1 Department of Public Health, Kinki University School of Medicine 3772 Ohno-Higashi, Osaka-Sayama, Osaka 5898511, Japan
2 Department of Public Health, Nara Medical University, Kashihara, Japan
3 Department of Welfare Promotion and Epidemiology, Toyama Medical and Pharmaceutical University, Toyama, Japan
4 Kagawa Nutrition University, Tokyo, Japan
5 Institute of Comprehensive Community Care, Tokyo, Japan
6 Kasukabe Shuuwa Hospital, Kasukabe, Japan
7 Second Department of Internal Medicine, Graduate School of Tokyo Medical and Dental University, Tokyo, Japan
Correspondence: Akemi Morita, Department of Public Health, Kinki University School of Medicine, 3772 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan. E-mail: akemi{at}med.kindai.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods Fifty women were randomly selected from each of the 5-year age stratified populations (1579 years) in each of the three municipalities examined, as a part of the Japanese population-based osteoporosis (JPOS) baseline study in 1996. In the study, BMD at the lumbar spine, hip, and distal forearm were measured using dual energy X-ray absorptiometry (DXA). Two polymorphisms were determined in the VDR gene locus identified by the restriction endonucleases ApaI and TaqI through a novel allele discrimination method using two different allele-specific fluorescence probes. The other VDR gene polymorphism was identified by the restriction endonuclease FokI using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Changes in BMD were determined in a follow-up study 3 years after the baseline study.
Results After the exclusion of women who had any medical or menstrual history affecting BMD, 1434 women were analysed. The annual per cent changes in BMD at the lumbar spine over 3 years in subjects with tt genotype in the TaqI polymorphism were different from other genotypes, both in the women who were premenopausal at the follow-up survey (F-premenopausal women) and in the women who were postmenopausal at the baseline survey (B-postmenopausal women). However, the effects of tt genotype on change in BMD were opposite in the two groups. In addition, these associations or tendencies were not observed at the different skeletal sites.
Conclusions This study revealed that none of the individual VDR gene polymorphisms displayed consistent association with baseline BMD or BMD change. Therefore, the effect of the VDR genotype on bone mass is negligible in Japanese women.
Accepted 6 May 2004
Osteoporotic fracture is one of the leading causes of disability in elderly people in developed countries, including Japan.13 The prevention of osteoporosis is of great importance, both in maintaining the quality of life of elderly people and in reducing medical expenditures for the treatment of fractures.46 Low bone mineral density (BMD) is the most prominent risk factor for osteoporotic fractures. Previous twin and family studies have suggested that genetic factors play an important role in regulating BMD.79 In 1994, Morrison et al. reported a strong relationship between BMD and restriction fragment length polymorphism (RFLP) based on BsmI endonuclease digestion at the vitamin D receptor (VDR) gene locus in Caucasian women.10 This report revealed the possibility that a single nucleotide polymorphism (SNP) may result in susceptibility to osteoporosis. Since then, a number of other candidate genes have been studied in relation to BMD or the pathogenesis of osteoporosis.1114 However, studies on VDR polymorphisms provide the greatest volume of data to date.
Four common RFLP have been detected: one each at the BsmI, ApaI, and TaqI restriction sites in intron 8 and exon 9,10 and one at the FokI restriction sites in the translation initiation site of the VDR gene.1517 Many studies have examined the associations between these polymorphisms and BMD at various skeletal sites with conflicting results.1821
There are several possible reasons for these discrepancies. First, the sample sizes of previous studies were too small, and consequently their statistical power was limited. Second, the samples of the previous studies were recruited on a voluntary basis, making self-selection biases inevitable. Third, some previous studies did not allow for confounding due to heterogeneity across the different allele groups in genetic background, age, menstrual status, body size, or due to lifestyle factors including dietary calcium intake, physical activity, and smoking habits.2224 Fourth, linkage disequilibrium may generate a false relationship between the VDR genotype and BMD, and this may exist only in some ethnic groups.25 Most studies on this topic have been performed on Caucasian people. It is important to clarify the effect of VDR genotypes on the BMD in other ethnic populations as well. There are fewer studies evaluating the association of both start codon polymorphism (FokI) and 3' end polymorphisms (BsmI, ApaI, and TaqI) simultaneously with BMD in Asian people26 than there are in Caucasian people.2730
To overcome these problems, we have launched a large-scale cohort study on representative samples of the Japanese female population, called the Japanese Population-based Osteoporosis (JPOS) Study. In this report, we present the associations of three previously reported polymorphisms at ApaI, TaqI, and FokI sites of the VDR gene with BMD and its change over time.
![]() |
Subjects and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Written informed consent regarding all the study procedures was obtained from each subject in advance.
BMD measurement
We measured the BMD of the subjects by dual energy X-ray absorptiometry (DXA) at the lumbar spine and right hip (QDR4500A, Hologic Inc., USA) in postero-anterior projection and the non-dominant distal forearm (pDXA, Norland/Stratec, USA/FRG) in both baseline and follow-up studies. If the subjects had a history of fractures or bone disease in the right hip or non-dominant forearm, the other side was scanned. Morphometry of the spine was performed with QDR4500A, in order to exclude from the analysis BMD data of subjects with fractures in the lumbar spine.
The densitometric raw data of QDR4500A taken at the appropriate region of interest (ROI) were analysed by one radiologist (YS) and confirmed by one physician (MI). We obtained the BMD (g/cm2) of the second through fourth lumbar vertebrae and of the femoral neck. The analysis of the densitometric data from pDXA was made automatically immediately after scanning and yielded the BMD of the distal 1/3 site of the radius and the ultradistal site of the forearm (the ultradistal ROI spans 10 mm of the lowest BMD region in the distal forearm).
The short-term precision of the in vivo BMD measurement, calculated from five measurements on different days for each of five volunteers, were 1.2%, 1.6%, and 1.2% (coefficient of variation: CV) for the spine, femoral neck, and distal 1/3 site of the radius, respectively. No remarkable drift in the BMD value of a spine or forearm phantom was observed throughout the study, the in vitro CV being 0.40% and 0.34%, respectively.31
The subjects underwent BMD measurement at baseline and follow-up studies. The change in BMD was expressed as the annualized percentage change from the baseline BMD.
Interviews
Detailed interviews were performed during both the baseline and follow-up studies by trained nurses according to a questionnaire which was delivered to and was completed by the subject beforehand. The questionnaire included questions regarding menstrual history and menopausal status, history and present status of gynaecological and other diseases or medications which may affect bone metabolism, and lifestyle factors such as smoking, drinking, exercise, and dietary habits.
Another interview was conducted by trained dietitians utilizing a validated food frequency questionnaire,32 in order to estimate the dietary calcium intake for each subject. This questionnaire mentioned 26 items of commonly eaten calcium-rich foods in Japan and formed the basis of an estimate of daily calcium intake.
According to the menstrual information obtained from the interviews, the subjects who menstruated regularly at the time of the baseline or follow-up surveys were judged to be premenopausal, while those who had entered menopause 6 months prior to either survey were grouped as postmenopausal women. Subjects with hysterectomy-induced menopause or whose age at menopause could not be determined were classified as postmenopausal women if they were >57 years at the time of either survey. In all, 95% of the Japanese women aged >57 are postmenopausal.31 Subjects younger than 57 years whose age at menopause could not be determined were excluded from the analysis stratified by menopausal status.
Body size and grip strength measurements
The height (cm) and weight (kg) of the subjects were measured with an automatic scale (Body Measure 2, Takei Kagaku Co., Tokyo, Japan). The body mass index (BMI in kg/m2) was calculated as weight (kg) divided by height (m) squared. The peak strength of the grip was measured with a strain gauge dynamometer (TKK5101, Takei Kagaku Co., Tokyo, Japan). We took several measurements and settled on the maximum value for each hand; the average of the maximum values for both hands was used as the representative grip strength of each subject.
Genotyping
Genomic DNA was extracted from peripheral blood leukocytes collected from the subjects at baseline in three municipalities. The FokI polymorphism in exon 2 of the VDR gene was detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method previously described by Gross et al.,15 with the primers shown in Table 1.
|
|
The sequences of the primers and probes used in this study are given in Table 1. The loci of the polymorphic sites and allele coding are shown in Figure 1A.
Statistics
Data are presented as mean ± standard error of mean (SEM). Differences in basic characteristics, baseline BMD, and change in BMD across the different genotype or haplotype groups were tested using non-paired student's t-test and analysis of variance (ANOVA), with Bonferroni's correction for multiple comparisons. Where necessary, further comparisons of BMD adjusted for confounding factor such as body size24,34 were performed by analysis of covariance (ANCOVA). All statistical analyses were performed using the SPSS system for personal computers (Release 10.0.7, SPSS Japan Inc., Tokyo, Japan).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Table 2 shows the basic characteristics of the subjects grouped according to menopausal status at baseline. The postmenopausal women were older, shorter in height, lighter in weight, lower in grip strength, and greater in BMI than the premenopausal women. The age at menarche and dietary calcium intake were lower in the premenopausal women than in the postmenopausal women. Baseline BMD values at the lumbar spine, femoral neck, distal 1/3 site of the radius, and the ultradistal site were higher in the premenopausal women than in the postmenopausal women.
|
|
|
VDR genotypes and baseline BMD
The mean ± SEM values of baseline BMD at the lumbar spine, femoral neck, distal 1/3 site of the radius, and the ultradistal site of the forearm for ApaI, TaqI, and FokI genotypes in pre- and post-menopausal women are shown in Table 5. The crude BMD at the distal 1/3 radius in the premenopausal subjects differed significantly among TaqI genotypes (the effect size of TaqI polymorphism calculated by ANOVA was 0.09). The mean BMD at this site was significantly higher for women with Tt genotype than for women with TT. This difference in BMD remained even after adjustment for age, height, and weight (the effect size of TaqI polymorphism calculated by ANCOVA was 0.10).
|
None of the individual polymorphisms defined by ApaI, TaqI and FokI restriction sites showed a consistent association with BMD over the skeletal sites measured in the present study.
After analysing three major genotypes (aaTT, AaTT, AaTt) assessed with ApaI and TaqI RFLP, we found that, in the premenopausal women, the crude BMD at the distal 1/3 radius in the subjects with AaTt genotype was significantly greater than that of the subjects with aaTT, while the BMD of the subjects with AaTT was intermediate (AaTt, 0.762 ± 0.006 n = 119; AaTT, 0.746 ± 0.006 n = 194; aaTT, 0.744 0.007 g/cm2 n = 389, P = 0.028; the effect size of these genotypes calculated by ANOVA was 0.10). Adjustment for age, height, and weight did not attenuate the significance of this association (P = 0.009; the effect size of these genotypes calculated by ANCOVA in was 0.12).
Subsequently we analysed the effect of the carrier status for ApaI and TaqI haplotype (AT or aT) on BMD. There was no significant effect on BMD according to these haplotypes at any of the sites measured in this study.
We added grip strength and years since menopause into the ANCOVA model but this did not materially alter the results. In further analysis, we adjusted only for age, height, and weight.
VDR genotypes and follow-up BMD
Among 1434 women included in these analyses, 976 subjects (68.1%) completed the follow-up study. There was no significant difference in baseline characteristics between the baseline subjects and the follow-up subjects except for age (47.4 ± 0.5 versus 50.3 ± 0.5 years old, P < 0.001) and height (152.9 ± 0.2 versus 152.2 ± 0.2 cm, P = 0.015). There was no significant difference in baseline BMD at any of the skeletal sites between the baseline subjects and the follow-up subjects.
We separated the follow-up subjects into two groups based on the menopausal status at baseline and follow-up. One group consisted of the subjects who had been premenopausal at follow-up, denoted as F-premenopausal women. The other group consisted of the subjects who already had been postmenopausal at baseline, denoted as B-postmenopausal women. The absolute values of the annual per cent changes in BMD during the 3 years of follow-up were smaller in the F-premenopausal women than in the B-postmenopausal women (Table 2).
In the subjects of the follow-up study, some fundamental baseline characteristics were significantly different between the genotypes in the B-postmenopausal women. The age at menarche was higher in the subjects with tt genotype than those with TT (17.2 ± 0.9 versus 15.5 ± 0.1 years old, P = 0.015). The baseline age was lower (63.8 ± 0.6 versus 66.2 ± 0.6 years old, P = 0.010), years since menopause was shorter (14.4 ± 0.7 versus 16.7 ± 0.6 years, P = 0.045) and grip strength was higher (23.0 ± 0.3 versus 21.5 ± 0.3 kg, P = 0.008) in the subjects with Ff genotype than those with FF.
Table 6 presents the annual per cent changes in BMD during the 3 years of follow-up in subjects with different TaqI genotypes. There were significant differences in BMD change at the lumbar spine among TaqI genotypes in both the F-premenopausal women and the B-postmenopausal women, as evaluated by ANOVA (each of the effect sizes of TaqI polymorphism calculated by ANOVA was 0.13). In the F-premenopausal women, bone loss at the lumbar spine in the subjects with tt genotype was significantly greater than that of subjects with Tt or TT. In contrast, the subjects with tt genotype in the B-postmenopausal women showed a tendency toward bone gain at the lumbar spine, compared with the subjects with Tt, who showed a tendency toward bone loss. There was a significant difference between the BMD change at the lumbar spine of the B-postmenopausal women with tt and with Tt. However, the number of subjects with tt genotype was very small (the number of eligible subjects for analysis of BMD at the lumbar spine was four for the F-premenopausal women and six for the B-postmenopausal women). Comparing BMD at the lumbar spine between subjects with TT and with Tt, there was no significant difference in BMD change in either the F-premenopausal women or the B-postmenopausal women.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Morrison et al. first showed that the polymorphism in the 3' end region of the VDR gene, as determined by the restriction enzymes BsmI, ApaI, and TaqI, was related to BMD.10 A great deal of attention was focused on this relationship, and these VDR polymorphisms were the most extensively studied genetic markers. However, the results from numerous subsequent studies on the association of these 3' end region polymorphisms with BMD have been highly inconsistent. Most of these studies were performed on Caucasian subjects. Even focusing solely on the existing studies of Asian people, we have found that the results of the association analyses are inconsistent.26,37,38,4145 Moreover, two meta-analysis studies have failed to yield consistent results46,47 and two recent large population-based studies on Caucasians have also shown controversial results.48,49 Various potential causes for this discrepancy in the results have been advocated, such as arbitrariness due to small sample size, biased samples due to convenience sampling, confounding due to heterogeneity in genetic background, age, menstrual status, body size, or lifestyle factors in the study population. In addition, population admixture and linkage disequilibrium may explain the discrepancy. These polymorphisms do not change the amino acid sequence and are probably only markers in linkage disequilibrium with another functional polymorphism. Different linkage disequilibriums may exist in different populations: for example, Ingress et al. reported that the strength of the linkage disequilibrium between BsmI polymorphisms at intron 8 and the length of the poly (A) string at the 3' end untranslated region in the VDR gene varied with ethnicity.25 Hence, this may cause different associations in different populations.
Arai et al. reported16 that the variant of the FokI polymorphic site at the start codon which lacks the first three amino acids interacted more efficiently with transcription factor IIB and possessed elevated transcriptional activity. They reported that this polymorphism was related to BMD. Recent studies50,51 indicate that the effect of the FokI polymorphisms on BMD could prove to be less than the effect proposed by the study of Arai et al. Further, a functional analysis suggested that FokI polymorphisms did not relate to the differences in functions such as DNA binding and transactivation activity.52
Although many polymorphisms in other candidate genes have been investigated, none of the contributions of these polymorphisms to the pathogenesis of osteoporosis has been found to be much larger than that originally reported by Morrison et al. It is difficult to conclude whether any individual polymorphism actually makes a significant contribution to the development of osteoporosis.
The extreme discordance of the results in a long series of studies on VDR polymorphisms, beginning with Morrison's reports, have provided us with an appropriate means of carrying on an association study for evaluating the effects of gene polymorphisms. To solve the problems in the design and analysis of the previous studies, it is necessary that: (1) the sample should be large enough to have sufficient statistical power; (2) the sample be representative of the study population; (3) the study design allow the control of confounding factors such as race, age, body size, and lifestyle; and (4) various instruments of measurement in the study have high reliability.
We performed this study in order to confirm the association of the VDR 3' region polymorphisms (ApaI and TaqI) and the start codon polymorphism (FokI) with BMD in Japanese women. Our present study has several advantages in design over the previous studies, as follows: (1) our sample consisted of more than 1400 subjects, sufficient for consolidating statistical power; (2) our sample was randomly selected from the general population in different region of Japan; and (3) our measurement of BMD and analysis of DNA was performed by highly reliable methods.
Still, the present study has some limitations. First, the study areas were not randomly selected from all the municipalities of Japan and did not include large cities. This might bias the study results. However, the frequencies of the genotypes were not significantly different from the results of previous studies on Japanese women.16,37,38 The height and weight of the subjects were similar to the Japanese average for the same ages.53 Therefore, it is likely that the characteristics of the present subjects were not substantially biased.
Second, the follow-up rate was not very high and there were small differences in the basic characteristics between the baseline subjects and follow-up subjects. This was caused by the low follow-up rate of the subjects under the age of 30, which might have biased the results for the F-premenopausal women. Practically, relatively small changes in BMD can be observed in the younger generation: therefore, the predicted result in the case of a high follow-up rate would not significantly differ from the observed result. The low follow-up rate resulted in few subjects with tt genotype. The effect size of the t-test on tt and Tt genotypes in the F-premenopausal women is <1.45 and it could not be detected at statistical power = 0.8 and alpha = 0.05. However, the actual effect size of each polymorphism of the three VDR genotypes calculated by ANOVA in this study was smaller than 0.14 in both premenopausal and postmenopausal women. Therefore, even if we got more subjects and statistically significant results at stronger power, the VDR polymorphisms would not be shown to have substantial effect on BMD in Japanese women.
Third, many genes have been reported to affect bone mass, and some of them have been thought to interact with the VDR genotype and thus to influence BMD.54,55 Furthermore, environmental factors such as calcium intake, physical activity, and smoking habits may confound the VDR genotypes. Particularly, the interaction of the VDR genotype with calcium intake or confounding effects connected to dietary calcium intake may explain the discrepancies in the results of the associations between the VDR genotypes and BMD in many studies.5658 In the present study, since the level of BMD adjusted for calcium intake was not different from crude BMD (data not shown), we did not perform further analysis to find out whether the differences in calcium intake caused a difference in the level of BMD over the VDR genotypes. The average calcium intake in our study subjects was similar to the Japanese average53 and to the results of previous studies on Japanese women at the respective ages.59 Therefore, it is considered that the results of our present study could reflect the effect of the VDR genotypes on BMD under the usual nutritional calcium intake conditions for Japanese women. In addition, other lifestyle factors such as physical activity and smoking habits did not differ greatly between the genotypes in our study (data not shown). Heterogeneity in these factors over the different allele groups was assumed to be small. We did not present further analyses of the interaction with or confounding with these lifestyle factors in this report, because these analyses would not materially alter our conclusion.
In this large-scale cohort study on representative samples of the Japanese female population, we evaluated the association between BMD and three VDR genotypes as follows: start codon polymorphism (FokI) does not have an independent effect on BMD, either in pre- or post-menopausal women; in 3' end polymorphisms (ApaI and TaqI), we detected some differences in BMD at the 1/3 radius among the TaqI genotypes and 3 major genotypes (aaTT, AaTT, AaTt) determined with both ApaI and TaqI. We also detected a difference in annual BMD changes in the lumbar spine among the TaqI genotypes. However, the effects of particular alleles or genotypes on BMD were mutually inconsistent. ApaI and TaqI polymorphisms in the VDR gene were not found to have consistent enough associations with BMD to enable the prediction of osteoporosis in either pre- or post-menopausal women. In conclusion, the effect of the previously reported VDR polymorphisms on bone mass is negligible in Japanese women. In future studies, the effects of other candidate genes on BMD and genegene or geneenvironment interactions should be researched.
KEY MESSAGES
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 Hosoda Y, Fujiwara S. The epidemiology of osteoporosis in Japan. J Epidemiol 1992;2(Suppl.):S20513.
3 Hashimoto T, Sakata K, Yoshimura N. Epidemiology of osteoporosis in Japan. Osteoporos Int 1997;7(Suppl.3):S99102.[Medline]
4 Koval KJ, Zuckerman JD. Functional recovery after fracture of the hip. Am J Bone Joint Surg 1994;76:75158.[Medline]
5 Kanis JA, Pitt FA. Epidemiology of osteoporosis. Bone 1992;13(Suppl.1):S715.[ISI][Medline]
6 Melton LJ. Hip fractures: a worldwide problem today and tomorrow. Bone 1993;14(Suppl.):S18.[ISI][Medline]
7 Pocock NA, Eisman JA, Hopper JL, Yeates MG, Sambrook PN, Ebert S. Genetic determinants of bone mass in adults: A twin study. J Clin Invest 1987;80:70610.[ISI][Medline]
8 Seeman E, Hopper JL, Bach LA et al. Reduced bone mass in daughters of women with osteoporosis. N Engl J Med 1989;320:55458.[Abstract]
9 Seeman E, Hopper JL, Young NR, Formina C, Goss P, Tsalamandris C. Do genetic factors explain associations between muscle strength, lean mass, and bone density? A twin study. Am J Physiol 1996;270:E32027.[ISI][Medline]
10 Morrison NA, Qi JC, Tokita A et al. Prediction of bone density from vitamin D receptor alleles. Nature 1994;367:28487 (Corrections) Nature 1997;387:106.[CrossRef][ISI][Medline]
11 Kobayashi S, Inoue S, Hosoi T, Ouchi Y, Shiraki M, Orimo H. Association of bone mineral density with polymorphism of the estrogen receptor gene. J Bone Miner Res 1996;11:30611.[ISI][Medline]
12 Shiraki M, Shiraki Y, Aoki C et al. Association of bone mineral density with apolipoprotein E phenotype. J Bone Miner Res 1997;12:143845.[ISI][Medline]
13 Grant SFA, Reid DM, Blake G, Herd R, Fogelman I, Ralston SH. Reduced bone density and osteoporotic vertebral fracture associated with a polymorphis Sp1 binding site in the collagen type I 1 gene. Nat Genet 1996;14:20305.[ISI][Medline]
14 Yamada Y, Miyauchi A, Goto J et al. Association of a polymorphism of the transforming growth factor-beta1 gene with genetic susceptibility to osteoporosis in postmenopausal Japanese women. J Bone Miner Res 1998;13:156976.[ISI][Medline]
15 Gross C, Eccleshall TR, Malloy PJ, Villa ML, Marcus R, Feldman D. The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women. J Bone Miner Res 1996;11:185055.[ISI][Medline]
16 Arai H, Miyamoto K, Taketani Y et al. A vitamin D receptor gene polymorphism in the translation initiation codon: effect on protein activity and relation to bone mineral density in Japanese women. J Bone Miner Res 1997;12:91521.[ISI][Medline]
17 Harris SS, Eccleshall TR, Gross C, Dawson-Hughes B, Feldman D. The vitamin D receptor start codon polymorphism (FokI) and bone mineral density in premenopausal American black and white women. J Bone Miner Res 1997;12:104348.[ISI][Medline]
18 Peacock M. Vitamin D receptor gene alleles and osteoporosis: a contrasting view. J Bone Miner Res 1995;10:129497.[ISI][Medline]
19 Fleet JC, Harris SS, Wood RJ, Dawson-Hughes B. The BsmI vitamin D receptor restriction fragment length polymorphism (BB) predicts low bone density in premenopausal black and white women. J Bone Miner Res 1995;10:98590.[ISI][Medline]
20 Garnero P, Borel O, Sornay-Rendu E, Delmas PD. Vitamin D receptor gene polymorphisms do not predict bone turnover and bone mass in healthy premenopausal women. J Bone Miner Res 1995;10:128388.[ISI][Medline]
21 Salamone LM, Glynn NW, Black DM et al. Determinants of premenopausal bone mineral density: the interplay of genetic and lifestyle factors. J Bone Miner Res 1996;11:155765.[ISI][Medline]
22 Cooper GS. Genetic studies of osteoporosis: What have we learned? J Bone Miner Res 1996;14:164648.
23 Gennari L, Becherini L, Masi L et al. Vitamin D receptor genotypes and intestinal calcium absorption in postmenopausal women. Calcif Tissue Int 1997;61:46063.[CrossRef][ISI][Medline]
24 Geusens P, Vandevyver C, Vanhoof J, Cassiman JJ, Boonen S, Raus J. Quadriceps and grip strength are related to vitamin D receptor genotype in elderly nonobese women. J Bone Miner Res 1997;12:208288.[ISI][Medline]
25 Ingles SA, Haile RW, Henderson BE et al. Strength of linkage disequilibrium between two vitamin D receptor markers in five ethnic groups: implications for association study. Cancer Epidemiol Biomarkers Prev 1997;6:9398.[Abstract]
26 Hayakawa Y, Yanagi H, Hara S et al. Genetic and environmental factors affecting peak bone mass in premenopausal Japanese women. Environ Health Prev Med 2001;6:17783.[CrossRef]
27 Ferrari S, Rizzoli R, Manen D, Slosman D, Bonjour JP. Vitamin D receptor gene start codon polymorphisms (FokI) and bone mineral density: interaction with age, dietary calcium, and 3-end region polymorphisms. J Bone Miner Res 1998;13:92530.[ISI][Medline]
28 Tao C, Yu T, Garnett S et al. Vitamin D receptor alleles predict growth and bone density in girls. Arch Dis Child 1998;79:48893.
29 Langdahl BL, Gravholt CH, Brixen K, Eriksen EF. Polymorphisms in the vitamin D receptor gene and bone mass, bone turnover and osteoporotic fractures. Eur J Clin Invest 2000;30:60817.[CrossRef][ISI][Medline]
30 Efstathiadou Z, Kranas V, Ioannidis JP, Georgiou I, Tsatsoulis A. The Sp1 COLIA1 gene polymorphism, and not vitamin D receptor or estrogen receptor gene polymorphisms, determines bone mineral density in postmenopausal Greek women. Osteoporos Int 2001;12:32631.[CrossRef][ISI][Medline]
31 Iki M, Kagamimori S, Kagawa Y, Matsuzaki T, Yoneshima H, Marumo F. Bone mineral density of the spine, hip and distal forearm in representative samples of the Japanese female population: Japanese Population-Based Osteoporosis (JPOS) Study. Osteoporos Int 2001;12:52937.[CrossRef][ISI][Medline]
32 Iki M, Kitayama F, Kusaka Y. Development of a food frequency questionnaire of measuring dietary calcium intake. Abstract book of 16th Int Cong Nutr. Montreal, Canada, 1997; 303.
33 Livak KJ. Allelic discrimination using fluorogenic probes and the 5 nuclease assay. Genet Anal 1999;14:14349.[CrossRef][ISI][Medline]
34 Barger-Lux MJ, Heaney RP, Hayes J, DeLuca HF, Johnson ML, Gong G. Vitamin D receptor gene polymorphism, bone mass, body size and vitamin D receptor density. Calcif Tissue Int 1995;57:16162.[ISI][Medline]
35 McCloskey EV, Spector TD, Eyres KS et al. The assessment of vertebral deformity: a method for use in population studies and clinical trials. Osteoporos Int 1993;3:13847.[ISI][Medline]
36 Nathan H. Osteophytes of the vertebral column. J Bone Joint Surg 1962;44-A:24369.
37 Yamagata Z, Miyamura T, Iijima S et al. Vitamin D receptor gene polymorphism and bone mineral density in healthy Japanese women. Lancet 1994;344(8928):1027.[ISI][Medline]
38 Tokita A, Matsumoto H, Morrison NA et al. Vitamin D receptor alleles, bone mineral density and turnover in premenopausal Japanese women. J Bone Miner Res 1996;11:100309.[ISI][Medline]
39 Riggs BL. Involutional osteoporosis. New Engl J Med 1986;314:167686.[ISI][Medline]
40 Consensus Development Conference. Diagnosis, prophylaxis and treatment of osteoporosis. Am J Med 1993;94:64650.[ISI][Medline]
41 Choi YM, Jun JK, Choe J et al. Association of the vitamin D receptor start codon polymorphism (FokI) with bone mineral density in postmenopausal Korean women. J Hum Genet 2000;45:28083.[CrossRef][ISI][Medline]
42 Lau EM, Young RP, Ho SC et al. Vitamin D receptor gene polymorphisms and bone mineral density in elderly Chinese men and women in Hong Kong. Osteoporos Int 1999;10:22630.[CrossRef][ISI][Medline]
43 Kim JG, Lim KS, Kim EK, Choi YM, Lee JY. Association of vitamin D receptor and estrogen receptor gene polymorphisms with bone mass in postmenopausal Korean women. Menopause 2001;8:22228.[CrossRef][ISI][Medline]
44 Iki M, Dohi Y, Yonemasu K et al. Vitamin D receptor haplotype and bone density in Japanese postmenopausal women. Osteoporos Int 1996;6(Suppl.1):S138.
45 Zhao J, Zhou X, Meng X et al. Polymorphisms of vitamin D receptor gene and its association with bone mineral density and osteocalcin in Chinese. Chin Med J (Engl) 1997;110:36671.[Medline]
46 Cooper GS, Umbach DM. Are vitamin D receptor polymorphisms associated with bone mineral density? A meta-analysis. J Bone Miner Res 1996;11:184149.[ISI][Medline]
47 Gong G, Stern HS, Cheng SC et al. The association of bone mineral density with vitamin D receptor gene polymorphisms. Osteoporos Int 1999;9:5564.[CrossRef][ISI][Medline]
48 Vandevyver C, Wylin T, Cassiman JJ, Raus J, Geusens P. Influence of the vitamin D receptor gene alleles on bone mineral density in postmenopausal and osteoporotic women. J Bone Miner Res 1997;12:24147.[ISI][Medline]
49 Uitterlinden AG, Weel AE, Burger H et al. Interaction between the vitamin D receptor gene and collagen type Ialpha1 gene in susceptibility for fracture. J Bone Miner Res 2001;16:37985.[ISI][Medline]
50 Eccleshall TR, Garnero P, Gross C, Delmas PD, Feldman D. Lack of correlation between start codon polymorphism of the vitamin D receptor gene and bone mineral density in premenopausal French women: the OFELY study. J Bone Miner Res 1998;13:3135.[ISI][Medline]
51 Zmuda JM, Cauley JA, Danielson ME, Theobald TM, Ferrell RE. Vitamin D receptor translation initiation codon polymorphism and markers of osteoporotic risk in older African-American women. Osteoporos Int 1999;9:21419.[CrossRef][ISI][Medline]
52 Gross C, Krishnan AV, Malloy PJ, Eccleshall TR, Zhao XY, Feldman D. The vitamin D receptor gene start codon polymorphism: a functional analysis of FokI variants. J Bone Miner Res 1998;13:169199.[ISI][Medline]
53 Statistics and Information Department, Japanese Ministry of Health and Welfare. Health and Welfare Statistics in Japan. Tokyo: Health and Welfare Statistics Association, 1998, pp. 6162.
54 Willing M, Sowers M, Aron D et al. Bone mineral density and its change in white women: estrogen and vitamin D receptor genotypes and their interaction. J Bone Miner Res 1998;13:695705.[ISI][Medline]
55 Gennari L, Becherini L, Masi L et al. Vitamin D and estrogen receptor allelic variants in Italian postmenopausal women: evidence of multiple gene contribution to bone mineral density. J Clin Endocrinol Metab 1998;83:93944.
56 Dawson-Hughes B, Harris SS, Finneran S. Calcium absorption on high and low calcium intakes in relation to vitamin D receptor genotype. J Clin Endocrinol Metab 1995;80:365761.[Abstract]
57 Kiel DP, Myers RH, Cupples LA et al. The BsmI vitamin D receptor restriction fragment length polymorphism (bb) influences the effect of calcium intake on bone mineral density. J Bone Miner Res 1997;12:104957.[ISI][Medline]
58 Ames S, Ellis K, Gunn S, Copeland K, Abrams S. Vitamin D receptor gene Fok1 polymorphism predicts calcium absorption and bone mineral density in children. J Bone Miner Res 1999;14:74046.[ISI][Medline]
59 Sinpo S, Cho S, Watanabe T et al. Estimate of mineral intakes using food composition tables vs. measures by inductively-coupled plasma mass spectrometry. Jpn J Hyg 1999;54:109.