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
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Abstract |
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Key words:
bone mineral density/estrogen receptor /haplotype/hormone replacement therapy/single nucleotide polymorphism
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Introduction |
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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., 1994). Some post-menopausal women respond strongly to HRT, whereas
8% who are compliant with therapy are nonetheless non-responders (Rosen and Kessenich, 1994
). This raises the possibility that genetic determinants as well as geneenvironment interactions might modulate bone responses to HRT in individual patients. The genotype of the estrogen receptor
(ER
) 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
genotype and BMD response to estrogen (Han et al., 1997
), although only two genotypes were analysed. The aim of this study was to determine whether single nucleotide polymorphisms (SNP) of the ER
gene contribute to the effect of HRT on lumbar spine BMD.
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Materials and methods |
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Bone densitometry
BMD, expressed as mass per unit area (g/cm2), was measured in the anteriorposterior plane of the lumber spine (L2L4), 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 (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 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 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
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.
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Results |
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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 (AG)/IVS4+4238 (T
G) estimated by means of SNPAlyze, GT and GG showed significant correlations; a lower P-value was obtained with haplotype GG. 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 GG [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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Discussion |
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In this study, we characterized a total of 18 SNP in the ER 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 HardyWeinberg 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., 2000a,b
; Fallin et al., 2001
). 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., 2000a
; Fallin et al., 2001
). 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)
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 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., 1997
; O'Neill et al., 1998
). Intronic enhancer regions that augment gene transcription have been reported for other genes (Scohy et al., 2000
); however, no such region has been described for ER
. 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, 2003
). Although the sequence around IVS6+14144 was not recognized as a conserved non-coding sequence [length > 80 bp with sequence identity >75% (Meisler, 2001
)] when compared with a mouse genomic sequence, it seemed to be relatively highly conserved with 5075% 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
or another gene that influences the effect of HRT on BMD. Additional studies are necessary to clarify the precise mechanisms by which ER
gene polymorphisms modulate the responsiveness of BMD to HRT.
In summary, our genetic analyses of ER 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
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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