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 BMD loci that reached genome-wide significance (GWS; P < 5 x 10(-8)), of which 13 map to regions not previously associated with this trait: 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 meta-analysis 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 many SNPs associated with BMD map to genes in signaling pathways with relevance to bone metabolism and highlight the complex genetic architecture that underlies osteoporosis and variation in BMD.

BACKGROUND

Osteoporosis is diagnosed by the measurement of bone mineral density, which is a highly heritable and multifactorial trait. We aimed to identify genetic loci that are associated with bone mineral density.

METHODS

In this genome-wide association study, we identified the most promising of 314 075 single nucleotide polymorphisms (SNPs) in 2094 women in a UK study. We then tested these SNPs for replication in 6463 people from three other cohorts in western Europe. We also investigated allelic expression in lymphoblast cell lines. We tested the association between the replicated SNPs and osteoporotic fractures with data from two studies.

RESULTS

We identified genome-wide evidence for an association between bone mineral density and two SNPs (p<5x10(-8)). The SNPs were rs4355801, on chromosome 8, near to the TNFRSF11B (osteoprotegerin) gene, and rs3736228, on chromosome 11 in the LRP5 (lipoprotein-receptor-related protein) gene. A non-synonymous SNP in the LRP5 gene was associated with decreased bone mineral density (rs3736228, p=6.3x10(-12) for lumbar spine and p=1.9x10(-4) for femoral neck) and an increased risk of both osteoporotic fractures (odds ratio [OR] 1.3, 95% CI 1.09-1.52, p=0.002) and osteoporosis (OR 1.3, 1.08-1.63, p=0.008). Three SNPs near the TNFRSF11B gene were associated with decreased bone mineral density (top SNP, rs4355801: p=7.6x10(-10) for lumbar spine and p=3.3x10(-8) for femoral neck) and increased risk of osteoporosis (OR 1.2, 95% CI 1.01-1.42, p=0.038). For carriers of the risk allele at rs4355801, expression of TNFRSF11B in lymphoblast cell lines was halved (p=3.0x10(-6)). 1883 (22%) of 8557 people were at least heterozygous for these risk alleles, and these alleles had a cumulative association with bone mineral density (trend p=2.3x10(-17)). The presence of both risk alleles increased the risk of osteoporotic fractures (OR 1.3, 1.08-1.63, p=0.006) and this effect was independent of bone mineral density.

CONCLUSIONS

Two gene variants of key biological proteins increase the risk of osteoporosis and osteoporotic fracture. The combined effect of these risk alleles on fractures is similar to that of most well-replicated environmental risk factors, and they are present in more than one in five white people, suggesting a potential role in screening.

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.

METHODS

Large-scale meta-analysis of genome-wide association data.

METHODS

5 international, multicenter, population-based studies.

METHODS

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.

METHODS

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.

CONCLUSIONS

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.

CONCLUSIONS

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.

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.

BACKGROUND

Juvenile Paget's disease, an autosomal recessive osteopathy, is characterized by rapidly remodeling woven bone, osteopenia, fractures, and progressive skeletal deformity. The molecular basis is not known. Osteoprotegerin deficiency could explain juvenile Paget's disease because osteoprotegerin suppresses bone turnover by functioning as a decoy receptor for osteoclast differentiation factor (also called RANK ligand).

METHODS

We evaluated two apparently unrelated Navajo patients with juvenile Paget's disease for defects in the gene encoding osteoprotegerin (TNFRSF11B) using polymerase-chain-reaction (PCR) amplification followed by direct sequencing and Southern blotting of genomic DNA. Genetic markers near TNFRSF11B were evaluated by both a PCR method that involved sequence-tagged site-content mapping of a deletion of TNFRSF11B and PCR spanning the DNA break points.

RESULTS

Both patients had a homozygous deletion of TNFRSF11B, with identical break points, on chromosome 8q24.2. The defect spans approximately 100 kb, but neighboring genes are intact. We found that serum levels of osteoprotegerin and soluble osteoclast differentiation factor were undetectable and markedly increased, respectively.

CONCLUSIONS

Juvenile Paget's disease can result from osteoprotegerin deficiency caused by homozygous deletion of TNFRSF11B.

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10(-8)) level: 14q24.2 (rs227425, P-value 3.98 × 10(-13), SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10(-9), CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n = 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis.

As the TNF and TNFR superfamilies have grown to more than two dozen combined members over the past 30 years, their involvement in interactions between immune cells, with regard to the events governing cellular differentiation, activation, and survival have been well established. The recently identified TNF superfamily cytokine, TRANCE (RANKL/OPGL/ODF/TNFSF11), which interacts with two receptors-one functional, TRANCE-R (RANK/TNFRSF11A), and one decoy, OPG (TNFRSF11B)-is a survival factor for activated dendritic cells, and may also be important for the maintenance of immune tolerance. TRANCE is also the key cytokine involved in osteoclast differentiation and activation, making TRANCE signaling crucial for proper bone homeostasis, and a potential therapeutic target in diseases such as osteoporosis, osteolytic metastatic cancer, arthritis, and periodontitis. Importantly, the positive role that TRANCE has in activating the immune system, appears to significantly contribute to pathologic bone loss. These observations have spurred intense study of the various ways in which the immune system can influence bone. Furthermore, TRANCE has also been demonstrated to play essential roles in the developmental processes leading to both lymph node formation, and the expansion and function of mammary glands during pregnancy and lactation. Thus, TRANCE is quickly emerging as a cytokine of significant importance to further understanding unique aspects of mammalian biology.

The osteoclast is the cell that resorbs bone. It has been known for many years that its formation and function are regulated by cells of the osteoblastic lineage. Recently the molecular basis for this regulation was identified; osteoblastic cells induce osteoclastic differentiation and resorptive activity through expression of tumour necrosis factor (TNF) activation-induced cytokine (TRANCE) (also known as RANKL, ODF, OPGL, and TNFSF11), a novel membrane-inserted member of the TNF superfamily. Osteoclastic regulation is assisted through secretion of an inhibitor, osteoprotegerin (OPG) (OCIF, TNFRSF11B), a soluble (decoy) receptor for TRANCE. Osteoclast formation and survival also depend on and are substantially enhanced by transforming growth factor-beta (TGF-beta), which is abundant in bone matrix. Surprisingly, not only TRANCE but also TNF-alpha can induce osteoclast formation in vitro from bone marrow-derived mononuclear phagocytes, especially in the presence of TGF-beta. Whether or not TNF-alpha does the same in vivo, its ability to generate osteoclasts in vitro has significant implications regarding the nature of osteoclasts and their relationship to other mononuclear phagocytes, and a possible wider role for TRANCE in macrophage pathobiology. A hypothesis is presented in which the osteoclast is a mononuclear phagocyte directed towards a debriding function by TGF-beta, activated for this function by TRANCE, and induced to become specifically osteoclastic by the characteristics of the substrate or signals from bone cells that betoken such characteristics.

In multiple myeloma, an overabundance of monoclonal plasma cells in the bone marrow induces localized osteolytic lesions that rarely heal due to increased bone resorption and suppressed bone formation. Matrix-embedded osteocytes comprise more than 95% of bone cells and are major regulators of osteoclast and osteoblast activity, but their contribution to multiple myeloma growth and bone disease is unknown. Here, we report that osteocytes in a mouse model of human MM physically interact with multiple myeloma cells in vivo, undergo caspase-3-dependent apoptosis, and express higher RANKL (TNFSF11) and sclerostin levels than osteocytes in control mice. Mechanistic studies revealed that osteocyte apoptosis was initiated by multiple myeloma cell-mediated activation of Notch signaling and was further amplified by multiple myeloma cell-secreted TNF. The induction of apoptosis increased osteocytic Rankl expression, the osteocytic Rankl/Opg (TNFRSF11B) ratio, and the ability of osteocytes to attract osteoclast precursors to induce local bone resorption. Furthermore, osteocytes in contact with multiple myeloma cells expressed high levels of Sost/sclerostin, leading to a reduction in Wnt signaling and subsequent inhibition of osteoblast differentiation. Importantly, direct contact between osteocytes and multiple myeloma cells reciprocally activated Notch signaling and increased Notch receptor expression, particularly Notch3 and 4, stimulating multiple myeloma cell growth. These studies reveal a previously unknown role for bidirectional Notch signaling that enhances MM growth and bone disease, suggesting that targeting osteocyte-multiple myeloma cell interactions through specific Notch receptor blockade may represent a promising treatment strategy in multiple myeloma.

Runx2 is a transcription factor that is essential for osteoblast differentiation and chondrocyte maturation. Ihh, expressed in prehypertrophic and hypertrophic chondrocytes, is required for the specification of Runx2+ osteoprogenitors in endochondral bone development. Runx2 induces Sp7, an essential transcription factor for osteoblast differentiation. Canonical Wnt signaling is also required for osteoblast differentiation. Runx2+ osteoprogenitors retain the capacity to differentiate into chondrocytes, and Sp7 and canonical Wnt signaling direct cells to osteoblasts, thereby inhibiting chondrocyte differentiation. The function of Runx2 after the commitment to osteoblasts remains controversial. Runx3 has a redundant function with Runx2 in chondrocyte maturation. Runx2 regulates the expression of Ihh, Col10a1, Spp1, Ibsp, Mmp13, and Vegfa in the respective layers in growth plates. Runx2 enhances chondrocyte proliferation through the induction of Ihh. Ihh induces Pthlh, which inhibits Runx2 and chondrocyte maturation, forming a negative feedback loop for chondrocyte maturation. Runx2 is one of the genes responsible for the pathogenesis of osteoarthritis (OA) because RUNX2 is up-regulated in chondrocytes in OA cartilage and a germline haplodeficiency or deletion of Runx2 in articular chondrocytes decelerates OA progression. Runx2 plays an important role in the bone metastasis of breast and prostate cancers by up-regulating Spp1, Ibsp, Mmp9, Mmp13, Vegfa, Tnfsf11, and Ihh expression and down-regulating Tnfrsf11b expression. Cbfb forms a heterodimer with Runx2 and is required for the efficient DNA binding of Runx2. Cbfb stabilizes Runx proteins at different levels among Runx family proteins by inhibiting their ubiquitination-mediated degradation. Cbfb plays more important roles in endochondral ossification than in intramembranous ossification.

Idiopathic hyperphosphatasia is an autosomal recessive bone disease characterized by deformities of long bones, kyphosis and acetabular protrusion, increasing in severity as affected children pass through adolescence. Biochemical and histological evidence indicate that there is extremely rapid bone turnover, with indices of both bone resorption and formation greatly increased. A genome-wide search, in a family with three children affected by idiopathic hyperphosphatasia, suggested linkage to a locus on the long arm of chromosome 8 (8q24). The gene TNFRSF11B encoding osteoprotegerin (OPG), which lies within this locus, was an obvious candidate, given the critical role of OPG in regulating osteoclast development. All three affected siblings were homozygous for a 3 bp inframe deletion in exon 3 of the TNFRSF11B gene, resulting in the loss of an aspartate residue. Their parents (who were first cousins) were heterozygous for the mutation. Recombinant wild-type and mutant OPG cDNAs were expressed in human epithelial kidney cells, and secreted OPG was collected from the conditioned medium. In vitro measurements of bone resorption showed that wild-type OPG suppressed bone resorption, whereas the mutant form did not, confirming this to be an inactivating mutation. This description of abnormal OPG function in humans expands the spectrum of genetic bone diseases arising from perturbations of the OPG/RANK-L/RANK system that regulates osteoclastogenesis.

Most genome-wide association (GWA) studies have focused on populations of European ancestry with limited assessment of the influence of the sequence variants on populations of other ethnicities. To determine whether markers that we have recently shown to associate with Bone Mineral Density (BMD) in Europeans also associate with BMD in East-Asians we analysed 50 markers from 23 genomic loci in samples from Korea (n = 1,397) and two Chinese Hong Kong sample sets (n = 3,869 and n = 785). Through this effort we identified fourteen loci that associated with BMD in East-Asian samples using a false discovery rate (FDR) of 0.05; 1p36 (ZBTB40, P = 4.3×10(-9)), 1p31 (GPR177, P = 0.00012), 3p22 (CTNNB1, P = 0.00013), 4q22 (MEPE, P = 0.0026), 5q14 (MEF2C, P = 1.3×10(-5)), 6q25 (ESR1, P = 0.0011), 7p14 (STARD3NL, P = 0.00025), 7q21 (FLJ42280, P = 0.00017), 8q24 (TNFRSF11B, P = 3.4×10(-5)), 11p15 (SOX6, P = 0.00033), 11q13 (LRP5, P = 0.0033), 13q14 (TNFSF11, P = 7.5×10(-5)), 16q24 (FOXL1, P = 0.0010) and 17q21 (SOST, P = 0.015). Our study marks an early effort towards the challenge of cataloguing bone density variants shared by many ethnicities by testing BMD variants that have been established in Europeans, in East-Asians.

A family history of hip fracture carries a twofold increased risk of fracture among descendants. Genetic factors indeed play a major role in the determination of bone mineral density (BMD) and osteoporosis risk. Multiple chromosomal loci have been mapped by linkage approaches which potentially carry hundreds of genes involved in the determination of bone mass and quality. Association studies based on candidate gene polymorphisms and subsequent meta-analyses, and the more recent genome-wide association studies (GWAS), have clearly identified a handful of genes associated with BMD and/or fragility fractures. Among them are genes coding for the LDL-receptor related protein 5 (LRP5), estrogen receptor alpha (ESR1) and osteoprotegerin, OPG (TNFRSf11b). However, the percentage of osteoporosis risk explained by any of these polymorphisms is small, indicating that most genetic risk factors remain to be discovered and/or that interaction with environmental factors needs further consideration.

In order to assess the contribution of polymorphisms in the RANKL (TNFSF11), RANK (TNFRSF11A) and OPG (TNFRSF11B) genes to variations in bone mineral density (BMD), a population-based cohort with 1,120 extreme low hip BMD cases or extreme high hip BMD controls was genotyped on five SNPs. We further explored the associations between these genetic variations and forearm BMDs by genotyping 266 offspring and 309 available parents from 160 nuclear families. A family-based association test was used. Significantly positive associations were found for A163G polymorphisms in the promoter regions of the OPG gene, a missense substitution in exon 7 (Ala192Val) of the RANK gene and rs9594782 SNP in the 5' UTR of the RANKL gene with BMD in men only. Men with TC/CC genotypes of the rs9594782 SNP had a 2.1 times higher risk of extremely low hip BMD (P = 0.004), and lower whole body BMD (P < 0.001). Subjects with the TC genotype of the Ala192Val polymorphism had a 40% reduced risk of having extremely low hip BMD (P < 0.01), and higher whole body BMD (P < 0.01). Subjects with the GG genotype of the A163G polymorphism had a 70% reduced risk of having extremely low hip BMD (P < 0.05), and higher whole body BMD (P < 0.01). Significant gene-gene interactions were also observed among the OPG, RANK and RANKL genes. Our findings suggest that genetic variation in genes involved in the RANKL/RANK/OPG bone remodeling pathway are strongly associated with BMD at different skeletal sites in adult men, but not in women.

Expression quantitative trait loci (eQTL) studies have established convincing relationships between genetic variants and gene expression. Most of these studies focused on the mean of gene expression level, but not the variance of gene expression level (i.e., gene expression variability). In the present study, we systematically explore genome-wide association between genetic variants and gene expression variability in humans. We adapt the double generalized linear model (dglm) to simultaneously fit the means and the variances of gene expression among the three possible genotypes of a biallelic SNP. The genomic loci showing significant association between the variances of gene expression and the genotypes are termed expression variability QTL (evQTL). Using a data set of gene expression in lymphoblastoid cell lines (LCLs) derived from 210 HapMap individuals, we identify cis-acting evQTL involving 218 distinct genes, among which 8 genes, ADCY1, CTNNA2, DAAM2, FERMT2, IL6, PLOD2, SNX7, and TNFRSF11B, are cross-validated using an extra expression data set of the same LCLs. We also identify ∼300 trans-acting evQTL between >13,000 common SNPs and 500 randomly selected representative genes. We employ two distinct scenarios, emphasizing single-SNP and multiple-SNP effects on expression variability, to explain the formation of evQTL. We argue that detecting evQTL may represent a novel method for effectively screening for genetic interactions, especially when the multiple-SNP influence on expression variability is implied. The implication of our results for revealing genetic mechanisms of gene expression variability is discussed.

Cathepsin K and MMP-9 are considered to be the most abundant proteases in osteoclasts. TRAP is a marker for osteoclasts, and there is increasing evidence of its proteolytic role in bone resorption. RANKL is a recently discovered regulator of osteoclast maturation and activity and induces expression of many genes. This study compared cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin gene expression in the proximal femur of patients with osteoarthritis with that of patients with femoral neck fracture. Fifty-six patients undergoing arthroplasty because of osteoarthritis or femoral neck fracture were included in the study. Total mRNA was extracted from the bone samples obtained from the intertrochanteric region of the proximal femur. Real-time RT-PCR was used to quantify CTSK (cathepsin K), MMP-9 (matrix metalloproteinase 9), ACP5 (TRAP), TNFSF11 (RANKL), TNFRSF11B (OPG), and BGLAP (osteocalcin) mRNAs. The levels of mRNAs coding for MMP-9 and osteocalcin indicated higher expression in the osteoarthritic group (P = 0.011, P = 0.001, respectively), whereas RANKL expression and the ratio RANKL/OPG were both significantly lower in the osteoarthritic group than in the fracture group. Expression of cathepsin K, MMP-9, and TRAP relative to RANKL was significantly higher in the osteoarthritic group. Ratios of all three proteolytic enzymes relative to formation marker osteocalcin were higher in the fracture group. Gene expression of cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin and the association between their mRNA levels pointed to higher bone resorption and bone formation in osteoarthritis, differences in balance between them, and differences in regulation of bone resorption in osteoarthritic and osteoporotic bone.

BACKGROUND

The primary goals of genome-wide association studies (GWAS) are to discover new molecular and biological pathways involved in the regulation of bone metabolism that can be leveraged for drug development. In addition, the identified genetic determinants may be used to enhance current risk factor profiles.

METHODS

There have been more than 40 published GWAS on skeletal phenotypes, predominantly focused on dual-energy x-ray absorptiometry-derived bone mineral density (BMD) of the hip and spine.

RESULTS

Sixty-six BMD loci have been replicated across all the published GWAS, confirming the highly polygenic nature of BMD variation. Only seven of the 66 previously reported genes (LRP5, SOST, ESR1, TNFRSF11B, TNFRSF11A, TNFSF11, PTH) from candidate gene association studies have been confirmed by GWAS. Among 59 novel BMD GWAS loci that have not been reported by previous candidate gene association studies, some have been shown to be involved in key biological pathways involving the skeleton, particularly Wnt signaling (AXIN1, LRP5, CTNNB1, DKK1, FOXC2, HOXC6, LRP4, MEF2C, PTHLH, RSPO3, SFRP4, TGFBR3, WLS, WNT3, WNT4, WNT5B, WNT16), bone development: ossification (CLCN7, CSF1, MEF2C, MEPE, PKDCC, PTHLH, RUNX2, SOX6, SOX9, SPP1, SP7), mesenchymal-stem-cell differentiation (FAM3C, MEF2C, RUNX2, SOX4, SOX9, SP7), osteoclast differentiation (JAG1, RUNX2), and TGF-signaling (FOXL1, SPTBN1, TGFBR3). There are still 30 BMD GWAS loci without prior molecular or biological evidence of their involvement in skeletal phenotypes. Other skeletal phenotypes that either have been or are being studied include hip geometry, bone ultrasound, quantitative computed tomography, high-resolution peripheral quantitative computed tomography, biochemical markers, and fractures such as vertebral, nonvertebral, hip, and forearm.

CONCLUSIONS

Although several challenges lie ahead as GWAS moves into the next generation, there are prospects of new discoveries in skeletal biology. This review integrates findings from previous GWAS and provides a roadmap for future directions building on current GWAS successes.

OBJECTIVE

Identify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process.

METHODS

Genome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions.

RESULTS

Among the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. TNFRSF11B and FRZB). Also several inflammatory genes such as CD55, PTGES and TNFAIP6, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of NGF in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score) but were independent of joint-site or sex.

CONCLUSIONS

We provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways.

The high mechanical mismatch between stiffness of silicon and metal microelectrodes and soft cortical tissue, induces strain at the neural interface which likely contributes to failure of the neural interface. However, little is known about the molecular outcomes of electrode induced low-magnitude strain (1-5%) on primary astrocytes, microglia and neurons. In this study we simulated brain micromotion at the electrode-brain interface by subjecting astrocytes, microglia and primary cortical neurons to low-magnitude cyclical strain using a biaxial stretch device, and investigated the molecular outcomes of induced strain in vitro. In addition, we explored the functional consequence of astrocytic and microglial strain on neural health, when they are themselves subjected to strain. Quantitative real-time PCR array (qRT-PCR Array) analysis of stretched astrocytes and microglia showed strain specific upregulation of an Interleukin receptor antagonist - IL-36Ra (previously IL-1F5), to ≈ 1018 and ≈ 236 fold respectively. Further, IL-36Ra gene expression remained unchanged in astrocytes and microglia treated with bacterial lipopolysaccharide (LPS) indicating that the observed upregulation in stretched astrocytes and microglia is potentially strain specific. Zymogram and western blot analysis revealed that mechanically strained astrocytes and microglia upregulated matrix metalloproteinases (MMPs) 2 and 9, and other markers of reactive gliosis such as glial fibrillary acidic protein (GFAP) and neurocan when compared to controls. Primary cortical neurons when stretched with and without IL-36Ra, showed a ≈ 400 fold downregulation of tumor necrosis factor receptor superfamily, member 11b (TNFRSF11b). Significant upregulation of members of the caspase cysteine proteinase family and other pro-apoptotic genes was also observed in the presence of IL-36Ra than in the absence of IL-36Ra. Adult rats when implanted with microwire electrodes showed upregulation of IL-36Ra (≈ 20 fold) and IL-1Ra (≈ 1500 fold) 3 days post-implantation (3 DPI), corroborating in vitro results, although these transcripts were drastically down regulated by ≈ 20 fold and ≈ 1488 fold relative to expression levels 3 DPI, at the end of 12 weeks post-implantation (12 WPI). These results demonstrate that IL receptor antagonists may be negatively contributing to neuronal health at acute time-points post-electrode implantation.

Inflammatory cross talk between perivascular adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We previously reported that human perivascular (PV) adipocytes exhibit a proinflammatory phenotype and less adipogenic differentiation than do subcutaneous (SQ) adipocytes. To gain a global view of the genomic basis of biologic differences between PV and SQ adipocytes, we performed genome-wide expression analyses to identify differentially expressed genes between adipocytes derived from human SQ vs. PV adipose tissues. Although >90% of well-expressed genes were similarly regulated, we identified a signature of 307 differentially expressed genes that were highly enriched for functions associated with the regulation of angiogenesis, vascular morphology, inflammation, and blood clotting. Of the 156 PV upregulated genes, 59 associate with angiogenesis, vascular biology, or inflammation, noteworthy of which include TNFRSF11B (osteoprotegerin), PLAT, TGFB1, THBS2, HIF1A, GATA6, and SERPINE1. Of 166 PV downregulated genes, 21 associated with vascular biology and inflammation, including ANGPT1, ANGPTL1, and VEGFC. Consistent with the emergent hypothesis that PV adipocytes differentially regulate angiogenesis and inflammation, cell culture-derived adipocyte-conditioned media from PV adipocytes strongly enhanced endothelial cell tubulogenesis and monocyte migration compared with media from SQ adipocytes. These findings demonstrate that PV adipocytes have the potential to significantly modulate vascular inflammatory crosstalk in the setting of atherosclerosis by their ability to signal to both endothelial and inflammatory cells.

OBJECTIVE

Identification of the genes responsible for systemic lupus erythematosus (SLE).

METHODS

All the exons and putative promoter regions of 53 candidate genes (TNFRSF6/Fas, TNFSF6/FasL, Fli1, TNFSF10/TRAIL, TNFSF12/TWEAK, Bcl-2, PTEN, FADD, TRADD, CDKN1A, TNFRSF1A/TNFR1, TNFRSF4/OX40, TNFSF4/OX40L, TNFSF5/CD40L, TNFSF13B/BAFF, ICOS, CTLA4, CD28, FYN, G2A, CR2, PTPRC/CD45, CD22, CD19, Lyn, PDCD1, PTPN6, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, CD3Z, DNASE1, APCS, MERTK, C3, C1QA, C1QB, C1QG, C2, MBL2, IGHM, IL-2, IL-4, IL-10, IFNG, TNFA, MAN2A1, TNFRSF11A/RANK, TNFRSF11B/OPG, TNFSF11/OPGL) were screened for single nucleotide polymorphisms (SNPs) and their association with SLE was assessed by case-control studies. A total of 509 cases and 964 controls of Japanese descent were enrolled.

RESULTS

A total of 316 SNPs was identified. When analysed in the Japanese population, the allele frequencies of T at rs7951 and G at rs2230201 of the C3 gene were 0.110 and 0.626, respectively, in SLE patients; significantly higher than the frequencies of 0.081 and 0.584, respectively, in controls [odds ratio (OR) = 1.40, 95% confidence interval (CI) = 1.05-1.86, P = 0.016 and OR=1.19, 95% CI = 1.01-1.41, P = 0.038, respectively]. The mean serum C3 level of carriers of the rs7951 T allele was significantly lower than that of non-carriers of the T allele in 87 SLE patients whose medical records were available (P = 0.0018).

CONCLUSIONS

rs7951 T allele of the C3 gene was significantly associated with SLE, and decreased serum level of C3 seems to be correlated with this allele.

Homozygous mutations in TNFRSF11B, the gene encoding osteoprotegerin, were found in affected members from six of nine families with idiopathic hyperphosphatasia. The severity of the phenotype was related to the predicted effects of the mutations on osteoprotegerin function.

BACKGROUND

Idiopathic hyperphosphatasia (IH) is a rare high bone turnover congenital bone disease in which affected children are normal at birth but develop progressive long bone deformities, fractures, vertebral collapse, skull enlargement, and deafness. There is, however, considerable phenotypic variation from presentation in infancy with severe progressive deformity through to presentation in late childhood with minimal deformity. Two recent reports have linked idiopathic hyperphosphatasia with deletion of, or mutation in, the TNFRSF11B gene that encodes osteoprotegerin (OPG), an important paracrine modulator of RANKL-mediated bone resorption.

METHODS

We studied subjects with a clinical diagnosis of IH and unaffected family members from nine unrelated families. Clinical, biochemical, and radiographic data were collected, and genomic DNA examined for mutations in TNFRSF11B. The relationship between the mutations, their predicted effects on OPG function, and the phenotype were then examined.

RESULTS

Of the nine families studied, affected subjects from six were homozygous for novel mutations in TNFRSF11B. Their parents were heterozygous, consistent with autosomal recessive inheritance. Four of the six mutations occurred in the cysteine-rich ligand-binding domain and are predicted to disrupt binding of OPG to RANKL. Missense mutations in the cysteine residues, predicted to cause major disruption to the ligand-binding region, were associated with a severe phenotype (deformity developing before 18 months age and severe disability), as was a large deletion mutation. Non-cysteine missense mutations in the ligand-binding domain were associated with an intermediate phenotype (deformity recognized around the age of 5 years and an increased rate of long bone fracture). An insertion/deletion mutation at the C-terminal end of the protein was associated with the mildest phenotype.

CONCLUSIONS

Mutations in TNFRSF11B account for the majority of, but not all, cases of IH, and there are distinct genotype-phenotype relationships.

High mammographic density is associated with a increased risk of breast cancer. We hypothesized that specific pathways exist that are associated with increased mammographic density, and may therefore be used to identify potential targets for chemoprevention. Histologically confirmed normal breast tissue was collected from women undergoing breast surgery who had available demographic data and mammograms for review. Women with low versus high mammographic breast density were compared. Differentially expressed genes using Affymetrix HG U133Plus2 chips were identified in dense versus non-dense tissue. Immunohistochemical analysis (IHC) of estrogen receptor, progesterone receptor, Ki67, and COX2 expression was performed. About 66 women were identified, 28 (42%) had high, and 38 (58%) had low mammographic density. About 73 genes had differential expression between normal breast tissue with high and low mammographic density (P < 0.001, fold change>> or = 1.5 with a low false discovery rate (<10%). Network and canonical pathway analysis indicated decreased TGFbeta signaling (TGFBR2, SOS, SMAD3, CD44 and TNFRSF11B) in dense breast tissue relative to non-dense breast. By IHC, only COX2 expression in the stroma was statistically significant on multivariate analysis. TGFbeta ligands are currently the only growth factors known to prevent mammary epithelial cell proliferation. TGFbeta signaling has been reported to be inhibited by COX-2, and these molecules are highly differentially expressed in individuals at high risk of developing breast cancer. These results strongly suggest that COX2 inhibition should be investigated for breast cancer prevention despite possible increase in cardiovascular risk.

Wnt/beta-catenin signaling is normally involved in embryonic development and tissue homeostasis, and its misregulation leads to several forms of cancer. We have reported that misregulated Wnt/beta-catenin signaling occurs in ovarian granulosa cell tumors (GCT) and have created the Catnb(flox(ex3)/+);Amhr2(cre/+) mouse model, which expresses a dominant-stable mutant of beta-catenin in granulosa cells and develops late-onset GCT. To study the mechanisms leading to GCT development, gene expression analysis was done using microarrays comparing Catnb(flox(ex3)/+);Amhr2(cre/+) ovaries bearing pretumoral lesions with control ovaries. Overexpressed genes identified in Catnb(flox(ex3)/+);Amhr2(cre/+) ovaries included the Wnt/beta-catenin signaling antagonists Wif1, Nkd1, Dkk4, and Axin2, consistent with the induction of negative feedback loops that counteract uncontrolled Wnt/beta-catenin signaling. Expression of the antagonists was localized to cells forming the pretumoral lesions but not to normal granulosa cells. Microarray analyses also revealed the ectopic expression of bone markers, including Ibsp, Cdkn1c, Bmp4, and Tnfrsf11b, as well as neuronal/neurosecretory cell markers, such as Cck, Amph, Pitx1, and Sp5. Increased expression of the gene encoding the cytokine pleiotrophin was also found in Catnb(flox(ex3)/+);Amhr2(cre/+) ovaries and GCT but was not associated with increased serum pleiotrophin levels. In situ hybridization analyses using GCT from Catnb(flox(ex3)/+);Amhr2(cre/+) mice revealed that Wnt/beta-catenin antagonists and neuronal markers localized to a particular cell population, whereas the bone markers localized to a distinct cell type associated with areas of osseous metaplasia. Together, these results suggest that misregulated Wnt/beta-catenin signaling alters the fate of granulosa cells and that the GCT that arise in Catnb(flox(ex3)/+);Amhr2(cre/+) mice result from the clonal expansion of metaplastic cells.