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.

Background-Vertebroplasty is used commonly to treat painful, osteoporotic vertebral compression fractures.

Uric acid is the end product of purine metabolism in humans and great apes, which have lost hepatic uricase activity, leading to uniquely high serum uric acid concentrations (200-500 microM) compared with other mammals (3-120 microM). About 70% of daily urate disposal occurs via the kidneys, and in 5-25% of the human population, impaired renal excretion leads to hyperuricemia. About 10% of people with hyperuricemia develop gout, an inflammatory arthritis that results from deposition of monosodium urate crystals in the joint. We have identified genetic variants within a transporter gene, SLC2A9, that explain 1.7-5.3% of the variance in serum uric acid concentrations, following a genome-wide association scan in a Croatian population sample. SLC2A9 variants were also associated with low fractional excretion of uric acid and/or gout in UK, Croatian and German population samples. SLC2A9 is a known fructose transporter, and we now show that it has strong uric acid transport activity in Xenopus laevis oocytes.

SUMMARY The extent to which low-frequency (minor allele frequency [MAF] between 1–5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is largely unknown. Bone mineral density (BMD) is highly heritable, is a major predictor of osteoporotic fractures and has been previously associated with common genetic variants1–8, and rare, population-specific, coding variants9. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n=2,882 from UK10K), whole-exome sequencing (n= 3,549), deep imputation of genotyped samples using a combined UK10K/1000Genomes reference panel (n=26,534), and de-novo replication genotyping (n= 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size 4-fold larger than the mean of previously reported common variants for lumbar spine BMD8 (rs11692564[T], MAF = 1.7%, replication effect size = +0.20 standard deviations [SD], Pmeta = 2×10−14), which was also associated with a decreased risk of fracture (OR = 0.85; P = 2×10−11; ncases = 98,742 and ncontrols = 409,511). Using an En1Cre/flox mouse model, we observed that conditional loss of En1 results in low bone mass, likely as a consequence of high bone turn-over. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817[T], MAF = 1.1%, replication effect size = +0.39 SD, Pmeta = 1×10−11). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Osteoporosis is a common disease with a strong genetic component, characterized by reduced bone mass and increased fracture risk. Current evidence suggests that the inheritance of bone mass is under polygenic control but the genes responsible are poorly defined. Type I collagen is the major protein of bone encoded by the COLIA1 and COLIA2 genes. While these are strong candidates for the genetic regulation of bone mass, no abnormality of either gene has so far been defined in osteoporosis. In this study, we describe a novel G-->T polymorphism in a regulatory region of COLIA1 at a recognition site for the transcription factor Sp1(7) that is significantly related to bone mass and osteoporotic fracture. G/T heterozygotes at the polymorphic Sp1 site (Ss) had significantly lower bone mineral density (BMD) than G/G homozygotes (SS) in two populations of British women and BMD was lower still in T/T homozygotes (ss). The unfavourable Ss and ss genotypes were over-represented in patients with severe osteoporosis and vertebral fractures (54%), as compared with controls (27%), equivalent to a relative risk of 2.97 (95% confidence interval 1.63-9.56) for vertebral fracture in individuals who carry the 's' allele. While the mechanisms that underlie this association remain to be defined, the COLIA1 Sp1 polymorphism appears to be an important marker for low bone mass and vertebral fracture, raising the possibility that genotyping at this site may be of value in identifying women who are at risk of osteoporosis.

Familial expansile osteolysis (FEO, MIM 174810) is a rare, autosomal dominant bone disorder characterized by focal areas of increased bone remodelling. The osteolytic lesions, which develop usually in the long bones during early adulthood, show increased osteoblast and osteoclast activity. Our previous linkage studies mapped the gene responsible for FEO to an interval of less than 5 cM between D18S64 and D18S51 on chromosome 18q21.2-21.3 in a large Northern Irish family. The gene encoding receptor activator of nuclear factor-kappa B (RANK; ref. 5), TNFRSF11A, maps to this region. RANK is essential in osteoclast formation. We identified two heterozygous insertion mutations in exon 1 of TNFRSF11A in affected members of four families with FEO or familial Paget disease of bone (PDB). One was a duplication of 18 bases and the other a duplication of 27 bases, both of which affected the signal peptide region of the RANK molecule. Expression of recombinant forms of the mutant RANK proteins revealed perturbations in expression levels and lack of normal cleavage of the signal peptide. Both mutations caused an increase in RANK-mediated nuclear factor-kappaB (NF-kappaB) signalling in vitro, consistent with the presence of an activating mutation.

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.

SUMMARYNitric oxide (NO) is a free radical which has important effects on bone cell function. The endothelial isoform of nitric oxide synthase (eNOS) is widely expressed in bone on a constitutive basis, whereas inducible NOS is only expressed in response to in¯ammatory stimuli. It is currently unclear whether neuronal NOS is expressed by bone cells. Pro-in¯ammatory cytokines such as IL-1 and TNF cause activation of the iNOS pathway in bone cells and NO derived from this pathway potentiates cytokine and in¯ammation induced bone loss. These actions of NO are relevant to the pathogenesis of osteoporosis in in¯ammatory diseases such as rheumatoid arthritis, which are characterized by increased NO production and cytokine activation. Interferon gamma is a particularly potent stimulator of NO production when combined with other cytokines, causing very high concentrations of NO to be produced. These high levels of NO inhibit bone resorption and formation and may act to suppress bone turnover in severe in¯ammation. The eNOS isoform seems to play a key role in regulating osteoblast activity and bone formation since eNOS knockout mice have osteoporosis due to defective bone formation. Other studies have indicated that the NO derived from the eNOS pathway acts as a mediator of the effects of oestrogen in bone. eNOS also mediates the effects of mechanical loading on the skeleton where it acts along with prostaglandins, to promote bone formation and suppress bone resorption. Pharmacological NO donors have been shown to increase bone mass in experimental animals and preliminary evidence suggests that these agents may also in¯uence bone turnover in man. These data indicate that the L-arginine/NO pathway represents a novel target for therapeutic intervention in the prevention and treatment of bone diseases.