Background
Bone mass increases during the growth period and peaks by young adulthood. Although the greatest gain in bone mass takes place during the accelerated growth in adolescence, bone mineral density (BMD) continues to increase for several years (yrs) later [
1].
The importance of peak bone mass as a determinant of osteoporosis and fractures later in life is supported by several studies. For instance, Hui et al. [
2] estimated that peak bone mass and postmenopausal bone loss contributed equally to bone status in 70-year-old women. Hernandez et al. [
3] estimated that a 10% increase in peak BMD may delay the development of osteoporosis by 13 yrs.
Low BMD is, indeed, a major determinant of osteoporotic fractures, even though environmental factors, such as dietary intake and physical activity play an important role in the BMD determination. From studies of monozygotic and dizygotic twins, inheritance was estimated to account for 60–80% of BMD in both men [
4] and women [
5]. Many other predictors of fragility fracture, bone turnover markers and skeletal geometry are also under genetic control. In the last two decades, an exceptionally wide range of candidate genes have been proposed as risk markers of osteoporosis outcomes [
6], but our ability to predict which patients are most likely to sustain low BMD and/or osteoporotic fractures based on genetic screening is still far to be complete.
Among the analyzed candidate genes are those encoding estrogen receptor α (
ESR1) and β (
ESR2). In particular, single nucleotide polymorphisms [
7] defined by the restriction enzymes
PvuII (
rs2234693, C/T) and
XbaI (
rs9340799, A/G) in the
ESR1 intron 1, and by
AluI (
rs4986938, A/G) in the 3'-untraslated region (3'
UTR) of
ESR2 exon 8 have been evaluated in more than 90 population-based studies, with inconclusive results. For their specific ethnic distribution, clinical predictability of estrogen receptor polymorphisms are strongly dependent on the analysis of homogenous populations [
8].
The purpose of this study was to relate skeletal traits such as height (Ht), BMD and bone turnover markers, measured in a large cohort of healthy premenopausal Caucasian women aged 20–50 yrs, to several genotyped polymorphisms in the ESR1 (rs2234693 and rs9340799) and ESR2 (rs4986938) loci.
Results
A total of 641 healthy premenopausal women meeting the inclusion and exclusion criteria were recruited from 20 investigative sites. Two women were excluded due to serum calcium >10.5 mg/dL and one patient for P1NP and OC levels three times above the upper normal range, who is now under investigation for suspected Paget disease of the bone. No other patients had abnormal values of serum phosphate, magnesium or creatinine (data not shown).
Serum FSH >30 IU/L was found in 18 women even though menstruating normally. They are considered per protocol as perimenopausal. Twelve subjects were on treatment with stable doses of thyroxine, 11 on antihyperthensive agents not associated with diuretics, 3 on serotonin-uptake inhibitors, and 2 on proton pump inhibitors. Eighty-three women were on oral contraceptive treatment [
9].
Genetic data were available for 573 enrolled subjects. Three non-Caucasian women were excluded from genetic analyses and their exclusion did not modify the results of the study (data not shown). Statistical analysis was performed on the remaining 570 Caucasian women who were not on treatment with known bone-active drugs.
The general characteristics of the study population on the basis of allelic variability for
ESR1 and
ESR2 loci are described in Table
1. According to [
12], genotype distributions of these two loci were found to be in HW equilibrium (data not shown), suggesting that the enrolled subjects represented a homogeneous genetic background. No difference was evidenced in
ESR1 and
ESR2 loci for all considered variables, though a tendency to on increased Ht and a delayed menarche age were detected for
ESR1 rs2234693 CC,
rs9340799 GG or
CCGG, and
ESR2 AA genotypes than for the opposites (
P > 0.05, data not shown). Moreover, the combination of
ESR1 CCGG plus
ESR2 AA-AG genotype was significantly taller (164.2 ± 6.06 cm) than
ESR1 TTAA plus
ESR2 GG genotype (161.7 ± 6.74 cm;
P = 0.044). When
ESR1 and
ESR2 genotypes were evaluated alone or in combination, no significant correlation was observed with weight, BMI, heart rate and blood pressure (
P > 0.05).
Table 1
Genotype variability for ESR1 and ESR2 loci in the 570 Caucasian women enrolled in the BONTURNO study.
ESR1 rs2234693
| |
CC (n [%]) | 123 (21.6) |
CT (n [%]) | 287 (50.3) |
TT (n [%]) | 160 (28.1) |
ESR1 rs9340799 | |
AA (n [%]) | 203 (35.6) |
AG (n [%]) | 272 (47.7) |
GG (n [%]) | 95 (16.7) |
ESR2 rs4986938
| |
AA (n [%]) | 92 (16.2) |
AG (n [%]) | 266 (46.7) |
GG (n [%]) | 212 (16.7) |
Regarding family history of osteoporosis (FHO) and of hip fracture (FHF), no significative association was found with single or combined analysis of
ESR1 polymorphisms. In subjects positive both for FHO and for FHF, the 3
ESR1 rs2234693 genotypes (but not the
rs9340799) were differently distributed than in subjects with double negative FHO-FHF (χ
2 = 10.957,
P < 0.01; Table
2), with odds ratio (OR) of founding
ESR1 CT-
TT genotypes in double positive FHO-FHF being 1.836 (95% CI 0.860–3.919,
P = 0.06). No significant association was detected combining both
ESR1 polymorphisms (
P > 0.05). Furthermore,
ESR2 rs4986938 genotypes correlated with FHF (χ
2 = 11.881,
P < 0.01) but not with FHO (
P > 0.05), as having at least one
ESR2 rs4986938 A allele correlated both with positive FHF (χ
2 = 11.550,
P < 0.001; OR 2.387, 95% CI 1.432–3.977) and with double positive FHO-FHF (χ
2 = 9.407,
P < 0.005; OR 2.871, 95% CI 8.804–35.403) (Table
2). No association was detected co-analyzing
ESR1 and
ESR2 genotypes (
P > 0.05).
Table 2
Family history of osteoporosis (FHO) and of hip fracture (FHF) regarding ESR1 and ESR2 genotypes.
| positive | negative |
ESR1 rs2234693
| | | | | | |
CC (n [%]) | 39 (17.6) | 18 (17.6) | 9 (14.7) | 77 (24.0) | 98 (22.1) | 68 (24.1) |
CT (n [%]) | 118 (53.1) | 50 (49.1) | 32 (52.5) | 158 (49.0) | 227 (51.2) | 141 (50.0) |
TT (n [%]) | 65 (29.3) | 34 (33.3) | 20 (32.8) | 87 (27.0) | 118 (26.7) | 73 (25.9) |
Total | 222 | 102 | 61 | 322 | 443 | 282 |
ESR2 rs4986938
| | | | | | |
AA (n [%]) | 36 (16.3) | 18 (18.2) | 12 (19.7) | 50 (15.8) | 68 (15.5) | 44 (15.8) |
AG (n [%]) | 104 (47.1) | 59 (59.6) | 38 (62.3) | 119 (37.5) | 193 (44.0) | 127 (45.5) |
GG (n [%]) | 81 (36.6) | 22 (22.2) | 11 (18.0) | 148 (46.7) | 178 (40.5) | 108 (38.7) |
Total | 221 | 99 | 61 | 317 | 439 | 279 |
The 570 enrolled women were divided in 3 age groups: from 20 to 30 yrs (class 1), from 31 to 40 yrs (class 2) and from 41 to 50 yrs (class 3) (Table
3). In class 1, subjects having at least one
ESR1 rs2234693 C allele (
i.e. CC and
CT genotypes) presented lower LS-BMD (1.023 ± 0.112 g/cm
2) and TH-BMD (0.927 ± 0.122 g/cm
2) than
TT genotypes, respectively (LS-BMD 1.077 ± 0.131 g/cm
2,
P = 0.0083; TH-BMD 0.969 ± 0.121 g/cm
2,
P = 0.0474). Similar but not significant trends were detected in classes 2 and 3. In class 3, TH-BMD (0.860 ± 0.111 g/cm
2) and FN-BMD (0.738 ± 0.108 g/cm
2) of
ESR2 rs4986938 AA genotype were lower than the opposite
GG ones (TH-BMD 0.923 ± 0.130 g/cm
2,
P = 0.0227; FN-BMD 0.798 ± 0.119 g/cm
2,
P = 0.0180). Regarding LS-, TH- or FN-BMD, no other signifivative differences were observed co-analyzing
ESR1 and
ESR2 loci.
Table 3
Mean age-class adjusted values (SD) of LS-, TH- and FN-BMD regarding ESR1 and ESR2 genotypes.
Subjects (n) | 158 | 192 | 219 |
Age range (yrs) | 20–30 | 31–40 | 41–50 |
BMD (g/cm2) |
LS
|
TH
|
FN
|
LS
|
TH
|
FN
|
LS
|
TH
|
FN
|
ESR1 rs2234693
| | | | | | | | | |
CC | 1.063 (0.134) | 0.941 (0.117) | 0.859 (0.150) | 1.075 (0.138) | 0.906 (0.098) | 0.821 (0.125) | 1.021 (0.122) | 0.902 (0.115) | 0.778 (0.122) |
CT | 1.025 (0.112) | 0.930 (0.120) | 0.845 (0.116) | 1.067 (0.122) | 0.906 (0.117) | 0.795 (0.118) | 1.041 (0.127) | 0.905 (0.127) | 0.789 (0.115) |
TT | 1.077 (0.131) | 0.969 (0.121) | 0.874 (0.129) | 1.059 (0.106) | 0.928 (0.105) | 0.813 (0.118) | 1.049 (0.125) | 0.904 (0.124) | 0.777 (0.122) |
ESR2 rs4986938
| | | | | | | | | |
AA | 1.039 (0.130) | 0.932 (0.110) | 0.846 (0.112) | 1.059 (0.123) | 0.922 (0.110) | 0.814 (0.122) | 1.044 (0.147) | 0.860 (0.111) | 0.738 (0.108) |
AG | 1.044 (0.126) | 0.935 (0.125) | 0.847 (0.131) | 1.076 (0.117) | 0.910 (0.106) | 0.804 (0.110) | 1.032 (0.128) | 0.900 (0.118) | 0.784 (0.118) |
GG | 1.056 (0.121) | 0.963 (0.122) | 0.870 (0.135) | 1.056 (0.125) | 0.911 (0.115) | 0.806 (0.132) | 1.046 (0.115) | 0.923 (0.130) | 0.798 (0.119) |
According to previously published data, oral contraceptive users and 18 women considered in perimenopausal phase for serum FSH levels >30 IU/mL were excluded from statistic analysis for bone turnover markers [
9]. Age class-adjusted levels of serum OC, CTX and P1NP did not segregate with
ESR1 and
ESR2 loci (
P > 0.05). Furthermore, no differences between
ESR1 and
ESR2 polymorphisms, were detected for serum age class-adjusted levels of calcium, phosphate and magnesium (
P > 0.05).
Discussion
Low BMD is a major risk factor for spine and proximal femur fractures [
13,
14]. In women, BMD in adulthood is largely determined by the amount of bone accumulated at the end of their skeletal growth (peak bone mass), their rate of bone loss after menopause when ovaries cease producing estrogens, and age-related bone loss. It has been well established with the study of twins, that peak bone mass is highly heritable with an estimated heritability between 0.50 and 0.80 [
15]. Conversely, published data on the heritability of bone loss at menopause are conflicting [
16‐
18]. Therefore, BMD is a trait that lends itself to studies designed to identify the genes underlying its normal variation [
16,
17,
19].
The past decade has seen an important increase in the use of association studies with candidate genes for the genetic analysis of complex traits such as BMD and/or fracture risk. Many genes have been examined for their association with normal BMD variation, which yields an ever-expanding candidate gene list. However, this approach has been largely criticized because of discrepancy in the results [
20,
21], often related to the small size of the enrolled cohorts. Moreover, most of the studies focused on postmenopausal female populations. In this view, the main purpose of the present study was to evaluate allelic influence of target genes, such as estrogen receptors, on inherited skeletal traits in a large and homogeneous population-based cohort of premenopausal healthy Caucasian women [
9].
For
ESR1 and
ESR2, two genes worldwide evaluated by independent research groups, the results obtained even if compelling for their involvement in BMD, osteoporosis, or fracture, are, however, not conclusive [
22]. Confounding factors encompassed ethnic-specific distribution of
ESR1 and
ESR2 polymorphisms [
8,
23,
24]. For example, in the SWAN study [
23] which enrolled 693 Caucasian participants (366 premenopausal women), specific associations of BMD with
ESR1 and
ESR2 genotypes varied according to race/ethnicity. Furthermore, 4 independent studies [
24] concluded that
ESR2 locus could be involved in FN-BMD in Caucasians, LS-BMD in Japanese postmenopausal women, and LS- and FN-BMD in Chinese premenopausal women. In addition, most of the human studies of genetic association with BMD have been cross-sectional, and only very few studies examined the association of the genotypes to BMD change within specific age ranges. For all these reasons the present study was aiming to compare the results obtained for
ESR1 and
ESR2 to other data obtained in age-equivalent studies in Caucasian women, examining the relation of
ESR1 and
ESR2 genes' polymorphisms.
Differently than for the
ESR2 locus [
25], allelic variants of
ESR1 gene were proposed to affect skeletal growth, through a genotype-dependent estrogen sensitivity at the growth cartilage, with the
ESR1 px haplotype being less sensitive to estrogen effects [
26]. The
ESR1 haplotype effect was supported by functional studies [
27,
28] and by multiple association analysis documented for this gene variant [
29‐
33]. For example, body Ht in pre- and postmenopausal women [
29] and estradiol levels in premenopausal women were lower [
30], with the number of copies of
ESR1 px haplotype in their genotype. Lorentzon et al. [
31] found an association between reduced Ht and
PvuII
T and
XbaI
A alleles, which corresponded to the
ESR1 px haplotype. Although this study was performed in adolescent boys [
31], it is in line with other findings in adult women. In 607 Caucasian women (aged 55–80 yrs) in whom vertebral fractures were excluded, Schuit et al. [
29] observed significant association between Ht and
ESR1 PvuII-
XbaI haplotypes. In contrast to [
32], a significant allele dose effect was observed for
ESR1 px haplotype, corresponding to a 0.9-cm decrease in Ht per allele copy (
P for trend = 0.02), extreme genotypes varied 1.8 cm. Boot et al. [
33] partially confirmed this allele dose-effect to some extent, as in girls heterozygous for
ESR1 px haplotype the Ht was higher than in those homozygous for the
ESR1 px haplotype. In our series, higher Ht was slightly (
P > 0.05) correlated with
ESR1 CC,
GG or
CCGG, and
ESR2 AA genotypes, while
ESR1 CCGG plus
ESR2 AA-AG genotype was significantly (2.5-cm) taller than the opposite genotype. As
ESR2 modulates
ESR1 transcriptional activity [
34], this novel biological interaction between
ESR2 and
ESR1 genotypes is not surprising.
Family history is a major risk factor for osteoporotic fractures [
35]. In white postmenopausal women, increased BMD-independent risk for vertebral (but not non-vertebral) fractures was found in
ESR1 px haplotype carriers [
26]. Moreover, the GENOMOS Consortium found a BMD-independent protective effect against vertebral fractures in
ESR1 XX homozygous individuals, while no effects on fracture risk were seen for
ESR1 PvuII polymorphism [
22]. Similarly to the InCHIANTI study [
36], we could not demonstrate any strong association between FHF and
ESR1 rs2234693 and
rs9340799 genotypes. However, our study might have had not enough power to detect any differences.
Variants of
ESR2 gene, alone and in interaction with
ESR1 genotypes influenced the fracture risk in postmenopausal women. Moron et al. [
37] suggested that
ESR2 rs4986938 (but not
ESR1 rs2234693) could have a role (
P = 0.04) in osteoporosis in Spanish postmenopausal women. Furthermore, they detected a joint effect of
ESR1 gene in osteoporosis modulating the penetrance of
ESR2 rs4986938 genotype [
37]. Rivadeneira et al. [
38] showed for the first time that white postmenopausal women (≥ 55 yrs of age) who are homozygous for a common intron 2–3'
UTR ESR2 haplotype allele have 40–80% increased risk of fragility and vertebral fracture. Interestingly, we also observed
ESR2 rs4986938 genotypes significantly correlated with FHF risk but not with FHO, suggesting
ESR2 variants may affect bone strength independently of BMD.
According to our findings, McGuigan et al. [
39] observed a modest association between
ESR1 PvuII genotypes and BMD at the hip (
P = 0.034) but not at the spine in 216 young Irish women (mean age 22.6 ± 1.6 yrs), with no differences regarding the
ESR1 XbaI locus [
39]. On the other hand, Valero et al. [
40] found no significant relations between FN- or LS-BMD with both
ESR1 PvuII and
XbaI loci in 194 older Caucasian women aged 22–45 yrs. Furthermore, a cross sectional study of
XbaI and BMD in women who were premenopausal and perimenopausal, did not confirm this association [
41]. Finally, in perimenopausal Caucasian women (older than 48.5 yrs) enrolled in the GENOMOS consortium, none of two
ESR1 intron 1 polymorphisms (
i.e. PvuII and
XbaI loci) or derived haplotypes had any statistically significant effect on BMD, with estimated differences between genetic contrasts being 0.01 g/cm
2 or less [
22]. Collectively, our findings and the published studies [
22,
39‐
41] make possible to support a significant effects of the
ESR1 rs2234693 (but not
rs9340799) locus on the BMD mainly in the young adult next to her achievement of bone peak mass.
Previous approaches have also suggested the role of
ESR2 in BMD within different ethnic backgrounds [
24]. No association between
ESR2 rs4986938 with LS- or FN-BMD were detected in 1291 Caucasian women (from 192 families) aged 33.2 ± 7.1 yrs (range 20–50 yrs) [
42]. Similarly, no associations between
ESR2 rs4986938 genotypes and Ht, LS-BMD and serum OC levels were detected in 147 healthy peri and postmenopausal Greek women (mean age 54 ± 7.9 yrs) [
25]. On the other hand, we detected significant BMD variations of the
ESR2 rs4986938 genotypes only in the later age group (i.e. 41–50 yrs old women). Together with
ESR1 rs2234693 data, this reinforces the hypothesis that
ESR1 and
ESR2 genes affect bone metabolism in precise and distinct age-sequential windows. Larger pre-planned analysis will be necessary to confirm our interpretation.
In conclusion, taken together, our findings indicated that, although the effect size may be small, allelic variations in
ESR1 and
ESR2 genes are associated with various and different bone traits (e.g. Ht, BMD and FHF risk) in normal premenopausal Caucasian subject. Furthermore, multiple genotype interactions were detected that reinforced the polygenic and complex character of skeletal system. In some cases however, the mean pattern of bone trait values for a gene polymorphism with evidence of association was not in agreement with previously published studies. Therefore, even though family history of fragility fractures is one of the risk factors [
35], we cannot recommend genetic testing for clinical use in humans to better identify population at risk for pathologic bone traits such as fragility fractures. However, as it has been shown for other diseases [
43], extended panels of several polymorphic markers could be used in the future, in addition to traditional risk factors, to evaluate the skeletal disorder risk in humans.
Competing interests
All authors participating to the BONTURNO study, and then to the preparation of this manuscript, did not have competing interests regarding the present data.
Authors' contributions
Both FM carried out the molecular genetic studies, performed the statistical analysis and drafted the manuscript. LM participated in the sequence alignment. GB, SM, GL and LM carried out the subject enrolment and their clinical evaluation. AP, SS and DM participated in the design of the study and in the subject data collection. MLB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.