Opticool: Cutting-edge transgenic optical tools.

Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.

Cell-specific occupancy dynamics between the pioneer-like factor Opa/ZIC and Ocelliless/OTX regulate early head development in embryos.

During development, embryonic patterning systems direct a set of initially uncommitted pluripotent cells to differentiate into a variety of cell types and tissues. A core network of transcription factors, such as Zelda/POU5F1, Odd-paired (Opa)/ZIC3 and Ocelliless (Oc)/OTX2, are conserved across animals. While Opa is essential for a second wave of zygotic activation after Zelda, it is unclear whether Opa drives head cell specification, in the Drosophila embryo. Our hypothesis is that Opa and Oc are interacting with distinct cis-regulatory regions for shaping cell fates in the embryonic head. Super-resolution microscopy and meta-analysis of single-cell RNAseq datasets show that opa's and oc's overlapping expression domains are dynamic in the head region, with both factors being simultaneously transcribed at the blastula stage. Additionally, analysis of single-embryo RNAseq data reveals a subgroup of Opa-bound genes to be Opa-independent in the cellularized embryo. Interrogation of these genes against Oc ChIPseq combined with in situ data, suggests that Opa is competing with Oc for the regulation of a subgroup of genes later in gastrulation. Specifically, we find that Oc binds to late, head-specific enhancers independently and activates them in a head-specific wave of zygotic transcription, suggesting distinct roles for Oc in the blastula and gastrula stages.

Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster.

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.

Odd-paired is a pioneer-like factor that coordinates with Zelda to control gene expression in embryos.

Pioneer factors such as Zelda (Zld) help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls the transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates expression through enhancer sog_Distal along the dorso-ventral axis, we hypothesized Opa's role is more general. Chromatin-immunoprecipitation (ChIP-seq) confirmed its in vivo binding to sog_Distal but also identified widespread binding throughout the genome, comparable to Zld. Furthermore, chromatin assays (ATAC-seq) demonstrate that Opa, like Zld, influences chromatin accessibility genome-wide at cellularization, suggesting both are pioneer factors with common as well as distinct targets. Lastly, embryos lacking opa exhibit widespread, late patterning defects spanning both axes. Collectively, these data suggest Opa is a general timing factor and likely late-acting pioneer factor that drives a secondary wave of zygotic gene expression.

Distinct Roles of Broadly Expressed Repressors Support Dynamic Enhancer Action and Change in Time.

How broadly expressed repressors regulate gene expression is incompletely understood. To gain insight, we investigated how Suppressor of Hairless-Su(H)-and Runt regulate expression of bone morphogenetic protein (BMP) antagonist short-gastrulation via the sog_Distal enhancer. A live imaging protocol was optimized to capture this enhancer's spatiotemporal output throughout the early Drosophila embryo, finding in this context that Runt regulates transcription initiation, Su(H) regulates transcription rate, and both factors control spatial expression. Furthermore, whereas Su(H) functions as a dedicated repressor, Runt temporally switches from repressor to activator. Our results demonstrate that broad repressors play temporally distinct roles and contribute to dynamic gene expression. Both Run and Su(H)'s ability to influence the spatiotemporal domains of gene expression may serve to counterbalance activators and function in this manner as important regulators of the maternal-to-zygotic transition in early embryos.

Failure in diagnosis and under-treatment of osteoporosis in elderly patients with fragility fractures.

We evaluated whether osteoporosis is adequately managed and treated in patients suffering from fragility fractures. Factors that influenced osteoporosis diagnosis and treatment rates were also assessed. To this end, patients with the principal diagnosis of low-energy hip, vertebral, or distal radius fractures were recruited for the study. Collected data included risk factors for osteoporosis, history of previous fractures, known history of osteoporosis, and osteoporosis treatment at the time of admission. The patients' prefracture risk profile was also assessed to determine whether osteoporosis could have been identified prior to the index fracture. We identified 308 patients with fragility fractures, including 214 hip, 41 vertebral, and 53 distal radius fractures. Overall, 238 patients (77.3%) had at least one risk factor for osteoporosis. Eighty-eight patients (28.6%) had sustained ≥ 1 prior fragility fractures in the past. However, only 79 patients (25.6%) were aware that they had osteoporosis and even fewer (66 patients, 21.4%) had been receiving osteoporosis treatment preceding the current admission. Anti-osteoporotic agents were more commonly prescribed in patients 66-75 years old (p = 0.008), with a family history of osteoporosis (p = 0.009) or history of a prior fragility fracture (p = 0.012). The treatment rate was higher in women than men (p = 0.026) and in patients with vertebral or multiple prior fractures compared to patients with prior hip fractures. The current study provides evidence that individuals who experience fragility fractures are not adequately managed for osteoporosis. Only few of the historically known risk factors for osteoporosis were adequately recognized and associated with osteoporosis evaluation and treatment.

Broadly expressed repressors integrate patterning across orthogonal axes in embryos.

The role of spatially localized repressors in supporting embryonic patterning is well appreciated, but, alternatively, the role ubiquitously expressed repressors play in this process is not well understood. We investigated the function of two broadly expressed repressors, Runt (Run) and Suppressor of Hairless [Su(H)], in patterning the Drosophila embryo. Previous studies have shown that Run and Su(H) regulate gene expression along anterior-posterior (AP) or dorsal-ventral (DV) axes, respectively, by spatially limiting activator action, but here we characterize a different role. Our data show that broadly expressed repressors silence particular enhancers within cis-regulatory systems, blocking their expression throughout the embryo fully but transiently, and, in this manner, regulate spatiotemporal outputs along both axes. Our results suggest that Run and Su(H) regulate the temporal action of enhancers and are not dedicated regulators of one axis but, instead, act coordinately to pattern both axes, AP and DV.

Estrogens antagonize RUNX2-mediated osteoblast-driven osteoclastogenesis through regulating RANKL membrane association.

In addition to its thoroughly investigated role in bone formation, the osteoblast master transcription factor RUNX2 also promotes osteoclastogenesis and bone resorption. Here we demonstrate that 17ß-estradiol (E2), strongly inhibits RUNX2-mediated osteoblast-driven osteoclastogenesis in co-cultures. Towards deciphering the underlying mechanism, we induced premature expression of RUNX2 in primary murine pre-osteoblasts, which resulted in robust differentiation of co-cultured splenocytes into mature osteoclasts. This was attributable to RUNX2-mediated increase in RANKL secretion, determined by ELISA, as well as to RUNX2-mediated increase in RANKL association with the osteoblast membrane, demonstrated using confocal fluorescence microscopy. The increased association with the osteoblast membrane was recapitulated by transiently expressed GFP-RANKL. E2 abolished the RUNX2-mediated increase in membrane-associated RANKL and GFP-RANKL, as well as the concomitant osteoclastogenesis. RUNX2-mediated RANKL cellular redistribution was attributable in part to a decrease in Opg expression, but E2 did not influence Opg expression either in the presence or absence of RUNX2. Diminution of RUNX2-mediated osteoclastogenesis by E2 occurred regardless of whether the pre-osteoclasts were derived from wild type or estrogen receptor alpha (ERα)-knockout mice, suggesting that activated ERα inhibited osteoblast-driven osteoclastogenesis by acting in osteoblasts, possibly targeting RUNX2. Indeed, microarray analysis demonstrated global attenuation of the RUNX2 response by E2, including abrogation of Pstpip2 expression, which likely plays a critical role in membrane trafficking. Finally, the selective ER modulators (SERMs) tamoxifen and raloxifene mimicked E2 in abrogating the stimulatory effect of osteoblastic RUNX2 on osteoclast differentiation in the co-culture assay. Thus, E2 antagonizes RUNX2-mediated RANKL trafficking and subsequent osteoclastogenesis. Targeting RUNX2 and/or downstream mechanisms that regulate RANKL trafficking may lead to the development of improved SERMs and possibly non-hormonal therapeutic approaches to high turnover bone disease.

The G5¹6T CYP2B6 germline polymorphism affects the risk of acute myeloid leukemia and is associated with specific chromosomal abnormalities.

The etiology of acute myeloid leukemia (AML) underlies the influence of genetic variants in candidate genes. The CYP2B6 enzyme detoxifies many genotoxic xenobiotics, protecting cells from oxidative damage. The CYP2B6 gene is subjected to a single-nucleotide polymorphism (G5¹6T) with heterozygotes (GT) and homozygotes (TT) presenting decreased enzymatic activity. This case-control study aimed to investigate the association of CYP2B6 G5¹6T polymorphism with the susceptibility of AML and its cytogenetic and clinical characteristics. Genotyping was performed on 619 AML patients and 430 healthy individuals using RCR-RFLP and a novel LightSNip assay. The major finding was a statistically higher frequency of the variant genotypes (GT and TT) in patients compared to the controls (GT:38.8% vs 29.8% and TT:9.3% vs 5.3% respectively) (p<0.001). More specifically, a significantly higher frequency of GT+TT genotypes in de novo AML patients (46.6%) and an immensely high frequency of TT in secondary AML (s-AML) (20.5%) were observed. The statistical analysis showed that the variant T allele was approximately 1.5-fold and 2.4-fold higher in de novo and s-AML respectively than controls. Concerning FAB subtypes, the T allele presented an almost 2-fold increased in AML-M2. Interestingly, a higher incidence of the TT genotype was observed in patients with abnormal karyotypes. In particular, positive correlations of the mutant allele were found in patients carrying specific chromosomal aberrations [-7/del(7q), -5/del(5q), +8, +21 or t(8;21)], complex or monosomal karyotypes. Finally, a strikingly higher frequency of TT genotype was also observed in patients stratified to the poor risk group. In conclusion, our results provide evidence for the involvement of the CYP2B6 polymorphism in AML susceptibility and suggest a possible role of the CYP2B6 genetic background on the development of specific chromosomal aberrations.

Expression profile of osteoprotegerin, RANK and RANKL genes in the femoral head of patients with avascular necrosis.

INTRODUCTION: Femoral head avascular necrosis (AVN) is a recalcitrant disease of the hip that leads to joint destruction. Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B (RANK) and RANK ligand (RANKL) regulate the balance between osteoclasts-osteoblasts. The expression of these genes affects the maturation and function of osteoblasts-osteoclasts and bone remodeling. In this study, we investigated the molecular pathways leading to AVN by studying the expression profile of OPG, RANK and RANKL genes. MATERIAL AND METHODS: Quantitative Real Time-PCR was performed for evaluation of OPG, RANK and RANKL expression. Analysis was based on parallel evaluation of mRNA and protein levels in normal/necrotic sites of 42 osteonecrotic femoral heads (FHs). OPG and RANKL protein levels were estimated by western blotting. RESULTS: The OPG mRNA levels were higher (insignificantly) in the necrotic than the normal site (p > 0.05). Although the expression of RANK and RANKL was significantly lower than OPG in both sites, RANK and RANKL mRNA levels were higher in the necrotic part than the normal (p < 0.05). Protein levels of OPG and RANKL showed no remarkable divergence. CONCLUSIONS: Our results indicate that differential expression mechanisms for OPG, RANK and RANKL that could play an important role in the progress of bone remodeling in the necrotic area, disturbing bone homeostasis. This finding may have an effect on the resulting bone destruction and the subsequent collapse of the hip joint.

Glucocorticoids antagonize RUNX2 during osteoblast differentiation in cultures of ST2 pluripotent mesenchymal cells.

The efficacy of glucocorticoids (GCs) in treating a wide range of autoimmune and inflammatory conditions is blemished by severe side effects, including osteoporosis. The chief mechanism leading to GC-induced osteoporosis is inhibition of bone formation, but the role of RUNX2, a master regulator of osteoblast differentiation and bone formation, has not been well studied. We assessed effects of the synthetic GC dexamethasone (dex) on transcription of RUNX2-stimulated genes during the differentiation of mesenchymal pluripotent cells into osteoblasts. Dex inhibited a RUNX2 reporter gene and attenuated locus-dependently RUNX2-driven expression of several endogenous target genes. The anti-RUNX2 activity of dex was not attributable to decreased RUNX2 expression, but rather to physical interaction between RUNX2 and the GC receptor (GR), demonstrated by co-immunoprecipitation assays and co-immunofluorescence imaging. Investigation of the RUNX2/GR interaction may lead to the development of bone-sparing GC treatment modalities for the management of autoimmune and inflammatory diseases.

De novo 393 kb microdeletion of 7p11.2 characterized by aCGH in a boy with psychomotor retardation and dysmorphic features.

We report on a 27 month old boy presenting with psychomotor delay and dysmorphic features, mainly mild facial asymmetry, prominent cup-shaped ears, long eyelashes, open mouth appearance and slight abnormalities of the hands and feet. Array comparative genomic hybridization revealed a 393 kb microdeletion in 7p11.2. We discuss the possible involvement of CHCHD2, GBAS, MRPS17, SEPT14 and PSPH on our patient's phenotype. Additionally, we studied the expression of two other genes deleted in the patient, CCT6A and SUMF2, for which there is scarce data in the literature. Based on current knowledge and the de novo occurrence of this finding in our proband we presume that the aberration is likely to be pathogenic in our case. However, a single gene disorder, elsewhere in the genome or in this very region cannot be ruled out. Further elucidation of the properties of this chromosomal region, as well as of the role of the genes involved will be needed in order to draw safe conclusions regarding the association of the chromosomal deletion with the patient's features.

CER1 gene variations associated with bone mineral density, bone markers, and early menopause in postmenopausal women.

BACKGROUND: Osteoporosis has a multifactorial pathogenesis characterized by a combination of low bone mass and increased fragility. In our study, we focused on the effects of polymorphisms in CER1 and DKK1 genes, recently reported as important susceptibility genes for osteoporosis, on bone mineral density (BMD) and bone markers in osteoporotic women. Our objective was to evaluate the effect of CER1 and DKK1 variations in 607 postmenopausal women. The entire DKK1 gene sequence and five selected CER1 SNPs were amplified and resequenced to assess whether there is a correlation between these genes and BMD, early menopause, and bone turnover markers in osteoporotic patients. RESULTS: Osteoporotic women seem to suffer menopause 2 years earlier than the control group. The entire DKK1 gene sequence analysis revealed six variations. There was no correlation between the six DKK1 variations and osteoporosis, in contrast to the five common CER1 variations that were significantly associated with BMD. Additionally, osteoporotic patients with rs3747532 and rs7022304 CER1 variations had significantly higher serum levels of parathyroid hormone and calcitonin and lower serum levels of osteocalcin and IGF-1. CONCLUSIONS: No significant association between the studied DKK1 variations and osteoporosis was found, while CER1 variations seem to play a significant role in the determination of osteoporosis and a potential predictive role, combined with bone markers, in postmenopausal osteoporotic women.

Bone morphogenetic proteins (BMPs) expression in the femoral heads of patients with avascular necrosis.

Avascular necrosis (AVN) is a disorder of the bone repair process which usually results in femoral head (FH) destruction. Bone morphogenetic proteins (BMPs) are the key proteins regulating bone remodelling and healing. BMPs gene expression levels were analyzed in the normal and necrotic sites of osteonecrotic FHs. Quantitative RT-PCR for BMP-2, -4, -6, -7 genes was performed in bone tissue samples from 47 osteonecrotic FHs. Protein levels of BMP-2, -4, -6 were estimated by Western Blot. Statistical analysis was performed using the Wilcoxon signed rank test. BMP-2 and BMP-6 mRNA levels were higher in the normal than the necrotic site (normal/necrotic: 16.8/6.8 and 1.75/1.64, respectively). On the contrary, BMP-4 mRNA levels were higher in the necrotic (0.75) than the normal (0.62), while BMP-7 mRNA levels were extremely low. At the protein level, BMP-2 continued to have a higher expression in the normal region (normal/necrotic: 0.67/0.64). BMP-4 and -6 were detected at higher levels in the necrotic site (normal/necrotic: 0.51/0.61 for BMP-4, 0.51/0.56 for BMP-6), while BMP-7 was not detectable. Different BMP levels between the normal and necrotic site, as well as discrepancies between the gene and protein expression pattern suggest a different regulation mechanism for BMPs between the two regions of FHs. The understanding of the expression pattern and the correlation of BMPs could lead to a more successful use in the prevention and treatment of AVN.

High frequency of NAD(P)H:quinone oxidoreductase 1 (NQO1) C(609)T germline polymorphism in MDS/AML with trisomy 8.

The NQO1 C(609)T germline polymorphism resulting in a lowering of enzyme activity may confer susceptibility to MDS. To assess this association, we performed a case-control study including 330 Greek patients with de novo MDS and 416 healthy donors, using a Real-Time PCR genotyping method. Focusing on cytogenetic aberrations most commonly found in MDS, we retrospectively genotyped 566 MDS/AML patients carrying -5/del(5q), -7/del(7q), +8, del(20q) and -Y. The case-control analysis revealed no differences in NQO1 genotype distribution. Interestingly, a 6-fold increased frequency of the homozygous variant genotype was observed among patients with isolated trisomy 8 (p<0.0001), suggesting that null NQO1 activity may influence the occurrence of +8 in MDS/AML.

Association of A(313)G glutathione S-transferase P1 germline polymorphism with susceptibility to de novo myelodysplastic syndrome.

Models for the pathogenesis of myelodysplastic syndrome (MDS) imply the role of individual genetic variations in genes involved in detoxification mechanisms. GSTP1 enzyme plays a key role in the biotransformation of a variety of carcinogens. The corresponding gene is subject to a single nucleotide polymorphism (A(313)G) leading to abolished enzyme activity. In order to evaluate whether the GSTP1 polymorphism influences MDS susceptibility, we conducted a case-control study comprising 310 de novo patients and 370 healthy controls using a real-time polymerase chain reaction (PCR) genotyping method. The GSTP1 gene status was also evaluated in relation to patients' characteristics and chromosomal abnormalities. A significantly higher incidence of the GSTP1 variant genotypes was observed in patients with MDS compared to controls (p < 0.0001). The results revealed increased frequencies of heterozygotes in patients younger than 60 years old and of homozygotes G/G in older patients (p = 0.007). Our results provide evidence for a pathogenetic role of the GSTP1 polymorphism in MDS risk, probably in an age-dependent manner.

Severe osteoporosis and mutation in NOTCH2 gene in a woman with Hajdu-Cheney syndrome.

Hajdu-Cheney syndrome (HCS) is a rare genetic disorder characterised by acro-osteolysis, skull deformation and generalised osteoporosis. Recently, truncating mutations in the last exon of NOTCH2, a protein-coding gene, were found to be responsible. We present the case of a young woman with HCS in whom clinical and radiologic diagnosis was confirmed with DNA tests.

Assessment of gene-by-sex interaction effect on bone mineral density.

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 expression quantitative trait loci (eQTL) analysis and bioinformatics network analysis. 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 8 cohorts. In a second stage, we followed up the 12 top single-nucleotide polymorphisms (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. 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 and p = 3.0 × 10(-5) ; female effect = -0.007 and p = 3.3 × 10(-2) ), and 11 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. Despite the large collaborative effort, no genome-wide significant evidence for gene-by-sex interaction was found to influence 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. © 2012 American Society for Bone and Mineral Research.

Novel sequence variations in the CER1 gene are strongly associated with low bone mineral density and risk of osteoporotic fracture in postmenopausal women.

Osteoporosis is a common skeletal disease characterized by a combination of low bone mass and increased fragility. In this case-control study, we investigated the possible association of two novel candidate genes, CER1 and TOB1, with bone mineral density (BMD) and fragility risk in 300 postmenopausal women of Hellenic origin. The entire CER1 and TOB1 gene sequences were amplified and resequenced to assess whether there is a correlation between these genes and BMD. We identified 26 variants in both genes. Statistical analysis did not reveal any correlation between TOB1 and osteoporosis. However, CER1 genetic analysis indicated that five polymorphisms, c.194C>G, c.507+506G>T, c.508-182A>G, c.531A>G, and c.*121T>C, were correlated, with a mean T score ≤-2.2. In particular, the greater number of vertebral fractures was found in patients with osteoporosis carrying the G allele of c.531A>G SNP (p = 0.015). When multiple logistic regression analysis was performed, only the c.507+506G>T polymorphism was independently associated with hip fractures or the presence of any fracture (OR = 6.95, p = 0.016, and OR = 5.33, p < 0.001, respectively). These results suggest that CER1 gene variations play a significant role in determining BMD and vertebral or hip fractures, which might be helpful in clinical practice to identify patients with increased fracture risk.

Genome-wide meta-analysis identifies 56 bone mineral density loci and reveals 14 loci associated with risk of fracture.

Bone mineral density (BMD) is the most widely used 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 BMD-associated markers for replication in 50,933 independent subjects and for association with risk of low-trauma fracture in 31,016 individuals with a history of fracture (cases) and 102,444 controls. We identified 56 loci (32 new) associated with BMD at genome-wide significance (P < 5 × 10(-8)). Several of these factors cluster within the RANK-RANKL-OPG, mesenchymal stem cell differentiation, endochondral ossification and Wnt signaling pathways. However, we also discovered loci that were localized to genes not known to have a role in bone biology. Fourteen BMD-associated loci were also associated with fracture risk (P < 5 × 10(-4), Bonferroni corrected), of which six reached P < 5 × 10(-8), including at 18p11.21 (FAM210A), 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.