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Androgen receptor (AR) gene microsatellite polymorphism in postmenopausal women: correlation to bone mineral density and susceptibility to osteoporosis
European Journal of Obstetrics & Gynecology and Reproductive Biology 107 (2003) 52±56
Androgen receptor (AR) gene microsatellite polymorphism in postmenopausal women: correlation to bone mineral density and susceptibility to osteoporosis Huey-Yi Chena,e, Wen-Chi Chenb, Mei-Chen Wuc, Fuu-Jen Tsaic,d,*, Chang-Hai Tsaid a
Department of Obstetrics and Gynaecology, China Medical College Hospital, School of Medicine, China Medical College, Taichung, Taiwan b Department of Urology, China Medical College Hospital, School of Medicine, China Medical College, Taichung, Taiwan c Department of Medical Genetics, China Medical College Hospital, School of Medicine, No. 2 Yu-Der Road, Taichung 404, Taiwan d Department of Pediatrics, China Medical College Hospital, School of Medicine, China Medical College, Taichung, Taiwan e Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan Received 8 November 2001; received in revised form 21 May 2002; accepted 8 August 2002
Abstract Objective: To investigate the correlation of the androgen receptor gene microsatellite polymorphism (CAG trinucleotide repeat polymorphism on exon 1) with bone mineral density and their relationship to osteoporosis in postmenopausal women. Study design: A number of 168 of 477 postmenopausal women were randomly recruited. The androgen receptor gene microsatellite polymorphism was determined using polymerase chain reaction-based microsatellite analysis. Bone mineral density of the lumbar spine and proximal femur was measured using dual-energy X-ray absorptiometry. Results: The AR genotype was classi®ed from ``9'' to ``32'' according to the number of CAG trinucleotide repeats they contained to represent ``signposts''. After adjustment for potential confounding factors such as age, height, weight, years since menopause, and daily calcium intake, subjects with genotype 20 (n 64) had lower bone mineral density values and a signi®cantly greater risk for osteoporosis (OR 4.2, 95% CI 1.0±17.2) when compared with subjects with genotype 20 (n 104) at the femoral neck. Conclusion: The present study suggests that the androgen receptor gene microsatellite polymorphism may be a candidate genetic marker for risk of osteoporosis in postmenopausal women. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Androgen receptor gene microsatellite polymorphism; Bone mineral density; Polymerase chain reaction; Postmenopausal women; Osteoporosis
1. Introduction Osteoporosis is a common disabling age-related disease characterized by reduced bone mineral density (BMD), disorganization of skeletal integrity and microarchitecture, and by an increased risk of fragility fractures [1]. BMD is under strong genetic control, where as much as 60% of the variance in BMD has been attributed to genetic factors [2]. The results of at least one twin study have further suggested that genetics in¯uence age-related bone loss [3]. The androgen receptor (AR), a member of the steroid receptor superfamily, is involved in various biological processes such as sexual differentiation, maturation and spermatogenesis [4]. The gene is located at chromosome Xq11-q12 region and has a polymorphic cytosine, adenine, *
Corresponding author. Tel.: 886-4-22052121x7080; fax: 886-4-22033295. E-mail address: [email protected] (F.-J. Tsai).
and guanine (CAG) microsatellite on exon 1 which codes for variable-length glutamine repeats in the N-terminal domain of the AR protein [5]. Expansion of the polyglutamine segment in the male leads to spinal bulbar muscular atrophy [6], a fatal neuromuscular disease associated with low virilization, oligospermia or azoospermia, and reduced fertility [7,8]. Relatively short polyglutamine tract changes have been linked to an increased risk [9] or an earlier age of onset of prostate cancer [10], an androgen-dependent tumor. Mitsumori et al. demonstrated that shorter CAG alleles may be a genetic factor that promotes the growth of benign prostatic hyperplasia [11]. Androgens have long been recognized to play an important role in the normal development and physiology of bone and have profound in¯uences on bone development and metabolism [12]. The gene encoding AR is an important candidate for the determination of osteoporotic risk. However, no investigator has demonstrated the relationship between osteoporosis and CAG repeats in the AR gene. The
0301-2115/02/$ ± see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 2 ) 0 0 3 1 5 - 9
H.-Y. Chen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 107 (2003) 52±56
estrogen receptor (ER) a gene microsatellite polymorphism has been shown to be associated with BMD in postmenopausal women [13,14]. In this study, we aimed to investigate the correlation between the AR gene microsatellite polymorphism and BMD and their relationship to the prevalence of osteoporosis in postmenopausal women. 2. Materials and methods 2.1. Participants Between 1 November 1999 and 4 September 2000, there were 515 postmenopausal women (at least 12 months after last menstrual period) who presented for osteoporotic consultation in our department. Of these, 38 who met any one of the following criteria were excluded: (1) had undergone bilateral ovariectomies or had natural menopause occurring before the age of 40; (2) took drugs or had a history of taking drugs which affected bone metabolism, such as glucocorticoids, thyroxin, antiepileptics, bisphosphonates, calcitonin, or hormone replacement therapy for more than 4 months; (3) weighed more than 100 kg; (4) had disease, such as primary hyperparathyroidism, hyperthyroidism, diabetes, cirrhosis, and kidney failure. The exclusion of subjects with speci®ed diseases was made on the basis of medical history, laboratory ®ndings or both. 168 of 477 eligible postmenopausal women were randomly recruited. The computer generated random numbers were used to determine which in the consecutive sequence of the cohort should be approached. For each woman, a detailed medical history was obtained and her dietary calcium intake was assessed using a sequential questionnaire that included foods accounting for the majority of dietary calcium [15]. This study was approved by the medical ethics committee of the China Medical College Hospital, and written informed consent was obtained from participants. 2.2. Polymerase chain reaction-based microsatellite analysis Polymerase chain reactions (PCRs) were carried out in a total volume of 50 ml, containing genomic DNA; 2±6 pmole of each primer; 1X Taq polymerase buffer (1.5 mM MgCl2); and 0.25 units of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, USA). The genomic DNA was prepared from peripheral blood using a DNA Extractor WB kit (Wako, Japan). The CAG region of the AR gene was ampli®ed by PCR. The primers used for PCR were designed as follows: upstream, 50 -TGCGCGAAGTGATCCAGAAC-30 ; downstream, 50 -CTTGGGGAGAACCATC CTCA-30 [11]. PCR ampli®cation was performed in a programmable thermal cycler GeneAmp PCR System 2400 (Applied Biosystems, Foster City, CA, USA). The cycling condition for the AR gene microsatellite polymorphism was set as follows: one cycle at 94 8C for 2 min, 35 cycles at
53
94 8C for 10 s, 60 8C for 45 s, and 72 8C for 20 s, and one ®nal cycle of extension at 72 8C for 10 min. Appropriate amounts of PCR products (0.75 ml) were mixed with 1.75 ml of a premixed buffer solution [formamide: loading buffer (blue dextran, 50 mg/ml; EDTA, 25 mM): standard 5:1:1]. Genescan-350 TAMRA (6-carboxy-tetramethylrhodamine, red) (Applied Biosystems, Foster City, CA, USA) was used as the reference standard. Electrophoresis was performed using a 6% denaturing polyacrylamide gel and an ABI Prism 377 DNA Sequencer. The sizes of the microsatellite-containing DNA fragments were measured using GeneScan Analysis 3.0 software (Applied Biosystems, Foster City, CA, USA). 2.3. Assessments of bone mineral density BMD of the lumbar spine (L2±L4) and the right femoral neck were measured using dual-energy X-ray absorptiometry (DPX-L densitometer, Lunar, Madison, Wis). Osteoporosis was de®ned according to the classi®cations of the World Health Organization [16]. The diagnosis of osteoporosis was made if a patient's BMD value was 2.5 S.D. or more than 2.5 S.D. below the mean of premenopausal Chinese women in Taiwan, at the lumbar spine or the right femoral neck [17]. 2.4. Statistical analysis BMD and other relevant clinical variables of the various genotypic groups were compared by Student's t-test. Multiple linear regression was used to adjust BMD values for confounding factors such as age, anthropometric variables, and calcium intake. Odds ratios (with 95% con®dence intervals) for the susceptibility to osteoporosis were calculated in the subjects with different AR genotypes using multivariate logistic regression model. All statistical tests were two-sided. A P-value less than 0.05 was considered statistically signi®cant. All calculations were performed by the Statistical Package for Social Sciences (SPSS for Windows, release 8.0, SPSS Inc., Chicago, IL, USA). 3. Results The size of the PCR products ranged in length from 168 base pairs (bp) (containing 9 CAG repeats with 141 bp of ampli®ed ¯anking sequences) to 237 bp (32 CAG repeats). Twenty-four alleles were classi®ed between ``allele 9'' and ``allele 32'' according to the number of trinucleotide repeats they contained. The frequency distribution of trinucleotide repeat polymorphism in the 168 postmenopausal women (336 chromosomes) is shown in Fig. 1, with one peak at allele 20 (201 bp). Presence or absence of alleles were denoted by a plus or minus sign after the allele number (e.g. genotype 9, genotype 9 ). We assessed the relationship between the AR genotype and BMD from genotype 9 to 32. Of these,
54
H.-Y. Chen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 107 (2003) 52±56
Fig. 1. The frequency distribution of the androgen receptor gene trinucleotide (CAG) repeat polymorphism in 168 postmenopausal women (336 chromosomes). PCR products ranged in length from 168 bp (9 repeats, allele 9) to 237 bp (32 repeats, allele 32).
only genotype 20 was found to be associated with susceptibility to osteoporosis. Table 1 shows anthropometric data, daily calcium intake, and BMD according to presence or lack of allele 20 (genotype 20; genotype 20 ). The two genotypic groups did not differ signi®cantly in age, height, Table 1 Characteristics of postmenopausal women according to presence or lack of allele 20 (genotype 20; genotype 20 )a Characteristic
AR CAG trinucleotide repeats genotype 20
Age (year) Height (cm) Weight (kg) Years since menopause Daily calcium intake (mg)
(n 104)
54.23 155.47 55.87 6.06 532
6.24 5.72 7.72 5.61 152
P-value
20 (n 64) 54.34 156.03 58.05 5.16 528
5.37 5.29 8.99 4.01 149
0.903 0.541 0.130 0.283 0.878
Bone mineral density (g/cm2) Lumbar spine Unadjusted Age-adjusted Adjustedb
1.00 0.16 1.00 0.01 0.99 0.02
0.97 0.16 0.97 0.02 0.95 0.02
0.287 0.287 0.176
Femoral neck Unadjusted Age-adjusted Adjustedb
0.81 0.11 0.80 0.01 0.80 0.01
0.78 0.11 0.79 0.01 0.78 0.01
0.246 0.321 0.264
a
Values are mean S:D:; statistical analysis with Student's t-test. Values are adjusted for age, height, weight, years since menopause, and daily calcium intake. b
weight, years since menopause, daily calcium intake, or BMD (unadjusted or age-adjusted). As shown in Table 1, BMD of the lumbar spine and the femoral neck for 168 postmenopausal women was adjusted for age, height, weight, years since menopause, and daily calcium intake. Subjects with genotype 20 had greater BMD than subjects with genotype 20 at the lumbar spine and at the femoral neck, but these differences did not reach statistical signi®cance. After adjustment for potential confounding factors including age, height, weight, years since menopause, and daily calcium intake in multivariate logistic regression model, genotype 20 and genotype 20 showed a signi®cant effect on the prevalence of osteoporosis at the femoral neck. Women with genotype 20 had a 4.2 times greater risk for osteoporosis than women with genotype 20 (P < 0:05) (Table 2). No correlations between mean number of CAG Table 2 Odds ratios of genotype 20 and genotype 20 showing the susceptibility to osteoporosis among postmenopausal women Genotype
20 20 a
Osteoporosis at lumbar spine, odds ratio (95% CI)a
Osteoporosis at femoral neck; odds ratio (95% CI)a
Age-adjusted
Multivariate
Age-adjusted
Multivariate
1.0 1.7 (0.7±3.9)
1.0 2.3 (0.8±6.9)
1.0 2.6 (0.9±7.7)
1.0 4.2* (1.0±17.2)
CI denotes confidence interval. Odds ratios have been adjusted for age, height, weight, years since menopause, and daily calcium intake. * P < 0:05.
H.-Y. Chen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 107 (2003) 52±56
55
Fig. 2. Bone mineral density of the lumbar spine (panel A) and femoral neck (panel B) in 168 postmenopausal women according to mean number of CAG trinucleotide repeats. The solid bar represents the upper 95% confidence limit. The number of subjects in each group is given inside the columns.
trinucleotide repeats and lumbar and femoral neck BMD values were observed (Figs. 2A and B). 4. Discussion Osteoporosis in postmenopausal women is a multifactorial disease in which genetic determinants are modulated by hormonal, environmental and nutritional factors. Many candidate genes have been implicated in the determination of BMD and in the pathogenesis of osteoporosis [14]. Although polymorphisms of candidate genes are not directly linked to a certain disease, they are useful tools in the study of multifactorial disorders [18,19]. Polymorphisms, such as those for the vitamin D receptor gene, ER a gene, and the
calcitonin receptor gene, have been related to BMD in some studies [13,14,20,21]. BMD values were confounded by age, weight, height, years since menopause, or daily calcium intake. The best way to show the relationship between BMD and AR gene microsatellite polymorphism is to use adjusted BMD values or to perform a multiple linear regression adjusted for age, weight, height, years since menopause, and daily calcium intake. Given the role of androgen in bone metabolism, the gene encoding AR is an important candidate for the determination of osteoporotic risk. Increased CAG repeat length for the AR is therefore likely to have an effect on bone metabolism and risk of osteoporosis. The AR genotype was classi®ed from ``9'' to ``32'' according to the number of CAG trinucleotide repeats they contained to represent ``signposts''. Our results
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H.-Y. Chen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 107 (2003) 52±56
found that subjects with genotype 20 had lower BMD values and a signi®cantly greater risk for osteoporosis (OR 4.2, 95% CI 1.0±17.2) when compared with subjects with genotype 20 at the femoral neck, after adjustment for potential confounding factors such as age, height, weight, years since menopause, and daily calcium intake. As such, a cutoff value of 20 repeats was considered to be appropriate for the analyses. The presence of AR has been described in human osteoblastic cells as well as in human osteoclasts, suggesting that androgen exerts direct effects on and plays important roles in bone cells [22]. The present study shows that the AR gene microsatellite polymorphism is associated with reduced BMD and predisposes women to osteoporosis at the femoral neck. Power analysis showed that the distribution of different AR genotypes on the development of osteoporosis in 168 postmenopausal women have a power of over 75%. We postulate that the AR gene microsatellite polymorphism may contribute to the pathogenesis of osteoporosis at the femoral neck in our population. The molecular mechanism underlying how bone mineralization is affected by the variation in the number of trinucleotide repeats is still unclear. However, decreased functional competence of AR with longer glutamine tracts may be attributed to reduced trans-activation [23], which could, in turn, reduce bone mineralization. Further research is needed to determine whether the different modulation of the AR gene in functionally different androgen target cells occurs at the gene transcription level by the use of distinct promoters, or by alternative splicing of large RNA precursors, or even a combination of these mechanisms. In conclusion, the results of our study suggest that the AR gene microsatellite polymorphism may be one of the candidate genetic markers responsible for osteoporosis in postmenopausal women. Further study of other possible genetic markers with regard to osteoporosis might provide new information to the genetic background underlying BMD in women. Acknowledgements The authors would like to thank the National Science Council of the Republic of China and the China Medical College Hospital for ®nancially supporting this research under Contract NSC 89-2314-B-039-023 and DMR-91-051. References [1] Keen RW, Kelly PJ. Genetic factors in osteoporosis: what are the implications for prevention and treatment? Drug Aging 1997;11: 333±7. [2] Pocock NA, Eisman JA, Hopper JL, et al. Genetic determinants of bone mass in adults. J Clin Invest 1987;80:706±10. [3] Kelly PJ, Nguyen TV, Hopper JL, et al. Changes in axial bone density with age: a twin study. J Bone Miner Res 1993;8:11±7.
[4] Wieacker PF, Knoke I, Jakubiczka S. Clinical and molecular aspects of androgen receptor defects. Exp Clin Endocrinol Diabetes 1998;106:446±53. [5] Edwards A, Hammond HA, Jin L, Caskey CT, Chakraborty R. Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 1992;12:241±53. [6] La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 1991;352:77±9. [7] Arbizu T, Santamaria J, Gomex JM, Quilez A, Serra JP. A family with adult spinal and bulbar muscular atrophy, X-linked inheritance and associated testicular failure. J Neurol Sci 1983;59:371±82. [8] Nagashima T, Seko K, Hirose K. Familial bulbo-spinal muscular atrophy associated testicular atrophy and sensory neuropathy (Kennedy-Alter-Sung syndrome): autopsy case report of two brothers. J Neurol Sci 1988;87:141±52. [9] Irvine RA, Yu MC, Ross RK, Coetzee GA. The CAG and GGC microsatellites of the androgen receptor gene are in linkage disequilibrium in men with prostate cancer. Cancer Res 1994; 54:2861±4. [10] Hardy DO, Scher HI, Bogenreider T, et al. Androgen receptor CAG repeat lengths in prostate cancer: correlation with age of onset. J Clin Endocrinol Metab 1996;81:4400±5. [11] Mitsumori K, Terai A, Oka H, et al. Androgen receptor CAG repeat length polymorphism in benign prostatic hyperplasia (BPH): correlation with adenoma growth. Prostate 1999;41:253±7. [12] Oursler MJ, Kassem M, Riggs BL, Spelsberg TC. Androgen. In: Marcus, Feldman, Kelsey, editors. Osteoporosis. New York: Academic Press; 1996. p. 237±60. [13] Sano M, Inoue S, Hosoi T, et al. Association of estrogen receptor nucleotide repeat polymorphism with osteoporosis. Biochem Biophys Res Commun 1995;217:378±83. [14] Becherini L, Gennari L, Masi L, et al. Evidence of a linkage disequilibrium between polymorphisms in the human estrogen receptor a gene and their relationship to bone mass variation in postmenopausal Italian women. Hum Mol Genet 2000;9:2043±50. [15] Pun KK, Wong FH, Lok T. Rapid postmenopausal loss of total body and regional bone mass in normal Southern Chinese females in Hong Kong. Osteoporos Int 1991;1:87±94. [16] Report of a World Health Organization Study Group: assessment of fracture risk and its application to screening for postmenopausal osteoporosis. WHO Technical Report Series 843. Geneva: WHO; 1994. [17] Tsai KS, Huang KM, Chieng PU, Su CT. Bone mineral density of normal Chinese women in Taiwan. Calcif Tissue Int 1991;48:161±6. [18] Anderson TI, Heimdal KR, Skrede M, Tveit K, Berg K, Borresen AL. Oestrogen receptore (ESR) polymorphisms and breast cancer susceptibility. Hum Genet 1994;94:665±70. [19] Lehrer SP, Schmutzler TK, Rabin JM, Schachter BS. An estrogen receptor genetic polymorphism and a history of spontaneous abortions: correlation in women with estrogen receptor positive breast cancer but not in women with estrogen receptor negative breast cancer or in women without cancer. Breast Cancer Res Treat 1993;26:175±80. [20] Morrison NA, Qi JC, Tokita A, et al. Prediction of bone density from vitamin D receptor alleles. Nature 1994;367:284±7. [21] Masi L, Becherini L, Gennari L, et al. Allelic variants of human calcitonin receptor: distribution and association with bone mass in postmenopausal Italian women. Biochem Biophys Res Comm 1998;245:622±6. [22] Pederson L, Kremer M, Judd J, et al. Androgens regulate bone resorption activity of isolated osteoclasts in vitro. Proc Natl Acad Sci USA 1999;96:505±10. [23] Tut TG, Ghadessy FJ, Trifiro MA, Pinsky L, Yong EL. Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impaired sperm production, and male infertility. J Clin Endocrinol Metab 1997;82:3777±82.
Androgen receptor (AR) gene microsatellite polymorphism in postmenopausal women: correlation to bone mineral density and susceptibility to osteoporosis Huey-Yi Chena,e, Wen-Chi Chenb, Mei-Chen Wuc, Fuu-Jen Tsaic,d,*, Chang-Hai Tsaid a
Department of Obstetrics and Gynaecology, China Medical College Hospital, School of Medicine, China Medical College, Taichung, Taiwan b Department of Urology, China Medical College Hospital, School of Medicine, China Medical College, Taichung, Taiwan c Department of Medical Genetics, China Medical College Hospital, School of Medicine, No. 2 Yu-Der Road, Taichung 404, Taiwan d Department of Pediatrics, China Medical College Hospital, School of Medicine, China Medical College, Taichung, Taiwan e Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan Received 8 November 2001; received in revised form 21 May 2002; accepted 8 August 2002
Abstract Objective: To investigate the correlation of the androgen receptor gene microsatellite polymorphism (CAG trinucleotide repeat polymorphism on exon 1) with bone mineral density and their relationship to osteoporosis in postmenopausal women. Study design: A number of 168 of 477 postmenopausal women were randomly recruited. The androgen receptor gene microsatellite polymorphism was determined using polymerase chain reaction-based microsatellite analysis. Bone mineral density of the lumbar spine and proximal femur was measured using dual-energy X-ray absorptiometry. Results: The AR genotype was classi®ed from ``9'' to ``32'' according to the number of CAG trinucleotide repeats they contained to represent ``signposts''. After adjustment for potential confounding factors such as age, height, weight, years since menopause, and daily calcium intake, subjects with genotype 20 (n 64) had lower bone mineral density values and a signi®cantly greater risk for osteoporosis (OR 4.2, 95% CI 1.0±17.2) when compared with subjects with genotype 20 (n 104) at the femoral neck. Conclusion: The present study suggests that the androgen receptor gene microsatellite polymorphism may be a candidate genetic marker for risk of osteoporosis in postmenopausal women. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Androgen receptor gene microsatellite polymorphism; Bone mineral density; Polymerase chain reaction; Postmenopausal women; Osteoporosis
1. Introduction Osteoporosis is a common disabling age-related disease characterized by reduced bone mineral density (BMD), disorganization of skeletal integrity and microarchitecture, and by an increased risk of fragility fractures [1]. BMD is under strong genetic control, where as much as 60% of the variance in BMD has been attributed to genetic factors [2]. The results of at least one twin study have further suggested that genetics in¯uence age-related bone loss [3]. The androgen receptor (AR), a member of the steroid receptor superfamily, is involved in various biological processes such as sexual differentiation, maturation and spermatogenesis [4]. The gene is located at chromosome Xq11-q12 region and has a polymorphic cytosine, adenine, *
Corresponding author. Tel.: 886-4-22052121x7080; fax: 886-4-22033295. E-mail address: [email protected] (F.-J. Tsai).
and guanine (CAG) microsatellite on exon 1 which codes for variable-length glutamine repeats in the N-terminal domain of the AR protein [5]. Expansion of the polyglutamine segment in the male leads to spinal bulbar muscular atrophy [6], a fatal neuromuscular disease associated with low virilization, oligospermia or azoospermia, and reduced fertility [7,8]. Relatively short polyglutamine tract changes have been linked to an increased risk [9] or an earlier age of onset of prostate cancer [10], an androgen-dependent tumor. Mitsumori et al. demonstrated that shorter CAG alleles may be a genetic factor that promotes the growth of benign prostatic hyperplasia [11]. Androgens have long been recognized to play an important role in the normal development and physiology of bone and have profound in¯uences on bone development and metabolism [12]. The gene encoding AR is an important candidate for the determination of osteoporotic risk. However, no investigator has demonstrated the relationship between osteoporosis and CAG repeats in the AR gene. The
0301-2115/02/$ ± see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 2 ) 0 0 3 1 5 - 9
H.-Y. Chen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 107 (2003) 52±56
estrogen receptor (ER) a gene microsatellite polymorphism has been shown to be associated with BMD in postmenopausal women [13,14]. In this study, we aimed to investigate the correlation between the AR gene microsatellite polymorphism and BMD and their relationship to the prevalence of osteoporosis in postmenopausal women. 2. Materials and methods 2.1. Participants Between 1 November 1999 and 4 September 2000, there were 515 postmenopausal women (at least 12 months after last menstrual period) who presented for osteoporotic consultation in our department. Of these, 38 who met any one of the following criteria were excluded: (1) had undergone bilateral ovariectomies or had natural menopause occurring before the age of 40; (2) took drugs or had a history of taking drugs which affected bone metabolism, such as glucocorticoids, thyroxin, antiepileptics, bisphosphonates, calcitonin, or hormone replacement therapy for more than 4 months; (3) weighed more than 100 kg; (4) had disease, such as primary hyperparathyroidism, hyperthyroidism, diabetes, cirrhosis, and kidney failure. The exclusion of subjects with speci®ed diseases was made on the basis of medical history, laboratory ®ndings or both. 168 of 477 eligible postmenopausal women were randomly recruited. The computer generated random numbers were used to determine which in the consecutive sequence of the cohort should be approached. For each woman, a detailed medical history was obtained and her dietary calcium intake was assessed using a sequential questionnaire that included foods accounting for the majority of dietary calcium [15]. This study was approved by the medical ethics committee of the China Medical College Hospital, and written informed consent was obtained from participants. 2.2. Polymerase chain reaction-based microsatellite analysis Polymerase chain reactions (PCRs) were carried out in a total volume of 50 ml, containing genomic DNA; 2±6 pmole of each primer; 1X Taq polymerase buffer (1.5 mM MgCl2); and 0.25 units of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, USA). The genomic DNA was prepared from peripheral blood using a DNA Extractor WB kit (Wako, Japan). The CAG region of the AR gene was ampli®ed by PCR. The primers used for PCR were designed as follows: upstream, 50 -TGCGCGAAGTGATCCAGAAC-30 ; downstream, 50 -CTTGGGGAGAACCATC CTCA-30 [11]. PCR ampli®cation was performed in a programmable thermal cycler GeneAmp PCR System 2400 (Applied Biosystems, Foster City, CA, USA). The cycling condition for the AR gene microsatellite polymorphism was set as follows: one cycle at 94 8C for 2 min, 35 cycles at
53
94 8C for 10 s, 60 8C for 45 s, and 72 8C for 20 s, and one ®nal cycle of extension at 72 8C for 10 min. Appropriate amounts of PCR products (0.75 ml) were mixed with 1.75 ml of a premixed buffer solution [formamide: loading buffer (blue dextran, 50 mg/ml; EDTA, 25 mM): standard 5:1:1]. Genescan-350 TAMRA (6-carboxy-tetramethylrhodamine, red) (Applied Biosystems, Foster City, CA, USA) was used as the reference standard. Electrophoresis was performed using a 6% denaturing polyacrylamide gel and an ABI Prism 377 DNA Sequencer. The sizes of the microsatellite-containing DNA fragments were measured using GeneScan Analysis 3.0 software (Applied Biosystems, Foster City, CA, USA). 2.3. Assessments of bone mineral density BMD of the lumbar spine (L2±L4) and the right femoral neck were measured using dual-energy X-ray absorptiometry (DPX-L densitometer, Lunar, Madison, Wis). Osteoporosis was de®ned according to the classi®cations of the World Health Organization [16]. The diagnosis of osteoporosis was made if a patient's BMD value was 2.5 S.D. or more than 2.5 S.D. below the mean of premenopausal Chinese women in Taiwan, at the lumbar spine or the right femoral neck [17]. 2.4. Statistical analysis BMD and other relevant clinical variables of the various genotypic groups were compared by Student's t-test. Multiple linear regression was used to adjust BMD values for confounding factors such as age, anthropometric variables, and calcium intake. Odds ratios (with 95% con®dence intervals) for the susceptibility to osteoporosis were calculated in the subjects with different AR genotypes using multivariate logistic regression model. All statistical tests were two-sided. A P-value less than 0.05 was considered statistically signi®cant. All calculations were performed by the Statistical Package for Social Sciences (SPSS for Windows, release 8.0, SPSS Inc., Chicago, IL, USA). 3. Results The size of the PCR products ranged in length from 168 base pairs (bp) (containing 9 CAG repeats with 141 bp of ampli®ed ¯anking sequences) to 237 bp (32 CAG repeats). Twenty-four alleles were classi®ed between ``allele 9'' and ``allele 32'' according to the number of trinucleotide repeats they contained. The frequency distribution of trinucleotide repeat polymorphism in the 168 postmenopausal women (336 chromosomes) is shown in Fig. 1, with one peak at allele 20 (201 bp). Presence or absence of alleles were denoted by a plus or minus sign after the allele number (e.g. genotype 9, genotype 9 ). We assessed the relationship between the AR genotype and BMD from genotype 9 to 32. Of these,
54
H.-Y. Chen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 107 (2003) 52±56
Fig. 1. The frequency distribution of the androgen receptor gene trinucleotide (CAG) repeat polymorphism in 168 postmenopausal women (336 chromosomes). PCR products ranged in length from 168 bp (9 repeats, allele 9) to 237 bp (32 repeats, allele 32).
only genotype 20 was found to be associated with susceptibility to osteoporosis. Table 1 shows anthropometric data, daily calcium intake, and BMD according to presence or lack of allele 20 (genotype 20; genotype 20 ). The two genotypic groups did not differ signi®cantly in age, height, Table 1 Characteristics of postmenopausal women according to presence or lack of allele 20 (genotype 20; genotype 20 )a Characteristic
AR CAG trinucleotide repeats genotype 20
Age (year) Height (cm) Weight (kg) Years since menopause Daily calcium intake (mg)
(n 104)
54.23 155.47 55.87 6.06 532
6.24 5.72 7.72 5.61 152
P-value
20 (n 64) 54.34 156.03 58.05 5.16 528
5.37 5.29 8.99 4.01 149
0.903 0.541 0.130 0.283 0.878
Bone mineral density (g/cm2) Lumbar spine Unadjusted Age-adjusted Adjustedb
1.00 0.16 1.00 0.01 0.99 0.02
0.97 0.16 0.97 0.02 0.95 0.02
0.287 0.287 0.176
Femoral neck Unadjusted Age-adjusted Adjustedb
0.81 0.11 0.80 0.01 0.80 0.01
0.78 0.11 0.79 0.01 0.78 0.01
0.246 0.321 0.264
a
Values are mean S:D:; statistical analysis with Student's t-test. Values are adjusted for age, height, weight, years since menopause, and daily calcium intake. b
weight, years since menopause, daily calcium intake, or BMD (unadjusted or age-adjusted). As shown in Table 1, BMD of the lumbar spine and the femoral neck for 168 postmenopausal women was adjusted for age, height, weight, years since menopause, and daily calcium intake. Subjects with genotype 20 had greater BMD than subjects with genotype 20 at the lumbar spine and at the femoral neck, but these differences did not reach statistical signi®cance. After adjustment for potential confounding factors including age, height, weight, years since menopause, and daily calcium intake in multivariate logistic regression model, genotype 20 and genotype 20 showed a signi®cant effect on the prevalence of osteoporosis at the femoral neck. Women with genotype 20 had a 4.2 times greater risk for osteoporosis than women with genotype 20 (P < 0:05) (Table 2). No correlations between mean number of CAG Table 2 Odds ratios of genotype 20 and genotype 20 showing the susceptibility to osteoporosis among postmenopausal women Genotype
20 20 a
Osteoporosis at lumbar spine, odds ratio (95% CI)a
Osteoporosis at femoral neck; odds ratio (95% CI)a
Age-adjusted
Multivariate
Age-adjusted
Multivariate
1.0 1.7 (0.7±3.9)
1.0 2.3 (0.8±6.9)
1.0 2.6 (0.9±7.7)
1.0 4.2* (1.0±17.2)
CI denotes confidence interval. Odds ratios have been adjusted for age, height, weight, years since menopause, and daily calcium intake. * P < 0:05.
H.-Y. Chen et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 107 (2003) 52±56
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Fig. 2. Bone mineral density of the lumbar spine (panel A) and femoral neck (panel B) in 168 postmenopausal women according to mean number of CAG trinucleotide repeats. The solid bar represents the upper 95% confidence limit. The number of subjects in each group is given inside the columns.
trinucleotide repeats and lumbar and femoral neck BMD values were observed (Figs. 2A and B). 4. Discussion Osteoporosis in postmenopausal women is a multifactorial disease in which genetic determinants are modulated by hormonal, environmental and nutritional factors. Many candidate genes have been implicated in the determination of BMD and in the pathogenesis of osteoporosis [14]. Although polymorphisms of candidate genes are not directly linked to a certain disease, they are useful tools in the study of multifactorial disorders [18,19]. Polymorphisms, such as those for the vitamin D receptor gene, ER a gene, and the
calcitonin receptor gene, have been related to BMD in some studies [13,14,20,21]. BMD values were confounded by age, weight, height, years since menopause, or daily calcium intake. The best way to show the relationship between BMD and AR gene microsatellite polymorphism is to use adjusted BMD values or to perform a multiple linear regression adjusted for age, weight, height, years since menopause, and daily calcium intake. Given the role of androgen in bone metabolism, the gene encoding AR is an important candidate for the determination of osteoporotic risk. Increased CAG repeat length for the AR is therefore likely to have an effect on bone metabolism and risk of osteoporosis. The AR genotype was classi®ed from ``9'' to ``32'' according to the number of CAG trinucleotide repeats they contained to represent ``signposts''. Our results
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found that subjects with genotype 20 had lower BMD values and a signi®cantly greater risk for osteoporosis (OR 4.2, 95% CI 1.0±17.2) when compared with subjects with genotype 20 at the femoral neck, after adjustment for potential confounding factors such as age, height, weight, years since menopause, and daily calcium intake. As such, a cutoff value of 20 repeats was considered to be appropriate for the analyses. The presence of AR has been described in human osteoblastic cells as well as in human osteoclasts, suggesting that androgen exerts direct effects on and plays important roles in bone cells [22]. The present study shows that the AR gene microsatellite polymorphism is associated with reduced BMD and predisposes women to osteoporosis at the femoral neck. Power analysis showed that the distribution of different AR genotypes on the development of osteoporosis in 168 postmenopausal women have a power of over 75%. We postulate that the AR gene microsatellite polymorphism may contribute to the pathogenesis of osteoporosis at the femoral neck in our population. The molecular mechanism underlying how bone mineralization is affected by the variation in the number of trinucleotide repeats is still unclear. However, decreased functional competence of AR with longer glutamine tracts may be attributed to reduced trans-activation [23], which could, in turn, reduce bone mineralization. Further research is needed to determine whether the different modulation of the AR gene in functionally different androgen target cells occurs at the gene transcription level by the use of distinct promoters, or by alternative splicing of large RNA precursors, or even a combination of these mechanisms. In conclusion, the results of our study suggest that the AR gene microsatellite polymorphism may be one of the candidate genetic markers responsible for osteoporosis in postmenopausal women. Further study of other possible genetic markers with regard to osteoporosis might provide new information to the genetic background underlying BMD in women. Acknowledgements The authors would like to thank the National Science Council of the Republic of China and the China Medical College Hospital for ®nancially supporting this research under Contract NSC 89-2314-B-039-023 and DMR-91-051. References [1] Keen RW, Kelly PJ. Genetic factors in osteoporosis: what are the implications for prevention and treatment? Drug Aging 1997;11: 333±7. [2] Pocock NA, Eisman JA, Hopper JL, et al. Genetic determinants of bone mass in adults. J Clin Invest 1987;80:706±10. [3] Kelly PJ, Nguyen TV, Hopper JL, et al. Changes in axial bone density with age: a twin study. J Bone Miner Res 1993;8:11±7.
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