Association between single nucleotide polymorphisms of estrogen receptor {alpha} gene and efficacy of HRT on bone mineral density in post-menopausal Japanese women

T. Yahata1, J. Quan, N. Tamura, H. Nagata, T. Kurabayashi and K. Tanaka

Department of Obstetrics & Gynecology, Niigata University School of Medicine, 1-757 Asahimachi-dori, Niigata 951-8510, Japan

1 To whom correspondence should be addressed. Email: yahatat{at}med.niigata-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Although HRT for post-menopausal women can protect against bone loss, variations in bone responses exist. We studied whether single nucleotide polymorphisms (SNP) of the estrogen receptor-{alpha} (ER{alpha}) gene contribute to the effect of HRT on lumbar spine bone mineral density (BMD). METHODS: Subjects were 84 post-menopausal women who had been taking HRT for 3 years to treat osteopenia or osteoporosis. Eighteen SNP in the ER{alpha} gene were characterized by a single nucleotide primer extension assay. RESULTS: Genotyping of the 84 individuals revealed that all SNP were quite common, the minor allele frequency being ≥20%. A SNP in intron 6 (IVS6+14144) was significantly associated with the response to HRT for the first 3 years after starting treatment (P=0.043, 0.025 and 0.032 for the first, second and third years respectively). Haplotype analysis revealed that a combination of SNP IVS6+14144 and IVS4+4238 was significantly correlated with the response to HRT; women with haplotype G–G (IVS6 14144–IVS4 4238) showed a significantly higher response (P=0.014, 0.043 and 0.010 for the first second and third year respectively). CONCLUSIONS: These results suggest that a specific SNP and the haplotype of the selected SNP could be used to predict the effect of HRT on lumbar BMD.

Key words: bone mineral density/estrogen receptor {alpha}/haplotype/hormone replacement therapy/single nucleotide polymorphism


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Osteoporosis is a multifactorial disease with a strong genetic component. Genetic factors are thought to be responsible for up to 80% of variations in bone mineral density (BMD) (Dequecker et al., 1987Go; Slemenda et al., 1991Go). A number of candidate genes such as the estrogen receptor and vitamin D receptor (VDR) genes are reportedly associated with osteoporosis (Yamagata et al., 1994Go; Krall et al., 1995Go; Kobayashi et al., 1996Go; Tokita et al., 1996Go; Mizunuma et al., 1997Go; Sainz et al., 1997Go; Salmen et al., 2000Go; Albagha et al., 2001Go; Katsunuma et al., 2002Go; Palomba et al., 2003Go) suggesting that polymorphisms might be useful genetic markers for predicting the risk of osteoporosis. In a previous study of Japanese women, we found a TaqI polymorphism of the VDR gene to be associated with the effect of HRT at 1 year on lumbar spine BMD (Kurabayashi et al., 1999Go, 2004Go).

Estrogen plays a significant role in bone metabolism, and its deficiency after menopause is the main reason for accelerated bone loss and development of post-menopausal osteoporosis, which are preventable by estrogen administration. Although overall, post-menopausal HRT is effective in prevention of bone loss, individual variations in bone response exist (Hassager et al., 1994Go). Some post-menopausal women respond strongly to HRT, whereas ~8% who are compliant with therapy are nonetheless non-responders (Rosen and Kessenich, 1994Go). This raises the possibility that genetic determinants as well as gene–environment interactions might modulate bone responses to HRT in individual patients. The genotype of the estrogen receptor {alpha} (ER{alpha}) gene, a direct target of estrogen, is the most probable genetic determinant. In a recent study of post-menopausal Korean women, no association was found between ER{alpha} genotype and BMD response to estrogen (Han et al., 1997Go), although only two genotypes were analysed. The aim of this study was to determine whether single nucleotide polymorphisms (SNP) of the ER{alpha} gene contribute to the effect of HRT on lumbar spine BMD.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
The subjects were 84 Japanese women, ranging in age from 40 to 64 years (51.0 ± 0.7 years, mean ± SEM), who had been taking HRT for 3 years to manage osteopenia or osteoporosis. The diagnoses of osteopenia and osteoporosis were based on the criteria recommended by the Japanese Society of Bone and Mineral Research; that is, an L2–L4 BMD of <80 and <70% of the young adult (20–44 years) mean respectively. All women had low serum estradiol levels (<20 pg/ml) and elevated serum FSH levels (>50 mIU/ml). Exclusion criteria were: a history of metabolic disease (including hyperparathyroidism, previously diagnosed osteoporosis, or non-traumatic vertebral fracture on baseline X-ray); chronic disease (uncontrolled hypo- or hyperthyroidism, liver disease or unstable cardiac disease); cancer or thromboembolic disease or a history of either; current or past treatment with glucocorticoids for > 6 months; current or previous HRT use within the past 3 months. No subjects smoked or drank alcohol, and none engaged in regular strenuous exercise. Furthermore, none had a history of illness or medical therapy, apart from HRT, that might affect bone turnover. The subjects were not genetically related. HRT was administered in a sequential regimen consisting of 0.625 mg of conjugated equine estrogen (CEE) for 24 days (days 1 to 24) and 5 mg of medroxyprogesterone acetate (MPA) for 10 days (days 15 to 24), or in a continuous regimen consisting of 0.625 mg of CEE and 2.5 mg of MPA for 28 days.

Bone densitometry
BMD, expressed as mass per unit area (g/cm2), was measured in the anterior–posterior plane of the lumber spine (L2–L4), using dual-energy X-ray absorptiometry (DXA) with a QDR-2000 analyser (Hologic Inc., USA); absorptiometries were examined by the same observer. The average coefficients of variation (CV) of phantom measurements of bone mineral content (BMC), bone area (BA) and BMD during the study period were 1.1, 0.7 and 0.6% respectively. In addition, with the control women, the CV of the in vivo precision of BMD between two measurements (mean interval: 2.6 ± 1.2 years) was 0.9%. There was no scanner drift during the study period. BMD changes ({Delta}BMD) were expressed as the percentage of BMD change compared to the pre-treatment baseline.

Biochemical markers
Peripheral blood samples were collected in the early morning after an overnight fast. Serum pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen was measured with a radioimmunoassay kit (Orion Diagnostica, Finland).

DNA isolation and genotyping
Peripheral blood samples were collected after obtaining informed consent from each subject. Genomic DNA was extracted from peripheral blood leukocytes using a DNA purification kit (QIAamp DNA Blood Mini kit; Qiagen, USA) according to the manufacturer's instructions. At the time of conducting this study, 18 ER{alpha} SNP were available in the Japanese SNP database, JSNP (http://www.snp.ims.u-tokyo.ac.jp/indexja.html). We analysed all these SNP. All PCR were performed on a Perkin Elmer GeneAmp 9700 system and the presence of amplicons was checked on agarose gels. A single nucleotide primer extension assay was carried out to analyse SNP using a SNaPshot Kit (Applied Biosystems, USA). The extended primers were analysed on an ABI 3100 (Applied Biosystems). The primer sequences for the PCR and primer extension reactions are available in JSNP database. Initial denaturation was performed at 95°C for 2 min, followed by 35 cycles each consisting of denaturation at 95°C for 30 s, annealing at 60°C and extension at 72°C for 1 min, followed by final extension at 72°C for 8 min. This study was approved by the Niigata University Human Investigation Committee.

Statistical analysis
Differences in baseline characteristics and {Delta}BMD among genotypes were tested using analyses of variance (ANOVA). Linkage disequilibrium (LD) of all possible two-way combinations of SNP with the absolute value of the correlation coefficient was tested using r2 statistics; r2>0.18 was considered significant LD. Estimations of haplotype frequency were performed using SNPAlyze (DYNACOM, Japan) based on an expectation maximization algorithm and the maximum-likelihood approach. The duplotype of each subject was also estimated using SNPAlyze. Quantitative associations between genotype and {Delta}BMD were analysed through analyses of variance (ANOVA), with post hoc regression analysis. Differences in baseline characteristics and absolute BMD value among genotypes were tested using ANOVA with Fisher's protected least significant difference (PLSD) test. All data are expressed as the mean ± SEM. Differences at P<0.05 were considered statistically significant. All data management and statistical computations were performed with StatView 4.0 (Abacus Concepts, USA) and SNPAlyze.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Molecular variants and LD
In this study, we characterized 18 SNP in the ER{alpha} gene from a total of 168 chromosomes from 84 post-menopausal women. Frequencies of variant SNP alleles ranged from 20 to 47%. Genotypic distribution of IVS6+14144 deviated from Hardy–Weinberg equilibrium. All other SNP sites were in Hardy–Weinberg equilibrium (Table I). Figure 1 displays the results of the LD assessment of each SNP in the ER{alpha} gene. Pair-wise LD between SNP was performed for all possible two-way comparisons, according to r2 statistics. Individual LD values varied within small genomic regions, and a strong LD (r2>0.18) was verified in 70 of 306 pairs of SNP.


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Table I. Genotypes and allele frequencies of 18 single nucleotide polymorphisms (SNPS) in the ER{alpha} gene in Japanese subjects

 


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Figure 1. Analysis of linkage disequilibrium for all possible two-way comparisons of the 18 single nucleotide polymorphisms in the ER{alpha} gene. Indices of linkage equilibrium (r2) are presented; r2>0.18 are highlighted in grey.

 
Single-locus analysis
To test whether specific genetic variants of the ER{alpha} gene might be involved in the response to HRT, we conducted linear regression of the correlation between the percentage change of lumbar BMD ({Delta}BMD) after HRT for each of the 18 SNP. Table II shows that SNP IVS6+14144 in intron 6 of the ER{alpha} gene demonstrated a significant association with lumbar BMD response for the first 3 years since starting HRT. Mean changes in the BMD of all subjects at years 1, 2 and 3 of treatment were 3.3 ± 0.4, 4.4 ± 0.6 and 4.2 ± 0.7% respectively. Subjects with the homo (variant) genotype (GG) of IVS6+141441 showed significantly higher {Delta}BMD (5.90, 7.99 and 7.60% at years 1, 2 and 3 respectively) with HRT than those with the hetero (GA) and homo (AA; wild type) genotypes (Table III). The absolute value of BMD also showed significant difference among different genotype groups at years 2 and 3 of treatment. Measurements of serum pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (a suggested marker of bone absorption) did not differ between the three genotypes, but they decreased significantly in patients with the GG genotype after 1 year of HRT (P<0.018) (Tables III and IV).


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Table II. Changes in lumbar bone mineral density (BMD) according to single nucleotide polymorphisms (SNP) of the ER{alpha} gene during 3 years of HRT

 

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Table III. Changes in bone mineral density (BMD) after HRT according to IVS+14144 genotypes

 

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Table IV. Baseline characteristics according to the IVS6+14144 genotype

 
Although IVS6+14144 showed LD with other SNP in introns 6 and 7, no other SNP showed a significant correlation with percentage changes in lumbar BMD. There were no significant differences in baseline characteristics with genotype IVS6+14144 (Table IV) or any of the other genotypes tested in this study (data not shown).

Haplotype analyses
Haplotype frequencies for various marker combinations were estimated via an expectation maximization algorithm. We tested all combinations of two to four loci including IVS6+14144 that showed a significant single-locus association. The combinations of IVS6+14144/IVS1+3688 and IVS6+14144/IVS4+4238 demonstrated a significant correlation with the effect of HRT on lumbar spine BMD. The best combination with the lowest P-value was obtained with the combination of IVS6+14144 and IVS4+4238 (P=0.004) 3 years after HRT. Among the four haplotypes of IVS6+14144 (A->G)/IVS4+4238 (T->G) estimated by means of SNPAlyze, G–T and G–G showed significant correlations; a lower P-value was obtained with haplotype G–G. To analyse the chromosomal dosage of distinctive haplotypes and the associated response to HRT, a duplotype in each individual was estimated with SNPAlyze. This analysis revealed that duplotypes including haplotype G–G [in IVS6+14144 (A->G) and IVS4+4238 (T->G)] showed a higher percentage change in lumbar BMD (P=0.014 at 1 year, P=0.043 at 2 years, and P=0.010 at 3 years after HRT) than the other duplotypes (Figure 2).



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Figure 2. Changes in bone mineral density ({Delta}BMD) after HRT according to duplotypes of IVS6+14144 and IVS4+4238. {Delta}BMD is plotted against the year after starting HRT. Closed squares and circles indicate the mean values of {Delta}BMD for+/–, which possess one G–G (IVS6+14144-IVS4+4238) allele, and –/–, which possess no G–G (IVS6+14144–IVS4+4238) allele, respectively. Error bars indicate SE.

 
Cross-species comparison of the sequence around IVS6+14144
To test if IVS6+14144 is within a conserved non-coding sequence, we compared human and mouse genomic DNA sequences. Although the VISTA Genome Browser output for human/mouse sequence comparisons of ER{alpha}did not identify the sequence around IVS6+14144 as a candidate conserved non-coding sequence, the sequence around IVS6+14144 was relatively highly conserved with 50–75% identity over 400 bp. Next, we compared cross-species sequences using the UCSC Genome Browser. Figure 3 displays alignments of human, mouse and rat genomic sequences around IVS6+14144 (human Ch6: 152350754). A nucleotide corresponding to IVS6+14144 (human Ch6: 152350754) was found to be conserved among these three species.



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Figure 3. Alignments of human/mouse/rat genomic DNA sequences. Custom sequence comparisons with the human, mouse or rat genome assembly were performed using the UCSC Genome Browser. Indicated regions of human, mouse and rat DNA sequences are aligned. Asterisks indicate a conserved nucleotide among the three species. The nucleotide corresponding to IVS6+14144 (human Chr6: 152427394) is highlighted in grey.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Allelic variants of the ER{alpha} gene might alter its expression or function and account for the genetic variability observed, especially in women. Associations between numerous naturally occurring sequence variations of ER{alpha} and several clinical phenotypes have been studied, including the age of menarche (Stavrou et al., 2002Go), risk of breast cancer (Andersen et al., 1994Go), risk of spontaneous abortion (Lehrer et al., 1990Go; Berkowits et al., 1994Go), lipid levels (Matsubara et al., 1997Go; Kikuchi et al., 2000Go; Herrington et al., 2002Go), and BMD (Kobayashi et al., 1996Go; Mizunuma et al., 1997Go; Salmen et al., 2000Go; Albagha et al., 2001Go; Katsunuma et al., 2002Go; Matsushita et al., 2004Go). Most reports have focused on restriction fragment length polymorphisms (RFLP) such as XbaI and PvuII restriction enzyme polymorphisms. In two small clinical trials studying the effects of estrogen on BMD (Den et al., 1998Go; Ongphiphadhanakul et al., 2000Go), PvuII polymorphisms were shown to be associated with the effect of estrogen on lumbar spine BMD; however, a study of 248 Korean women found no such association (Han et al., 1997Go); information on these various SNP is available. Rather than focusing on these limited indirect marker variations, we chose to analyse multiple SNP spanning the ER{alpha} gene and available in the Japanese SNP database, JSNP (Hirakawa et al., 2002Go).

In this study, we characterized a total of 18 SNP in the ER{alpha} gene of 84 post-menopausal Japanese women and showed an association between a polymorphism in intron 6 (IVS6+14144) and the rate of change in BMD during HRT totalling 3 years. The genotype frequency distribution of IVS+14144 deviated from the Hardy–Weinberg equilibrium because of a minor homozygote excess. Possibly, IVS+14144 is an important candidate SNP prediposing to osteopenia or osteoporosis although the baseline BMD did not show significant difference between different genotypes in this study. HRT was associated with a positive change in lumbar BMD in those with the IVS6+14144 (GG) genotype; a strong response was seen with the GG genotype compared to the GA and AA genotypes.

The utility of SNP-based genetic association analyses for complex diseases is provided in previous studies (Martin et al., 2000aGo,bGo; Fallin et al., 2001Go). Single locus analyses have some utility in detecting associations between a disease and genotype, and it has also been suggested that a multilocus approach might be more powerful (Martin et al., 2000aGo; Fallin et al., 2001Go). By evaluating haplotypes, rather than conducting single-locus tests of association, the loss of information attributable to biallelic rather than multiallelic loci can be overcome, and possibly improved. Fallin et al. (2001)Go showed that an association between SNP and disease status could be detected via haplotype methods using SNP surrounding the functional allele even if the functional allele was not typed. We tested all combinations of two to four loci including the IVS6+14144 and showing a significant single-locus association. The combinations of IVS6+14144 and IVS4+4238 displayed significant correlations with the effect of HRT on lumbar spine BMD. Genetic variation, which influences BMD responses to HRT, might therefore be linked to this haplotype.

The molecular mechanisms by which the IVS6+14144 SNP genotype or IVS6 14144/IVS4+4238 haplotype might influence the effect of HRT on BMD remain uncertain. No SNP with amino acid substitutions have been reported for ER{alpha} genes. All SNP analysed in this study were in the exon or intron and showed no amino acid changes. They might influence alternative splicing, splicing efficiency, or mRNA turnover, resulting in significant changes in gene function; such mechanisms have been reported for other disease-causing genes (Gotoda et al., 1997Go; O'Neill et al., 1998Go). Intronic enhancer regions that augment gene transcription have been reported for other genes (Scohy et al., 2000Go); however, no such region has been described for ER{alpha}. By comparing genomic DNA sequences from diverse species using comparative sequence-based visualization tools and databases, functional elements might be recognized on the basis of their evolutionary conservation (Pennacchio and Rubin, 2003Go). Although the sequence around IVS6+14144 was not recognized as a conserved non-coding sequence [length > 80 bp with sequence identity >75% (Meisler, 2001Go)] when compared with a mouse genomic sequence, it seemed to be relatively highly conserved with 50–75% identity over 400 bp around IVS6+14144. Interestingly, a nucleotide corresponding to IVS6+14144 (human Ch6: 152350754) was shown to be conserved among human, mouse and rat genomes. These results raise the possibility that the non-coding sequence might be involved in gene regulation; for example, transcription, DNA replication, chromosomal pairing, and chromosome condensation. Another possibility is that the SNP analysed in this study are in disequilibrium with a polymorphism of ER{alpha} or another gene that influences the effect of HRT on BMD. Additional studies are necessary to clarify the precise mechanisms by which ER{alpha} gene polymorphisms modulate the responsiveness of BMD to HRT.

In summary, our genetic analyses of ER{alpha} variations, haplotype, and correlations between these features with regard to the effect of HRT on lumbar spine BMD, suggest that selected SNP in the ER{alpha} gene might act as a marker of drug response. Analysis of ER gene SNP might prove useful for selection of HRT for management of osteopenia or osteoporosis in post-menopausal women.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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