Introduction
The sympathetic nervous system (SNS) influences bone remodeling in rodents by signaling through the beta-2 adrenergic receptor (B2AR) on the osteoblast. Osteoblast-specific deletion of the B2AR decreased bone resorption and led to an increase in bone mineral density [
1]. To date, it is uncertain whether this mechanism also exists in humans. Retrospective observational studies in humans on the association of beta-blockers and fracture risk reported conflicting results, yet a recent meta-analysis showed a reduction in fracture risk with a relative risk around 0.85 [
2].
The B2AR is a G-protein coupled receptor (GPCR). Binding of a ligand to the receptor leads to activation of the enzyme adenylate cyclase and generation of cyclic adenosine monophosphate (cAMP). Ligand binding also leads to phosphorylation of GPCR kinases which promotes downregulation of the receptor [
3]. The intronless B2AR gene is localized on chromosome 5q31-32 and encodes for a protein of 413 amino acids. In 1993, the first single nucleotide polymorphism (SNP) in the B2AR gene was reported [
4], and since then, many more SNPs have been identified. Four SNPs are non-synonymous and three of these are in the coding region of the gene [
5] on amino acid positions 16 (Arg16Gly; rs1042713), 27 (Gln27Glu; rs1042714), and 164 (Thr164Ile; rs1800888). The SNP on position 164 has not been found in humans homozygously and has a low minor allele frequency (<5 %) [
6]. The B2AR gene is highly evolutionary conserved, the mouse and human gene are 88 % similar (comparison based on amino acids), and this also applies to the region surrounding the polymorphisms.
In vitro [
7,
8] and in vivo [
9] experiments have shown that the Thr164Ile polymorphism lowers the binding affinity and activation of cAMP, leading to a signaling defect of the receptor. For the Arg16Gly and Gln27Glu polymorphisms, the Gly16 variant was shown to be more susceptible to agonist-induced downregulation whereas the Glu27 is more resistant. The Gly16 variant dominates the phenotype when both variants are present (reviewed in [
10‐
13]). Clinical research has extensively shown an effect of B2AR gene polymorphisms in cardiovascular therapy [
14] and asthma [
15,
16], and there are reports of B2AR gene polymorphisms influencing obesity [
17], healthy aging [
18], bacterial meningitis [
19], and Graves’ disease [
20].
Since these studies show that the function of the B2AR can be influenced by polymorphisms, we hypothesized that these polymorphisms in the B2AR gene could also have an effect on bone metabolism. To examine the role of beta-adrenergic signaling in human bone metabolism, we investigated the association of these polymorphisms in the B2AR gene with fracture risk in the Utrecht Cardiovascular Pharmacogenetics (UCP) cohort and the Rotterdam Study cohorts. We also investigated the association with bone mineral density (BMD) using the meta-analysis of genome-wide association (GWA) data of the GEFOS consortium.
Discussion
In both the Utrecht Cardiovascular Pharmacogenetics and the Rotterdam Study cohorts, there were no associations of polymorphisms in the beta-2 adrenergic receptor with fracture risk. In the GEFOS consortium, there was also no association of B2AR alleles with bone mineral density.
The B2AR is involved in bone metabolism in rodents. B2AR knockout mice have an increased bone mass and treatment with beta-blockers or beta-agonists increases and decreases bone mass respectively [
1,
33‐
36]. The B2AR gene is highly evolutionary conserved; the mouse and human genes are 88 % similar (comparison based on amino acids), and this also applies to the region surrounding the polymorphisms (
http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg19&position=chr5%3A148206156-148208197&hgsid=406908755_GVOKV49jJeWv0lNk5TyiXyon0z8p). In humans, the role of the B2AR in bone metabolism remains speculative. Epidemiological studies investigating the association between beta-blocker treatment and fracture risk presented inconclusive results, although a recent meta-analysis showed some fracture reduction with beta-blocker treatment [
2]. Two recent studies support a role of the SNS in the regulation of human bone metabolism. We showed that bone resorption is increased in pheochromocytoma patients and normalized following adrenalectomy [
37]. Pheochromocytomas are tumors of the adrenal gland, producing catecholamines, the natural ligands of adrenergic receptors. Farr et al. showed that sympathetic activity, as measured with microneurography, was inversely associated with trabecular microstructure and bone strength in women [
38]. Although both studies indicated that the SNS is involved in the regulation of human bone metabolism, they provided no information on the type of adrenergic receptor involved in the signal transduction from the SNS to bone cells. In mice, knockout of adrenergic receptors, including beta-1, beta-2 [
39], beta-3 [
40], alpha-2A, and alpha-2C [
41], results in distinct bone phenotypes, but the bone cell-specific effect has only been demonstrated for the B2AR. The type of adrenergic receptor involved in human bone metabolism remains largely unknown. Our data do not support a critical role for the B2AR.
Polymorphisms in the B2AR gene are functionally relevant in vitro and in vivo [
12] in diverse (patho)physiological processes. Mitchell et al. examined the genetics of heterotopic ossification, a skeletal complication in trauma and surgery patients, and identified an increased risk with a polymorphism (rs1042714) in the B2AR gene [
42]. This suggests an effect of polymorphisms in the B2AR gene on bone metabolism. A genome-wide association study on bone mineral density has been performed, and the identified loci have been tested for an association with fracture risk, but the B2AR gene was not identified as a significant contributor in this study [
28]. However, this does not completely exclude the possibility that polymorphisms in the B2AR gene are involved in bone metabolism. Liu et al. clearly showed in a set of elegant theoretical analyses using simulation studies that the power to identify certain loci depends, among others, on the number of causal SNPs, the effect size of the SNP, and the degree of heterogeneity of the studies [
43]. Considering that osteoporosis is a multifactorial disease with probably many causal SNPs differing in effect size [
44] and a considerable amount of heterogeneity in the GWAS populations, it remains possible that polymorphisms in the B2AR gene play a role in bone metabolism without being identified in a GWAS analysis. Therefore, candidate gene studies such as ours, based on sound animal experimental evidence, are still important to perform and report.
There are several limitations to this study. Subjects in the UCP cohort had an increased cardiovascular risk, and many were receiving beta-blockers (79 % of controls and 58 % of cases), which may have blurred an effect of the polymorphisms. Since 90 % of the beta-blockers prescribed were beta-1 selective with a low affinity for the beta-2 adrenergic receptor, the effect on the association between polymorphisms and fracture risk is probably limited. In addition, the lack of association did not change by adjusting the analysis for the use of beta-blockers (Table
3). In the UCP cohort, vertebral fractures seem underrepresented. This is probably due to the relatively young age of the cohort (mean 58 years) and the definition of fracture based on hospital discharge code; vertebral fracture patients are not routinely admitted to the hospital and mostly treated as outpatients. The Rotterdam Study cohorts, in contrast, contain many fracture patients diagnosed based on interviewing, status reviews, and imaging. The follow-up of the Rotterdam Study I comprises over 20 years of follow-up of inhabitants aged >55 years whereas the follow-up of the Rotterdam Study II participants started in 2000; this explains the difference in fracture rate between these populations. The Rotterdam Study III population, started in 2006 at a younger age of 45, underwent standard X-ray examinations of vertebral bodies and thus contains only vertebral fractures, underestimating the true fracture rate. However, vertebral fractures are highly representative of osteoporotic fractures, and the association results for this cohort are not different from the other two cohorts.
Overall, the populations of the UCP and Rotterdam Study were comparable, population-based prospective cohorts of middle-aged subjects. Therefore, in combination, the two cohorts provide a relevant population with an adequate sample size to investigate the hypothesis. However, some information on subject characteristics, such as vitamin D levels, smoking, BMI, prior fracture, and alcoholism, was not fully available in both cohorts, and therefore, we could not perform these secondary analyses. In addition, medication use was determined at baseline in the Rotterdam Study, and with over 20 years of follow-up, this could be very different at the time of fracture; therefore, we did not perform a medication-adjusted analysis for the Rotterdam Study. In the UCP cohort, medication use was determined as present if used during follow-up; therefore, all medication was included. This seems a reliable method for patients suffering from chronic diseases such as hypertension, diabetes, and epilepsy, which generally are long-term medication users, but could influence the results for incident medications such as sleep medication, known to increase fracture risk. However, we did observe a higher percentage of sleep medication use in cases, and considering the crude and adjusted odds ratio’s for the associations between fracture and the polymorphisms, an effect of medication does not seem very likely.
The Rotterdam Study included prevalent radiographic vertebral fractures (diagnosed at the start of the study, so there is difficulty in defining follow-up time) and incident fractures (not only clinical fractures occurring during the course of the study but also radiographic vertebral fractures at the follow-up visit after 4 to 7 years, of which a significant proportion is asymptomatic, and thus, determining the exact time of fracture is impossible). Therefore, we chose to analyze the data of the Rotterdam Study as a case–control study. In addition, application of a Cox proportional hazard model to imputed SNP data requires special methods. The UCP cohort included incident fractures, occurring during the course of the study with accurate assessment of the fracture date from the hospital discharge database. Therefore, we re-analyzed the data of the UCP cohort using a Cox proportional hazard model, but this did not change the results.
Finally, although we did not observe any effect of polymorphisms in the B2AR gene on fracture risk and bone mineral density, this does not exclude the possibility of an effect of these polymorphisms through interaction with other, as yet, unknown genes. The original studies in mice were performed in one strain of mice, with the same genetic background. In humans of course, this is very different and it is difficult to control for variations in genetic background possibly affecting the effect of the gene on the outcome. We did analyze haplotypes, which could account for some genetic background variance; however, this did not change the results.
Furthermore, according to the GWAS studies on fracture, fracture risk is probably poly-genetically determined, and thus, interaction of the B2AR gene with other genes is possible. However, the animal studies showed profound influence on bone remodeling by the B2AR itself, and we know that the B2AR is highly conserved among humans and mice. In addition, we know that polymorphisms in the B2AR by itself influence receptor function. Therefore, if the B2AR influences human bone remodeling, we expected at least some effect of these polymorphisms on fracture risk and bone mineral density.
During the last decade, several lines of research have shown a role of the beta-2 adrenergic receptor in bone metabolism in rodents. In humans, there is convincing evidence that the sympathetic nervous system influences bone remodeling, although the exact mechanism is still unclear. In the present study, we aimed to elucidate the role of the beta-2 adrenergic receptor by investigating the effect of polymorphisms on fracture risk in two population-based cohorts and on bone mineral density using GWA data, but we did not find an association. For the future, the challenge remains to identify the exact pathway from the sympathetic nervous system to bone and to unravel this complex mechanism.