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Estrogen Receptor Regulates Area-Adjusted Bone Mineral Content in Late Pubertal Girls

J. H. Tobias, C. D. Steer, C. Vilario-Güell and M. A. Brown

Clinical Science at South Bristol (J.H.T.), Community Medicine (C.D.S.), University of Bristol, Bristol BS2 8HW, United Kingdom; Botnar Research Centre (C.V.-G., M.A.B.), University of Oxford, Oxford BS2 8HW, United Kingdom; and Centre for Immunology and Cancer Research (M.A.B.), University of Queensland, Queensland 4102, Australia

Address all correspondence and requests for reprints to: Dr. J. Tobias, Rheumatology Unit, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom. E-mail: Jon.Tobias{at}bristol.ac.uk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Whether the action of estrogen in skeletal development depends on estrogen receptor as encoded by the ESR1 gene is unknown.

Objectives: The aim of this study was to establish whether the gain in area-adjusted bone mineral content (ABMC) in girls occurs in late puberty and to examine whether the magnitude of this gain is related to ESR1 polymorphisms.

Design: We conducted a cross-sectional analysis.

Setting: The study involved the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based prospective study.

Participants: Participants included 3097 11-yr-olds with DNA samples, dual x-ray absorptiometry measurements, and pubertal stage information.

Outcomes: Outcome measures included separate prespecified analyses in boys and girls of the relationship between ABMC derived from total body dual x-ray absorptiometry scans and Tanner stage and of the interaction between ABMC, Tanner stage, and ESR1 polymorphisms.

Results: Total body less head and spinal ABMC were higher in girls in Tanner stages 4 and 5, compared with those in Tanner stages 1, 2, and 3. In contrast, height increased throughout puberty. No differences were observed in ABMC according to Tanner stage in boys. For rs2234693 (PvuII) and rs9340799 (XbaI) polymorphisms, differences in spinal ABMC in late puberty were 2-fold greater in girls who were homozygous for the C and G alleles, respectively (P = 0.001). For rs7757956, the difference in total body less head ABMC in late puberty was 50% less in individuals homozygous or heterozygous for the A allele (P = 0.006).

Conclusions: Gains in ABMC in late pubertal girls are strongly associated with ESR1 polymorphisms, suggesting that estrogen contributes to this process via an estrogen receptor -dependent pathway.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
ESTROGEN EXERTS AN important protective effect on the skeleton, as illustrated by the reduction in bone mass that occurs after the loss of estrogen production at the menopause, and its prevention by estrogen replacement therapy (1). In cortical bone, estrogen acts to reduce intracortical bone remodeling to suppress endocortical resorption and to stimulate endosteal formation; in cancellous bone, estrogen improves trabecular bone remodeling balance through inhibition of osteoclastic resorption and stimulation of osteoblastic bone formation (1). These actions are together expected to increase bone mineral density (BMD) as measured by methods such as dual x-ray absorptiometry (DXA). In addition, estrogen exerts important inhibitory effects on skeletal growth. For example, estrogen acts to suppress longitudinal growth (2) to reduce radial expansion of long bones through inhibition of periosteal bone formation in women (3) but may stimulate radial bone growth in males (4).

Amenorrhea, for example, caused by excessive exercise, is associated with a decrease in peak bone mass (5), presumably caused by lack of estrogen’s positive effects on BMD described. These effects become manifest in late pubertal girls, when estrogen levels rise to adult levels, reaching a peak soon before menarche (6). For example, spinal bone mineral apparent density (BMAD) is increased in late pubertal girls (7) (8); increased cortical thickness has been observed in metacarpal radiographs from adolescent girls (9); femoral midshaft "volumetric" BMD derived from DXA measurements appears to increase after the menarche (10). In a longitudinal study using tibial peripheral quantitative computed tomography. Wang et al. (6) found that cortical thickness increased in pubertal girls attributable to suppression of endosteal resorption and stimulation of endosteal apposition after menarche. In contrast, Kontulainen et al. (11) found no evidence of endosteal apposition in late pubertal girls using the same technique; and in both sexes, the gain in cortical bone mass largely resulted from periosteal expansion.

The case report of a male subject who developed excessive stature and reduced bone mass in association with a nonfunctioning mutation of the estrogen receptor (ER) gene (ESR1) suggests that estrogen also contributes to skeletal development in males (12). This link between ER and bone mass is consistent with results of our previous studies of male ER-null mice, in which stimulation of trabecular bone formation by estrogen was found to be deficient (13). Several previous studies have explored the role of ER in bone in human populations by analyzing relationships between ESR1 polymorphisms and skeletal phenotypes as measured by DXA. In a meta-analysis involving 18,917 individuals in eight European centers, ESR1 polymorphisms were related to fracture risk in adults, but not BMD (14). In terms of the role of ER in bone development, in a small group of 147 children, adolescents, and young adults, rs2234693 (PvuII) and rs9340799 (XbaI) polymorphisms were associated with lumbar spine BMD and BMAD (15).

Therefore, although ER is likely to contribute to effects of estrogen on BMD in late puberty, evidence for this conclusion is largely based on animal models. In the present investigation, we explored the role of ER in mediating effects of estrogen on skeletal development in late puberty by performing genetic association studies in a large well-characterized cohort of children. In particular, we wished to determine whether the magnitude of the gain in BMD in late pubertal girls is influenced by ESR1 gene polymorphisms by examining whether an interaction exists between BMD, Tanner stage, and ESR1 polymorphisms in girls. Because conventional "areal" BMD measurements may be difficult to interpret because they are not fully corrected for skeletal size, we based our analyses on results for area-adjusted bone mineral content (ABMC) to provide a more accurate estimate for volumetric BMD.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Study population

The Avon Longitudinal Study of Parents and Children (ALSPAC) is a geographically based cohort that recruited pregnant women residing in Avon with an expected date of delivery between April 1, 1991 and December 31, 1992; 14,541 pregnancies were initially enrolled, with 14,062 children born. This represented 80–90% of the eligible population (see www.alspac.bris.ac.uk for further details on the ALSPAC cohort) (16). Of these births, 13,988 were alive at 12 months. The present study is based on results for total body DXA scans obtained at a research clinic to which the whole cohort was invited at mean age of 11.8 yr. Ethical approval was obtained from the ALSPAC Law & Ethics Committee and local research ethics committees. Parental consent and child’s assent were obtained for all measurements made.

Genotyping

Genotyping was performed by Kbiosciences (Hoddesdon, UK), using DNA extracted as previously described from cord/peripheral blood from all available children (n = 9804) (17). Based on suggestive association data with DXA measures in a previous study of 11 tag single nucleotide polymorphisms (SNPs) in ESR1 in the ALSPAC children-in-focus group (a 10% subsample of the cohort studied in more detail in early childhood) at age 9 yr (our unpublished observations), four ESR1 SNPs were selected for further study in the whole cohort. Because genotypes of one of these four SNPs, rs2228480, were not in Hardy-Weinberg equilibrium (P = 0.007), it was excluded from further analysis. Genotype frequencies for the three remaining SNPs on which this study is based are shown in Table 1. The frequency of genotyping failures for these three SNPs ranged from 6.4–8.6%. Error rates as measured by analysis of duplicate samples were less than 1%. Haplotypes were analyzed using the program WHAP, which used the EM-algorithm to determine haplotype frequencies (http://www.genome.wi.mit.edu/shaun/whap/). Subjects for whom the probability of correct assignment of haplotype was less than 0.95 were excluded. Only haplotypes with frequency greater than 5% were analyzed.

The present analysis is based on DXA measures obtained on attendance at the age 11 yr screening visit (mean age, 11.8 yr). Of the 7159 children who attended, 7057 underwent a whole-body DXA scan, which was performed using a Lunar Prodigy (Lunar, Madison, WI) with pediatric scanning software. At the same time, sitting and standing height were measured using a Harpenden Stadiometer, as was weight using a Tanita Body Fat Analyser (Tanita Corp., Arlington Heights, IL). After exclusion of scans with anomalies (e.g. artifacts caused by movement or jewelry), scans were available for 7006 children. These were evaluated and reanalyzed as necessary to ensure that borders between adjacent subregions were optimally placed. DXA results were expressed as total body less head (TBLH) bone area, bone mineral content (BMC), and "areal" BMD. A more rigorous method for adjusting for skeletal size (ABMC) was also derived to provide a measure of volumetric BMD, by using linear regression to adjust BMC for bone area (18). Because separate lumbar spine scans were not obtained, DXA variables for the spine were derived from spine subregional analysis of whole-body scans. Results were only included in children with no evidence of spinal curvature, based on findings of our previous validation study (19). Coefficients of variation for TBLH BMC and BMD were 2.6% and 0.9%, respectively, and for spinal BMC and BMD 4.9% and 3.3%, respectively.

Other variables

Gender was obtained from birth notifications. At the time of the DXA scan and measurement of the anthropometric variables, the child’s age was calculated from the date of birth and date of attendance at the research clinic. Puberty was assessed by self-completion questionnaires using diagrams based on Tanner staging of pubic hair distribution for boys, and pubic hair and breast development for girls. In view of the major influence of puberty on DXA-derived parameters, the present study was based on the subgroup of children in whom pubertal stage information was available within 16 wk of the age-11.8-yr clinic visit.

Statistical analysis

Age, height, weight, and the DXA variables were normally distributed; comparisons were made between Tanner stages using F-tests for boys and girls separately. Associations between each ESR1 polymorphism and DXA outcome (i.e. TBLH and spinal BMC, BMD, area, and ABMC) were initially examined using linear regression to test different hypotheses concerning genotype effects. To test for a rare allele recessive/common allele-dominant effect, results for common allele homozygotes and heterozygotes were combined and compared with rare allele homozygotes; to test for a rare allele dominant/common allele recessive effect, those homozygous for the common allele were compared with combined results for heterozygotes and rare allele homozygotes. To explore possible gene-dose effects, the three genotypes were examined separately. Analyses, which were performed in boys and girls separately, were adjusted for age, pubertal stage, height, and weight. To take account of multiple comparisons, P value for statistical significance for these initial analyses was taken as 0.01 (assuming two independent genetic effects and two independent phenotypes). To examine whether an interaction exists between ABMC, puberty, and ESR1 polymorphisms in girls as specified in our original hypothesis, terms for interactions with gender and/or puberty were added to linear regression analyses between TBLH/spinal ABMC and ESR1 polymorphisms. The latter were entered into these analyses based on recessive/dominant genetic models as described, along with other variables used in adjustment (i.e. age, height, and weight).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Of the 9804 children with available genotyping results shown in Table 1, DXA data were available at age 11 in 5754 for TBLH, and 2979 for spine, for whom matching pubertal stage information was available in 3098 and 1616 children, respectively. Height, weight, and DXA results at the total body and spine for the latter two subgroups, which form the basis of the present study, are shown according to Tanner stage for boys (Table 2) and girls (Table 3). The majority of boys were in Tanner stage 1 and 2, whereas girls were predominantly in Tanner stages 2, 3, and 4. TBLH and spinal BMC, area, and BMD varied according to Tanner stage in both sexes. In contrast, TBLH and spinal ABMC differed by Tanner stage in girls, but not boys. TBLH and spinal area increased according to pubertal stage in girls, whereas ABMC appeared to remain unchanged until late puberty.

Associations were examined between DXA parameters and ESR1 polymorphisms shown in Table 1. In girls, intron 1 polymorphisms were related to ABMC in the dominant genetic model (Table 4), with rs2234693 and rs9340799 showing strongest associations with spinal and TBLH ABMC, respectively. In addition, there was evidence of a weak association between intron 1 polymorphisms and spinal area. To investigate whether the apparent increase in ABMC in late pubertal girls shown in Table 3 is related to ESR1 polymorphisms, interactions between ABMC, ESR1 genotype, and Tanner stage were examined. Both intron 1 polymorphisms showed strong interactions with puberty in relation to spine ABMC in girls but not boys, whereas no interaction was observed for TBLH ABMC (Table 5). The proportion of girls with a given genotype was unrelated to Tanner stage (Table 6), suggesting that ESR1 genotype does not affect the timing of puberty.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Mean (+95% confidence interval) TBLH and spine ABMC according to Tanner stage and genotype in girls (n = 1814 for TBLH, and 932 for spine). P value is for puberty interaction (see Table 5). White bars, Tanner stages 1, 2, and 3 combined; gray bars, Tanner stage 4; black bars, Tanner stage 5 (TBLH), or Tanner stages 4 and 5 combined (spine). For rs7757956, heterozygotes and rare homozygotes were combined due to small numbers (see Table 6 for numbers per group).

No associations were observed between rs7757956 and ABMC in any genetic model. However, using a recessive genetic model, this polymorphism was related to spinal area in girls (Table 4). There was a strong interaction between rs7757956, Tanner stage, and TBLH ABMC in girls but not boys, but no interaction was seen for spinal ABMC (Table 5). The proportion of girls with a given genotype was unrelated to Tanner stage (Table 6) (TA and AA genotypes were combined because of low frequency of the AA genotype). The difference in TBLH ABMC between early and late pubertal girls was approximately 50% less in girls with one or two copies of the rare allele of rs7757956 (i.e. TA and AA genotypes) (Fig. 1). For example, in girls with the TT genotype, TBLH ABMC was 1201 ± 3 vs. 1277 ± 13 in those in Tanner stages 1–3 and 5, respectively (difference = 76); in contrast, in girls with the TA/AA genotype, TBLH ABMC was 1211 ± 6 vs. 1254 ± 19 in those in stages 1–3 and 5, respectively (difference = 43) (mean g + 95% confidence interval).

Haplotype analysis

The extent of linkage disequilibrium (LD) between these three ESR1 SNPs was subsequently explored by haplotype analysis. As expected, the two intron 1 SNPs showed a high level of LD (D = 0.80), whereas there was no evidence of linkage between either intron 1 SNP or rs7757956 (D < 0.05). We then investigated whether the difference in spinal ABMC according to Tanner stage in girls is influenced by haplotypes formed from rs2234693 and rs9340799. After analyses based on the five most common haplotypes (frequencies > 0.05), P values for the interaction between haplotype, pubertal stage, and spinal ABMC were 0.1, 0.009, and 0.01 in boys, girls, and boys and girls combined, respectively (results adjusted for age, height, and weight; n = 1518). Finally, we examined mean spinal ABMC in girls according to haplotype and Tanner stage (Fig. 2). As expected, individuals who were homozygous or heterozygous for the CG haplotype showed greater differences in spinal ABMC according to Tanner stage, with the former showing the largest differences.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Mean (+95% confidence interval) spine ABMC according to Tanner stage and haplotypes in girls (n = 965). White bars, Tanner stages 1, 2, and 3 combined; black bars, Tanner stages 4 and 5 combined. Number of Tanner stage 1–3 girls: 196 (TA/TA), 82 (TA/CA), 261 (TA/CG), 55 (CA/CG), and 92 (CG/CG). Number of Tanner stage 4–5 girls: 40 (TA/TA), 23 (TA/CA), 77 (TA/CG), 13 (CA/CG), and 26 (CG/CG).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

To explore the role of estrogen in changes in ABMC in late pubertal girls, we examined whether these are influenced by ESR1 polymorphisms. Intron 1 polymorphisms, rs2234693 and rs9340799, were associated with ABMC in girls as a whole. However, an interaction was evident with puberty, such that the difference in spinal ABMC in late puberty was approximately 2-fold greater in girls who were homozygous for the C and G alleles of rs2234693 and rs9340799, respectively. Haplotype analyses supported these findings, because an equivalent difference in spinal ABMC was observed according to puberty in girls homozygous for the CG haplotype.

An association was also seen between the intron 4 polymorphism, rs7757956, and spinal area in girls as a whole. However, a distinct genetic effect was evident in terms of interactions with puberty, because the difference in TBLH ABMC according to puberty was reduced by approximately 50% in girls heterozygous or homozygous for the A allele. No overall association was observed between rs7757956 and TBLH ABMC in girls, whereas the A allele appeared to reduce the gain in TBLH ABMC with puberty, ABMC in pre-pubertal and early-pubertal girls with this allele appeared to be increased. These observations raise the intriguing possibility that the rs7757956 polymorphism, or a polymorphism in LD with it, enhances and reduces ESR1 function in the presence of low and high estrogen levels, respectively.

Taken together, our observations that suggest that the gain in ABMC in late pubertal girls is strongly influenced by ESR1 polymorphisms provide further support for the hypothesis that this aspect of skeletal development is mediated at least in part by ER. This suggestion that ESR1 genotype modulates skeletal sensitivity to estrogen exposure is consistent with previous findings. For example, the protective effect of hormone replacement therapy on fracture risk in postmenopausal women has been found to relate to rs2234693 genotype (20). Similarly, an interaction has previously been reported between rate of bone loss in men, bioavailable levels of estradiol, and rs2234693 and rs9340799 genotypes (21). Although it is possible that the associations which we observed were chance events, a P value cutoff for significance of 0.01 was used to adjust for multiple genetic influences and phenotypic outcomes in the analysis. In view of the low P values observed, particularly in the case of interaction tests involving intron 1 polymorphisms, our results are unlikely to have been affected by more conservative approaches for adjusting for multiple outcomes.

To our knowledge, this is the first reported association between rs7757956 and any phenotype. Linkage disequilibrium analysis confirmed that rs7757956 is not in LD with either intron 1 SNP, and therefore represents an independent genetic effect. This investigation also provides the first evidence that rs2234693 and rs9340799 affect the skeletal response to puberty. In previous studies of the relationship between ESR1 polymorphisms and DXA measurements in children, a positive relationship was found between the CG haplotype and spinal BMAD, in 57 boys and 90 girls aged 4.3–19.9 yr (15). However, it is unclear whether an influence of CG haplotype on gain in spinal ABMC in girls, as observed in the present study, contributed to this association. In a study of 448 boys and girls aged 4.5–6.5 yr, no association was found between DXA values and rs2234693 and rs9340799 (22), consistent with the present findings that suggest these polymorphisms exert their major effect during puberty. However, two association studies between ESR1 polymorphisms and DXA in childhood reported discrepant findings to our results, but were considerably smaller: in 245 10- to 13-yr-old girls, no association was observed between rs2234693 and rs9340799 polymorphisms and BMD (although there was an apparent interaction with physical activity) (23); in 90 boys with mean age 16.9 yr, rs2234693 and rs9340799 were associated with spinal BMD adjusted for skeletal size based on a cylindrical model, but the direction of association was opposite to that reported here (24).

Despite an overall association between ESR1 intron 1 polymorphisms and spinal ABMC in girls, there was little or no association with BMD, which is consistent with a previous study based on a wide range of adult populations (14). The weakness of any association with BMD may reflect the fact that there was also evidence of an opposing association between ESR1 intron 1 polymorphisms and spine area; because areal BMD as measured conventionally reflects both bone size and volumetric BMD, a larger but less dense skeleton may have an equivalent areal BMD to one that is smaller but denser. Associations between ESR1 intron 1 polymorphisms and spine area in childhood that we found may be particularly relevant to the future risk of osteoporotic fracture, in light of previous evidence that vertebral cross-sectional area is inversely related the risk of vertebral fracture (25). Consistent with this suggestion, ESR1 intron 1 alleles associated with reduced spine area in children in the present study were found to be associated with an increased risk of vertebral fracture in adults (14).

This study represents by far the largest genetic association study of the relationship between ESR1 polymorphisms and DXA values in childhood. Furthermore, the 11-yr-old girls who provided the basis for our results were evenly distributed across the different Tanner stages, reflecting the normal distribution of age of puberty onset; this provided an opportunity to examine interactions between DXA parameters, puberty, and ESR1 polymorphisms using a cross-sectional study design. In terms of weaknesses, although we aimed to examine whether ESR1 polymorphisms influence gains in ABMC during puberty, this question was addressed based on cross-sectional as opposed to longitudinal data. In addition, it was necessary to obtain spinal DXA parameters from whole-body scans, rather than from lumbar spine scans, which is likely to have introduced greater measurement error. Furthermore, although we reported associations with ABMC, the validity of this term as a measure of volumetric BMD remains to be established.

In conclusion, we investigated the role played by ER in skeletal development during puberty by examining relationships between DXA parameters, pubertal stage, and ESR1 polymorphisms. We found that TBLH and spinal ABMC differed according to puberty in a manner suggestive of an estrogenic response, because differences were only observed in late pubertal girls, when estrogen levels rise toward adult levels. We then investigated whether this response is mediated by ER by examining whether differences in ABMC in late puberty are associated with ESR1 polymorphisms. Interestingly, the difference in TBLH ABMC in late pubertal girls was approximately 50% less in those homozygous or heterozygous for the rs7757956 A allele. However, in girls homozygous for C and G alleles of rs2234693 and rs9340799, respectively, or for the CG haplotype, differences in spinal ABMC in late puberty were approximately 2-fold greater. Taken together, these findings suggest that estrogen plays an important role in skeletal development in girls, by acting to increase ABMC of both trabecular and cortical bone during late puberty via an ER-dependent pathway.

	Acknowledgments

 
We are extremely grateful to all the families who took part in this study, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists, and nurses.

	Footnotes

 
Grants or fellowships supporting the writing of this paper: The UK Medical Research Council, the Wellcome Trust, and the University of Bristol provided core support for ALSPAC. This publication is the responsibility of J.H.T. and C.D.S., who also serve as guarantors for the contents of this article. This research was specifically funded by grants from the Wellcome Trust and National Osteoporosis Society.

First Published Online November 14, 2006

Abbreviations: ABMC, Area-adjusted bone mineral content; BMAD, bone mineral apparent density; BMD, bone mineral density; DXA, dual x-ray absorptiometry; LD, linkage disequilibrium; SNP, single nucleotide polymorphism; TBLH, total body less head.

Received July 17, 2006.

Accepted November 6, 2006.

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