⫽ 92)
Controls (n ⫽ 65)
59.3 ⫾ 1.02 154.7 ⫾ 0.64 57.7 ⫾ 1.21 24.1 ⫾ 0.46 50.6 ⫾ 0.46 207.9 ⫾ 3.58 117.6 ⫾ 6.64 58.7 ⫾ 1.74 39.7 (25/92) 29.6 (21/92) 24.3 (18/92)
57.4 ⫾ 1.38 156.1 ⫾ 0.74 57.1 ⫾ 1.16 23.5 ⫾ 0.50 49.2 ⫾ 0.50 208.7 ⫾ 3.81 89.4 ⫾ 5.35* 67.9 ⫾ 2.12* 22.6 (12/65) 8.3 (5/65)† 8.3 (5/65)†
BMI ⫽ body mass index; T-CHO ⫽ total cholesterol; TG ⫽ triglyceride; HDL-C ⫽ high-density lipoprotein cholesterol. Data are presented as mean ⫾ standard error of the mean. * P ⬍ .01. † P ⬍ .05.
differences in age, height, weight, BMI, age at menopause, serum total cholesterol level, or the presence of hypertension between cases and controls. Significant differences in the presence of diabetes mellitus, nulliparity, serum triglyceride level, and HDL cholesterol level were observed between the two groups (P ⬍ .05, P ⬍ .05, P ⬍ .01, P ⬍ .01, respectively). Nonadjusted (univariable) logistic regression analysis demonstrated the significant associations of diabetes mellitus (no/yes), serum triglyceride level (continuous), HDL cholesterol level (continuous), nulliparity (no/yes), and menopausal status (pre-/postmenopausal) with endometrial cancer. Nonadjusted ORs were 3.55 (95% CI 1.26, 9.98) for diabetes mellitus, 1.01 (95% CI 1.00, 1.02) for serum triglyceride level, 0.97 (95% CI 0.95, 0.99) for
serum HDL cholesterol level, 2.92 (95% CI 1.02, 8.32) for nulliparity, and 3.96 (95% CI 1.86, 8.41) for menopausal status. Nonadjusted (univariable) ORs of the Pvu II genotypes Pp and PP for endometrial cancer did not significantly differ from those of the reference group pp: ORs were 1.78 (95% CI 0.72, 4.41) for Pp and 0.92 (95% CI 0.35, 2.41) for PP. However, after multiple adjustment including diabetes mellitus (no/yes), serum triglyceride level (continuous), serum HDL cholesterol level (continuous), nulliparity (no/yes), and menopausal status (pre-/ postmenopausal) into the logistic regression analysis models, the PP genotype was associated with a significantly decreased risk for endometrial cancer (OR 0.23; 95% CI 0.07, 0.82; P ⬍ .05) (Table 2). Furthermore, when BMI (continuous) and hypertension (no/yes), which are well-known risk factors for endometrial cancer, were added into the analysis models, the OR of PP was again significant (OR 0.22; 95% CI 0.06, 0.82; P ⬍ .05). The OR of Pp was 1.01 (95% CI 0.33, 3.13) even after multiple adjustment (Table 2). Nonadjusted (univariable) ORs for the Xba I genotypes Xx and XX did not significantly differ compared with the reference group xx: ORs were 0.92 (95% CI 0.45, 1.88) for Xx and 0.57 (95% CI 0.24, 1.40) for XX. However, after multiple adjustments including diabetes mellitus (no/yes), serum triglyceride level (continuous), serum HDL cholesterol level (continuous), nulliparity (no/yes), and menopausal status (pre-/postmenopausal) into the logistic regression analysis models, the XX genotype was associated with a significantly decreased risk for endometrial cancer (OR 0.26; 95% CI 0.09, 0.79; P ⬍ .05) (Table 2). Furthermore, when BMI (continu-
Table 2. Genotype Distribution Among Endometrial Cancer Cases and Controls of Pvu II and Xba I Polymorphisms of the Estrogen Receptor ␣ and Mva I Polymorphism of the 3-Adrenergic Receptor OR (95% CI)* Genotype Pvu II pp Pp PP Xba I xx Xx XX Mva I Trp/Trp Trp/Arg, Arg/Arg
Cases/controls
Nonadjusted
Multivariable†
13/12 54/28 25/25
1 (referent) 1.78 (0.72, 4.41) 0.92 (0.35, 2.41)
1 (referent) 1.01 (0.33, 3.13) 0.23 (0.07, 0.82)‡
39/24 39/26 14/15
1 (referent) 0.92 (0.45, 1.88) 0.57 (0.24, 1.40)
1 (referent) 0.60 (0.25, 1.45) 0.26 (0.09, 0.79)‡
74/53 18/12
1 (referent) 0.70 (0.26, 1.42)
1 (referent) 0.55 (0.20, 1.51)
OR ⫽ odds ratio; CI ⫽ confidence interval; DM ⫽ diabetes mellitus. Other abbreviations as in Table 1. * Genotypes compared with indicated references. † Multivariable, adjusted simultaneously for DM (no/yes), serum TG level (continuous), serum HDL-C level (continuous), nulliparity (no/yes), and menopausal status (pre-/postmenopausal). ‡ P ⬍ .05.
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ous) and hypertension (no/yes), which were variables known for association with endometrial cancer, were added into the analysis models, the OR of XX was again significant (OR 0.26; 95% CI 0.08, 0.80; P ⬍ .05). Even after multiple adjustment, Xx was not associated with a decreased risk for endometrial cancer (OR 0.60; 95% CI 0.25, 1.45) (Table 2). Nonadjusted (univariable) OR of the 3-adrenergic receptor Mva I genotype (combined Trp/Arg and Arg/ Arg) did not significantly differ compared with the reference group Trp/Trp (OR 0.70; 95% CI 0.26, 1.42). Even after multiple adjustment was performed by including endometrial cancer-associated variables in the logistic regression models, there was no significant association between the 3-adrenergic receptor gene variants and endometrial cancer risk (OR 0.55; 95% CI 0.20, 1.51).
DISCUSSION In the present study, we investigated the association of estrogen receptor ␣ and 3-adrenergic receptor polymorphisms with endometrial cancer risk along with endometrial cancer-associated factors, such as obesity, diabetes mellitus, hypertension, nulliparity, and menopausal status. Serum lipid levels were also investigated in conjunction with obesity and diabetes mellitus. We found significant differences in the presence of diabetes mellitus, nulliparity, serum triglyceride level, and HDL cholesterol level between the case and control groups. We also found significant ORs of diabetes mellitus, serum triglyceride level, HDL cholesterol level, nulliparity, and menopausal status for endometrial cancer risk. Crude ORs of PP and XX for endometrial cancer risk were not significant compared with pp or xx, respectively. However, inclusion of other covariates in the model (namely diabetes mellitus, serum triglyceride level, serum HDL cholesterol level, nulliparity, and menopausal status), which were found to be associated with endometrial cancer, changed the estimated risks; PP and XX genotypes were associated with a significantly decreased risk of endometrial cancer. These results suggest that the estrogen receptor ␣ gene may play some role in decreasing the susceptibility to endometrial cancer through interactions with other endometrial cancer-predisposing factors, such as diabetes mellitus, serum triglyceride level, HDL cholesterol level, nulliparity, and menopausal status. The Pvu II and Xba I RFLP sites are located in intron, but not in the coding domains of the estrogen receptor gene. The possible mechanism by which the variant in intron affects the susceptibility to endometrial cancer is altered receptor function attributed to differential splic-
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ing of messenger RNA. In this context, it is of interest that an association of a variant of exon of estrogen receptor ␣ gene with endometrial cancer risk was found. Sasaki et al reported a protective effect of a variant of codon 10 (T3 C) in exon 1 of the estrogen receptor ␣ gene.17 They discussed that the alterations in the coding domain of estrogen receptor ␣ could be linked with altered tissue responsiveness to estrogens, thus affecting the physiologic and pathologic process controlled by estrogens. Other related findings are polymorphisms in genes involved in steroid hormone biosynthesis. One such candidate is a single (T3 C) nucleotide change in the 5⬘ region of CYP 17, a gene encoding a cytochrome P450 enzyme, P450c17 ␣. Haiman et al reported that a variant of CYP 17 decreased endometrial cancer risk, and that the variant had a weak effect on endogenous estrogen levels among postmenopausal women.18 Berstein et al19 confirmed the protective effect of CYP 17, which had been reported by Haiman et al.18 Interestingly, they suggested an untraditional (nonsteroidal) pathway for the protective effect of the CYP 17 variant gene against endometrial cancer. The variant of the 3-adrenergic receptor (Trp64Arg) has been noted as one of the genetic markers for susceptibility to diabetes mellitus and obesity.8 –14 Diabetes mellitus and obesity are well-known predisposing factors for endometrial cancer.15 Recently, Huang et al reported a possible association between 3-adrenergic receptor polymorphism and susceptibility to breast cancer.20 Both mammary glands and endometrium are target organs of estrogen, and obesity is a common risk factor for both cancers. Therefore, we examined the association of 3-adrenergic receptor polymorphism with endometrial cancer risk. However, we could not obtain a significant association of 3-adrenergic receptor variants with endometrial cancer risk in our samples. It is known that the frequency of the Trp64Arg missense mutation of the 3-adrenergic receptor differs between races.8 –10 The allelic frequency of the mutation of 0.31 in Pima Indians is the highest, and the association of 3-adrenergic receptor polymorphism with the onset of noninsulin-dependent diabetes mellitus in Pima Indians was reported. Ueda et al reported that the frequency of the Arg allele was about 0.20 in their study in which samples were from three different districts in Japan,21 whereas the frequency of the Arg allele in the present study was 0.10, suggesting that the frequency may also have a regional difference inside Japan. The lower allelic frequency of the mutation in our samples may have led to the absence of an association between the 3-adrenergic receptor polymorphism and endometrial cancer risk in the present study.
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We found that serum triglyceride and HDL cholesterol levels were significantly different between cases and controls. Weight, BMI, and the frequency of variations in the 3-adrenergic receptor gene, which influence lipid metabolism, did not differ between the two groups. Therefore, estrogen receptor gene variations might be associated with serum lipid levels, or it might simply be a secondary phenomenon attributed to the cancer itself. In fact, there is a report that cancer patients tend to have dyslipidemia.22 The present study has certain limitations. First, the sample size in the present study was relatively small. Second, our study was a hospital-based, case– control study, but not a population-based study, and the samples were from the Kagoshima prefecture only. However, genotype frequencies for the Pvu II and Xba I estrogen receptor ␣ polymorphisms in our study samples were not very different from those in other reports in Japan.23,24 In conclusion, the present exploratory analysis suggests an association between estrogen receptor ␣ polymorphisms and endometrial cancer risk in people living in the Kagoshima prefecture, Japan. Further extensive studies all over Japan are warranted to obtain more accurate findings.
9.
10.
11.
12.
13.
14. REFERENCES 1. Imamura T, Arima T, Kato H, Miyamoto S, Sasazuki T, Wake N. Chromosomal deletions and K-RAS gene mutations in human endometrial carcinomas. Int J Cancer 1992;51:47–52. 2. Honda T, Kato H, Imamura T, Gima T, Nishida J, Sasaki M, et al. Involvement of p53 gene mutations in human endometrial carcinomas. Int J Cancer 1993;53:9963–7. 3. Gima T, Kato H, Honda T, Imamura T, Sasazuki T, Wake N. DCC gene alteration in human endometrial carcinomas. Int J Cancer 1994;57:480–5. 4. Yaich L, Dupont WD, Cavener DC, Parl FF. Analysis of the Pvu II restriction fragment-length polymorphism and exon structure of the estrogen receptor gene in breast cancer and peripheral blood. Cancer Res 1992;52:77–83. 5. Weiderpass E, Persson I, Melhus H, Wedren S, Kindmark A, Baron JA. Estrogen receptor gene ␣ polymorphisms and endometrial cancer risk. Carcinogenesis 2000;21: 623–7. 6. Krief S, Lonnqvist F, Raimbault S, Baude B, Van Spronsen A, Arner P, et al. Tissue distribution of  3-adrenergic receptor mRNA in man. J Clin Invest 1993;91:344–9. 7. Strosberg AD. Association of beta 3-adrenoreceptor polymorphism with obesity and diabetes: Current status. Trends Pharmacol Sci 1997;18:449–54. 8. Walston J, Silver K, Bogardus C, Knowler WC, Celi FS, Austin S, et al. Time of onset of non-insulin-dependent
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diabetes mellitus and genetic variation in the beta 3-adrenergic receptor gene. N Engl J Med 1995;333:343–7. Clement K, Vaisse C, Manning BS, Basdevant A, GuyGrand B, Ruiz J, et al. Genetic variation in the beta 3-adrenergic receptor and an increased capacity to gain weight in patients with morbid obesity. N Engl J Med 1995;333:352–4. Widen E, Lehto M, Kanninen T, Walston J, Shuldiner AR, Groop LC. Association of a polymorphism in the beta 3-adrenergic-receptor gene with features of the insulin resistance syndrome in Finns. N Engl J Med 1995;333: 348–51. Yoshida T, Sakane N, Umekawa T, Sakai M, Takahashi T, Kondo M. Mutation of beta 3-adrenergic receptor gene and response to treatment of obesity. Lancet 1995; 346(8987):1433–4. Sakane N, Yoshida T, Yoshioka K, Nakamura Y, Umekawa T, Kogure A, et al. Beta 3-adrenoreceptor gene polymorphism: A newly identified risk factor for proliferative retinopathy in NIDDM patients. Diabetes 1997;46: 1633–6. Sakane N, Yoshida T, Umekawa T, Kogure A, Takakura Y, Kondo M. Effects of Trp64Arg mutation in the beta 3-adrenergic receptor gene on weight loss, body fat distribution, glycemic control, and insulin resistance in obese type 2 diabetic patients. Diabetes Care 1997;20:1887–90. Sakane N, Yoshida T, Umekawa T, Kondo M, Sakai Y, Takahashi T. Beta 3-adrenergic-receptor polymorphism: A genetic marker for visceral fat obesity and the insulin resistance syndrome. Diabetologia 1997;40:200–4. Giannone R, Bernorio R, Poli M. Risk factors of endometrial carcinoma. Minerva Ginecol 1993;45:361–4. Shihara N, Yasuda K, Moritani K, Ue H, Adachi T, Tanaka H, et al. The association between Trp64Arg polymorphism of the  3-adrenergic receptor and autonomic nervous system activity. J Clin Endocrinol Metab 1999;84: 1623–7. Sasaki M, Tanaka Y, Kaneuchi M, Sakuragi N, Dahiya R. Polymorphisms of estrogen receptor ␣ gene in endometrial cancer. Biochem Biophys Res Commun 2002;297:558–64. Haiman CA, Hankinson SF, Colditz GA, Hunter DJ, Vivo ID. A polymorphism in CYP17 and endometrial cancer risk. Cancer Res 2001;61:3955–60. Berstein LM, Imyanitov EN, Gamajunova VB, Kovalevskij AJ, Kuligina ES, Belogubova EV, et al. CYP17 genetic polymorphism in endometrial cancer: Are only steroids involved? Cancer Lett 2002;180:47–53. Huang XEN, Hamajima N, Saito T, Matsuo K, Mizutani M, Iwata H, et al. Possible association of beta2- and beta3-adrenergic receptor gene polymorphisms with susceptibility to breast cancer. Breast Cancer Res 2001;3: 264–9. Ueda K, Tanizawa Y, Oota Y, Inoue H, Kizuki N, Inoue H, et al. Prevalence of the Trp64Arg missense mutation of
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the  3-adrenergic receptor gene in Japanese subjects. Metabolism 1997;46:199–202. 22. Ostroumova MN, Kovalenko IG, Bershtein LM, Tsyrlina EV, Dilman VM. Characteristics of dyslipidemia in cancer patients. Vopr Onkol 1986;32:34–43. 23. Katsumata K, Nishizawa K, Unno A, Fujita Y, Tokita A. Association of gene polymorphisms and bone density in Japanese girls. J Bone Miner Metab 2002;20:164–9. 24. Kobayashi N, Fujino T, Shirogane T, Furuta I, Kobamatsu Y, Yaegashi M, et al. Estrogen receptor alpha polymor-
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phism as a genetic marker for bone loss, vertebral fractures and susceptibility to estrogen. Maturitas 2002;41:193–201. Address reprint requests to: Ichiro Iwamoto, MD, Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan; E-mail: [email protected]. Received November 27, 2002. Received in revised form March 14, 2003. Accepted April 17, 2003.
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Endometrial cancer is one of the most common gynecologic cancers, and its incidence is increasing in Japan, similarly to colorectal and breast cancers. In general, cancers develop as a result of the combination of several genetic causes and environmental stimuli. There have been many reports regarding the genetic changes of somatic cells in endometrial cancer; mutational activation of oncogene1 and inactivation of tumor suppression genes2,3 are examples. Apart from genetic changes in somatic cells, germline mutation or polymorphism has become another target From the Department of Obstetrics and Gynecology and School of Health Sciences, Faculty of Medicine, Kagoshima University, Kagoshima, Japan.
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for investigating the causes of cancer. Endometrial cancer is an estrogen-dependent neoplasm, and estrogen exerts its effects by binding to its receptor. The estrogen receptor ␣ gene is located on chromosome 6, and it consists of eight exons and seven introns. An estrogen receptor ␣ polymorphism was found in intron 1. The polymorphism results from a point mutation (T3 C). There are two restriction sites, Pvu II and Xba I; the Pvu II restriction fragment length polymorphism (RFLP) site is located in intron, 1400 base pair (bp) upstream of exon 2, and the Xba I RFLP site is approximately 50 bp apart from the Pvu II site.4 Recently, Weiderpass et al5 reported that variants of the estrogen receptor ␣ gene might be associated with an altered risk of endometrial cancer in the Swedish population. They calculated odds ratios (ORs) using multivariable logistic regression analysis; the presence of endometrial cancer was the dependent variable, with age, parity, age at last birth, smoking, diabetes mellitus, hypertension, body mass index (BMI), use of contraceptives, menopausal status, age at menopause, and estrogen receptor ␣ variants being independent variables. The 3-adrenergic receptor gene is mainly expressed in brown adipose tissues,6 and its major role is thought to be regulation of the resting metabolic rate. A knockout mouse for the 3-adrenergic receptor gene showed increased body weight and fat stores.7 Thus, the 3-adrenergic receptor gene is thought to be one of the key genes for obesity and insulin resistance. Walston et al identified a missense mutation in the 3-adrenergic receptor gene that resulted in the replacement of tryptophan at position 64 by arginine (Trp64Arg),8 and reported an association of genetic variation in the 3-adrenergic receptor with the onset of noninsulin-dependent diabetes mellitus in Pima Indians.8 In the same issue of the journal, the association of the 3-adrenergic receptor gene variation with obesity9 and insulin-resistance syndrome10 was reported. Thereafter, many reports regarding the relationships between the 3-adrenergic receptor gene mutation (Trp64Arg) and obesity, diabetes mellitus, and body fat
VOL. 102, NO. 3, SEPTEMBER 2003 © 2003 by The American College of Obstetricians and Gynecologists. Published by Elsevier.
0029-7844/03/$30.00 doi:10.1016/S0029-7844(03)00578-7
distribution have been reported.11–14 Obesity and diabetes mellitus are well-known predisposing factors for endometrial cancer.15 In the present study, we investigated the association of estrogen receptor ␣ and 3-adrenergic receptor polymorphisms with endometrial cancer risk in Japanese women along with the endometrial cancer-associated factors, such as obesity, diabetes mellitus, hypertension, nulliparity, menopausal status, and serum lipids levels. MATERIALS AND METHODS Ninety-two patients with endometrial cancer (78 menopausal women), who had been referred to the Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan, between 1997 and 2000, were consecutively recruited in this study. All patients underwent an operation, and the diagnosis of endometrial cancer was confirmed by the histopathology of the extirpated uterus in Kagoshima University. They all agreed to participate in this study. Control subjects who were at their peri-or postmenopausal state were recruited in this study during the same period. They came to the Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, for periodic gynecologic checkup. They fulfilled the following recruitment criteria: no abnormal genital bleeding and no abdominal thickening of the endometrium observed by transvaginal ultrasonography. Control subjects were not randomly selected, but were recruited consecutively. A total of 65 control subjects (38 postmenopausal women) were asked to participate in this study, and they all agreed. None of them had ever received hormone replacement therapy and none of them had smoking. Age, height, and weight were measured. Also, BMI was calculated by the equation: weight (kg)/(height [m])2. Fasting blood was drawn, and serum total cholesterol level, triglyceride level, and high-density lipoprotein (HDL) cholesterol level were measured. Diabetes mellitus and hypertension met the diagnostic criteria of the World Health Organization. This study was conducted following our institutional guidelines. Oral or written informed consent was obtained from each subject before study entry. Genomic deoxyribonucleic acid (DNA) was extracted from blood, and DNA analysis was carried out to identify RFLPs. Polymerase chain reaction (PCR) amplification of estrogen receptor ␣ using specific primers4 was conducted as follows: denaturation at 94C for 30 seconds, annealing at 62C for 20 seconds, and polymerase extension at 72C for 90 seconds. The PCR amplification was per-
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formed through 30 cycles. After amplification, the PCR products were digested with restriction enzyme Pvu II or Xba I. After digestion, fragments obtained were separated on a 1.5% agarose gel. Genotypes for Pvu II polymorphism were defined as follows: no restriction site for the P allele and fragments of 850 and 450 bp for the p allele. Genotyping for Xba I was as follows: no restriction site for the X allele and fragments of 950 and 400 bp for the x allele. The specific primers of 3-adrenergic receptor16 were used as described in previous studies. The PCR amplification consisted of the following steps: denaturation at 94C for 30 seconds, annealing at 61C for 30 seconds, and polymerase extension at 72C for 30 seconds. The PCR was performed through 30 cycles. The PCR products were digested with the restriction enzyme Mva I. After digestion, fragments obtained were separated on a 3% agarose gel. Genotypes were defined as follows: fragments of 99, 62, 30, 12, and 7 bp for the Trp allele, and eliminating one of the above fragments, yielding a novel 161-bp fragment for the Arg allele. Differences in the means of age, height, weight, BMI, serum total cholesterol level, triglyceride level, and HDL cholesterol level between patients with endometrial cancer and control women were evaluated by two-tailed unpaired t test. The prevalence of hypertension, diabetes mellitus, and nulliparity was compared between groups by 2 test. We calculated the ORs and 95% confidence intervals (CIs) using logistic regression analysis. Genotype frequencies for the Pvu II, Xba I, and Mva I polymorphisms were compared using subjects homozygous for p, x, and Trp, respectively, which had previously been reported as the most common allele as the reference. As the number of Arg/Arg genotype was very small in the present study, we examined the association of Mva I polymorphism with endometrial cancer risk, combining Arg/Arg and Trp/Arg into one group. We first estimated unadjusted (univariable) ORs for estrogen receptor ␣ and 3-adrenergic receptor genotypes along with clinical characteristics. We subsequently included the variables that were found to be associated with endometrial cancer in the present study into the logistic regression models, and calculated the multivariable ORs of Pvu II, Xba I, and Mva I polymorphisms for endometrial cancer risk. All calculations were performed with a Macintosh microcomputer using StatView-j 5 (SAS Institute Inc., Cary, NC). Significance was recognized at P ⬍ .05. RESULTS Table 1 shows the clinical characteristics of the endometrial cancer cases and controls. There were no significant
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Table 1. Clinical Characteristics of the Subjects
Age (y) Height (cm) Weight (kg) BMI (kg/m2) Age at menopause (y) T-CHO (mg/dL) TG (mg/dL) HDL-C (mg/dL) Hypertension (%) Diabetes mellitus (%) Nulliparity (%)
Cases (n ⫽ 92)
Controls (n ⫽ 65)
59.3 ⫾ 1.02 154.7 ⫾ 0.64 57.7 ⫾ 1.21 24.1 ⫾ 0.46 50.6 ⫾ 0.46 207.9 ⫾ 3.58 117.6 ⫾ 6.64 58.7 ⫾ 1.74 39.7 (25/92) 29.6 (21/92) 24.3 (18/92)
57.4 ⫾ 1.38 156.1 ⫾ 0.74 57.1 ⫾ 1.16 23.5 ⫾ 0.50 49.2 ⫾ 0.50 208.7 ⫾ 3.81 89.4 ⫾ 5.35* 67.9 ⫾ 2.12* 22.6 (12/65) 8.3 (5/65)† 8.3 (5/65)†
BMI ⫽ body mass index; T-CHO ⫽ total cholesterol; TG ⫽ triglyceride; HDL-C ⫽ high-density lipoprotein cholesterol. Data are presented as mean ⫾ standard error of the mean. * P ⬍ .01. † P ⬍ .05.
differences in age, height, weight, BMI, age at menopause, serum total cholesterol level, or the presence of hypertension between cases and controls. Significant differences in the presence of diabetes mellitus, nulliparity, serum triglyceride level, and HDL cholesterol level were observed between the two groups (P ⬍ .05, P ⬍ .05, P ⬍ .01, P ⬍ .01, respectively). Nonadjusted (univariable) logistic regression analysis demonstrated the significant associations of diabetes mellitus (no/yes), serum triglyceride level (continuous), HDL cholesterol level (continuous), nulliparity (no/yes), and menopausal status (pre-/postmenopausal) with endometrial cancer. Nonadjusted ORs were 3.55 (95% CI 1.26, 9.98) for diabetes mellitus, 1.01 (95% CI 1.00, 1.02) for serum triglyceride level, 0.97 (95% CI 0.95, 0.99) for
serum HDL cholesterol level, 2.92 (95% CI 1.02, 8.32) for nulliparity, and 3.96 (95% CI 1.86, 8.41) for menopausal status. Nonadjusted (univariable) ORs of the Pvu II genotypes Pp and PP for endometrial cancer did not significantly differ from those of the reference group pp: ORs were 1.78 (95% CI 0.72, 4.41) for Pp and 0.92 (95% CI 0.35, 2.41) for PP. However, after multiple adjustment including diabetes mellitus (no/yes), serum triglyceride level (continuous), serum HDL cholesterol level (continuous), nulliparity (no/yes), and menopausal status (pre-/ postmenopausal) into the logistic regression analysis models, the PP genotype was associated with a significantly decreased risk for endometrial cancer (OR 0.23; 95% CI 0.07, 0.82; P ⬍ .05) (Table 2). Furthermore, when BMI (continuous) and hypertension (no/yes), which are well-known risk factors for endometrial cancer, were added into the analysis models, the OR of PP was again significant (OR 0.22; 95% CI 0.06, 0.82; P ⬍ .05). The OR of Pp was 1.01 (95% CI 0.33, 3.13) even after multiple adjustment (Table 2). Nonadjusted (univariable) ORs for the Xba I genotypes Xx and XX did not significantly differ compared with the reference group xx: ORs were 0.92 (95% CI 0.45, 1.88) for Xx and 0.57 (95% CI 0.24, 1.40) for XX. However, after multiple adjustments including diabetes mellitus (no/yes), serum triglyceride level (continuous), serum HDL cholesterol level (continuous), nulliparity (no/yes), and menopausal status (pre-/postmenopausal) into the logistic regression analysis models, the XX genotype was associated with a significantly decreased risk for endometrial cancer (OR 0.26; 95% CI 0.09, 0.79; P ⬍ .05) (Table 2). Furthermore, when BMI (continu-
Table 2. Genotype Distribution Among Endometrial Cancer Cases and Controls of Pvu II and Xba I Polymorphisms of the Estrogen Receptor ␣ and Mva I Polymorphism of the 3-Adrenergic Receptor OR (95% CI)* Genotype Pvu II pp Pp PP Xba I xx Xx XX Mva I Trp/Trp Trp/Arg, Arg/Arg
Cases/controls
Nonadjusted
Multivariable†
13/12 54/28 25/25
1 (referent) 1.78 (0.72, 4.41) 0.92 (0.35, 2.41)
1 (referent) 1.01 (0.33, 3.13) 0.23 (0.07, 0.82)‡
39/24 39/26 14/15
1 (referent) 0.92 (0.45, 1.88) 0.57 (0.24, 1.40)
1 (referent) 0.60 (0.25, 1.45) 0.26 (0.09, 0.79)‡
74/53 18/12
1 (referent) 0.70 (0.26, 1.42)
1 (referent) 0.55 (0.20, 1.51)
OR ⫽ odds ratio; CI ⫽ confidence interval; DM ⫽ diabetes mellitus. Other abbreviations as in Table 1. * Genotypes compared with indicated references. † Multivariable, adjusted simultaneously for DM (no/yes), serum TG level (continuous), serum HDL-C level (continuous), nulliparity (no/yes), and menopausal status (pre-/postmenopausal). ‡ P ⬍ .05.
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ous) and hypertension (no/yes), which were variables known for association with endometrial cancer, were added into the analysis models, the OR of XX was again significant (OR 0.26; 95% CI 0.08, 0.80; P ⬍ .05). Even after multiple adjustment, Xx was not associated with a decreased risk for endometrial cancer (OR 0.60; 95% CI 0.25, 1.45) (Table 2). Nonadjusted (univariable) OR of the 3-adrenergic receptor Mva I genotype (combined Trp/Arg and Arg/ Arg) did not significantly differ compared with the reference group Trp/Trp (OR 0.70; 95% CI 0.26, 1.42). Even after multiple adjustment was performed by including endometrial cancer-associated variables in the logistic regression models, there was no significant association between the 3-adrenergic receptor gene variants and endometrial cancer risk (OR 0.55; 95% CI 0.20, 1.51).
DISCUSSION In the present study, we investigated the association of estrogen receptor ␣ and 3-adrenergic receptor polymorphisms with endometrial cancer risk along with endometrial cancer-associated factors, such as obesity, diabetes mellitus, hypertension, nulliparity, and menopausal status. Serum lipid levels were also investigated in conjunction with obesity and diabetes mellitus. We found significant differences in the presence of diabetes mellitus, nulliparity, serum triglyceride level, and HDL cholesterol level between the case and control groups. We also found significant ORs of diabetes mellitus, serum triglyceride level, HDL cholesterol level, nulliparity, and menopausal status for endometrial cancer risk. Crude ORs of PP and XX for endometrial cancer risk were not significant compared with pp or xx, respectively. However, inclusion of other covariates in the model (namely diabetes mellitus, serum triglyceride level, serum HDL cholesterol level, nulliparity, and menopausal status), which were found to be associated with endometrial cancer, changed the estimated risks; PP and XX genotypes were associated with a significantly decreased risk of endometrial cancer. These results suggest that the estrogen receptor ␣ gene may play some role in decreasing the susceptibility to endometrial cancer through interactions with other endometrial cancer-predisposing factors, such as diabetes mellitus, serum triglyceride level, HDL cholesterol level, nulliparity, and menopausal status. The Pvu II and Xba I RFLP sites are located in intron, but not in the coding domains of the estrogen receptor gene. The possible mechanism by which the variant in intron affects the susceptibility to endometrial cancer is altered receptor function attributed to differential splic-
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ing of messenger RNA. In this context, it is of interest that an association of a variant of exon of estrogen receptor ␣ gene with endometrial cancer risk was found. Sasaki et al reported a protective effect of a variant of codon 10 (T3 C) in exon 1 of the estrogen receptor ␣ gene.17 They discussed that the alterations in the coding domain of estrogen receptor ␣ could be linked with altered tissue responsiveness to estrogens, thus affecting the physiologic and pathologic process controlled by estrogens. Other related findings are polymorphisms in genes involved in steroid hormone biosynthesis. One such candidate is a single (T3 C) nucleotide change in the 5⬘ region of CYP 17, a gene encoding a cytochrome P450 enzyme, P450c17 ␣. Haiman et al reported that a variant of CYP 17 decreased endometrial cancer risk, and that the variant had a weak effect on endogenous estrogen levels among postmenopausal women.18 Berstein et al19 confirmed the protective effect of CYP 17, which had been reported by Haiman et al.18 Interestingly, they suggested an untraditional (nonsteroidal) pathway for the protective effect of the CYP 17 variant gene against endometrial cancer. The variant of the 3-adrenergic receptor (Trp64Arg) has been noted as one of the genetic markers for susceptibility to diabetes mellitus and obesity.8 –14 Diabetes mellitus and obesity are well-known predisposing factors for endometrial cancer.15 Recently, Huang et al reported a possible association between 3-adrenergic receptor polymorphism and susceptibility to breast cancer.20 Both mammary glands and endometrium are target organs of estrogen, and obesity is a common risk factor for both cancers. Therefore, we examined the association of 3-adrenergic receptor polymorphism with endometrial cancer risk. However, we could not obtain a significant association of 3-adrenergic receptor variants with endometrial cancer risk in our samples. It is known that the frequency of the Trp64Arg missense mutation of the 3-adrenergic receptor differs between races.8 –10 The allelic frequency of the mutation of 0.31 in Pima Indians is the highest, and the association of 3-adrenergic receptor polymorphism with the onset of noninsulin-dependent diabetes mellitus in Pima Indians was reported. Ueda et al reported that the frequency of the Arg allele was about 0.20 in their study in which samples were from three different districts in Japan,21 whereas the frequency of the Arg allele in the present study was 0.10, suggesting that the frequency may also have a regional difference inside Japan. The lower allelic frequency of the mutation in our samples may have led to the absence of an association between the 3-adrenergic receptor polymorphism and endometrial cancer risk in the present study.
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We found that serum triglyceride and HDL cholesterol levels were significantly different between cases and controls. Weight, BMI, and the frequency of variations in the 3-adrenergic receptor gene, which influence lipid metabolism, did not differ between the two groups. Therefore, estrogen receptor gene variations might be associated with serum lipid levels, or it might simply be a secondary phenomenon attributed to the cancer itself. In fact, there is a report that cancer patients tend to have dyslipidemia.22 The present study has certain limitations. First, the sample size in the present study was relatively small. Second, our study was a hospital-based, case– control study, but not a population-based study, and the samples were from the Kagoshima prefecture only. However, genotype frequencies for the Pvu II and Xba I estrogen receptor ␣ polymorphisms in our study samples were not very different from those in other reports in Japan.23,24 In conclusion, the present exploratory analysis suggests an association between estrogen receptor ␣ polymorphisms and endometrial cancer risk in people living in the Kagoshima prefecture, Japan. Further extensive studies all over Japan are warranted to obtain more accurate findings.
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the  3-adrenergic receptor gene in Japanese subjects. Metabolism 1997;46:199–202. 22. Ostroumova MN, Kovalenko IG, Bershtein LM, Tsyrlina EV, Dilman VM. Characteristics of dyslipidemia in cancer patients. Vopr Onkol 1986;32:34–43. 23. Katsumata K, Nishizawa K, Unno A, Fujita Y, Tokita A. Association of gene polymorphisms and bone density in Japanese girls. J Bone Miner Metab 2002;20:164–9. 24. Kobayashi N, Fujino T, Shirogane T, Furuta I, Kobamatsu Y, Yaegashi M, et al. Estrogen receptor alpha polymor-
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phism as a genetic marker for bone loss, vertebral fractures and susceptibility to estrogen. Maturitas 2002;41:193–201. Address reprint requests to: Ichiro Iwamoto, MD, Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan; E-mail: [email protected]. Received November 27, 2002. Received in revised form March 14, 2003. Accepted April 17, 2003.
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