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Original Studies |
Sports Medicine Unit, Department of Orthopedics (M.L., R.L., P.N.), Department of Geriatric Medicine (P.N.), Department of Gynecology and Obstetrics (T.B.), and Department of Musculoskeletal Research, National Institute for Working Life (R.L.), Umeå University, 901 85 Umeå, Sweden
Address correspondence and requests for reprints to: Peter Nordström, M.D., Ph.D., Sports Medicine Unit, Department of Orthopedics, Umeå University, 901 85 Umeå, Sweden.
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Abstract |
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gene polymorphism and estradiol on height and
bone density during and after puberty in males. Using the restriction
enzymes XbaI and PvuII, the allelic
variants XX, Xx, xx, PP, Pp, and pp were identified in 90 Caucasian
boys 16.9 ± 0.3 yr of age (mean ± SD). Bone
mineral density (BMD; g/cm2) of the total body, head,
femoral neck, and lumbar spine was measured using dual-energy x-ray
absorptiometry. Volumetric BMD (vBMD; mg/cm3) was estimated
for the spine. The XbaI or PvuII
genotypes were not related to the levels of estradiol, and the levels
of estradiol were not related to BMD (P > 0.05).
The xx allelic variant was associated with a higher spine vBMD than the
Xx allelic variant (361 vs. 340 mg/cm3,
P = 0.04). In a multivariate analysis including
pubertal development, physical activity, and body weight, the
XbaI genotype independently predicted total body BMD,
head BMD, and spine vBMD (P < 0.05). The
PvuII genotype independently predicted spine vBMD
(pp > PP, P = 0.01). The 20 boys with the PP
allelic variant were found to have a greater body height than the other
70 boys (182 cm vs. 179 cm, P =
0.03). At a 2-yr follow-up the XbaI genotype was still
independently related to total body BMD, head BMD, and spine vBMD. In
conclusion, estrogen receptor gene polymorphism is related to bone
density and height during late puberty and at attainment of peak bone
density in young men. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsReferences |
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Estrogen is known to be important for preservation of bone mass
in females during menopause (10). More recently,
Kobayashi et al. (11) demonstrated that polymorphism of the
estrogen receptor gene was related to BMD of the lumbar spine and
total body in Japanese postmenopausal women. Riggs et al.(12) suggested that estrogen deficiency also
contributes to the continuous bone loss in men, and quite recently a
relationship was demonstrated between estrogen receptor gene
polymorphism and spine BMD in a cohort of men 20–79 yr of age (13).
Furthermore, recent case reports suggest that estrogen may influence
bone accumulation in males. Smith et al. (14) reported
osteoporosis in a 28-yr-old man with estrogen resistance due to a
disruptive mutation of the estrogen receptor gene. Two other case
reports (15, 16) found increased bone mass from treatment with estrogen
in young adult males with null homozygous mutations of the gene for the
p-450 aromatase, which is required for the conversion of androgens to
estrogen. Administration of estrogens increased BMD and stopped linear
growth by fusion of the growth plates. The purpose of the present study
was to investigate whether polymorphism in the estrogen receptor
gene and estradiol levels were related to bone density and height
during late puberty and to peak bone mass in healthy adolescent
Caucasian males.
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Subjects and Methods |
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From advertisement and information in schools and local sports clubs, 96 healthy Caucasian boys participated in a longitudinal study investigating the influence of puberty on bone density at our Department. Of these boys, 92 could be reached and were asked to participate in the present study, and 91 boys volunteered. One of these boys was excluded because it was judged he had not passed the pubertal growth spurt, leaving 90 boys (age 16.9 ± 0.3 yr) for the present study. None of the subjects had any disease or medication known to affect bone metabolism. Weight and height were measured using standardized equipment. The same physician divided the subjects into different pubertal stages according to Tanner (17). All participants were judged to have passed the pubertal growth spurt period and were at least Tanner stage 4, based on at least two measurements of weight and height during 1 yr and also development of axillary and pubertal hair growth and growth of beard. Using a questionnaire, the average amount of weight-bearing physical activity per week was assessed during the last year. A follow-up was conducted 2.4 ± 0.7 yr later with new measurements of bone density. Eighty-eight of the 90 boys volunteered to participate, and all these boys had past puberty (Tanner 5). The two boys who could not be followed up were not different in physical characteristics or bone density (mean total body BMD, 1.227 g/cm2) than the rest of the boys. Informed written consent was given by all the participants, and the study protocol was approved by the Ethical Committee of the Medical Faculty, Umeå University.
Techniques for estimating bone density
BMD (g/cm2) of the total body, head, femoral neck, and BMD, the bone mineral content (BMC, grams), the bone area, and height of the lumbar spine were measured using a Lunar Corp. DPX-L (Lunar Corp., Madison, WI) dual-energy x-ray absorptiometer, software version 1.3y. Others have previously discussed the precision of this method in detail (18, 19). The coefficient of variation (CV) value (SD/mean) for repeated measurements is 0.7–2.0% in our laboratory, depending on application. Because areal BMD (g/cm2) is affected by the bone size, volumetric BMD (vBMD; mg/cm3) was also estimated for the lumbar spine (20). It was then assumed that this site is cylindrical in shape. The volume of this cylinder can be estimated from the area and height. The vBMD (mg/cm3) is then estimated as (BMC/volume) x 1000 (mg/cm3).
Estradiol measurements
The Double Antibody Estradiol procedure (Diagnostic Products Corp., Los Angeles, CA) was used to determine the levels of estradiol in whole blood. The method is an antiestradiol antiserum raised in rabbit. The radioactive ligand was 125I-labeled estradiol. Two samples from each subject were analyzed and the mean from these samples was then used in further analysis. The samples were analyzed according to the manufactures manual, within assay CV was 5% and between assay CV 5.5%. The minimal detectable concentration of estradiol in this assay was 1.4 pg/mL. However, we analyzed frozen whole blood instead of serum. To assure the accuracy and precision of the results, we analyzed estradiol in both frozen whole blood and serum in 10 girls of the same age. The correlation coefficient was 0.823, P = 0.003 between whole blood and serum measurements (mean ± SE, 67 ± 12 and 77 ± 20 pg/mL, respectively, no significant difference).
Genomic DNA analysis
Genomic DNA from the 90 boys was isolated from EDTA-stabilized blood, using the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI). Genomic DNA (40 ng) was amplified (11) in a 100-µL reaction mixture consisting of 0.4 µM forward primer (5'- CTGCCACCCTATCTGTATCTTTTCCTATTCTCC-3'), 0.4 µM reverse primer (5'-TCTTTCTCTGCCACCCTGGCGTCGATTATCTGA-3'), 0.2 mM each of dATP, dCTP, dGTP, dTTP, 1x PCR buffer, and 3.5 U Taq polymerase (Roche Molecular Biochemicals, Stockholm, Sweden). PCR was carried out in 30 cycles of 94 C for 30 sec, 61 C for 40 sec, and 72 C for 90 sec (Peltier Thermocycler; MJ Research, Inc., Watertown, MA).
The 1.3-kb product, a part of intron 1 and exon 2 of the estrogen
receptor gene (11), was purified using the
PCR Clean Up Kit (Roche Molecular Biochemicals), cleaved
with an excess of XbaI or PvuII restriction
endonucleases (Roche Molecular Biochemicals),
electrophoresed, and analyzed on 1.0% agarose gel. Genotypes for
PvuII and XbaI polymorphisms were termed PP, Pp,
and pp and XX, Xx, and xx, respectively. Uppercase letters represent
absence, and lowercase letters represent presence of restriction
sites.
Statistical analysis
Differences in bone density and physical characteristics between the three groups defined by the estrogen receptor genotypes were investigated using an ANOVA, with Bonferroni’s correction for multiple comparisons. The independent contribution of the estrogen receptor genotypes to bone density was also investigated using a multiple regression analysis. The estrogen receptor genotypes, weight, pubertal stage, and physical activity were used as explanatory variables. Since the relationship between bone density and the different estrogen receptor genotypes cannot be assumed to be linear, the estrogen receptor genotypes were first transformed into two Dummy variables. For the XbaI genotype the first Dummy variable was constructed to test whether XX > Xx, and the second was constructed to test whether xx > Xx. For the PvuII genotype the first Dummy variable was constructed to test whether pp > Pp, and the second was constructed to test whether pp > PP. Bivariate correlations were calculated using Pearson’s coefficient of correlations. The SPSS, Inc. package for PC was used for the statistical analysis. A P value less than 0.05 was considered significant.
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Results |
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The 90 boys studied were 16.9 ± 0.3-yr-old (mean ± SD). The restriction fragment length polymorphisms were coded as X or x (XbaI) and P or p (PvuII). The frequency of the different estrogen receptor allelic variants in the 17-yr-old cohort were 8.9% (XX), 40.0% (Xx), 51.1% (xx), 22.2% (PP), 44.4% (Pp), and 33.3% (pp). The different genotypes were found to be in Hardy-Weinburg equilibrium.
Genotypes, anthropometric characteristics, and bone density
Physical characteristics, levels of estradiol, and bone
density for all subjects and subgroups according to the XbaI
and PvuII allelic variants are presented in Tables 1
and 2.
The 40 boys with the Pp allelic variant were found to be older than the
boys with the PP allelic variant (Table 2). Adjustment, using linear
regression equations, for this difference in age did not change the
significance of any of the results in Table 2. The boys with the xx
allelic variant were found to have significantly higher spine vBMD
compared with the boys with the Xx allelic variant (mean difference, 21
mg/cm3; P < 0.05; Table 1).
There was a tendency toward a significant influence of the
PvuII genotype on body height (P = 0.09),
and the 20 boys with the PP allelic variant were found to have a
significantly greater body height than the other 70 boys (182 cm
vs. 179 cm, P = 0.03).
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The independent contribution to bone density by the estrogen receptor
genotypes, weight, pubertal stage, and physical activity were
investigated using multiple regression (Table 3). The different allelic variants were
then transformed into two Dummy variables, as described previously. The
XbaI genotype was then found to independently predict total
body BMD, head BMD, and spine vBMD, and the PvuII genotype
was found to independently predict spine vBMD (Table 3).
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and 2). Using bivariate correlations, the
levels of estradiol did not predict BMD of any site (r =
-0.05–0.07, P > 0.05).
Eighty-eight of the 90 boys could be followed up after
2.4 ± 0.7 yr (mean ± SD). Spine vBMD was still
associated with the XbaI genotype in this cohort
(xx>Xx, P = 0.04). Independent predictors of
bone density were estimated as in the 17-yr-old cohort. The
XbaI allelic variants were then found to predict total body
BMD (xx>Xx, P = 0.007; XX>Xx, P
= 0.04), head BMD (XX>Xx, P = 0.02), and spine vBMD
(xx>Xx, P = 0.01). The boys with the PP genotype were
still found to have a significantly greater body height than the rest
of the boys (183 cm vs. 180 cm, P = 0.02).
The predictors of the increase in BMD (
BMD) per year were also
analyzed. Body weight and pubertal development predicted total body
BMD and head BMD, and physical activity predicted spine BMD
(r = 0.24, P = 0.03) and spine vBMD (r =
0.23, P = 0.03). The PvuII and
XbaI genotypes were not related to the increase in bone
density.
Discussion
The main purpose of the present study was to evaluate the
influence of estrogen receptor gene polymorphism and estradiol
on BMD in adolescent boys. Since areal BMD
(g/cm2) is a measure influenced by the bone size,
vBMD (mg/cm3) was estimated for the spine
(20, 21). We found that the XbaI
allelic variants predicted the estimated volumetric bone density of the
lumbar spine at both 17 and 19 yr of age. Besides genetic factors, body
weight, pubertal development, and physical activity have previously
been demonstrated to be important determinants of bone density in
children and adolescents (22, 23, 24). In the present
study, all of these parameters were found to predict at least one BMD
site in a multiple regression analysis. When including these factors in
such a multivariate analysis, the XbaI genotypes were found
to independently predict total body BMD, head BMD, and spine vBMD in
the 17-yr-old cohort. However, the XbaI genotypes were not
associated with femoral neck BMD. Perhaps the greater influence of
estrogen on bone metabolism in cancellous bone, rather than on cortical
bone, could explain why no association between the XbaI
genotypes and femoral neck BMD, consisting mainly of cortical bone, was
found (25).
There was no relationship between the bone gain seen after 2 yr and the estrogen receptor genotypes. Furthermore, the difference between the extreme allelic variants did not change when comparing the 17-yr-old cohort and the 19-yr-old cohort (data not shown). This indicates that estrogen receptor gene polymorphism may influence bone development predominantly before late puberty in males. However, the XbaI allelic variants were still independently related to BMD at 19 yr of age when all boys had passed puberty and attained their approximate peak bone mass (26, 27).
Riggs et al. (12) recently suggested that estrogen deficiency might substantially contribute to the continuous bone loss not only in women but also in men. Theoretically, the association between the estrogen receptor genotypes and bone density found in the present study may primarily be related to levels of estradiol and a possible effect of estrogens on the growing bone. However, in our population, the estradiol levels were not related to the bone density of any site. This was rather expected considering we studied a very homogeneous group of young males with small differences in age.
To our knowledge, only one previous study has investigated the association between estrogen receptor polymorphism and bone density in adult men. Accordingly, Ongphiphadhanakul et al. (13) examined the association between estrogen receptor polymorphism defined by the restriction enzyme PvuII and bone density in 81 Thai men, 20–79 yr of age (13). Presence of the P allele was associated with a higher BMD of the lumbar spine. The results also demonstrated estradiol, but not testosterone, to be independently associated with BMD at several sites. In women, Kobayashi et al. (11) found an association between polymorphism of the estrogen receptor gene and BMD in the total body and lumbar spine in postmenopausal women. It should be noted, however, that these differences were found when combining the XbaI and PvuII genotypes. Willing et al. (28) demonstrated an influence of both XbaI and PvuII polymorphism on BMD of the lumbar spine in pre- and perimenopausal women. Another study was unable to find an association between estrogen receptor polymorphism and bone density in an elderly population (29), whereas a recent study (30) found an association present only before menopause.
The knowledge concerning the influence of estrogen on peak bone density in males is mainly based on case reports and some animal studies. Accordingly, Korash (31) demonstrated decreased BMD in female, as well as male, estrogen receptor knockout mice. Carani et al. (15) investigated a man with a mutation of the P-450 aromatase gene, which is required for the conversion of androgens to estrogen. Treatment with estrogen increased bone growth and maturation whereas androgens did not, suggesting that development of peak bone mass in men may depend mainly on the influence of estrogen, rather than androgens. In humans about 40% of peak skeletal mass accumulates during the peripubertal period (26, 27). Theoretically, a true influence of the estrogen receptor gene on bone accumulation would then be most obvious during this period. In the present study, there was no significant relationship between the annual increase in bone density after puberty and the estrogen receptor allelic variants.
The case reports investigating the influence of estrogen deficiency on bone in men found delayed bone maturation and increased body height besides osteopenia (14, 15, 16). In the men with aromatase deficiency, administration of estrogens stopped linear growth by fusing the growth plates (15, 16). This was not the case for the man with the estrogen receptor mutation (14). This indicates that the effect of the administrated estrogen on linear growth was mediated via the estrogen receptor. Estrogen receptors have also been detected in human growth plates (32). We, therefore, hypothesized that estrogen receptor gene polymorphism might be related to body height in adolescent men. Interestingly, absence of the p allele was associated with a significantly greater body height at both 17 and 19 yr of age in the present study.
The mechanism by which estrogen and the estrogen receptor exerts its
actions on bone mass has yet to be determined in detail. It has been
demonstrated in vitro that androgen as well as estrogen
receptors are present in male human osteoblasts (33), whereas
only estrogen receptors have been found in osteoclastic cells (34). It
has been suggested that 17 ß-estradiol may lower osteoclast
resorption of bone by influencing production and activation of
transforming growth factor-ß (35). Estrogen has also been
demonstrated to inhibit osteoclastogenesis by reducing interleukin-6
production in bone marrow stromal cells (36). The
polymorphic sites defined by restriction enzymes PvuII and
XbaI are both located in the first intron of the estrogen
receptor gene (11). The mechanism of how
these genetic differences might influence BMD is unknown. Intronic
sequences have been reported to contain regulatory elements for
transcription and splicing, giving rise to varying messenger RNA levels
and different isoforms of mature messenger RNA, respectively
(37, 38). Whether this is the case for the
estrogen receptor polymorphisms remains to be determined.
In the present study we could not demonstrate a gene dose-response relationship between the XbaI allelic variants and BMD, and the heterozygotic allelic variant was generally associated with the lowest bone density. We have no reasonable explanation for this result. Some previous studies have found dose-response relationship with bone density (28), whereas others have not (11, 13).
In conclusion, the present study suggests that estrogen receptor gene polymorphism may influence the development of bone density in adolescent Caucasian boys. Our results also indicate that estrogen receptor polymorphism may influence the accumulation of bone density predominantly before late puberty. However, the XbaI genotype remained significantly related to the attained peak bone mass. Given the relative small material studied and the few subjects defined by the XX allelic variant, additional research is needed to confirm these results. The results of the present study also suggests that estrogen receptor gene polymorphism might influence the development of body height in men.
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Acknowledgments |
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Footnotes |
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Received July 19, 1999.
Revised October 4, 1999.
Accepted October 10, 1999.
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References |
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in human and rabbit skeletal
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