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1.  Meta-Analysis of Genome-Wide Scans Provides Evidence for Sex- and Site-Specific Regulation of Bone Mass 
Several genome-wide scans have been performed to detect loci that regulate BMD, but these have yielded inconsistent results, with limited replication of linkage peaks in different studies. In an effort to improve statistical power for detection of these loci, we performed a meta-analysis of genome-wide scans in which spine or hip BMD were studied. Evidence was gained to suggest that several chromosomal loci regulate BMD in a site-specific and sex-specific manner.
Introduction
BMD is a heritable trait and an important predictor of osteoporotic fracture risk. Several genome-wide scans have been performed in an attempt to detect loci that regulate BMD, but there has been limited replication of linkage peaks between studies. In an attempt to resolve these inconsistencies, we conducted a collaborative meta-analysis of genome-wide linkage scans in which femoral neck BMD (FN-BMD) or lumbar spine BMD (LS-BMD) had been studied.
Materials and Methods
Data were accumulated from nine genome-wide scans involving 11,842 subjects. Data were analyzed separately for LS-BMD and FN-BMD and by sex. For each study, genomic bins of 30 cM were defined and ranked according to the maximum LOD score they contained. While various densitometers were used in different studies, the ranking approach that we used means that the results are not confounded by the fact that different measurement devices were used. Significance for high average rank and heterogeneity was obtained through Monte Carlo testing.
Results
For LS-BMD, the quantitative trait locus (QTL) with greatest significance was on chromosome 1p13.3-q23.3 (p = 0.004), but this exhibited high heterogeneity and the effect was specific for women. Other significant LS-BMD QTLs were on chromosomes 12q24.31-qter, 3p25.3-p22.1, 11p12-q13.3, and 1q32-q42.3, including one on 18p11-q12.3 that had not been detected by individual studies. For FN-BMD, the strongest QTL was on chromosome 9q31.1-q33.3 (p = 0.002). Other significant QTLs were identified on chromosomes 17p12-q21.33, 14q13.1-q24.1, 9q21.32-q31.1, and 5q14.3-q23.2. There was no correlation in average ranks of bins between men and women and the loci that regulated BMD in men and women and at different sites were largely distinct.
Conclusions
This large-scale meta-analysis provided evidence for replication of several QTLs identified in previous studies and also identified a QTL on chromosome 18p11-q12.3, which had not been detected by individual studies. However, despite the large sample size, none of the individual loci identified reached genome-wide significance.
doi:10.1359/jbmr.060806
PMCID: PMC4016811  PMID: 17228994
osteoporosis; BMD; linkage; meta-analysis; genome search; genome scan
2.  Assessment of Gene-by-Sex Interaction Effect on Bone Mineral Density 
Liu, Ching-Ti | Estrada, Karol | Yerges-Armstrong, Laura M. | Amin, Najaf | Evangelou, Evangelos | Li, Guo | Minster, Ryan L. | Carless, Melanie A. | Kammerer, Candace M. | Oei, Ling | Zhou, Yanhua | Alonso, Nerea | Dailiana, Zoe | Eriksson, Joel | García-Giralt, Natalia | Giroux, Sylvie | Husted, Lise Bjerre | Khusainova, Rita I. | Koromila, Theodora | Kung, Annie WaiChee | Lewis, Joshua R. | Masi, Laura | Mencej-Bedrac, Simona | Nogues, Xavier | Patel, Millan S. | Prezelj, Janez | Richards, J Brent | Sham, Pak Chung | Spector, Timothy | Vandenput, Liesbeth | Xiao, Su-Mei | Zheng, Hou-Feng | Zhu, Kun | Balcells, Susana | Brandi, Maria Luisa | Frost, Morten | Goltzman, David | González-Macías, Jesús | Karlsson, Magnus | Khusnutdinova, Elza K. | Kollia, Panagoula | Langdahl, Bente Lomholt | Ljunggren, Östen | Lorentzon, Mattias | Marc, Janja | Mellström, Dan | Ohlsson, Claes | Olmos, José M. | Ralston, Stuart H. | Riancho, José A. | Rousseau, François | Urreizti, Roser | Van Hul, Wim | Zarrabeitia, María T. | Castano-Betancourt, Martha | Demissie, Serkalem | Grundberg, Elin | Herrera, Lizbeth | Kwan, Tony | Medina-Gómez, Carolina | Pastinen, Tomi | Sigurdsson, Gunnar | Thorleifsson, Gudmar | vanMeurs, Joyce B.J. | Blangero, John | Hofman, Albert | Liu, Yongmei | Mitchell, Braxton D. | O’Connell, Jeffrey R. | Oostra, Ben A. | Rotter, Jerome I | Stefansson, Kari | Streeten, Elizabeth A. | Styrkarsdottir, Unnur | Thorsteinsdottir, Unnur | Tylavsky, Frances A. | Uitterlinden, Andre | Cauley, Jane A. | Harris, Tamara B. | Ioannidis, John P.A. | Psaty, Bruce M. | Robbins, John A | Zillikens, M. Carola | vanDuijn, Cornelia M. | Prince, Richard L. | Karasik, David | Rivadeneira, Fernando | Kiel, Douglas P. | Cupples, L. Adrienne | Hsu, Yi-Hsiang
Background
Sexual dimorphism in various bone phenotypes, including bone mineral density (BMD), is widely observed; however the extent to which genes explain these sex differences is unclear. To identify variants with different effects by sex, we examined gene-by-sex autosomal interactions genome-wide, and performed eQTL analysis and bioinformatics network analysis.
Methods
We conducted an autosomal genome-wide meta-analysis of gene-by-sex interaction on lumbar spine (LS-) and femoral neck (FN-) BMD, in 25,353 individuals from eight cohorts. In a second stage, we followed up the 12 top SNPs (P<1×10−5) in an additional set of 24,763 individuals. Gene-by-sex interaction and sex-specific effects were examined in these 12 SNPs.
Results
We detected one novel genome-wide significant interaction associated with LS-BMD at the Chr3p26.1-p25.1 locus, near the GRM7 gene (male effect = 0.02 & p-value = 3.0×10−5; female effect = −0.007 & p-value=3.3×10−2) and eleven suggestive loci associated with either FN- or LS-BMD in discovery cohorts. However, there was no evidence for genome-wide significant (P<5×10−8) gene-by-sex interaction in the joint analysis of discovery and replication cohorts.
Conclusion
Despite the large collaborative effort, no genome-wide significant evidence for gene-by-sex interaction was found influencing BMD variation in this screen of autosomal markers. If they exist, gene-by-sex interactions for BMD probably have weak effects, accounting for less than 0.08% of the variation in these traits per implicated SNP.
doi:10.1002/jbmr.1679
PMCID: PMC3447125  PMID: 22692763
gene-by-sex; interaction; BMD; association; aging
3.  Meta-analysis of genome-wide association studies confirms a susceptibility locus for knee osteoarthritis on chromosome 7q22 
Evangelou, Evangelos | Valdes, Ana M. | Kerkhof, Hanneke J.M | Styrkarsdottir, Unnur | Zhu, YanYan | Meulenbelt, Ingrid | Lories, Rik J. | Karassa, Fotini B. | Tylzanowski, Przemko | Bos, Steffan D. | Akune, Toru | Arden, Nigel K. | Carr, Andrew | Chapman, Kay | Cupples, L. Adrienne | Dai, Jin | Deloukas, Panos | Doherty, Michael | Doherty, Sally | Engstrom, Gunnar | Gonzalez, Antonio | Halldorsson, Bjarni V. | Hammond, Christina L. | Hart, Deborah J. | Helgadottir, Hafdis | Hofman, Albert | Ikegawa, Shiro | Ingvarsson, Thorvaldur | Jiang, Qing | Jonsson, Helgi | Kaprio, Jaakko | Kawaguchi, Hiroshi | Kisand, Kalle | Kloppenburg, Margreet | Kujala, Urho M. | Lohmander, L. Stefan | Loughlin, John | Luyten, Frank P. | Mabuchi, Akihiko | McCaskie, Andrew | Nakajima, Masahiro | Nilsson, Peter M. | Nishida, Nao | Ollier, William E.R. | Panoutsopoulou, Kalliope | van de Putte, Tom | Ralston, Stuart H. | Rivadeneira, Fernado | Saarela, Janna | Schulte-Merker, Stefan | Slagboom, P. Eline | Sudo, Akihiro | Tamm, Agu | Tamm, Ann | Thorleifsson, Gudmar | Thorsteinsdottir, Unnur | Tsezou, Aspasia | Wallis, Gillian A. | Wilkinson, J. Mark | Yoshimura, Noriko | Zeggini, Eleftheria | Zhai, Guangju | Zhang, Feng | Jonsdottir, Ingileif | Uitterlinden, Andre G. | Felson, David T | van Meurs, Joyce B. | Stefansson, Kari | Ioannidis, John P.A. | Spector, Timothy D.
Annals of the rheumatic diseases  2010;70(2):349-355.
Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in the elderly. It is characterized by changes in joint structure including degeneration of the articular cartilage and its etiology is multifactorial with a strong postulated genetic component. We performed a meta-analysis of four genome-wide association (GWA) studies of 2,371 knee OA cases and 35,909 controls in Caucasian populations. Replication of the top hits was attempted with data from additional ten replication datasets. With a cumulative sample size of 6,709 cases and 44,439 controls, we identified one genome-wide significant locus on chromosome 7q22 for knee OA (rs4730250, p-value=9.2×10−9), thereby confirming its role as a susceptibility locus for OA. The associated signal is located within a large (500kb) linkage disequilibrium (LD) block that contains six genes; PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, beta), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like), and BCAP29 (the B-cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.
doi:10.1136/ard.2010.132787
PMCID: PMC3615180  PMID: 21068099
4.  Genome-wide meta-analysis identifies 56 bone mineral density loci and reveals 14 loci associated with risk of fracture 
Estrada, Karol | Styrkarsdottir, Unnur | Evangelou, Evangelos | Hsu, Yi-Hsiang | Duncan, Emma L | Ntzani, Evangelia E | Oei, Ling | Albagha, Omar M E | Amin, Najaf | Kemp, John P | Koller, Daniel L | Li, Guo | Liu, Ching-Ti | Minster, Ryan L | Moayyeri, Alireza | Vandenput, Liesbeth | Willner, Dana | Xiao, Su-Mei | Yerges-Armstrong, Laura M | Zheng, Hou-Feng | Alonso, Nerea | Eriksson, Joel | Kammerer, Candace M | Kaptoge, Stephen K | Leo, Paul J | Thorleifsson, Gudmar | Wilson, Scott G | Wilson, James F | Aalto, Ville | Alen, Markku | Aragaki, Aaron K | Aspelund, Thor | Center, Jacqueline R | Dailiana, Zoe | Duggan, David J | Garcia, Melissa | Garcia-Giralt, Natàlia | Giroux, Sylvie | Hallmans, Göran | Hocking, Lynne J | Husted, Lise Bjerre | Jameson, Karen A | Khusainova, Rita | Kim, Ghi Su | Kooperberg, Charles | Koromila, Theodora | Kruk, Marcin | Laaksonen, Marika | Lacroix, Andrea Z | Lee, Seung Hun | Leung, Ping C | Lewis, Joshua R | Masi, Laura | Mencej-Bedrac, Simona | Nguyen, Tuan V | Nogues, Xavier | Patel, Millan S | Prezelj, Janez | Rose, Lynda M | Scollen, Serena | Siggeirsdottir, Kristin | Smith, Albert V | Svensson, Olle | Trompet, Stella | Trummer, Olivia | van Schoor, Natasja M | Woo, Jean | Zhu, Kun | Balcells, Susana | Brandi, Maria Luisa | Buckley, Brendan M | Cheng, Sulin | Christiansen, Claus | Cooper, Cyrus | Dedoussis, George | Ford, Ian | Frost, Morten | Goltzman, David | González-Macías, Jesús | Kähönen, Mika | Karlsson, Magnus | Khusnutdinova, Elza | Koh, Jung-Min | Kollia, Panagoula | Langdahl, Bente Lomholt | Leslie, William D | Lips, Paul | Ljunggren, Östen | Lorenc, Roman S | Marc, Janja | Mellström, Dan | Obermayer-Pietsch, Barbara | Olmos, José M | Pettersson-Kymmer, Ulrika | Reid, David M | Riancho, José A | Ridker, Paul M | Rousseau, François | Slagboom, P Eline | Tang, Nelson LS | Urreizti, Roser | Van Hul, Wim | Viikari, Jorma | Zarrabeitia, María T | Aulchenko, Yurii S | Castano-Betancourt, Martha | Grundberg, Elin | Herrera, Lizbeth | Ingvarsson, Thorvaldur | Johannsdottir, Hrefna | Kwan, Tony | Li, Rui | Luben, Robert | Medina-Gómez, Carolina | Palsson, Stefan Th | Reppe, Sjur | Rotter, Jerome I | Sigurdsson, Gunnar | van Meurs, Joyce B J | Verlaan, Dominique | Williams, Frances MK | Wood, Andrew R | Zhou, Yanhua | Gautvik, Kaare M | Pastinen, Tomi | Raychaudhuri, Soumya | Cauley, Jane A | Chasman, Daniel I | Clark, Graeme R | Cummings, Steven R | Danoy, Patrick | Dennison, Elaine M | Eastell, Richard | Eisman, John A | Gudnason, Vilmundur | Hofman, Albert | Jackson, Rebecca D | Jones, Graeme | Jukema, J Wouter | Khaw, Kay-Tee | Lehtimäki, Terho | Liu, Yongmei | Lorentzon, Mattias | McCloskey, Eugene | Mitchell, Braxton D | Nandakumar, Kannabiran | Nicholson, Geoffrey C | Oostra, Ben A | Peacock, Munro | Pols, Huibert A P | Prince, Richard L | Raitakari, Olli | Reid, Ian R | Robbins, John | Sambrook, Philip N | Sham, Pak Chung | Shuldiner, Alan R | Tylavsky, Frances A | van Duijn, Cornelia M | Wareham, Nick J | Cupples, L Adrienne | Econs, Michael J | Evans, David M | Harris, Tamara B | Kung, Annie Wai Chee | Psaty, Bruce M | Reeve, Jonathan | Spector, Timothy D | Streeten, Elizabeth A | Zillikens, M Carola | Thorsteinsdottir, Unnur | Ohlsson, Claes | Karasik, David | Richards, J Brent | Brown, Matthew A | Stefansson, Kari | Uitterlinden, André G | Ralston, Stuart H | Ioannidis, John P A | Kiel, Douglas P | Rivadeneira, Fernando
Nature genetics  2012;44(5):491-501.
Bone mineral density (BMD) is the most important predictor of fracture risk. We performed the largest meta-analysis to date on lumbar spine and femoral neck BMD, including 17 genome-wide association studies and 32,961 individuals of European and East Asian ancestry. We tested the top-associated BMD markers for replication in 50,933 independent subjects and for risk of low-trauma fracture in 31,016 cases and 102,444 controls. We identified 56 loci (32 novel)associated with BMD atgenome-wide significant level (P<5×10−8). Several of these factors cluster within the RANK-RANKL-OPG, mesenchymal-stem-cell differentiation, endochondral ossification and the Wnt signalling pathways. However, we also discovered loci containing genes not known to play a role in bone biology. Fourteen BMD loci were also associated with fracture risk (P<5×10−4, Bonferroni corrected), of which six reached P<5×10−8 including: 18p11.21 (C18orf19), 7q21.3 (SLC25A13), 11q13.2 (LRP5), 4q22.1 (MEPE), 2p16.2 (SPTBN1) and 10q21.1 (DKK1). These findings shed light on the genetic architecture and pathophysiological mechanisms underlying BMD variation and fracture susceptibility.
doi:10.1038/ng.2249
PMCID: PMC3338864  PMID: 22504420
5.  Glutamine Repeat Variants in Human RUNX2 Associated with Decreased Femoral Neck BMD, Broadband Ultrasound Attenuation and Target Gene Transactivation 
PLoS ONE  2012;7(8):e42617.
RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (−0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.
doi:10.1371/journal.pone.0042617
PMCID: PMC3418257  PMID: 22912713
6.  A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality 
Journal of Clinical Investigation  2001;107(7):899-907.
Osteoporosis is a common disease with a strong genetic component. We previously described a polymorphic Sp1 binding site in the COL1A1 gene that has been associated with osteoporosis in several populations. Here we explore the molecular mechanisms underlying this association. A meta-analysis showed significant associations between COL1A1 “s” alleles and bone mineral density (BMD), body mass index (BMI), and osteoporotic fractures. The association with fracture was stronger than expected on the basis of the observed differences in BMD and BMI, suggesting an additional effect on bone strength. Gel shift assays showed increased binding affinity of the “s” allele for Sp1 protein, and primary RNA transcripts derived from the “s” allele were approximately three times more abundant than “S” allele–derived transcripts in “Ss” heterozygotes. Collagen produced from osteoblasts cultured from “Ss” heterozygotes had an increased ratio of α1(I) protein relative to α2(I), and this was accompanied by an increased ratio of COL1A1 mRNA relative to COL1A2. Finally, the yield strength of bone derived from “Ss” individuals was reduced when compared with bone derived from “SS” subjects. We conclude that the COL1A1 Sp1 polymorphism is a functional genetic variant that predisposes to osteoporosis by complex mechanisms involving changes in bone mass and bone quality.
PMCID: PMC199568  PMID: 11285309
7.  Large-Scale Analysis of Association Between LRP5 and LRP6 Variants and Osteoporosis 
Jama  2008;299(11):1277-1290.
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 × 10−8), as was the Val1330 allele (n = 24 812; 14-mg/cm2 lower BMD per Val1330 copy; P=2.6 × 10−9). Similar effects were observed for femoral neck BMD, with a decrease of 11 mg/cm2 (P =3.8 × 10−5) and 8 mg/cm2 (P=5.0×10−6) 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.
doi:10.1001/jama.299.11.1277
PMCID: PMC3282142  PMID: 18349089
10.  A Class III Semaphorin (Sema3e) Inhibits Mouse Osteoblast Migration and Decreases Osteoclast Formation In Vitro 
Calcified Tissue International  2012;90(2):151-162.
Originally identified as axonal guidance cues, semaphorins are expressed throughout many different tissues and regulate numerous non-neuronal processes. We demonstrate that most class III semaphorins are expressed in mouse osteoblasts and are differentially regulated by cell growth and differentiation: Sema3d expression is increased and Sema3e expression decreased during proliferation in culture, while expression of Sema3a is unaffected by cell density but increases in cultures of mineralizing osteoblasts. Expression of Sema3a, -3e, and -3d is also differentially regulated by osteogenic stimuli; inhibition of GSK3β decreased expression of Sema3a and -3e, while 1,25-(OH)2D3 increased expression of Sema3e. Parathyroid hormone had no effect on expression of Sema3a, -3b, or -3d. Osteoblasts, macrophages, and osteoclasts express the Sema3e receptor PlexinD1, suggesting an autocrine and paracrine role for Sema3e. No effects of recombinant Sema3e on osteoblast proliferation, differentiation, or mineralization were observed; but Sema3e did inhibit the migration of osteoblasts in a wound-healing assay. The formation of multinucleated, tartrate-resistant acid phosphatase–positive osteoclasts was decreased by 81% in cultures of mouse bone marrow macrophages incubated with 200 ng/mL Sema3e. Correspondingly, decreased expression of osteoclast markers (Itgb3, Acp5, Cd51, Nfatc1, CalcR, and Ctsk) was observed by qPCR in macrophage cultures differentiated in the presence of Sema3e. Our results demonstrate that class III semaphorins are expressed by osteoblasts and differentially regulated by differentiation, mineralization, and osteogenic stimuli. Sema3e is a novel inhibitor of osteoclast formation in vitro and may play a role in maintaining local bone homeostasis, potentially acting as a coupling factor between osteoclasts and osteoblasts.
doi:10.1007/s00223-011-9560-7
PMCID: PMC3271215  PMID: 22227882
Macrophage; Plexin; Mineralization; Vitamin D3; Migration
11.  Role of cannabinoids in the regulation of bone remodeling 
The endocannabinoid system plays a key role in regulating a variety of physiological processes such as appetite control and energy balance, pain perception, and immune responses. Recent studies have implicated the endocannabinoid system in the regulation of bone cell activity and bone remodeling. These studies showed that endogenous cannabinoid ligands, cannabinoid receptors, and the enzymes responsible for ligand synthesis and breakdown all play important roles in bone mass and in the regulation of bone disease. These findings suggest that the endocannabinoid pathway could be of value as a therapeutic target for the prevention and treatment of bone diseases. Here, we review the role of the skeletal endocannabinoid system in the regulation of bone remodeling in health and disease.
doi:10.3389/fendo.2012.00136
PMCID: PMC3499879  PMID: 23181053
cannabinoid; Bone; CB1; CB2; GPR55; Osteoblasts; Osteoclasts; Adipocytes
12.  Twenty bone mineral density loci identified by large-scale meta-analysis of genome-wide association studies 
Nature genetics  2009;41(11):1199-1206.
Bone mineral density (BMD) is a heritable complex trait used in the clinical diagnosis of osteoporosis and the assessment of fracture risk. We performed meta-analysis of five genome-wide association studies of femoral neck and lumbar spine BMD in 19,195 subjects of Northern European descent. We identified 20 loci reaching genome-wide significance (GWS; P<5×10−8), of which 13 map to new regions including 1p31.3 (GPR177), 2p21 (SPTBN1), 3p22 (CTNNB1), 4q21.1 (MEPE), 5q14 (MEF2C), 7p14 (STARD3NL), 7q21.3 (FLJ42280), 11p11.2 (LRP4; ARHGAP1; F2), 11p14.1 (DCDC5), 11p15 (SOX6), 16q24 (FOXL1), 17q21 (HDAC5) and 17q12 (CRHR1). The metaanalysis also confirmed at GWS level, seven known BMD loci on 1p36 (ZBTB40), 6q25 (ESR1), 8q24 (TNFRSF11B), 11q13.4 (LRP5), 12q13 (SP7), 13q14 (TNFSF11), and 18q21 (TNFRSF11A). The numerous SNPs associated with BMD map to genes in signaling pathways with relevance to bone metabolism, and highlight the complex genetic architecture underlying osteoporosis and BMD variation.
doi:10.1038/ng.446
PMCID: PMC2783489  PMID: 19801982
13.  Collaborative Meta-analysis: Associations of 150 Candidate Genes With Osteoporosis and Osteoporotic Fracture 
Annals of internal medicine  2009;151(8):528-537.
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 TN-FSF11) 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.
Primary Funding Source
European Union, Netherlands Organisation for Scientific Research, Research Institute for Diseases in the Elderly, Netherlands Genomics Initiative, Wellcome Trust, National Institutes of Health, deCODE Genetics, and Canadian Institutes of Health Research.
PMCID: PMC2842981  PMID: 19841454
14.  Protocol for stage 2 of the GaP study (genetic testing acceptability for Paget's disease of bone): A questionnaire study to investigate whether relatives of people with Paget's disease would accept genetic testing and preventive treatment if they were available 
Background
Paget's disease of bone (PDB) disrupts normal bone architecture and causes pain, deformity, deafness, osteoarthritis, and fractures. Genetic factors play a role in PDB and genetic tests are now conducted for research purposes. It is thus timely to investigate the potential for a clinical programme of genetic testing and preventative treatment for people who have a family history of PDB. This study examines the beliefs of relatives of people with PDB. It focuses particularly on illness and treatment representations as predictors of the acceptability and uptake of potential clinical programmes. Illness representations are examined using Leventhal's Common Sense Self-Regulation Model while cognitions about treatment behaviours (acceptance of testing and treatment uptake) are conceptualised within the Theory of Planned Behaviour.
Methods/Design
A postal questionnaire of non-affected relatives of people with Paget's disease. The sample will include relatives of Paget's patients with a family history of Paget's disease and relatives of Paget's patients without a family history of Paget's disease. The questionnaire will explore whether a range of factors relate to acceptability of a programme of genetic testing and preventive treatment in relatives of Paget's disease sufferers. The questionnaire will include several measures: illness representations (as measured by the Brief Illness Perceptions Questionnaire); treatment representations (as measured by Theory of Planned Behaviour-based question items, informed by a prior interview elicitation study); descriptive and demographic details; and questions exploring family environment and beliefs of other important people.
Data will also be collected from family members who have been diagnosed with Paget's disease to describe the disease presentation and its distribution within a family.
Discussion
The answers to these measures will inform the feasibility of a programme of genetic testing and preventive treatment for individuals who are at a high risk of developing Paget's disease because they carry an appropriate genetic mutation. They will also contribute to theoretical and empirical approaches to predicting diagnostic and treatment behaviours from the combined theoretical models.
doi:10.1186/1472-6963-8-116
PMCID: PMC2442429  PMID: 18510762
15.  ER Stress-Mediated Apoptosis in a New Mouse Model of Osteogenesis imperfecta 
PLoS Genetics  2008;4(2):e7.
Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures, and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here, we describe a new mouse model for Osteogenesis imperfecta termed Aga2 (abnormal gait 2) that was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability, including reduced bone mass, multiple fractures, and early lethality. The causal gene was mapped to Chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1) gene as the cause of the disorder. Aga2 heterozygous animals had markedly increased bone turnover and a disrupted native collagen network. Further studies showed that abnormal proα1(I) chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an endoplasmic reticulum stress-specific unfolded protein response involving upregulation of BiP, Hsp47, and Gadd153 with caspases-12 and −3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for Osteogenesis imperfecta, and identified a role for intracellular modulation of the endoplasmic reticulum stress-associated unfolded protein response machinery toward osteoblast apoptosis during the pathogenesis of disease.
Author Summary
Osteogenesis imperfecta (OI) is a heterogeneous collection of connective tissue disorders typically caused by mutations in the COL1A1/2 genes that encode the chains of type I collagen, the principle structural protein of bone. Phenotypic expression in OI depends on the nature of the mutation, causing a clinical heterogeneity ranging from a mild risk of fractures to perinatal lethality. Here, we describe a new OI mouse model with a dominant mutation in the terminal C-propeptide domain of Col1a1 generated using the N-ethyl-N-nitrosourea (ENU) mutagenesis strategy. Heterozygous animals developed severe-to-lethal phenotypes that were associated with endoplasmic reticulum stress, and caspases-12 and −3 activation within calvarial osteoblasts. We provide evidence for endoplasmic reticulum stress–associated apoptosis as a key component in the pathogenesis of disease.
doi:10.1371/journal.pgen.0040007
PMCID: PMC2222924  PMID: 18248096
16.  Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors 
Nature medicine  2005;11(7):774-779.
Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and to the development of new treatments. Here we show that mice with inactivation of cannabinoid type 1 (CB1) receptors have increased bone mass and are protected from ovariectomy induced bone loss. Pharmacological antagonists of CB1 and CB2 receptors prevented ovariectomy induced bone loss in vivo and caused osteoclast inhibition in vitro by promoting osteoclast apoptosis and inhibiting production of several osteoclast survival factors. These studies show that the CB1 receptor plays a role in the regulation of bone mass and ovariectomy induced bone loss and that CB1 and CB2 selective cannabinoid receptor antagonists are a novel class of osteoclast inhibitors that may be of value in the treatment of osteoporosis and other bone diseases.
doi:10.1038/nm1255
PMCID: PMC1430341  PMID: 15908955
17.  Generation Scotland: the Scottish Family Health Study; a new resource for researching genes and heritability 
BMC Medical Genetics  2006;7:74.
Background
Generation Scotland: the Scottish Family Health Study aims to identify genetic variants accounting for variation in levels of quantitative traits underlying the major common complex diseases (such as cardiovascular disease, cognitive decline, mental illness) in Scotland.
Methods/Design
Generation Scotland will recruit a family-based cohort of up to 50,000 individuals (comprising siblings and parent-offspring groups) across Scotland. It will be a six-year programme, beginning in Glasgow and Tayside in the first two years (Phase 1) before extending to other parts of Scotland in the remaining four years (Phase 2). In Phase 1, individuals aged between 35 and 55 years, living in the East and West of Scotland will be invited to participate, along with at least one (and preferably more) siblings and any other first degree relatives aged 18 or over. The total initial sample size will be 15,000 and it is planned that this will increase to 50,000 in Phase 2. All participants will be asked to contribute blood samples from which DNA will be extracted and stored for future investigation. The information from the DNA, along with answers to a life-style and medical history questionnaire, clinical and biochemical measurements taken at the time of donation, and subsequent health developments over the life course (traced through electronic health records) will be stored and used for research purposes. In addition, a detailed public consultation process will begin that will allow respondents' views to shape and develop the study. This is an important aspect to the research, and forms the continuation of a long-term parallel engagement process.
Discussion
As well as gene identification, the family-based study design will allow measurement of the heritability and familial aggregation of relevant quantitative traits, and the study of how genetic effects may vary by parent-of-origin. Long-term potential outcomes of this research include the targeting of disease prevention and treatment, and the development of screening tools based on the new genetic information. This study approach is complementary to other population-based genetic epidemiology studies, such as UK Biobank, which are established primarily to characterise genes and genetic risk in the population.
doi:10.1186/1471-2350-7-74
PMCID: PMC1592477  PMID: 17014726
18.  Protocol for stage 1 of the GaP study (Genetic testing acceptability for Paget's disease of bone): an interview study about genetic testing and preventive treatment: would relatives of people with Paget's disease want testing and treatment if they were available? 
Background
Paget's disease of bone (PDB) is characterised by focal increases in bone turnover, affecting one or more bones throughout the skeleton. This disrupts normal bone architecture and causes pain, deformity, deafness, osteoarthritis, and fractures.
Genetic factors are recognised to play a role in PDB and it is now possible to carry out genetic tests for research. In view of this, it is timely to investigate the clinical potential for a programme of genetic testing and preventative treatment for people who have a family history of PDB, to prevent or delay the development of PDB.
Evidence from non-genetic conditions, that have effective treatments, demonstrates that patients' beliefs may affect the acceptability and uptake of treatment. Two groups of beliefs (illness and treatment representations) are likely to be influential.
Illness representations describe how people see their illness, as outlined in Leventhal's Self-Regulation Model. Treatment representations describe how people perceive potential treatment for their disease. People offered a programme of genetic testing and treatment will develop their own treatment representations based on what is offered, but the beliefs rather than the objective programme of treatment are likely to determine their willingness to participate. The Theory of Planned Behaviour is a theoretical model that predicts behaviours from people's beliefs about the consequences, social pressures and perceived control over the behaviour, including uptake of treatment.
Methods/design
This study aims to examine the acceptability of genetic testing, followed by preventative treatment, to relatives of people with PDB. We aim to interview people with Paget's disease, and their families, from the UK. Our research questions are:
1. What do individuals with Paget's disease think would influence the involvement of their relatives in a programme of genetic testing and preventative treatment?
2. What do relatives of Paget's disease sufferers think would influence them in accepting an offer of a programme of genetic testing and preventative treatment?
Discussion
Our research will be informed by relevant psychological theory: primarily the Self-Regulation Model and the Theory of Planned Behaviour. The results of these interviews will inform the development of a separate questionnaire-based study to explore these research questions in greater detail.
doi:10.1186/1472-6963-6-71
PMCID: PMC1513223  PMID: 16762063
20.  Large-Scale Evidence for the Effect of the COLIA1 Sp1 Polymorphism on Osteoporosis Outcomes: The GENOMOS Study  
PLoS Medicine  2006;3(4):e90.
Background
Osteoporosis and fracture risk are considered to be under genetic control. Extensive work is being performed to identify the exact genetic variants that determine this risk. Previous work has suggested that a G/T polymorphism affecting an Sp1 binding site in the COLIA1 gene is a genetic marker for low bone mineral density (BMD) and osteoporotic fracture, but there have been no very-large-scale studies of COLIA1 alleles in relation to these phenotypes.
Methods and Findings
Here we evaluated the role of COLIA1 Sp1 alleles as a predictor of BMD and fracture in a multicenter study involving 20,786 individuals from several European countries. At the femoral neck, the average (95% confidence interval [CI]) BMD values were 25 mg/cm 2 (CI, 16 to 34 mg/cm 2) lower in TT homozygotes than the other genotype groups ( p < 0.001), and a similar difference was observed at the lumbar spine; 21 mg/cm 2 (CI, 1 to 42 mg/cm 2), ( p = 0.039). These associations were unaltered after adjustment for potential confounding factors. There was no association with fracture overall (odds ratio [OR] = 1.01 [CI, 0.95 to 1.08]) in either unadjusted or adjusted analyses, but there was a non-significant trend for association with vertebral fracture and a nominally significant association with incident vertebral fractures in females (OR = 1.33 [CI, 1.00 to 1.77]) that was independent of BMD, and unaltered in adjusted analyses.
Conclusions
Allowing for the inevitable heterogeneity between participating teams, this study—which to our knowledge is the largest ever performed in the field of osteoporosis genetics for a single gene—demonstrates that the COLIA1 Sp1 polymorphism is associated with reduced BMD and could predispose to incident vertebral fractures in women, independent of BMD. The associations we observed were modest however, demonstrating the importance of conducting studies that are adequately powered to detect and quantify the effects of common genetic variants on complex diseases.
A large collaborative European study finds only weak links between a much studied potential genetic risk factor and bone mineral density or fracture risk.
doi:10.1371/journal.pmed.0030090
PMCID: PMC1370920  PMID: 16475872
21.  Signal peptide mutations in RANK prevent downstream activation of NF-κB 
Journal of Bone and Mineral Research  2011;26(8):1926-1938.
Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF-κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF-κB occurred in HEK293 cells overexpressing wild-type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild-type RANK demonstrated ligand-dependent activation of NF-κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild-type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins. © 2011 American Society for Bone and Mineral Research
doi:10.1002/jbmr.399
PMCID: PMC3378713  PMID: 21472776
RANK; FAMILIAL EXPANSILE OSTEOLYSIS; EARLY-ONSET PAGET'S DISEASE; EXPANSILE SKELETAL HYPERPHOSPHATASIA; OSTEOCLAST; NFκB
22.  No evidence of an association between mitochondrial DNA variants and osteoarthritis in 7393 cases and 5122 controls 
Annals of the Rheumatic Diseases  2012;72(1):136-139.
Objectives
Osteoarthritis (OA) has a complex aetiology with a strong genetic component. Genome-wide association studies implicate several nuclear genes in the aetiology, but a major component of the heritability has yet to be defined at the molecular level. Initial studies implicate maternally inherited variants of mitochondrial DNA (mtDNA) in subgroups of patients with OA based on gender and specific joint involvement, but these findings have not been replicated.
Methods
The authors studied 138 maternally inherited mtDNA variants genotyped in a two cohort genetic association study across a total of 7393 OA cases from the arcOGEN consortium and 5122 controls genotyped in the Wellcome Trust Case Control consortium 2 study.
Results
Following data quality control we examined 48 mtDNA variants that were common in cohort 1 and cohort 2, and found no association with OA. None of the phenotypic subgroups previously associated with mtDNA haplogroups were associated in this study.
Conclusions
We were not able to replicate previously published findings in the largest mtDNA association study to date. The evidence linking OA to mtDNA is not compelling at present.
doi:10.1136/annrheumdis-2012-201932
PMCID: PMC3551219  PMID: 22984172
Gene Polymorphism; Osteoarthritis; Pharmacogenetics

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