LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response.

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.

Identification of Transcripts with Shared Roles in the Pathogenesis of Postmenopausal Osteoporosis and Cardiovascular Disease.

Epidemiological evidence suggests existing comorbidity between postmenopausal osteoporosis (OP) and cardiovascular disease (CVD), but identification of possible shared genes is lacking. The skeletal global transcriptomes were analyzed in trans-iliac bone biopsies (n = 84) from clinically well-characterized postmenopausal women (50 to 86 years) without clinical CVD using microchips and RNA sequencing. One thousand transcripts highly correlated with areal bone mineral density (aBMD) were further analyzed using bioinformatics, and common genes overlapping with CVD and associated biological mechanisms, pathways and functions were identified. Fifty genes (45 mRNAs, 5 miRNAs) were discovered with established roles in oxidative stress, inflammatory response, endothelial function, fibrosis, dyslipidemia and osteoblastogenesis/calcification. These pleiotropic genes with possible CVD comorbidity functions were also present in transcriptomes of microvascular endothelial cells and cardiomyocytes and were differentially expressed between healthy and osteoporotic women with fragility fractures. The results were supported by a genetic pleiotropy-informed conditional False Discovery Rate approach identifying any overlap in single nucleotide polymorphisms (SNPs) within several genes encoding aBMD- and CVD-associated transcripts. The study provides transcriptional and genomic evidence for genes of importance for both BMD regulation and CVD risk in a large collection of postmenopausal bone biopsies. Most of the transcripts identified in the CVD risk categories have no previously recognized roles in OP pathogenesis and provide novel avenues for exploring the mechanistic basis for the biological association between CVD and OP.

Bone mineral density loci specific to the skull portray potential pleiotropic effects on craniosynostosis.

Skull bone mineral density (SK-BMD) provides a suitable trait for the discovery of key genes in bone biology, particularly to intramembranous ossification, not captured at other skeletal sites. We perform a genome-wide association meta-analysis (n ~ 43,800) of SK-BMD, identifying 59 loci, collectively explaining 12.5% of the trait variance. Association signals cluster within gene-sets involved in skeletal development and osteoporosis. Among the four novel loci (ZIC1, PRKAR1A, AZIN1/ATP6V1C1, GLRX3), there are factors implicated in intramembranous ossification and as we show, inherent to craniosynostosis processes. Functional follow-up in zebrafish confirms the importance of ZIC1 on cranial suture patterning. Likewise, we observe abnormal cranial bone initiation that culminates in ectopic sutures and reduced BMD in mosaic atp6v1c1 knockouts. Mosaic prkar1a knockouts present asymmetric bone growth and, conversely, elevated BMD. In light of this evidence linking SK-BMD loci to craniofacial abnormalities, our study provides new insight into the pathophysiology, diagnosis and treatment of skeletal diseases.

Lowering of Circulating Sclerostin May Increase Risk of Atherosclerosis and Its Risk Factors: Evidence From a Genome-Wide Association Meta-Analysis Followed by Mendelian Randomization.

OBJECTIVE: In this study, we aimed to establish the causal effects of lowering sclerostin, target of the antiosteoporosis drug romosozumab, on atherosclerosis and its risk factors. METHODS: A genome-wide association study meta-analysis was performed of circulating sclerostin levels in 33,961 European individuals. Mendelian randomization (MR) was used to predict the causal effects of sclerostin lowering on 15 atherosclerosis-related diseases and risk factors. RESULTS: We found that 18 conditionally independent variants were associated with circulating sclerostin. Of these, 1 cis signal in SOST and 3 trans signals in B4GALNT3, RIN3, and SERPINA1 regions showed directionally opposite signals for sclerostin levels and estimated bone mineral density. Variants with these 4 regions were selected as genetic instruments. MR using 5 correlated cis-SNPs suggested that lower sclerostin increased the risk of type 2 diabetes mellitus (DM) (odds ratio [OR] 1.32 [95% confidence interval (95% CI) 1.03-1.69]) and myocardial infarction (MI) (OR 1.35 [95% CI 1.01-1.79]); sclerostin lowering was also suggested to increase the extent of coronary artery calcification (CAC) (ß = 0.24 [95% CI 0.02-0.45]). MR using both cis and trans instruments suggested that lower sclerostin increased hypertension risk (OR 1.09 [95% CI 1.04-1.15]), but otherwise had attenuated effects. CONCLUSION: This study provides genetic evidence to suggest that lower levels of sclerostin may increase the risk of hypertension, type 2 DM, MI, and the extent of CAC. Taken together, these findings underscore the requirement for strategies to mitigate potential adverse effects of romosozumab treatment on atherosclerosis and its related risk factors.

Social stratification reflected in bone mineral density and stature: Spectral imaging and osteoarchaeological findings from medieval Norway.

This study presents skeletal material from five medieval burial sites in Eastern Norway, confined to one royal burial church, one Dominican monastery, and three burial sites representing parish populations. We combine osteological analysis and Dual Energy X-Ray Absorptiometry, studying the remains of 227 individuals (102 females and 125 males) employing young, middle, and old adult age categories. The aim is to assess bone mineral density as a skeletal indicator of socioeconomic status including stature as a variable. We detected that socioeconomic status significantly affected bone mineral density and stature. Individuals of high status had higher bone mineral density (0.07 g/cm2, p = 0.003) and taller stature (1.85 cm, p = 0.017) than individuals from the parish population. We detected no significant relationship between young adult bone mineral density and socioeconomic status (p = 0.127 and 0.059 for females and males, respectively). For males, high young adult bone mineral density and stature varied concordantly in both status groups. In contrast, females of high status were significantly taller than females in the parish population (p = 0.011). Our findings indicate quite different conditions during growth and puberty for the two groups of females. The age-related pattern of bone variation also portrayed quite different trajectories for the two socioeconomic status groups of both sexes. We discuss sociocultural practices (living conditions during childhood and puberty, as well as nutritional and lifestyle factors in adult life), possibly explaining the differences in bone mineral density between the high-status and parish population groups. The observation of greater differences in bone mineral density and stature for females than males in the medieval society of Norway is also further discussed.

Heavy-load exercise in older adults activates vasculogenesis and has a stronger impact on muscle gene expression than in young adults.

BACKGROUND: A striking effect of old age is the involuntary loss of muscle mass and strength leading to sarcopenia and reduced physiological functions. However, effects of heavy-load exercise in older adults on diseases and functions as predicted by changes in muscle gene expression have been inadequately studied. METHODS: Thigh muscle global transcriptional activity (transcriptome) was analyzed in cohorts of older and younger adults before and after 12-13 weeks heavy-load strength exercise using Affymetrix microarrays. Three age groups, similarly trained, were compared: younger adults (age 24 ± 4 years), older adults of average age 70 years (Oslo cohort) and above 80 years (old BSU cohort). To increase statistical strength, one of the older cohorts was used for validation. Ingenuity Pathway analysis (IPA) was used to identify predicted biological effects of a gene set that changed expression after exercise, and Principal Component Analysis (PCA) was used to visualize differences in muscle gene expressen between cohorts and individual participants as well as overall changes upon exercise. RESULTS: Younger adults, showed few transcriptome changes, but a marked, significant impact was observed in persons of average age 70 years and even more so in persons above 80 years. The 249 transcripts positively or negatively altered in both cohorts of older adults (q-value < 0.1) were submitted to gene set enrichment analysis using IPA. The transcripts predicted increase in several aspects of "vascularization and muscle contractions", whereas functions associated with negative health effects were reduced, e.g., "Glucose metabolism disorder" and "Disorder of blood pressure". Several genes that changed expression after intervention were confirmed at the genome level by containing single nucleotide variants associated with handgrip strength and muscle expression levels, e.g., CYP4B1 (p = 9.2E-20), NOTCH4 (p = 9.7E-8), and FZD4 (p = 5.3E-7). PCA of the 249 genes indicated a differential pattern of muscle gene expression in young and elderly. However, after exercise the expression patterns in both young and old BSU cohorts were changed in the same direction for the vast majority of participants. CONCLUSIONS: The positive impact of heavy-load strength training on the transcriptome increased markedly with age. The identified molecular changes translate to improved vascularization and muscular strength, suggesting highly beneficial health effects for older adults.

In silico discovery of blood cell macromolecular associations.

BACKGROUND: Physical molecular interactions are the basis of intracellular signalling and gene regulatory networks, and comprehensive, accessible databases are needed for their discovery. Highly correlated transcripts may reflect important functional associations, but identification of such associations from primary data are cumbersome. We have constructed and adapted a user-friendly web application to discover and identify putative macromolecular associations in human peripheral blood based on significant correlations at the transcriptional level. METHODS: The blood transcriptome was characterized by quantification of 17,328 RNA species, including 341 mature microRNAs in 105 clinically well-characterized postmenopausal women. Intercorrelation of detected transcripts signal levels generated a matrix with > 150 million correlations recognizing the human blood RNA interactome. The correlations with calculated adjusted p-values were made easily accessible by a novel web application. RESULTS: We found that significant transcript correlations within the giant matrix reflect experimentally documented interactions involving select ubiquitous blood relevant transcription factors (CREB1, GATA1, and the glucocorticoid receptor (GR, NR3C1)). Their responsive genes recapitulated up to 91% of these as significant correlations, and were replicated in an independent cohort of 1204 individual blood samples from the Framingham Heart Study. Furthermore, experimentally documented mRNAs/miRNA associations were also reproduced in the matrix, and their predicted functional co-expression described. The blood transcript web application is available at http://app.uio.no/med/klinmed/correlation-browser/blood/index.php and works on all commonly used internet browsers. CONCLUSIONS: Using in silico analyses and a novel web application, we found that correlated blood transcripts across 105 postmenopausal women reflected experimentally proven molecular associations. Furthermore, the associations were reproduced in a much larger and more heterogeneous cohort and should therefore be generally representative. The web application lends itself to be a useful hypothesis generating tool for identification of regulatory mechanisms in complex biological data sets.

Mechanical-Stress-Related Epigenetic Regulation of ZIC1 Transcription Factor in the Etiology of Postmenopausal Osteoporosis.

Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc finger of the cerebellum 1 (ZIC1) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between ZIC1 mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher ZIC1 expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between ZIC1 mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score). ZIC1 promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with ZIC1 promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.

Lipocalin 2 serum levels correlate with age and bone turnover biomarkers in healthy subjects but not in postmenopausal osteoporotic women.

PURPOSE: Lipocalin 2 (LCN2) is an adipokine involved in many physiological functions, including bone metabolism. We previously demonstrated its implication in mouse models of mechanical unloading-induced osteoporosis and in a cohort of bed rest volunteers. We therefore aimed at studying its involvement in postmenopausal osteoporosis. METHODS: We measured serum LCN2 and correlated its levels to Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1), Tartrate Resistant Acid Phosphatase 5B (TRAcP5B), sclerostin, urinary N-terminal telopeptide of type I collagen (NTX), serum C-terminal telopeptide of type I collagen (CTX), parathyroid hormone and vitamin K by ELISA performed in a cohort of younger (50-65 years) and older (66-90 years) osteoporotic women in comparison to healthy subjects. A cohort of male healthy and osteoarthritic patients was also included. Sobel mediation analysis was used to test indirect associations among age, LCN2 and DKK1 or NTX. RESULTS: LCN2 levels were unchanged in osteoporotic and in osteoarthritis patients when compared to healthy subjects and did not correlate with BMD. However, serum LCN2 correlated with age in healthy women (R = 0.44; P = 0.003) and men (R = 0.5; P = 0.001) and serum concentrations of DKK1 (R = 0.47; P = 0.003) and urinary NTX (R = 0.34; P = 0.04). Sobel mediation analysis showed that LCN2 mediates an indirect relationship between age and DKK1 (P = 0.02), but not with NTX, in healthy subjects. CONCLUSIONS: Taken together, the results suggest a hitherto unknown association between LCN2, DKK1 and age in healthy individuals, but not in postmenopausal osteoporotic women.

Postmenopausal osteoporosis is a musculoskeletal disease with a common genetic trait which responds to strength training: a translational intervention study.

BACKGROUND: Clinical evidence suggests that body muscle mass is positively associated with bone mass, of significance for the elderly population at risk of osteoporosis (OP). Furthermore, muscle and bone interact mechanically and functionally, via local interactions as well as remotely via secreted components. Thus, it was of interest to compare muscle transcriptomes in postmenopausal OP and healthy women, and study effects of strength training on the muscle transcriptome, muscle stress proteins and bone mineral density (BMD). METHODS: Skeletal muscle histological and genetic properties were compared in postmenopausal healthy (n = 18) and OP (n = 17) women before and after heavy-load strength training for 13-15 weeks. The cohorts were of similar age and body mass index without interfering diseases, medication or difference in lifestyle factors. Muscle biopsies obtained before and after intervention were studied histologically, and stress proteins and transcriptomes analyzed. RESULTS: The OP women showed distinct muscle transcription profiles when compared with healthy women and had higher levels of the stress proteins HSP70 and α-ß-crystalline. A set of 12 muscle transcripts, including ACSS3, FZD4, GNAI1 and IGF1, were differentially expressed before and after intervention (false discovery rate ⩽0.10, p ⩽0.001), and their corresponding bone transcripts were associated with BMD. Experimental data underline and describe the functionality of these genes in bone biology. OP women had 8% (p <0.01) higher proportion of type I fibres, but muscle fibre cross-sectional area did not differ. Muscle strength increased in both groups (p <0.01). CONCLUSIONS: Postmenopausal healthy and OP women have distinct muscle transcriptomes [messenger ribonucleic acids (mRNAs) and microRNAs] that are modulated by strength training, translating into key protein alterations and muscle fibre changes. The function of common skeletal muscle and bone genes in postmenopausal OP is suggestive of a shared disease trait.

Distinct Subsets of Noncoding RNAs Are Strongly Associated With BMD and Fracture, Studied in Weight-Bearing and Non-Weight-Bearing Human Bone.

We investigated mechanisms resulting in low bone mineral density (BMD) and susceptibility to fracture by comparing noncoding RNAs (ncRNAs) in biopsies of non-weight-bearing (NWB) iliac (n = 84) and weight bearing (WB) femoral (n = 18) postmenopausal bone across BMDs varying from normal (T-score > -1.0) to osteoporotic (T-score ≤ -2.5). Global bone ncRNA concentrations were determined by PCR and microchip analyses. Association with BMD or fracture, adjusted by age and body mass index, were calculated using linear and logistic regression and least absolute shrinkage and selection operator (Lasso) analysis. At 10% false discovery rate (FDR), 75 iliac bone ncRNAs and 94 femoral bone ncRNAs were associated with total hip BMD. Eight of the ncRNAs were common for the two sites, but five of them (miR-484, miR-328-3p, miR-27a-5p, miR-28-3p, and miR-409-3p) correlated positively to BMD in femoral bone, but negatively in iliac bone. Of predicted pathways recognized in bone metabolism, ECM-receptor interaction and proteoglycans in cancer emerged at both sites, whereas fatty acid metabolism and focal adhesion were only identified in iliac bone. Lasso analysis and cross-validations identified sets of nine bone ncRNAs correlating strongly with adjusted total hip BMD in both femoral and iliac bone. Twenty-eight iliac ncRNAs were associated with risk of fracture (FDR < 0.1). The small nucleolar RNAs, RNU44 and RNU48, have a function in stabilization of ribosomal RNAs (rRNAs), and their association with fracture and BMD suggest that aberrant processing of rRNAs may be involved in development of osteoporosis. Cis-eQTL (expressed quantitative trait loci) analysis of the iliac bone biopsies identified two loci associated with microRNAs (miRNAs), one previously identified in a heel-BMD genomewide association study (GWAS). In this comprehensive investigation of the skeletal genetic background in postmenopausal women, we identified functional bone ncRNAs associated to fracture and BMD, representing distinct subsets in WB and NWB skeletal sites. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Mendelian Randomization Analysis Reveals a Causal Influence of Circulating Sclerostin Levels on Bone Mineral Density and Fractures.

In bone, sclerostin is mainly osteocyte-derived and plays an important local role in adaptive responses to mechanical loading. Whether circulating levels of sclerostin also play a functional role is currently unclear, which we aimed to examine by two-sample Mendelian randomization (MR). A genetic instrument for circulating sclerostin, derived from a genomewide association study (GWAS) meta-analysis of serum sclerostin in 10,584 European-descent individuals, was examined in relation to femoral neck bone mineral density (BMD; n = 32,744) in GEFOS and estimated bone mineral density (eBMD) by heel ultrasound (n = 426,824) and fracture risk (n = 426,795) in UK Biobank. Our GWAS identified two novel serum sclerostin loci, B4GALNT3 (standard deviation [SD]) change in sclerostin per A allele (ß = 0.20, p = 4.6 × 10-49 ) and GALNT1 (ß = 0.11 per G allele, p = 4.4 × 10-11 ). B4GALNT3 is an N-acetyl-galactosaminyltransferase, adding a terminal LacdiNAc disaccharide to target glycocoproteins, found to be predominantly expressed in kidney, whereas GALNT1 is an enzyme causing mucin-type O-linked glycosylation. Using these two single-nucleotide polymorphisms (SNPs) as genetic instruments, MR revealed an inverse causal relationship between serum sclerostin and femoral neck BMD (ß = -0.12, 95% confidence interval [CI] -0.20 to -0.05) and eBMD (ß = -0.12, 95% CI -0.14 to -0.10), and a positive relationship with fracture risk (ß = 0.11, 95% CI 0.01 to 0.21). Colocalization analysis demonstrated common genetic signals within the B4GALNT3 locus for higher sclerostin, lower eBMD, and greater B4GALNT3 expression in arterial tissue (probability >99%). Our findings suggest that higher sclerostin levels are causally related to lower BMD and greater fracture risk. Hence, strategies for reducing circulating sclerostin, for example by targeting glycosylation enzymes as suggested by our GWAS results, may prove valuable in treating osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.

Meta-Analysis of Genomewide Association Studies Reveals Genetic Variants for Hip Bone Geometry.

Hip geometry is an important predictor of fracture. We performed a meta-analysis of GWAS studies in adults to identify genetic variants that are associated with proximal femur geometry phenotypes. We analyzed four phenotypes: (i) femoral neck length; (ii) neck-shaft angle; (iii) femoral neck width, and (iv) femoral neck section modulus, estimated from DXA scans using algorithms of hip structure analysis. In the Discovery stage, 10 cohort studies were included in the fixed-effect meta-analysis, with up to 18,719 men and women ages 16 to 93 years. Association analyses were performed with ∼2.5 million polymorphisms under an additive model adjusted for age, body mass index, and height. Replication analyses of meta-GWAS significant loci (at adjusted genomewide significance [GWS], threshold p ≤ 2.6 × 10-8 ) were performed in seven additional cohorts in silico. We looked up SNPs associated in our analysis, for association with height, bone mineral density (BMD), and fracture. In meta-analysis (combined Discovery and Replication stages), GWS associations were found at 5p15 (IRX1 and ADAMTS16); 5q35 near FGFR4; at 12p11 (in CCDC91); 11q13 (near LRP5 and PPP6R3 (rs7102273)). Several hip geometry signals overlapped with BMD, including LRP5 (chr. 11). Chr. 11 SNP rs7102273 was associated with any-type fracture (p = 7.5 × 10-5 ). We used bone transcriptome data and discovered several significant eQTLs, including rs7102273 and PPP6R3 expression (p = 0.0007), and rs6556301 (intergenic, chr.5 near FGFR4) and PDLIM7 expression (p = 0.005). In conclusion, we found associations between several genes and hip geometry measures that explained 12% to 22% of heritability at different sites. The results provide a defined set of genes related to biological pathways relevant to BMD and etiology of bone fragility. © 2019 American Society for Bone and Mineral Research.

Integrated analysis of DNA-methylation and gene expression using high-dimensional penalized regression: a cohort study on bone mineral density in postmenopausal women.

BACKGROUND: Using high-dimensional penalized regression we studied genome-wide DNA-methylation in bone biopsies of 80 postmenopausal women in relation to their bone mineral density (BMD). The women showed BMD varying from severely osteoporotic to normal. Global gene expression data from the same individuals was available, and since DNA-methylation often affects gene expression, the overall aim of this paper was to include both of these omics data sets into an integrated analysis. METHODS: The classical penalized regression uses one penalty, but we incorporated individual penalties for each of the DNA-methylation sites. These individual penalties were guided by the strength of association between DNA-methylations and gene transcript levels. DNA-methylations that were highly associated to one or more transcripts got lower penalties and were therefore favored compared to DNA-methylations showing less association to expression. Because of the complex pathways and interactions among genes, we investigated both the association between DNA-methylations and their corresponding cis gene, as well as the association between DNA-methylations and trans-located genes. Two integrating penalized methods were used: first, an adaptive group-regularized ridge regression, and secondly, variable selection was performed through a modified version of the weighted lasso. RESULTS: When information from gene expressions was integrated, predictive performance was considerably improved, in terms of predictive mean square error, compared to classical penalized regression without data integration. We found a 14.7% improvement in the ridge regression case and a 17% improvement for the lasso case. Our version of the weighted lasso with data integration found a list of 22 interesting methylation sites. Several corresponded to genes that are known to be important in bone formation. Using BMD as response and these 22 methylation sites as covariates, least square regression analyses resulted in R2=0.726, comparable to an average R2=0.438 for 10000 randomly selected groups of DNA-methylations with group size 22. CONCLUSIONS: Two recent types of penalized regression methods were adapted to integrate DNA-methylation and their association to gene expression in the analysis of bone mineral density. In both cases predictions clearly benefit from including the additional information on gene expressions.

Identification of a novel locus on chromosome 2q13, which predisposes to clinical vertebral fractures independently of bone density.

OBJECTIVES: To identify genetic determinants of susceptibility to clinical vertebral fractures, which is an important complication of osteoporosis. METHODS: Here we conduct a genome-wide association study in 1553 postmenopausal women with clinical vertebral fractures and 4340 controls, with a two-stage replication involving 1028 cases and 3762 controls. Potentially causal variants were identified using expression quantitative trait loci (eQTL) data from transiliac bone biopsies and bioinformatic studies. RESULTS: A locus tagged by rs10190845 was identified on chromosome 2q13, which was significantly associated with clinical vertebral fracture (P=1.04×10-9) with a large effect size (OR 1.74, 95% CI 1.06 to 2.6). Bioinformatic analysis of this locus identified several potentially functional SNPs that are associated with expression of the positional candidate genes TTL (tubulin tyrosine ligase) and SLC20A1 (solute carrier family 20 member 1). Three other suggestive loci were identified on chromosomes 1p31, 11q12 and 15q11. All these loci were novel and had not previously been associated with bone mineral density or clinical fractures. CONCLUSION: We have identified a novel genetic variant that is associated with clinical vertebral fractures by mechanisms that are independent of BMD. Further studies are now in progress to validate this association and evaluate the underlying mechanism.

Genetic Polymorphism of miR-196a-2 is Associated with Bone Mineral Density (BMD).

MicroRNAs (miRNAs) are small non-coding RNA molecules that post-transcriptionally regulate the translation of messenger RNAs. Given the crucial role of miRNAs in gene expression, genetic variants within miRNA-related sequences may affect miRNA function and contribute to disease risk. Osteoporosis is characterized by reduced bone mass, and bone mineral density (BMD) is a major diagnostic proxy to assess osteoporosis risk. Here, we aimed to identify miRNAs that are involved in BMD using data from recent genome-wide association studies (GWAS) on femoral neck, lumbar spine and forearm BMD. Of 242 miRNA-variants available in the GWAS data, we found rs11614913:C > T in the precursor miR-196a-2 to be significantly associated with femoral neck-BMD (p-value = 9.9 × 10-7, ß = -0.038) and lumbar spine-BMD (p-value = 3.2 × 10-11, ß = -0.061). Furthermore, our sensitivity analyses using the Rotterdam study data showed a sex-specific association of rs11614913 with BMD only in women. Subsequently, we highlighted a number of miR-196a-2 target genes, expressed in bone and associated with BMD, that may mediate the miRNA function in BMD. Collectively, our results suggest that miR-196a-2 may contribute to variations in BMD level. Further biological investigations will give more insights into the mechanisms by which miR-196a-2 control expression of BMD-related genes.

Bivariate genome-wide association meta-analysis of pediatric musculoskeletal traits reveals pleiotropic effects at the SREBF1/TOM1L2 locus.

Bone mineral density is known to be a heritable, polygenic trait whereas genetic variants contributing to lean mass variation remain largely unknown. We estimated the shared SNP heritability and performed a bivariate GWAS meta-analysis of total-body lean mass (TB-LM) and total-body less head bone mineral density (TBLH-BMD) regions in 10,414 children. The estimated SNP heritability is 43% (95% CI: 34-52%) for TBLH-BMD, and 39% (95% CI: 30-48%) for TB-LM, with a shared genetic component of 43% (95% CI: 29-56%). We identify variants with pleiotropic effects in eight loci, including seven established bone mineral density loci: WNT4, GALNT3, MEPE, CPED1/WNT16, TNFSF11, RIN3, and PPP6R3/LRP5. Variants in the TOM1L2/SREBF1 locus exert opposing effects TB-LM and TBLH-BMD, and have a stronger association with the former trait. We show that SREBF1 is expressed in murine and human osteoblasts, as well as in human muscle tissue. This is the first bivariate GWAS meta-analysis to demonstrate genetic factors with pleiotropic effects on bone mineral density and lean mass.Bone mineral density and lean skeletal mass are heritable traits. Here, Medina-Gomez and colleagues perform bivariate GWAS analyses of total body lean mass and bone mass density in children, and show genetic loci with pleiotropic effects on both traits.

Distinct DNA methylation profiles in bone and blood of osteoporotic and healthy postmenopausal women.

DNA methylation affects expression of associated genes and may contribute to the missing genetic effects from genome-wide association studies of osteoporosis. To improve insight into the mechanisms of postmenopausal osteoporosis, we combined transcript profiling with DNA methylation analyses in bone. RNA and DNA were isolated from 84 bone biopsies of postmenopausal donors varying markedly in bone mineral density (BMD). In all, 2529 CpGs in the top 100 genes most significantly associated with BMD were analyzed. The methylation levels at 63 CpGs differed significantly between healthy and osteoporotic women at 10% false discovery rate (FDR). Five of these CpGs at 5% FDR could explain 14% of BMD variation. To test whether blood DNA methylation reflect the situation in bone (as shown for other tissues), an independent cohort was selected and BMD association was demonstrated in blood for 13 of the 63 CpGs. Four transcripts representing inhibitors of bone metabolism-MEPE, SOST, WIF1, and DKK1-showed correlation to a high number of methylated CpGs, at 5% FDR. Our results link DNA methylation to the genetic influence modifying the skeleton, and the data suggest a complex interaction between CpG methylation and gene regulation. This is the first study in the hitherto largest number of postmenopausal women to demonstrate a strong association among bone CpG methylation, transcript levels, and BMD/fracture. This new insight may have implications for evaluation of osteoporosis stage and susceptibility.

Omics analysis of human bone to identify genes and molecular networks regulating skeletal remodeling in health and disease.

The skeleton is a metabolically active organ throughout life where specific bone cell activity and paracrine/endocrine factors regulate its morphogenesis and remodeling. In recent years, an increasing number of reports have used multi-omics technologies to characterize subsets of bone biological molecular networks. The skeleton is affected by primary and secondary disease, lifestyle and many drugs. Therefore, to obtain relevant and reliable data from well characterized patient and control cohorts are vital. Here we provide a brief overview of omics studies performed on human bone, of which our own studies performed on trans-iliacal bone biopsies from postmenopausal women with osteoporosis (OP) and healthy controls are among the first and largest. Most other studies have been performed on smaller groups of patients, undergoing hip replacement for osteoarthritis (OA) or fracture, and without healthy controls. The major findings emerging from the combined studies are: 1. Unstressed and stressed bone show profoundly different gene expression reflecting differences in bone turnover and remodeling and 2. Omics analyses comparing healthy/OP and control/OA cohorts reveal characteristic changes in transcriptomics, epigenomics (DNA methylation), proteomics and metabolomics. These studies, together with genome-wide association studies, in vitro observations and transgenic animal models have identified a number of genes and gene products that act via Wnt and other signaling systems and are highly associated to bone density and fracture. Future challenge is to understand the functional interactions between bone-related molecular networks and their significance in OP and OA pathogenesis, and also how the genomic architecture is affected in health and disease.

Novel Genetic Variants Associated With Increased Vertebral Volumetric BMD, Reduced Vertebral Fracture Risk, and Increased Expression of SLC1A3 and EPHB2.

Genome-wide association studies (GWASs) have revealed numerous loci for areal bone mineral density (aBMD). We completed the first GWAS meta-analysis (n = 15,275) of lumbar spine volumetric BMD (vBMD) measured by quantitative computed tomography (QCT), allowing for examination of the trabecular bone compartment. SNPs that were significantly associated with vBMD were also examined in two GWAS meta-analyses to determine associations with morphometric vertebral fracture (n = 21,701) and clinical vertebral fracture (n = 5893). Expression quantitative trait locus (eQTL) analyses of iliac crest biopsies were performed in 84 postmenopausal women, and murine osteoblast expression of genes implicated by eQTL or by proximity to vBMD-associated SNPs was examined. We identified significant vBMD associations with five loci, including: 1p36.12, containing WNT4 and ZBTB40; 8q24, containing TNFRSF11B; and 13q14, containing AKAP11 and TNFSF11. Two loci (5p13 and 1p36.12) also contained associations with radiographic and clinical vertebral fracture, respectively. In 5p13, rs2468531 (minor allele frequency [MAF] = 3%) was associated with higher vBMD (ß = 0.22, p = 1.9 × 10-8 ) and decreased risk of radiographic vertebral fracture (odds ratio [OR] = 0.75; false discovery rate [FDR] p = 0.01). In 1p36.12, rs12742784 (MAF = 21%) was associated with higher vBMD (ß = 0.09, p = 1.2 × 10-10 ) and decreased risk of clinical vertebral fracture (OR = 0.82; FDR p = 7.4 × 10-4 ). Both SNPs are noncoding and were associated with increased mRNA expression levels in human bone biopsies: rs2468531 with SLC1A3 (ß = 0.28, FDR p = 0.01, involved in glutamate signaling and osteogenic response to mechanical loading) and rs12742784 with EPHB2 (ß = 0.12, FDR p = 1.7 × 10-3 , functions in bone-related ephrin signaling). Both genes are expressed in murine osteoblasts. This is the first study to link SLC1A3 and EPHB2 to clinically relevant vertebral osteoporosis phenotypes. These results may help elucidate vertebral bone biology and novel approaches to reducing vertebral fracture incidence. © 2016 American Society for Bone and Mineral Research.