An integration of genome-wide association study and gene expression profiling to prioritize the discovery of novel susceptibility Loci for osteoporosis-related traits.

Osteoporosis is a complex disorder and commonly leads to fractures in elderly persons. Genome-wide association studies (GWAS) have become an unbiased approach to identify variations in the genome that potentially affect health. However, the genetic variants identified so far only explain a small proportion of the heritability for complex traits. Due to the modest genetic effect size and inadequate power, true association signals may not be revealed based on a stringent genome-wide significance threshold. Here, we take advantage of SNP and transcript arrays and integrate GWAS and expression signature profiling relevant to the skeletal system in cellular and animal models to prioritize the discovery of novel candidate genes for osteoporosis-related traits, including bone mineral density (BMD) at the lumbar spine (LS) and femoral neck (FN), as well as geometric indices of the hip (femoral neck-shaft angle, NSA; femoral neck length, NL; and narrow-neck width, NW). A two-stage meta-analysis of GWAS from 7,633 Caucasian women and 3,657 men, revealed three novel loci associated with osteoporosis-related traits, including chromosome 1p13.2 (RAP1A, p = 3.6x10(-8)), 2q11.2 (TBC1D8), and 18q11.2 (OSBPL1A), and confirmed a previously reported region near TNFRSF11B/OPG gene. We also prioritized 16 suggestive genome-wide significant candidate genes based on their potential involvement in skeletal metabolism. Among them, 3 candidate genes were associated with BMD in women. Notably, 2 out of these 3 genes (GPR177, p = 2.6x10(-13); SOX6, p = 6.4x10(-10)) associated with BMD in women have been successfully replicated in a large-scale meta-analysis of BMD, but none of the non-prioritized candidates (associated with BMD) did. Our results support the concept of our prioritization strategy. In the absence of direct biological support for identified genes, we highlighted the efficiency of subsequent functional characterization using publicly available expression profiling relevant to the skeletal system in cellular or whole animal models to prioritize candidate genes for further functional validation.

Meta-analysis of genome-wide scans for human adult stature identifies novel Loci and associations with measures of skeletal frame size.

Recent genome-wide (GW) scans have identified several independent loci affecting human stature, but their contribution through the different skeletal components of height is still poorly understood. We carried out a genome-wide scan in 12,611 participants, followed by replication in an additional 7,187 individuals, and identified 17 genomic regions with GW-significant association with height. Of these, two are entirely novel (rs11809207 in CATSPER4, combined P-value = 6.1x10(-8) and rs910316 in TMED10, P-value = 1.4x10(-7)) and two had previously been described with weak statistical support (rs10472828 in NPR3, P-value = 3x10(-7) and rs849141 in JAZF1, P-value = 3.2x10(-11)). One locus (rs1182188 at GNA12) identifies the first height eQTL. We also assessed the contribution of height loci to the upper- (trunk) and lower-body (hip axis and femur) skeletal components of height. We find evidence for several loci associated with trunk length (including rs6570507 in GPR126, P-value = 4x10(-5) and rs6817306 in LCORL, P-value = 4x10(-4)), hip axis length (including rs6830062 at LCORL, P-value = 4.8x10(-4) and rs4911494 at UQCC, P-value = 1.9x10(-4)), and femur length (including rs710841 at PRKG2, P-value = 2.4x10(-5) and rs10946808 at HIST1H1D, P-value = 6.4x10(-6)). Finally, we used conditional analyses to explore a possible differential contribution of the height loci to these different skeletal size measurements. In addition to validating four novel loci controlling adult stature, our study represents the first effort to assess the contribution of genetic loci to three skeletal components of height. Further statistical tests in larger numbers of individuals will be required to verify if the height loci affect height preferentially through these subcomponents of height.

Common genetic variation in the Estrogen Receptor Beta (ESR2) gene and osteoarthritis: results of a meta-analysis.

BACKGROUND: The objective of this study was to examine the relationship between common genetic variation of the ESR2 gene and osteoarthritis. METHODS: In the discovery study, the Rotterdam Study-I, 7 single nucleotide polymorphisms (SNPs) were genotyped and tested for association with hip (284 cases, 2772 controls), knee (665 cases, 2075 controls), and hand OA (874 cases, 2184 controls) using an additive model. In the replication stage one SNP (rs1256031) was tested in an additional 2080 hip, 1318 knee and 557 hand OA cases and 4001, 2631 and 1699 controls respectively. Fixed- and random-effects meta-analyses were performed over the complete dataset including 2364 hip, 1983 knee and 1431 hand OA cases and approximately 6000 controls. RESULTS: The C allele of rs1256031 was associated with a 36% increased odds of hip OA in women of the Rotterdam Study-I (OR 1.36, 95% CI 1.08-1.70, p = 0.009). Haplotype analysis and analysis of knee- and hand OA did not give additional information. With the replication studies, the meta-analysis did not show a significant effect of this SNP on hip OA in the total population (OR 1.06, 95% CI 0.99-1.15, p = 0.10). Stratification according to gender did not change the results. In this study, we had 80% power to detect an odds ratio of at least 1.14 for hip OA (α = 0.05). CONCLUSION: This study showed that common genetic variation in the ESR2 gene is not likely to influence the risk of osteoarthritis with effects smaller than a 13% increase.

The role of body mass index, insulin, and adiponectin in the relation between fat distribution and bone mineral density.

Despite the positive association between body mass index (BMI) and bone mineral density (BMD) and content (BMC), the role of fat distribution in BMD/BMC remains unclear. We examined relationships between BMD/BMC and various measurements of fat distribution and studied the role of BMI, insulin, and adiponectin in these relations. Using a cross-sectional investigation of 2631 participants from the Erasmus Rucphen Family study, we studied associations between BMD (using dual-energy X-ray absorptiometry (DXA]) at the hip, lumbar spine, total body (BMD and BMC), and fat distribution by the waist-to-hip ratio (WHR), waist-to-thigh ratio (WTR), and DXA-based trunk-to-leg fat ratio and android-to-gynoid fat ratio. Analyses were stratified by gender and median age (48.0 years in women and 49.2 years in men) and were performed with and without adjustment for BMI, fasting insulin, and adiponectin. Using linear regression (adjusting for age, height, smoking, and use of alcohol), most relationships between fat distribution and BMD and BMC were positive, except for WTR. After BMI adjustment, most correlations were negative except for trunk-to-leg fat ratio in both genders. No consistent influence of age or menopausal status was found. Insulin and adiponectin levels did not explain either positive or negative associations. In conclusion, positive associations between android fat distribution and BMD/BMC are explained by higher BMI but not by higher insulin and/or lower adiponectin levels. Inverse associations after adjustment for BMI suggest that android fat deposition as measured by the WHR, WTR, and DXA-based android-to-gynoid fat ratio is not beneficial and possibly even deleterious for bone.

Collaborative meta-analysis: associations of 150 candidate genes with osteoporosis and osteoporotic fracture.

BACKGROUND: Osteoporosis is a highly heritable trait. Many candidate genes have been proposed as being involved in regulating bone mineral density (BMD). Few of these findings have been replicated in independent studies. OBJECTIVE: To assess the relationship between BMD and fracture and all common single-nucleotide polymorphisms (SNPs) in previously proposed osteoporosis candidate genes. DESIGN: Large-scale meta-analysis of genome-wide association data. SETTING: 5 international, multicenter, population-based studies. PARTICIPANTS: Data on BMD were obtained from 19 195 participants (14 277 women) from 5 populations of European origin. Data on fracture were obtained from a prospective cohort (n = 5974) from the Netherlands. MEASUREMENTS: Systematic literature review using the Human Genome Epidemiology Navigator identified autosomal genes previously evaluated for association with osteoporosis. We explored the common SNPs arising from the haplotype map of the human genome (HapMap) across all these genes. BMD at the femoral neck and lumbar spine was measured by dual-energy x-ray absorptiometry. Fractures were defined as clinically apparent, site-specific, validated nonvertebral and vertebral low-energy fractures. RESULTS: 150 candidate genes were identified and 36 016 SNPs in these loci were assessed. SNPs from 9 gene loci (ESR1, LRP4, ITGA1, LRP5, SOST, SPP1, TNFRSF11A, TNFRSF11B, and TNFSF11) were associated with BMD at either site. For most genes, no SNP was statistically significant. For statistically significant SNPs (n = 241), effect sizes ranged from 0.04 to 0.18 SD per allele. SNPs from the LRP5, SOST, SPP1, and TNFRSF11A loci were significantly associated with fracture risk; odds ratios ranged from 1.13 to 1.43 per allele. These effects on fracture were partially independent of BMD at SPP1 and SOST. LIMITATION: Only common polymorphisms in linkage disequilibrium with SNPs in HapMap could be assessed, and previously reported associations for SNPs in some candidate genes could not be excluded. CONCLUSION: In this large-scale collaborative genome-wide meta-analysis, 9 of 150 candidate genes were associated with regulation of BMD, 4 of which also significantly affected risk for fracture. However, most candidate genes had no consistent association with BMD.

Vitamin D binding protein genotype and osteoporosis.

Osteoporosis is a bone disease leading to an increased fracture risk. It is considered a complex multifactorial genetic disorder with interaction of environmental and genetic factors. As a candidate gene for osteoporosis, we studied vitamin D binding protein (DBP, or group-specific component, Gc), which binds to and transports vitamin D to target tissues to maintain calcium homeostasis through the vitamin D endocrine system. DBP can also be converted to DBP-macrophage activating factor (DBP-MAF), which mediates bone resorption by directly activating osteoclasts. We summarized the genetic linkage structure of the DBP gene. We genotyped two single-nucleotide polymorphisms (SNPs, rs7041 = Glu416Asp and rs4588 = Thr420Lys) in 6,181 elderly Caucasians and investigated interactions of the DBP genotype with vitamin D receptor (VDR) genotype and dietary calcium intake in relation to fracture risk. Haplotypes of the DBP SNPs correspond to protein variations referred to as Gc1s (haplotype 1), Gc2 (haplotype 2), and Gc1f (haplotype3). In a subgroup of 1,312 subjects, DBP genotype was found to be associated with increased and decreased serum 25-(OH)D(3) for haplotype 1 (P = 3 x 10(-4)) and haplotype 2 (P = 3 x 10(-6)), respectively. Similar associations were observed for 1,25-(OH)(2)D(3). The DBP genotype was not significantly associated with fracture risk in the entire study population. Yet, we observed interaction between DBP and VDR haplotypes in determining fracture risk. In the DBP haplotype 1-carrier group, subjects of homozygous VDR block 5-haplotype 1 had 33% increased fracture risk compared to noncarriers (P = 0.005). In a subgroup with dietary calcium intake <1.09 g/day, the hazard ratio (95% confidence interval) for fracture risk of DBP hap1-homozygote versus noncarrier was 1.47 (1.06-2.05). All associations were independent of age and gender. Our study demonstrated that the genetic effect of the DBP gene on fracture risk appears only in combination with other genetic and environmental risk factors for bone metabolism.

Osmomediated natriuresis in humans: the role of vasopressin and tubular calcium sensing.

BACKGROUND: The aim was to investigate the unknown mechanism of osmomediated natriuresis. This is the phenomenon by which hypertonic saline (HS) produces a larger natriuresis than isotonic saline (IS), despite the same sodium content. METHODS: Seven healthy volunteers first received HS and then IS (both 3.85 mmol sodium/kg). To investigate the role of calcium metabolism, four patients received HS, two with an activating mutation (ADH) and two with an inactivating mutation (FHH) of the calcium-sensing receptor (CaSR). RESULTS: In healthy volunteers, HS produced mild hypernatraemia, a 4-fold rise in vasopressin (to 2.2 +/- 0.85 pg/mL) and a 3-fold rise in natriuresis compared with a 1.5-fold rise with IS (P = 0.002). This confirmed osmomediated natriuresis. HS caused calciuresis to increase 1.4-fold and then reduced it 1.4-fold, whereas IS failed to increase calciuresis and caused it to fall 3.7-fold (P = 0.05). Natriuresis and calciuresis in ADH patients were similar to healthy volunteers receiving HS, whereas a blunted response was seen in FHH patients. Patient vasopressin levels did not exceed 1.3 pg/mL and changes from baseline were variable. In one FHH patient, a 3-fold rise in vasopressin did not prevent the blunted natriuresis and calciuresis. In one ADH patient, natriuresis and calciuresis were similar to healthy volunteers despite a 1.7-fold fall in vasopressin. CONCLUSIONS: Our data suggest that not only vasopressin (possibly via its V1a receptor), but also the CaSR (which is sensitive to high sodium concentrations) may play a role in osmomediated natriuresis. These results shed new light on osmomediated natriuresis and suggest roles for the CaSR beyond calcium regulation.

Vitamin D status, bone mineral density, and the development of radiographic osteoarthritis of the knee: The Rotterdam Study.

OBJECTIVE: To study the association between baseline vitamin D status, bone mineral density (BMD), and the development of radiographic osteoarthritis (ROA) of the knee in a large population-based cohort of men and women. METHODS: A sample of 1248 subjects (728 women and 520 men) was drawn from the Rotterdam Study, a prospective population-based cohort study of the elderly. At baseline, vitamin D dietary intake was determined, and BMD and 25-hydroxy vitamin D (25(OH)D) serum levels were measured. After a mean follow-up time of 6.5 years incidence and progression of knee ROA of was assessed. RESULTS: The mean vitamin D intake in our study population was 64 IU/d and the mean 25(OH)D level 66 nmol/L. Vitamin D levels were associated with baseline BMD, particularly in subjects with baseline knee ROA. Progressive ROA occurred in 5.1% of the participants in the highest tertile of vitamin D intake against 12.6% in the lowest tertile, resulting in an adjusted odds ratio of 7.7 (95% CI: 1.3-43.5). Both intake and levels of 25(OH)D were not significantly related to incident ROA. However, we found a significant interaction between vitamin D intake and BMD in the association with incident knee ROA (P = 0.03): in subjects with low lumbar spine BMD at baseline we observe an increasing incidence of knee ROA with decreasing vitamin D intake and serum levels. CONCLUSIONS: Low dietary vitamin D intake increases the risk of progression of knee ROA. Particularly in subjects with low baseline BMD, vitamin D status seems to influence the incidence and progression of knee ROA. Thus, improving the vitamin D status in the elderly could protect against the development and worsening of knee OA, especially in those with low BMD.

The RIZ Pro704 insertion-deletion polymorphism, bone mineral density and fracture risk: the Rotterdam study.

Estrogens play a major role in the maintenance of bone and bone strength, and they exert their effects via estrogen receptors. Recently, an estrogen receptor alpha (ESR1) specific co-activator, retinoblastoma-interacting zinc-finger protein (RIZ1, 1p36), was shown to strongly enhance ESR1 function in vitro. The same study showed that a Proline insertion-deletion polymorphism at amino acid position 704 (Pro704 ins/del) in the RIZ1 gene was associated with heel BMD in young Swedish women. We tested the relation between the RIZ1 Pro704 ins/del polymorphism and BMD and fracture risk in Caucasian elderly men and women of the Rotterdam study. We also examined whether estradiol levels (measured in a subset) or genetic variation in ESR1 influenced this relation. In 2424 men and 3517 women from the Rotterdam study, RIZ1 genotypes were determined and associations with BMD (lumbar spine and femoral neck) and fracture risk were analysed. We recorded 374 vertebral fractures at baseline and during 6.4+/-0.4 (SD) years of follow-up, and 1219 incident non-vertebral fractures during 7.4+/-3.3 (SD) years of follow-up. The allele frequency of the Pro704 insertion was 41%, the genotype distribution was in Hardy-Weinberg Equilibrium (P=0.94). We found no association of this polymorphism with BMD or fracture risk. Stratification for gender, estradiol levels or interaction with ESR1 risk haplotype did not change these results. In conclusion, in this large study we observed no association of the RIZ1 Pro704 insertion-deletion polymorphism with BMD or fracture risk. This suggests this polymorphism to play a minor role, if any, as a genetic determinant of osteoporosis in elderly subjects.

Large-scale analysis of association between polymorphisms in the transforming growth factor beta 1 gene (TGFB1) and osteoporosis: the GENOMOS study.

INTRODUCTION: The TGFB1 gene which encodes transforming growth factor beta 1, is a strong candidate for susceptibility to osteoporosis and several studies have reported associations between bone mineral density (BMD), osteoporotic fractures and polymorphisms of TGFB1, although these studies have yielded conflicting results. METHODS: We investigated associations between TGFB1 polymorphisms and BMD and fracture in the GENOMOS study: a prospective multicenter study involving 10 European research studies including a total of 28,924 participants. Genotyping was conducted for known TGFB1 polymorphisms at the following sites: G-1639-A (G-800-A, rs1800468), C-1348-T (C-509-T, rs1800469), T29-C (Leu10Pro, rs1982073), G74-C (Arg25Pro, rs1800471) and C788-T (Thr263Ile, rs1800472). These polymorphisms were genotyped prospectively and methodology was standardized across research centers. Genotypes and haplotypes were related to BMD at the lumbar sine and femoral neck and fractures. RESULTS: There were no significant differences in either women or men at either skeletal site for any of the examined polymorphisms with the possible exception of a weak association with reduced BMD (-12 mg/cm2) in men with the T-1348 allele (p<0.05). None of the haplotypes was associated with BMD and none of the polymorphisms or haplotypes significantly affected overall risk of fractures, however, the odds ratio for incident vertebral fracture in carriers of the rare T788 allele was 1.64 (95% CI: 1.09-2.64), p<0.05. CONCLUSIONS: This study indicates that polymorphic variation in the TGFB1 gene does not play a major role in regulating BMD or susceptibility to fractures. The weak associations we observed between the C-1348-T and lumbar spine BMD in men and between C788-T and risk of incident vertebral fractures are of interest but could be chance findings and will need replication in future studies.

Effects of serum TSH and FT4 levels and the TSHR-Asp727Glu polymorphism on bone: the Rotterdam Study.

BACKGROUND: TSH and thyroid hormone may have independent effects on bone. In this study we investigated the association of TSH and free T4 (FT4) with different bone parameters in human subjects. TSH and FT4 are known to be associated with body mass index (BMI) and a higher BMI gives a higher bone mineral density (BMD). Thus, we aimed to determine whether the effects of TSH and FT4 on bone are mediated by BMI. As TSH exerts its biological effect through the TSH receptor (TSHR), the TSHR gene might be a candidate gene affecting bone mass. The TSHR-Asp727Glu polymorphism is associated with lower TSH levels. We therefore examined the association of this polymorphism with bone parameters. METHOD: Genotypes were determined by Taqman assay in 4934 elderly Caucasian men and women of the Rotterdam Study, of whom BMD and bone geometry data were available. Serum thyroid parameters were available in a random set of 1327 subjects. RESULTS: Femoral neck BMD as well as narrow neck BMD and cortical thickness increased with serum TSH. However, FT4 was more strongly and negatively associated with bone parameters. Regression models showed BMI-dependent and -independent effects of both TSH and FT4 on bone. Carriers of the TSHR-Glu(727) allele had a 2.3% higher femoral neck BMD. CONCLUSION: In line with the effect of TSH on bone in mice, serum TSH shows a positive trend with BMD in human subjects, a finding that is strengthened by the association between the TSHR-Asp727Glu polymorphism and femoral neck BMD. However, serum FT4 has a much greater influence on bone than TSH.

Estrogen receptors alpha and beta and the risk of open-angle glaucoma: the Rotterdam Study.

OBJECTIVE: To investigate whether polymorphisms in the estrogen receptor alpha (ESR1) and beta (ESR2) genes were a risk factor for open-angle glaucoma (OAG). METHODS: Participants 55 years and older from the population-based Rotterdam Study underwent, at baseline and at follow-up, the same ophthalmic examination, including visual field screening and stereo optic disc photography. A diagnosis of OAG was based on an algorithm using optic disc measures and visual field loss. Haplotypes of the ESR1 and ESR2 genes were determined. RESULTS: We diagnosed incident OAG in 87 of 3842 participants (2.3%) at risk after a mean follow-up of 6.5 years. We could not detect any association with ESR1 haplotypes. Haplotype 1 of ESR2 showed a 3.6-fold (95% confidence interval, 1.4-9.2) higher risk of incident OAG in men. In women, no association was found between ESR2 and incident OAG. CONCLUSION: Polymorphisms in the ESR1 gene are unrelated to OAG, but ESR2 polymorphisms seem to lead to increased risk of OAG in men.

The activin A-follistatin system: potent regulator of human extracellular matrix mineralization.

Bone quality is an important determinant of osteoporosis, and proper osteoblast differentiation plays an important role in the control and maintenance of bone quality. We investigated the impact of activin signaling on human osteoblast differentiation, extracellular matrix formation, and mineralization. Activins belong to the transforming growth factor-beta superfamily and activin A treatment strongly inhibited mineralization in osteoblast cultures, whereas the activin antagonist follistatin increased mineralization. Osteoblasts produced activin A and follistatin in a differentiation-dependent manner, leading to autocrine regulation of extracellular matrix formation and mineralization. In addition, mineralization in a vascular smooth muscle cell-based model for pathological calcification was inhibited. Comparative activin A and follistatin gene expression profiling showed that activin signaling changes the expression of a specific range of extracellular matrix proteins prior to the onset of mineralization, leading to a matrix composition with reduced or no mineralizing capacity. These findings demonstrate the regulation of osteoblast differentiation and matrix mineralization by the activin A-follistatin system, providing the possibility to control bone quality as well as pathological calcifications such as atherosclerosis by using activin A, follistatin, or analogs thereof.

Selective serotonin reuptake inhibiting antidepressants are associated with an increased risk of nonvertebral fractures.

BACKGROUND: Fractures related to osteoporosis and falling constitute a major health problem in the elderly population. Exposure to antidepressants is associated with an increased risk of falls and fractures, but most previous studies incriminate tricyclic antidepressants (TCAs) rather than selective serotonin reuptake inhibitors (SSRIs). OBJECTIVE: To examine the association between antidepressants, including TCAs, SSRIs, and other antidepressants and the risk of nonvertebral fractures in elderly. DESIGN: Prospective population-based cohort study. SETTING: The Rotterdam Study, consisting of 7983 individuals aged 55 years and older. PARTICIPANTS: All persons from the Rotterdam Study. RESULTS: One thousand two hundred nineteen persons experienced a nonvertebral fracture, 25 during TCA use and 18 during SSRI use. After adjustment for age, sex, lower-limb disability, and depression, the risk of nonvertebral fracture was 2.35 (95% confidence interval, 1.32-4.18) for current users of SSRIs compared with nonusers of antidepressants. Multiple adjusting for many possible risk factors did not affect the association. To deal with potential confounding by indication, we subsequently restricted the analysis to antidepressant users (n = 1217). Compared with past users of TCAs or SSRIs, current users of SSRIs had a 2.07-fold (95% confidence interval, 1.23-3.50) increased risk of fracture, which further increased with prolonged use. In this analysis, depressive state at baseline and during follow-up did not play a role, suggesting absence of confounding by indication. The use of TCAs was associated with an increased fracture risk that decreased with prolonged use. CONCLUSIONS: Not only users of TCAs but also of SSRIs have a significantly increased risk of nonvertebral fractures, in SSRI users especially after prolonged use. Despite fewer early adverse effects of SSRIs, physicians treating elderly depressive patients should be aware of the unfavorable long-term consequence of SSRIs on fracture risk.

Estrogen receptor alpha gene variation is associated with risk of myocardial infarction in more than seven thousand men from five cohorts.

Understanding the mechanisms by which estrogens affect cardiovascular disease risk, including the role of variation in the gene for estrogen receptor alpha (ESR1), may be key to new treatment strategies. We investigated whether the CC genotype at ESR1 c.454-397T>C is associated with increased risk among men. Study of more than 7000 whites in 5 cohorts from 4 countries provided evidence that genotype CC, present in roughly 20% of individuals, is a risk factor for nonfatal acute myocardial infarction (odds ratio=1.44; P<0.0001), after adjustment for established cardiovascular risk factors. After exclusion of younger subjects from 2 cohorts, because of age interaction, the odds ratio increased (to 1.63).

Inappropriate benzodiazepine use in older adults and the risk of fracture.

AIMS: The Beers criteria for prescribing in elderly are well known and used for many drug utilization studies. We investigated the clinical value of the Beers criteria for benzodiazepine use, notably the association between inappropriate use and risk of fracture. METHODS: We performed a nested case-control study within the Rotterdam Study, a population-based cohort study in 7983 elderly. The proportion of 'inappropriate' benzodiazepine use according to the Beers criteria was compared between fracture patients and controls. 'Inappropriate' use for elderly implies use of some long-acting benzodiazepines and some intermediate/short-acting ones exceeding a suggested maximum daily dose. Also, alternative criteria were applied to compare the risk of fracture. Cases were defined as persons with incident fracture between 1991 and 2002 who were current benzodiazepine users on the fracture date. Controls were matched on fracture date and were also current benzodiazepine users. RESULTS: The risk of fracture in 'inappropriate' benzodiazepine users according to the Beers criteria was not significantly different from 'appropriate' users [odds ratio (OR) 1.07, 95% confidence interval (CI) 0.72, 1.60]. However, a significantly higher risk of fracture was found in 'high dose' users and a longer duration of use (14-90 days), irrespective of the type of benzodiazepine (OR 3.45, 95% CI 1.38, 8.59). CONCLUSIONS: These findings suggest that inappropriate benzodiazepine use according to the Beers criteria is not associated with increased risk of fracture. Daily dose and longer duration of use (>14 days) is associated with higher risk of fracture, irrespective of the type of benzodiazepine prescribed.

Cost effectiveness of withdrawal of fall-risk-increasing drugs in geriatric outpatients.

BACKGROUND: Withdrawal of fall-risk-increasing drugs has been proven to be effective in older persons. However, given the enormous rise in healthcare costs in recent decades, the effect of such withdrawals on healthcare costs also needs to be considered. METHOD: Within a common geriatric outpatient population, patients with a history of falls were assessed for falls risk (n = 139). Fall-risk-increasing drugs were withdrawn when appropriate (n = 75). All participants had a 2-month follow-up for fall incidents. The number of prevented falls was calculated using a loglinear regression model. The savings on health expenditures as a result of prevented injuries (estimated from a literature review) and reduced consumption of pharmaceuticals were compared with the intervention costs. RESULTS: After adjustment for confounders, drug withdrawal resulted in a falls risk reduction of 0.89 (95% CI 0.33, 0.98) per patient compared with the non-withdrawal group. Net cost savings were euro1691 (95% CI 662, 2181) per patient in the cohort. This resulted in a cost saving of euro491 (95% CI 465, 497) per prevented fall. CONCLUSION: Withdrawal of fall-risk-increasing drugs generates significant cost savings. Extrapolation of these findings to a national scale results in an estimated reduction of euro60 million in healthcare expenditures, that is, 15% of fall-related health costs.

Large-scale analysis of association between LRP5 and LRP6 variants and osteoporosis.

CONTEXT: Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene cause rare syndromes characterized by altered bone mineral density (BMD). More common LRP5 variants may affect osteoporosis risk in the general population. OBJECTIVE: To generate large-scale evidence on whether 2 common variants of LRP5 (Val667Met, Ala1330Val) and 1 variant of LRP6 (Ile1062Val) are associated with BMD and fracture risk. DESIGN AND SETTING: Prospective, multicenter, collaborative study of individual-level data on 37,534 individuals from 18 participating teams in Europe and North America. Data were collected between September 2004 and January 2007; analysis of the collected data was performed between February and May 2007. Bone mineral density was assessed by dual-energy x-ray absorptiometry. Fractures were identified via questionnaire, medical records, or radiographic documentation; incident fracture data were available for some cohorts, ascertained via routine surveillance methods, including radiographic examination for vertebral fractures. MAIN OUTCOME MEASURES: Bone mineral density of the lumbar spine and femoral neck; prevalence of all fractures and vertebral fractures. RESULTS: The Met667 allele of LRP5 was associated with reduced lumbar spine BMD (n = 25,052 [number of participants with available data]; 20-mg/cm2 lower BMD per Met667 allele copy; P = 3.3 x 10(-8)), as was the Val1330 allele (n = 24,812; 14-mg/cm2 lower BMD per Val1330 copy; P = 2.6 x 10(-9)). Similar effects were observed for femoral neck BMD, with a decrease of 11 mg/cm2 (P = 3.8 x 10(-5)) and 8 mg/cm2 (P = 5.0 x 10(-6)) for the Met667 and Val1330 alleles, respectively (n = 25 193). Findings were consistent across studies for both LRP5 alleles. Both alleles were associated with vertebral fractures (odds ratio [OR], 1.26; 95% confidence interval [CI], 1.08-1.47 for Met667 [2001 fractures among 20 488 individuals] and OR, 1.12; 95% CI, 1.01-1.24 for Val1330 [1988 fractures among 20,096 individuals]). Risk of all fractures was also increased with Met667 (OR, 1.14; 95% CI, 1.05-1.24 per allele [7876 fractures among 31,435 individuals)]) and Val1330 (OR, 1.06; 95% CI, 1.01-1.12 per allele [7802 fractures among 31 199 individuals]). Effects were similar when adjustments were made for age, weight, height, menopausal status, and use of hormone therapy. Fracture risks were partly attenuated by adjustment for BMD. Haplotype analysis indicated that Met667 and Val1330 variants both independently affected BMD. The LRP6 Ile1062Val polymorphism was not associated with any osteoporosis phenotype. All aforementioned associations except that between Val1330 and all fractures and vertebral fractures remained significant after multiple-comparison adjustments. CONCLUSIONS: Common LRP5 variants are consistently associated with BMD and fracture risk across different white populations. The magnitude of the effect is modest. LRP5 may be the first gene to reach a genome-wide significance level (a conservative level of significance [herein, unadjusted P < 10(-7)] that accounts for the many possible comparisons in the human genome) for a phenotype related to osteoporosis.

The interleukin-6-174 G/C promoter polymorphism and arterial stiffness; the Rotterdam Study.

Arterial stiffness normally increases with age and has been established as a precursor of cardiovascular disease. Interleukin-6 is a pleiotropic inflammatory cytokine with an important role in the inflammatory cascade, such as up-regulation of C-reactive protein (CRP). The interleukin-6-174-G/C promoter polymorphism appears to influence levels of inflammatory markers, which have been shown to be associated with arterial stiffness. We studied the association of this polymorphism with levels of interleukin-6 and CRP and with arterial stiffness. The study (n=3849) was embedded in the Rotterdam Study, a prospective, population-based study. Analyses on the association between the -174-G/C polymorphism and pulse wave velocity, distensibility coefficient, and pulse pressure were performed using analyses of variance. Analyses on the levels of inflammatory markers and arterial stiffness were performed using linear regression analyses. Analyses were adjusted for age, sex, mean arterial pressure, heart rate, known cardiovascular risk factors, and atherosclerosis. We found pulse wave velocity to be 0.35 m/s higher for CC-homozygotes vs. wildtype GG-homozygotes (p = 0.018) with evidence for an allele-dose effect (p trend = 0.013), and a similar pattern for pulse pressure (p trend = 0.041). No apparent consistent association with the distensibility coefficient was found. CRP levels were associated with pulse wave velocity (p = 0.007). In conclusion, the interleukin-6-174 G/C polymorphism is associated with increased arterial stiffness and pulse pressure.

The catechol-O-methyltransferase Met158 low-activity allele and association with nonvertebral fracture risk in elderly men.

CONTEXT: Because sex steroids play an important role in bone development, variants in genes encoding proteins involved in estrogen synthesis and metabolism could contribute to interindividual variation in bone parameters and fracture risk. An example is catechol-O-methyltransferase (COMT), an estrogen-degrading enzyme involved in inactivation of catechol-estrogens. Its gene contains a functional valine to methionine substitution at codon 158. OBJECTIVE: The aim of our study was to determine whether this polymorphism is associated with bone parameters and fracture risk in elderly subjects. METHODS: COMT genotypes were determined using TaqMan allelic discrimination in 2515 men and 3554 women from the Rotterdam Study, a population-based cohort study of individuals aged 55 and older. Associations with bone mineral density (BMD) and bone loss were analyzed using ANOVA or analysis of covariance, whereas fracture risk was analyzed using Cox's proportional hazard regression analysis. COMT mRNA expression in three osteoblastic cell lines (SaOS, MG63, and SVHFO) was analyzed by RT-PCR. RESULTS: Male carriers of the Met(158) allele had an increased risk for osteoporotic fractures (hazard ratio = 1.6; 95% confidence interval, 1.0-2.4) and for fragility fractures (hazard ratio = 2.7; 95% confidence interval, 1.3-5.9), with evidence for a dominant effect. Adjustments for age, height, weight, and BMD did not change the risk estimates. In women, this association was weaker and not significant. BMD was not significantly associated with the variant in either men or women. COMT mRNA was expressed in all three osteoblastic cell lines tested. CONCLUSION: The COMT Val158Met polymorphism is associated with fracture risk in elderly men, through a mechanism independent of BMD.