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Keywords:

  • association;
  • polymorphisms;
  • cytokines;
  • osteoporosis;
  • estrogens

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Appendix 1
  9. REFERENCES

Lifestyle and dietary factors may influence the association of IL-6 polymorphisms with bone mass. In 1574 unrelated men and women from the Framingham Offspring Cohort, we observed significant hip BMD differences between IL-6 −174 genotypes only in older women, those without estrogens, and those with a poor calcium intake. Hence, association of IL-6 polymorphisms with BMD may be limited to discrete population subgroups.

Introduction: Interleukin (IL)-6 plays a central role in the pathogenesis of osteoporosis. Two functional variants in the IL-6 promoter have previously been associated with IL-6 expression, bone resorption levels, and BMD in late postmenopausal women, but results were conflicting in different populations. We hypothesized that the association between IL-6 promoter alleles and BMD may be affected by interactions with lifestyle and dietary factors known to influence bone turnover.

Materials and Methods: Among the Offspring Cohort of the Framingham Heart Study, 1574 unrelated men and women were genotyped for IL-6 −572 and −174 alleles. Interaction analyses with years since menopause, estrogen status, physical activity, smoking, dietary calcium, vitamin D, and alcohol intake were based on BMD measurements at the hip.

Results and Conclusions: In models that considered only the main effects of IL-6 polymorphisms, no significant association with BMD was observed in either gender. In contrast, p values (0.003–0.096 by ANOVA) suggestive of an interaction between IL-6 −174 genotypes and years since menopause, estrogen status, dietary calcium, and vitamin D intake were observed in women (n = 819). In turn, BMD was significantly lower with genotype −174 GG compared with CC, and intermediate with GC, in women who were more than 15 years past menopause and in those without estrogens or with calcium intake <940 mg/day. In estrogen-deficient women with poor calcium intake, BMD differences between genotypes CC and GG were 10.2% at femoral neck (p = 0.012), 12.0% at trochanter (p = 0.012), and 16.8% at Ward's area (p = 0.0014). In contrast, no such interactions were observed in men (n = 755). In conclusion, IL-6 genetic variation was prominently associated with hip BMD in late postmenopausal women, those without estrogen replacement therapy, and those with inadequate calcium intake. In contrast, IL-6 polymorphisms are unlikely to be significant determinants of bone mass in other women or men.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Appendix 1
  9. REFERENCES

INTERLEUKIN-6 (IL-6) is a pleiotropic cytokine playing a central role in bone turnover.(1) IL-6 produced by osteoblasts and monocytes from the bone marrow promotes osteoclast differentiation and activation.(1)IL-6 gene expression is tightly regulated by several hormones, cytokines, and their transcription factors. Among them, IL-1 and TNF-α activate, whereas estradiol (E2) and glucocorticoids repress, IL-6 gene transcription.(2) Hence, increased IL-1/TNF-α production, such as in rheumatoid arthritis, and decreased estrogen synthesis, as occurs in postmenopausal women, enhances IL-6 expression, bone resorption, and bone loss. Moreover, IL-6 expression in bone is triggered by parathyroid hormone and may therefore be implicated in the age-related bone loss associated with poor calcium and vitamin D intake.(2, 3)

Several allelic variants have been identified in the IL-6 gene promoter region.(4, 5) Among them, a common G>C polymorphism at position −174 involves a DNA-binding site for NF-IL-6, a transcription factor that transduces estradiol activity on IL-6 gene expression, whereas another rarer G>C polymorphism at position −573 is located in close vicinity to glucocorticoid response elements (GRE). There is some evidence that these polymorphisms produce functional variants in that the −174 C allele results in lower, whereas the −573 ITALIC>C allele results in higher, stimulated IL-6 promoter activity in vitro.(4, 6) In turn, IL-6 promoter polymorphisms have been associated with circulating levels of IL-6,(4) biochemical markers of IL-6 activity (C-reactive protein [CRP]), and bone resorption (C-terminal cross-linking of type 1 collagen [CTx]).(6, 7) Moreover, some studies have suggested an association of IL-6 promoter polymorphisms with BMD in late postmenopausal women,(6-8) whereas two other studies reported an association with peak bone mass.(9, 10) Several linkage studies have also identified the IL-6 gene locus in relation to BMD in postmenopausal women(11) and in families of osteoporotic probands,(12, 13) but not in young sib pairs.(14) Various factors may have contributed to these apparently discordant results among studies, including differences in the genetic background and age structure of the populations, in the BMD sites analyzed, and more specifically, failure to take into account specific factors that modulate IL-6 gene expression, such as estrogens and dietary calcium.

The Framingham Osteoporosis Study is an epidemiological cohort study that was initiated in 1988 to identify new risk factors for age-related bone loss in both men and women.(15, 16) In addition to sex steroid deficiency and the aging process, this and other population-based studies have contributed to the understanding of several lifestyle and environmental factors, particularly nutrients, on the maintenance of bone mass.(16, 17) In this study, we hypothesized that the association of IL-6 genetic variation with BMD could be influenced by factors known to modulate IL-6 gene expression and/or bone turnover, including age, estrogens, and dietary calcium.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Appendix 1
  9. REFERENCES

Framingham Study subjects and outcome variable

In 1971, the Framingham Offspring Study (FOS) was initiated with intent to evaluate, among other goals, the role of genetic factors in the etiology of coronary artery disease and included a total sample size of 5124 subjects.(18) The Offspring Cohort is comprised of adult offspring (and their spouses) of couples from the Original Framingham Cohort, and 96.4% of the Offspring Cohort are whites with origins in Eastern and Western Europe (see Appendix 1 for details). These Offspring Cohort members participated in FOS at either their examination cycle 6 or 7, as described elsewhere in detail.(15, 16) Informed consent was obtained from participants of each Cohort before entry into the study, which was approved by the Boston University Institutional Review Board for Human subjects Research.

The participants underwent bone densitometry of the femoral neck, trochanter, and Ward's area by DXA using a Lunar DPX-L device to measure BMD (g/cm2) in 1996-2000. The CVs in normal subjects for the DPX-L were 1.7% (femoral neck), 2.5% (trochanter), and 4.1% (Ward's area).(16)

Interaction factors and potential confounders

Potentially confounding variables measured at the time of BMD measurement were obtained for each participant, along with overall medical history. Details of these measurements have been published previously.(16, 19) These variables included age, sex, height, weight, body mass index (BMI), alcohol intake, caffeine consumption, calcium and vitamin D intake, smoking status, physical activity, and for women, estrogen use.

Age squared was considered in the models to account for potential nonlinear age effects.(20) Weight and height were measured using standard methods.(16, 19) BMI was calculated in kg/m2. Dietary intakes of calcium and vitamin D (including supplements) were assessed using the 126-item Willett food frequency questionnaire.(21) Total alcohol consumption was calculated based on a published equation using self-report of the intake of beer, wine, and mixed drinks per week, providing alcohol equivalents in ounces per week.(22) Smoking was assessed at the time of BMD measurement as current cigarette smoker, former smoker, or never smoker.(23) Physical activity was examined using the Physical Activity Scale for Elderly questionnaire.(24) For postmenopausal women, estrogen use was evaluated as current use, former use, and never used oral estrogen, patch, or cream. Subsequently, postmenopausal women currently on ERT and premenopausal women together were considered as one group of estrogen-repleted (versus estrogen-deficient) women.

Determination of IL-6 gene promoter alleles

DNA was available from 1811 biologically unrelated subjects from the Offspring cohort. Among these, 1574 subjects for whom BMD and dietary and lifestyle questionnaire data were also available were included in this study.

To directly determine IL-6 −572/−174 haplotypes, four pairs of fluorescence-labeled primers based on the sequence of all the possible haplotypes(5) were designed and used in allele-specific PCR reactions. The PCR results were analyzed, and the genotypes were determined based on the presence or absence of a peak corresponding to a particular haplotype using an ABI3700 device with an internal ROX labeled size standard and Genotyper 3.7 software. Primer pairs were as follows: IL6-C/C, 5′-GGCCAGGCAGTTCTACAACAGCCC-3′, 5′-TGCAATGTGACGTCCTTTAGCATG-3′; IL6-C/G, 5′-GGCCAGGCAGTTCTACAACAGCCC-3′, 5′-TGCAATGTGACGTCCTTTAGCATC-3′;IL6-G/C, 5′-GGCCAGGCAGTTCTACAACAGCCG-3′,5′-TGCAATGTGACGTCCTTTAGCATG-3′; IL6-G/G, 5′-GGCCAGGCAGTTCTACAACAGCCG-3′, 5′-TGCAATGTGACGTCCTTTAGCATC-3′.

To confirm calls by direct genotyping, two single nucleotide polymorphism (SNP) assays, one for each SNP at position −572 and −174, were designed and validated. SNP detection involves amplification of appropriate chromosomal regions by PCR and a primer extension reaction using a dideoxynucleoside that is complementary to one of the SNP alleles. The final length of the primer product depends on which allele is present and is scored using a Sequenom mass spectrometer run in high throughput mode.(25) The PCR primers for the −572 assay were 5′-ACGTTGGATGTCTTCTGTGTTCTGGCTCTC-3′ and 5′-ACGTTGGATGACGCCTTGAAG-3′, and the extension primer was 5′-TAACTGCACTTCTGGCTCTCCCTGTGAG-3′. For the −174 assay, the PCR primers were 5′-ACGTTGGATGAGCCTCAATGACGACCTAAG-3′ and 5′-ACGTTGGATGGATTGTGCAATGTGACGTCC-3′, and the extension primer was 5′-CCCCCTAGTTGTGTCTTGCC-3′. All reactions used protocols standardized by Sequenom for use by this machine.

Statistical analysis

Genotype frequencies were tested for Hardy-Weinberg equilibrium and linkage disequilibrium between polymorphisms at positions −572 and −174 was tested using a conventional statistical approach.(26) All following analyses were conducted separately in men and women. Initially, genotype groups were compared with regard to potential confounders using ANOVA for continuous variables and χ2 statistics for categorical variables. However, because of the presence of only two homozygotes −572 CC in this population, the latter were subsequently omitted from the analysis, and differences in continuous variables between IL-6 −572 genotypes GG and GC were analyzed by two-tailed Student's t-test. Statistical significance was considered to be at the 0.05 level for all comparisons by ANOVA and Student's t-test.

We performed analysis of covariance (ANCOVA) to compare BMD levels between genotype or haplotype groups, using several models that control for confounding variables. In the simplest case, BMD values were adjusted for age and by gender. A second model used a limited number of covariates (age, height, and BMI in males, with addition of estrogen replacement therapy in females), whereas a maximal model included all potential confounders listed above.

Cut-off levels to produce dichotomized covariates for interaction analysis were based either on the median value of the covariate by gender in our population (calcium intake, vitamin D intake, physical activity), on the presence/absence of a known risk factor for osteoporosis (estrogen status, smoking, and alcohol consumption), or in the case of years since menopause, on a value (15 YSM) that was estimated necessary to produce significant BMD differences between IL-6 genotypes according to a previous study(7) and which also produced two subgroups of comparable size. Interaction terms of IL-6 genotype with these covariates were included in two-factor ANOVA, together with the main effects of genotypes and covariates. Additional ANCOVAs were performed by entering only the covariates in the regression models, and R2s from these reduced models were compared with those from the models testing for interaction to determine the contribution of interaction term to the variation in bone phenotypes. If interaction was both substantial and reached a predetermined significance level at p < 0.099, it was considered suggestive for further analysis. In such a case, the study population was stratified according to the dichotomized covariate and least-squares mean BMD adjusted for potential confounders were compared using ANOVA or ANCOVA between genotypes within each covariate subgroup.

The SAS/STAT component (release 8.1) of the SAS system (SAS Institute, Cary, NC, USA) was used for all analyses.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Appendix 1
  9. REFERENCES

The demographic and clinical characteristics of the 1574 Framingham Offspring Cohort subjects in this study are reported in Table 1. Mean age was ∼60 years in both men and women. Cessation of ovarian function had occurred in 83% of all women, and close to one-third of these women were currently using estrogens.

Table Table 1.. Clinical Characteristics of Subjects From the Framingham Offspring Cohort
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The prevalence of the less common C allele was 5.6% at position −572 and 38.9% at position −174 (based on 3122 chromosomes in 816 women and 745 men, i.e., 99.2% of the original sample, for whom both alleles could be unambiguously determined), with the −572 C allele virtually always occurring on the −174 G allele (test for linkage disequilibrium, D′ = 0.987, p < 0.05; Table 2). Genotype distribution (GG, GC, and CC) was in Hardy-Weinberg equilibrium, and the two subjects who were homozygotes −572 CC were not included in subsequent analyses.

Table Table 2.. IL-6 Allelic Frequency*
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Body size and hip BMD measurements by IL-6 genotypes are shown in Table 3. There were no statistically significant differences between IL-6 genotypes or haplotypes for BMD at the femoral neck, trochanter, or Ward's region in either males or females. We further examined the association between IL-6 −174 genotypes and hip BMD in women who were 15 years or more past menopause compared with younger menopausal women, as previously suggested.(7)p values suggestive of an interaction between IL-6 −174 genotypes and years since menopause were found for all three regions of the hip (Table 4). Thus, in contrast to women within 15 years of menopause, in women 15+ years past menopause, BMD was significantly lower with genotype GG and intermediate with GC compared with genotype CC (femoral neck, +8.4%; trochanter, +10.2%; Ward's area, +12.6% for CC versus GG; Fig. 1). Of note, CC women 15+ years since menopause seemed to have a mean BMD at femoral neck and trochanter comparable with younger menopausal women (Fig. 1). Other variables including age, body size, and environmental/lifestyle factors did not differ among genotypes within subgroups for years since menopause (data not shown). In contrast, no significant interaction of age (below/above 60 and/or 65 years) with IL-6 polymorphisms could be detected in men (data not shown).

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Figure FIG. 1.. Hip BMD levels according to IL-6 −174 genotypes and years since menopause. The number of CC, GC, and GG women was, respectively, 40, 154, and 111 in women <15 years since menopause and 40, 148, and 121 in women >15 years since menopause. Significant p values for differences among genotypes, as shown, were obtained by ANOVA using BMD values adjusted for age and by gender. Further adjustment for height, BMI, and other potential confounders (maximal model) did not alter these results (femoral neck, p = 0.018; trochanter, p = 0.019; and Ward's area, p = 0.022 in women >15 years since menopause). Results are expressed as group means, with error bars indicating SE.

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Table Table 3.. Clinical and BMC Measurements According to IL-6 Genotypes*
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Table Table 4.. Interaction Terms of IL-6 −174 Genotypes With Nongenetic Factors on Hip BMD in Women*
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Potential interactions affecting BMD at specific regions of the hip were also suggested to occur between IL-6 −174 genotypes and estrogen status, dietary calcium and vitamin D intake, and physical activity (Table 4). Subsequently, BMD was found to be significantly lower at all hip sites in −174 GG compared with the other genotypes among postmenopausal women without estrogens, but not in estrogen-repleted women (Table 5). Similar findings were obtained at the Ward's area among women whose calcium intake was below, but not above, the median of 941 mg/day (Table 5). In addition, absence of estrogens and poor calcium intake had additive effects, because in postmenopausal women with both osteoporosis risk factors, BMD in CC was 16.8%, 12.0%, and 10.2% higher compared with GG at, respectively, the Ward's area, trochanter, and femoral neck (all p < 0.05, Fig. 2). None of the other covariates listed in Table 1 significantly differed among IL-6 genotypes within this group (data not shown). Eventually, interactions with years since menopause, estrogen status, and dietary calcium intake contributed 1.7% to the variance in BMD among all women, in addition to the contribution of all other factors included in the maximal model (R2 = 0.292 and 0.309 with and without interactions, respectively).

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Figure FIG. 2.. Hip BMD levels according to IL-6 −174 genotypes, estrogen status, and dietary calcium intake. The number of CC, GC, and GG women in the group of postmenopausal women without ERT and with low calcium intake (E−/Ca−) was 21, 99, and 73, whereas in the estrogen-replete, high calcium intake group (E+/Ca+), it was 21, 103, and 96, respectively. Significant p values for differences among genotypes (by ANOVA) are shown. After further adjustment for height, BMI, and other potential confounders (maximal model), significant differences remained at the femoral neck and Ward's area in the E−/Ca− group (femoral neck, p = 0.049; Ward's area, p = 0.006; and trochanter, p = 0.067). Results are expressed as group means, with error bars indicating SE.

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Table Table 5.. Hip BMD According to IL-6 Genotypes in Women With Osteoporosis Risk Factors
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In contrast, no significant BMD differences between genotypes were found according to vitamin D intake or physical activity subgroups (data not shown). Because we tested seven potential interactions at three BMD sites (i.e., 21 analyses), at the pre-established level of significance for positive interactions (p ≤ 0.099), we expected two false positive results among the seven significant p values for interaction observed (Table 4). In addition, we expected only one false positive result among the eight significant p values observed for assoication (at p < 0.05 for 18 tests in Figs. 1 and 2 and Table 5). Moreover, significant interactions were exclusively observed in women; none occurred in men (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Appendix 1
  9. REFERENCES

In this study from the Framingham Offspring Cohort, we made three major observations regarding the relationship between IL-6 genetic variation and hip BMD. First, there were no significant associations between IL-6 −174 or −572 genotypes and hip BMD in the population as a whole, whose mean age was close to 60 years. In contrast, a significant association with IL-6 −174 variants was detected in late postmenopausal women, starting 15 years after menopause (Fig. 1), as opposed to younger women or men. Several studies have identified the IL-6 gene locus as linked to BMD in postmenopausal women(8, 11) and in families of osteoporotic probands.(12, 13) Moreover, it has previously been suggested that at least 10 years since menopause may be required before differences in bone resorption levels between IL-6 genotypes −174 CC and GG eventually translate into significant differences in bone mass.(6, 7) In keeping with these observations, our results further indicate that IL-6 polymorphisms are associated with hip BMD mostly in elderly women, those carrying the IL-6 −174 genotype CC having a lower risk of developing osteoporosis with advancing age. Our observations are also consistent with the absence of linkage between the IL-6 gene locus and peak bone mass in a large cohort of young sib pairs,(14) but contrast with two other studies reporting association with peak bone mass in smaller cohorts of premenopausal women and young males.(9, 10) Taken together, these results seem to suggest that association of IL-6 alleles with BMD may predominantly occur in elderly women, whereas their association with peak bone mass in women or men is less clear. Although it may seem surprising that some genetic factors have an increasing influence on bone mass with aging, similar observations were previously reported concerning the Col1A1 alleles.(27) Moreover, we previously reported that a number of environmental and lifestyle factors, including caffeine, physical activity, serum 25(OH)D levels, and calcium intake, did not significantly affect bone loss in elderly women from the Original Framingham Cohort; only weight loss and estrogens did.(17) Thus, environmental factors alone are unlikely to explain all of the variance for bone loss observed in the elderly.

In our study, the association between IL-6 and hip BMD in women also depended on interactions with factors known to regulate bone turnover, particularly estrogens and dietary calcium intake.(3, 28, 29) The latter factor alone influenced the association of IL-6 −174 genotypes with BMD mostly at the Ward's area. This observation may be questionable at first, because the CV for measurements at Ward's area is larger than at the femoral neck, thereby potentially decreasing the ability to detect significant differences among groups. However, skeletal sites containing a higher proportion of trabecular bone, such as the Ward's area and the trochanter region, seem to be more sensitive to increased bone turnover, and in turn, to estrogen and calcium therapy.(28) Moreover, low calcium intake and estrogen deficiency had additive effects on BMD differences among genotypes in all three regions of the hip (Fig. 2), indicating that the IL-6 −174 GG genotype is a potential risk factor for osteoporosis among those women who are otherwise unprotected against postmenopausal bone loss.

It could be argued that at least some of the interactions and associations found in this study were spurious, considering that multiple comparisons were performed. However, there are several arguments to support the consistency of these results rather than attributing them to chance. First, this is the largest population-based association study with IL-6 genotypes performed so far, at least to our knowledge, and even specific subgroups in which these associations were found had a larger number of subjects than most previously published cohorts with IL-6 genotypes. Second, we hypothesized a priori that the interactions observed in this study would occur, based on the known biology of IL-6 in bone, which is stimulated in absence of estrogens and also in response to elevated parathyroid hormone levels consecutive to poor calcium (and vitamin D) intake, as occurs with increasing age.(2, 3) Moreover, IL-6 −174 alleles affect a binding site for NF-IL-6, a transcription factor that interacts with the estrogen receptor to repress IL-6 transcription.(4, 5) The Framingham Offspring Cohort seemed particularly suitable to test this hypothesis, because the mean age of the sample was 60 years, and the age range was nearly 25 years, including a large proportion (83%) of postmenopausal women and women on hormone replacement therapy (HRT; 32%; Table 1). Moreover, the cohort was evaluated for dietary calcium and vitamin D intake using validated food frequency instruments, among other variables. Third, to minimize the number of analyses performed, cut-offs for all the interaction factors that we examined were prespecified and not subject to additional cut-point testing. Fourth, all significant interactions appeared in women, but none appeared in men. If these results were because of chance alone, we would not expect to see them only in one gender. Fifth, similar trends for an association between IL-6 −174 genotypes and BMD prominently at trabecular bone sites, namely at the trochanter and lumbar spine, have previously been reported in an independent cohort of older, estrogen-deficient women.(6, 7)

A number of candidate genes have started to emerge as potential susceptibility factors for osteoporosis.(30) Among these, functional variants shown to alter the level of protein expression and activity, such as IL-6 promoter and collagen 1α1 (Col1A1) Sp1 alleles, seem to provide the most consistent results.(7, 27, 31, 32) Furthermore, these results depend on the homogeneity of the study cohort in terms of age, ethnicity, and genetic background; an accurate assessment of confounding factors; a systematic analysis of their potential interaction with genetic variants; and eventually, on the accuracy of the methods used for genetic testing. Our study fulfilled most of these criteria, including probing for IL-6 polymorphisms using two independent and highly accurate, high-throughput methods.(25) Nevertheless, our study has several limitations. First, its cross-sectional design precluded direct assessment of the rate of bone loss associated with IL-6 polymorphisms. Moreover, in the absence of lateral lumbar spine DXA measurements, we were not able to accurately evaluate the potential association of IL-6 genotypes with vertebral bone mass. Indeed, older women (in whom associations with hip BMD were mostly observed) are also prone to osteoarthritis at the spine, which is known to decrease the accuracy of PA DXA measurements at this site. In addition, IL-6 may be implicated in the pathophysiology of osteoarthritis itself. Nevertheless, as the study participants age and return for next examination cycles (every 4 years), it will eventually become possible to directly investigate whether changes in hip BMD are indeed related to IL-6 genetic variation. Second, because of the rather young mean age of the Framingham Offspring Cohort, the actual prevalence of osteoporosis-related fractures in our population was too low to be analyzed in relation to IL-6 polymorphisms. However, the steady increase in fracture risk associated with declining BMD is well established,(33) and detectable differences in the incidence of osteoporotic fractures could therefore be anticipated among IL-6 genotypes with increasing age of the sample. Third, patients were not randomized to receive calcium and/or estrogens based on their IL-6 genotypic profile. It remains to be directly demonstrated whether the efficacy of these antiresorptive agents will be greater among postmenopausal women carrying genotype −174 GG.

Despite these limitations, the Framingham Study Cohorts have previously proven to be useful in identifying important genetic and nongenetic risk factors for cardiovascular diseases and osteoporosis.(17, 18, 34, 35) Considering the implications of both IL-6 and estrogens in these diseases, we hope that our findings will prompt further investigations to determine the role of IL-6 genetic variation on the occurrence of osteoporotic fractures in elderly womenAppendix 1..

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Appendix 1
  9. REFERENCES

This study was supported by the Framingham Heart Study (supported by National Institutes of Health/NHLBI Contract N01-HC-38038) and National Institute of Arthritis, Musculoskeletal and Skin Diseases and National Institute on Aging Grant RO1 AR/AG 41398. We thank the Framingham Study members who participated in this study as well as the study coordinators who contributed to the success of this work. We also thank Dr Katie Tucker for help with food frequency questionnaire data.

Appendix 1

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Appendix 1
  9. REFERENCES
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Figure Appendix 1.. Ethnic Breakdown of Framingham Offspring (Self Report of “Country of Origin”)

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REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Appendix 1
  9. REFERENCES
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