Dr Cauley has received support from Eli Lilly & Co., Merck & Co., Pfizer, and Novartis and is on the speaker's bureau for Eli Lilly & Co. and Merck & Co. All other authors have no conflict of interest
Association of the G-174C Variant in the Interleukin-6 Promoter Region With Bone Loss and Fracture Risk in Older Women
Article first published online: 12 JUL 2004
Copyright © 2004 ASBMR
Journal of Bone and Mineral Research
Volume 19, Issue 10, pages 1612–1618, October 2004
How to Cite
Moffett, S. P., Zmuda, J. M., Cauley, J. A., Stone, K. L., Nevitt, M. C., Ensrud, K. E., Hillier, T. A., Hochberg, M. C., Joslyn, G., Morin, P. and Cummings, S. R. (2004), Association of the G-174C Variant in the Interleukin-6 Promoter Region With Bone Loss and Fracture Risk in Older Women. J Bone Miner Res, 19: 1612–1618. doi: 10.1359/JBMR.040707
- Issue published online: 2 DEC 2009
- Article first published online: 12 JUL 2004
- Manuscript Accepted: 18 MAY 2004
- Manuscript Revised: 19 FEB 2004
- Manuscript Received: 16 MAY 2003
- interleukin 6;
We analyzed the association between the IL-6 G-174C polymorphism and osteoporosis phenotypes in 3376 older women. Women with the C/C genotype had a significantly slower rate of decline in hip BMD and a 33% lower risk of wrist fracture than women with the G/G genotype. Variation at the IL-6 locus may contribute to the genetic susceptibility to bone fragility.
Introduction: Interleukin 6 (IL-6) promotes osteoclast formation and bone resorption. The C allele of the G-174C polymorphism in the IL-6 promoter region has been related to lower gene transcription and plasma IL-6 levels.
Materials and Methods: In this study, we evaluated the relationship between the IL-6 G-174C polymorphism and BMD, the rate of decline in BMD, and the risk of fracture in 3376 women 65 years of age and older participating in the Study of Osteoporotic Fractures. BMD was measured at the distal and proximal radius using single photon absorptiometry and at the hip using DXA. Hip BMD was measured again an average of 3.5 years later. Incident fractures over an average of 10.8 years of follow-up were confirmed by physician adjudication of radiology reports.
Results: Distal and proximal radius BMD was lowest among women with the G/G genotype, intermediate in the heterozygotes, and highest in women with the C/C genotype (p = 0.016 and p = 0.049, respectively), although the differences between the genotypes were small. While there were no differences by genotype with initial hip BMD, women with the C/C genotype experienced a slower rate of decline in total hip and femoral neck BMD compared with women with the G/G genotype (p = 0.004 and p = 0.029, respectively). Women with the C/C genotype also had 33% lower risk of wrist fracture compared with women with the G/G genotype, independent of age, body mass index, estrogen use, and study center (RR, 0.67; 95% CI, 0.45, 1.00; p = 0.048), whereas heterozygous women had a more intermediate risk (RR, 0.85; 95% CI, 0.65, 1.12; p = 0.256). No association was found between genotype and risk of hip or all non-spine fractures.
Conclusions: These results suggest that the IL-6 G-174C promoter polymorphism may be a genetic marker for bone loss and wrist fracture among older women.
Osteoporosis is a major clinical and public health problem among older women because of the considerable morbidity and health care costs resulting from osteoporotic fractures.(1) There is convincing evidence from family and twin studies of a strong hereditary component to osteoporosis. These studies indicate that the genetic contribution to normal variation in adult bone mass may be as high as 80% and that genetic factors remain important even into the eighth decade of life.(2–5)
Although less well studied, there is also evidence of a heritable component for age-related bone loss.(6) A substantial heritability of bone resorption and formation indices among older adults and the hormones regulating these processes also indicates that age-related bone loss has genetic foundations.(7–11) Furthermore, women with a maternal history of hip fracture have a 2-fold greater risk of hip fracture than those without a history of such fractures.(12) Although these studies establish an important genetic influence on bone metabolism and osteoporotic fracture risk, the genes involved and the magnitude of their effects are not well defined.
The natural decline in estrogen production after menopause leads to increased bone resorption in part because of increased production of pro-inflammatory cytokines, such as interleukin 6 (IL-6), which are normally suppressed by estrogen.(13)), ((14) Increased levels of IL-6 within the bone microenvironment promote the differentiation of osteoclast precursor cells into mature osteoclasts, which increase bone resorption.(15–17)
Dinucleotide repeat polymorphisms at the IL-6 locus have been associated with or linked to BMD in postmenopausal, but not premenopausal, women, suggesting that allelic variants of IL-6 may influence osteoporosis susceptibility.(18–20) The promoter region of the IL-6 gene contains a common G-174C polymorphism, where the C allele has been associated with decreased promoter activity and plasma IL-6 levels.(21)), ((22) This promoter variant has been associated with bone metabolism and BMD in several recent studies.(23–26) In this study, we further evaluated the relationship between the G-174C polymorphism in the IL-6 promoter region and BMD, the rate of decline in hip BMD, and the risk of fracture in a large community-based cohort of older women.
MATERIALS AND METHODS
All women were participants in the Study of Osteoporotic Fractures (SOF), a prospective study of 9704 community-dwelling white women who were at least 65 years of age at study entry. Women were recruited for SOF between 1986 and 1988 from population-based listings, such as voter registration lists, at four clinical centers in the United States: Baltimore, MD; Minneapolis, MN; Monongahela Valley, PA; and Portland, OR.(27) Women were excluded from the study if they reported a bilateral hip replacement or were unable to walk unassisted. Black women were excluded because of their low risk of hip fracture. The institutional review boards at each institution approved the study. All women provided written informed consent at entry into the study and at each clinical examination.
In 1989–1990, buffy coat specimens were collected in participants who returned for a second examination. In addition, whole blood samples were collected during 1997–1998 in women who had not previously provided samples. The present analysis was completed using samples (either buffy coat or whole blood) collected from the Baltimore, Minneapolis, and Monongahela Valley clinics. Among the 6975 participants who provided samples, genotyping was performed in a subsample of 3376 women. The demographic characteristics of the women in this subsample were similar to the entire SOF cohort.
Distal and proximal radial BMD was measured at baseline using single-photon absorptiometry using the Osteon Osteoanalyzer (Dove Medical Group, Los Angeles, CA, USA). Total hip BMD and subregions were measured by DXA (QDR 1000; Hologic, Bedford, MA, USA) at a second clinic visit in 1989–1990. Hip BMD measurements were repeated an average of 3.5 years later at a follow-up visit. Mean CVs between centers were 0.4% for BMD of the distal radius, 0.5% for the proximal radius, and 1.2% for the femoral neck for two research staff who visited all centers.(28) Details of these methods and quality control procedures have been reported elsewhere.(28)), ((29)
Details of our method for identifying fractures have been published previously.(30) Briefly, we contact participants every 4 months by postcard or telephone to ask whether they had sustained a fracture. More than 98% of these contacts were completed. All fractures are adjudicated by radiographic report. We excluded fractures that occurred because of major trauma such as motor vehicle accidents. We analyzed nonvertebral fractures as a group and also analyzed hip and distal forearm fractures separately.
Body weight was measured (after removal of shoes and heavy outer clothing) using a balance beam scale. Height was measured without shoes using a Harpenden stadiometer (Holtain, Dyved, UK). Height and weight were used to calculate body mass index (BMI; kg/m2). Family history of fractures, falls during the past year, reported health status, smoking status, and physical activity were assessed by a questionnaire, which was reviewed with the participant by a trained interviewer. A modified Paffenbarger questionnaire was used to assess physical activity, expressed as kilocalories expended per week.(31) Oral estrogen and calcium supplement use was confirmed by asking participants to bring the containers to their clinic visit.
Women were genotyped for the IL-6 promoter G-174C polymorphism using the 5′ exonuclease assay.(32) The assay is based on PCR and uses the 5′ exonuclease activity of TaqDNA polymerase. Allele-specific oligonucleotide probes are added to the PCR reaction and are cleaved by the 5′ exonuclease activity of Taq polymerase only if genomic DNA includes the corresponding variant of the polymorphism. The probes are labeled at the 5′ end with the reporter dye (FAM or TET) and at the 3′ end with the quencher dye (TAMRA). When the probes are degraded by Taq polymerase, the genotypes can be determined by measuring the increase in one or both of the fluorescent reporter dyes. The PCR primers used were 5′-AGCCTCAATGACGACCTAAGCT-3′ in the forward direction and 5′-TTGGAAACCTTATTAAGATTGTGCA-3′ in the reverse direction. The probe used for the G allele was 5′-AGTTGTGTCTTGCGATGCTAAAGGACGT-3′ and for the C allele was 5′-AGTTGTGTCTTGCCATGCTAAAGGACGT-3′. The PCR reaction mixtures included 20 ng of genomic DNA in a 10-μ;l reaction volume and the following concentrations of other reagents: probes (100 nM each), primers (900 nM each), and 1× TaqMan PCR Master Mix (PE Applied Biosystems, Foster City, CA, USA). PCR cycling conditions consisted of a preincubation period of 2 minutes at 50°C, an initial denaturation period of 10 minutes at 95°C, an annealing period of 30 s at 62°C, and a denaturation period of 30 s at 95°C for 40 cycles. We used the ABI Prism 7700 Sequence Detector (PE Applied Biosystems) for data acquisition.
Allele frequencies were estimated by the gene counting method, and departures from Hardy-Weinberg equilibrium were tested using a χ2 test. We compared characteristics of women by genotype using ANOVA for continuous measures and χ2 tests for categorical variables. We analyzed BMD and rates of change in BMD by genotype using analysis of covariance. To determine the relationship between genotype and subsequent fracture risk, we used Cox proportional hazards models to estimate hazard ratios (HRs) and 95% CIs for the associations between IL-6 genotype and fracture (SAS version 8.0). We adjusted BMD and fracture models for age and co-variates that differed significantly by genotype. Population stratification has been cited as a potential cause of unreplicated and false-positive associations in candidate gene studies; thus, we also adjusted models for study center.(33) No adjustments were made for multiple comparisons.
The frequency of the IL-6–174 allele in the SOF cohort was 0.429 and was similar to the frequencies observed in other studies of whites.(34) Genotype frequencies did not deviate from the expectations of Hardy-Weinberg equilibrium. Thirty-three percent of the women carried the G/G genotype, 48% were heterozygous, and 19% had the C/C genotype.
Table 1 shows descriptive characteristics of the SOF women by genotype for the IL-6 G-174C polymorphism. The average age for each genotype group was 73 years. Despite similar initial body weight, women with the C/C genotype lost significantly less body weight than those with the G/G genotype. Use of estrogen therapy also differed significantly by genotype, where women with the C/C genotype were more likely to take estrogen compared with those with the G/G genotype. No statistically significant differences in unadjusted BMD were found between the genotypes at the femoral neck, total hip, distal radius, or proximal radius.
Distal and proximal radius BMD were associated with the IL-6 G-174C genotype when adjusted for age, BMI, estrogen use, and study center (Table 2). BMD was lowest among women with the G/G genotype, intermediate in the heterozygotes, and highest in women with the C/C genotype. Although statistically significant, the absolute difference between homozygous genotypes was small. For example, the difference in BMD at the distal radius between women with the G/G and C/C genotypes was 0.01 g/cm2 or 0.12 SD. Similar findings were observed at the proximal radius. Hip BMD also tended to be lowest in women with the G/G genotype, but these differences were not statistically significant. When the analysis was repeated after excluding women on hormone therapy, the magnitude and direction of the effect was similar to the results from the entire study sample. For example, in the women not on hormone therapy, the genotypic means (SEM) were G/G = 0.347 (0.002), G/C = 0.351 (0.002), and C/C = 0.355 (0.003) for the distal radius (p = 0.10) and G/G = 0.621 (0.003), G/C = 0.627 (0.002), and C/C = 0.631 (0.004) for the proximal radius (p = 0.08).
We found a significant association between IL-6 genotype and the annualized percentage rate of change in hip BMD (Table 3). For example, women with the C/C genotype experienced about one-half the rate of decline in total hip BMD compared with women with the G/G genotype, whereas heterozygous women had a more intermediate rate of decline (G/G, −0.48 ± 0.05%/year; G/C, −0.42 ± 0.04%/year; C/C, −0.22 ± 0.06%/year; p = 0.004). Also, the association between IL-6 genotype and the annualized percentage rate of change in total hip BMD remained significant after excluding women on hormone therapy (G/G, −0.36 ± 0.03%/year; G/C, −0.29 ± 0.03%/year; C/C, −0.20 ± 0.04%/year; p = 0.022). We found similar bone loss patterns by genotype at the femoral neck (p = 0.029). These associations were independent of age, baseline BMD, BMI, weight change during follow-up, estrogen use, and study center. Similar results were obtained in unadjusted analyses. We also found similar genotype related patterns of hip bone loss expressed as an annualized absolute rate of change (p = 0.007).
There were 248 incident wrist fractures during an average follow-up of 10.8 ± 3.1 years. The risk of wrist fracture decreased by 17% per copy of the IL-6 C allele independent of age, BMI, estrogen use, and study center (RR), 0.83; 95% Confidence Interval (CI), 0.68, 0.99; p = 0.043] (Table 4). Women with the C/C genotype had 33% lower risk of wrist fracture compared with women with the G/G genotype independent of age, BMI, estrogen use and study center (RR, 0.67; 95% CI, 0.45, 1.00; p = 0.048), whereas heterozygous women had a more intermediate and nonsignificantly lower risk (RR, 0.85; 95% CI, 0.65, 1.12; p = 0.256). The results were similar after adjusting for BMD, but the CIs were wider and not statistically significant (Table 4). Exclusion of women on hormone therapy showed a similar effect of the C/C genotype versus the G/G genotype on wrist fracture (RR, 0.68; 95% CI, 0.46, 1.02; p = 0.064). In contrast, we found no significant association between IL-6 genotype and the risk of hip or all non-spine fractures. For example, compared with the referent group of women with the G/G genotype, those with the C/G or C/C genotypes had similar risk for hip fracture (RR, 0.98; 95% CI, 0.82, 1.17; p = 0.799).
This study examined the association between a G-174C polymorphism in the IL-6 promoter region and BMD, rate of decline in BMD, and fracture risk in a large cohort of older white women. We found that women with the C/C genotype have statistically significantly higher forearm BMD and a slower rate of decline in hip BMD than women with the G/G genotype. These women also had a 33% lower risk of wrist fractures compared with the other genotype groups. These associations were statistically independent of potential confounding factors.
Our findings are consistent with the known effects of IL-6 on bone resorption and bone loss after menopause.(35) Our results are also consistent with several smaller studies showing that the −174 C/C genotype is associated with lower biochemical indices of bone resorption and with higher BMD.(23–26) We extend the findings of these previous reports by showing that the −174 C/C genotype is also associated with a lower rate of hip bone loss and a lower incidence of wrist fractures among older women. These findings suggest that allelic variation at the IL-6 locus may play a role in the genetic susceptibility to skeletal fragility among older women.
The IL-6 G-174C polymorphism was associated with wrist but not hip or all non-spine fractures. The epidemiologic patterns for wrist and hip fractures are known to differ.(12)), ((36)), ((37) For example, in contrast to hip fractures, most aspects of lifestyle and general health are very similar in women who suffer a wrist fracture compared with those who do not.(12)), ((36)), ((37) Studies in inbred strains of mice suggest that genetic regulation of bone strength is skeletal site specific.(38) Our findings are consistent with this possibility.
The functional significance of the G-174C polymorphism is uncertain. The G-174C polymorphism lies within a potential DNA binding site for NF-1 in a known regulatory region and may directly alter IL-6 gene expression.(22)), ((39) Indeed, in vitro studies of the IL-6 G-174C polymorphism indicate that constructs containing the C allele have ∼40% lower promoter activity than those with the G allele.(22) This reduced transcription is mirrored in lower plasma levels of IL-6 among subjects with the −174 C/C genotype.(22) However, several other variants have been identified in the promoter region of IL-6, including G-597A, G-572C, and a minisatellite (-373AnTn). These polymorphisms are all in linkage disequilibrium with the G-174C variant and have been shown to interact with one another to alter transcription of IL-6.(21) Indeed, the G-572C variant and −572/−174 haplotype have been associated with indices of bone resorption and BMD among postmenopausal women.(25) Thus, it will be important to define the relationship between IL-6 promoter haplotypes and BMD, bone loss, and fracture risk in future studies.
We also noted associations between the IL-6 G-174C polymorphism and BMI and weight loss during follow-up in this study. The association with weight loss, but not BMI, remained significant even after adjusting for study center, age, and current estrogen use. Others have reported an association between the IL-6 G-174C promoter variant and obesity, where the -174G allele was found to be more common among thinner subjects.(40) These associations are consistent with a role of IL-6 in body weight regulation. For example, adipose tissue is a significant source of circulating IL-6, and plasma levels of IL-6 correlate positively with BMI.(41–43) Moreover, mice lacking the IL-6 gene develop mature-onset obesity, and low doses of IL-6 administered centrally decrease body weight and adipose tissue mass in rodents.(44)), ((45) These observations have led to the hypothesis that IL-6 may be a mediator of feedback regulation from adipose tissue to the hypothalamus, similar to the adipostat hormone, leptin.(45) Nevertheless, associations between the G-174C polymorphism and BMD and fracture risk were independent of BMI in this study. The association between the G-174C polymorphism and hip bone loss was also independent of weight change during follow-up.
Women with the C/C genotype were significantly more likely to use estrogen therapy compared with women with the G/G genotype in this analysis, independent of age, BMI, or study center. However, genotype-related differences in estrogen use did not explain our BMD, bone loss, or fracture findings. IL-6 is a potent stimulator of aromatase, which converts androgens to estrogens, and increases in IL-6 with menopause and aging may account for the increased peripheral aromatase activity among older women.(46) Our findings require confirmation, but raise the possibility that genetically mediated variation in IL-6 might influence patterns of hormone use among older women by altering endogenous estrogen levels.(47) A polymorphism in cytochrome P450c17α (CYP17), another regulator of endogenous estrogen production, has also been associated with hormone use.(48)
Our study has several strengths, which include being a large, prospective cohort with excellent follow-up rates and adjudicated fractures over an extended period of time. There are also several limitations to our study. Participants in this analysis were community-dwelling white women 65 years of age and older, and our findings may not be generalizable to women of other races, men, or institutionalized subjects. Population stratification is an often cited limitation of candidate gene studies because population stratification can contribute to false-positive associations and an inability to reproduce findings.(33) Women in this study were recruited from several clinical centers in the United States, and any resultant population stratification could have, in principle, produced false-positive results. However, adjusting for study center had no effect on our findings. Small sample size and low statistical power is also a frequent limitation of candidate gene association studies.(49) Our study included over 3000 women and had adequate statistical power to detect even modest associations. On the other hand, the significance of associations that we have reported are not adjusted for the number of statistical tests, and thus some of the positive associations may be false positives. There is currently no consensus on the ideal method for adjusting the probability of an observation occurring by chance for multiple testing. While the Bonferroni approach is probably the best-known procedure for controlling the type I error rate, this method is very conservative and results in substantial losses of power. The Bonferroni correction would also constitute an overadjustment, because the phenotypes assessed in this study are not independent. It is also unclear whether the number of tests that one corrects for should reflect the number completed to date or the number one might undertake. Nonetheless, readers should interpret the significance of our results in light of the number of statistical tests completed and consistency of findings across other studies to identify the associations that are most convincing. Our findings, especially for wrist fracture, should also be replicated in other populations.
In summary, community-dwelling older women with the IL-6-174 C/C genotype have higher BMD, slower age-related declines in BMD, and a decreased risk of wrist fracture. The lower incidence of wrist fracture among women with the C/C genotype was attenuated somewhat by adjusting for BMD, suggesting that the altered risk may be explained by changes in BMD. The IL-6 G-174C polymorphism was not related to hip or all non-spine fractures in the SOF cohort. Our findings require confirmation in other populations but suggest that the IL-6 promoter polymorphism, or a closely linked allelic variant, may be a genetic marker for skeletal fragility among older women.
This study was supported by Public Health Service Grants AG05407, AR35582, AG05394, AR35584, and AR35583. SM was supported by NIA T32 AG00181–13.
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