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Large-scale human exome sequencing can identify rare protein-coding variants with a large impact on complex traits such as body adiposity. We sequenced the exomes of 645,626 individuals from the United Kingdom, the United States, and Mexico and estimated associations of rare coding variants with body mass index (BMI). We identified 16 genes with an exome-wide significant association with BMI, including those encoding five brain-expressed G protein-coupled receptors (CALCR, MC4R, GIPR, GPR151, and GPR75). Protein-truncating variants in GPR75 were observed in ~4/10,000 sequenced individuals and were associated with 1.8 kilograms per square meter lower BMI and 54% lower odds of obesity in the heterozygous state. Knock out of Gpr75 in mice resulted in resistance to weight gain and improved glycemic control in a high-fat diet model. Inhibition of GPR75 may provide a therapeutic strategy for obesity.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare disorder, characterized by progressive heterotopic ossification (HO) and painful soft-tissue inflammatory flare-ups. This was a post hoc analysis from a phase 2 (NCT03188666) trial in which adults with FOP received intravenous anti-activin A antibody garetosmab 10 mg/kg or placebo every 4 wk over 28 wk (Period 1), followed by a 28-wk open-label treatment and extension (Periods 2 and 3). Here we describe flare-ups, their relationship to new HO lesions, and the impact of garetosmab on flare-ups. Volume of new HO lesions was measured by CT. Patient-reported flare-ups were defined by any 2 of the following: new onset of pain, swelling, joint stiffness, decrease in movement, or perceived presence of HO. Flare-ups were experienced by 71% (17/24) of placebo-treated patients, 59% (10/17) of whom developed a new HO lesion irrespective of flare-up location; 24% of flare-ups location-matched new HO lesions. Twenty-nine new HO lesions occurred in the placebo cohort by week 28, of which 12 (41%) occurred in the same location as new or ongoing flare-ups. A higher volume of newly formed heterotopic bone (week 28) occurred in placebo-treated patients who had experienced a prior flare-up vs those without (median [Q1:Q3] of 16.6 [12.0:31.1] vs 3.2 cm3). Garetosmab was previously shown to decrease patient-reported flare-up frequency in Period 1; here, garetosmab reduced the median (Q1:Q3) duration of patient-reported flares (15.0 [6.0:82.0] vs 48.0 [15.0:1.00] d) and the severity of flare-ups vs placebo. Frequency of corticosteroid use was numerically reduced in those treated with garetosmab (40.0%) vs placebo (58.3%). In this analysis, 71% of placebo-treated adults with FOP experienced flare-ups over 28 wk, which were associated with an increased volume of newly formed heterotopic bone. Garetosmab reduced the severity and duration of flare-ups, with effects sustained during the entire trial. Lay Summary: Fibrodysplasia ossificans progressiva (FOP) is a very rare genetic disorder caused by mutations in the ACVR1 gene. People with FOP experience growth of new bone in places where bone does not usually develop. Soft tissues (like skeletal muscles) and connective tissues (like tendons and ligaments) are gradually replaced by bone beyond the normal skeleton—a process called heterotopic ossification (HO). People with FOP experience flare-ups, which are painful swellings of the soft tissues. In this clinical study in people with FOP, we looked at the number of flareups, whether flareups were linked to new HO lesions, and the impact of garetosmab (a monoclonal antibody) on flareups. At random, about half the patients received placebo, or inactive drug, with the other half receiving garetosmab, the study drug. Of the patients who received placebo, 71% had flare-ups and 59% percent of those who had flare-ups also had a new HO lesion, which was not always related to the location of the flare-up. We have previously shown that garetosmab reduces the number of flareups patients report. In this study, we show that garetosmab reduces the length and pain severity of flare-ups too. The treatment effects were maintained for the whole study. [ABSTRACT FROM AUTHOR]

Anterior cruciate ligament (ACL) injuries often result in post-traumatic osteoarthritis (PTOA). To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced transcriptional changes in knee joints of three mouse strains with varying susceptibility to OA: STR/ort (highly susceptible), C57BL/6J (moderately susceptible) and super-healer MRL/MpJ (not susceptible). Right knee joints of the mice were injured using a non-invasive tibial compression injury model and global gene expression was quantified before and at 1-day, 1-week, and 2-weeks post-injury using RNA-seq. Following injury, injured and uninjured joints of STR/ort and injured C57BL/6J joints displayed significant cartilage degeneration while MRL/MpJ had little cartilage damage. Gene expression analysis suggested that prolonged inflammation and elevated catabolic activity in STR/ort injured joints, compared to the other two strains may be responsible for the severe PTOA phenotype observed in this strain. MRL/MpJ had the lowest expression values for several inflammatory cytokines and catabolic enzymes activated in response to ACL injury. Furthermore, we identified several genes highly expressed in MRL/MpJ compared to the other two strains including B4galnt2 and Tpsab1 which may contribute to enhanced healing in the MRL/MpJ. Overall, this study has increased our knowledge of early molecular changes associated with PTOA development.

Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ∼900,000 knee injuries in the United States, which account for ∼12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post-injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOSTTG ) and knockout (Sost-/- ) mice. Here we report that SOSTTG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post-injury compared with wild-type (WT) controls and Sost-/- . In addition, SOSTTG built ∼50% and ∼65% less osteophyte volume than WT and Sost-/- , respectively. Quantification of metalloproteinase (MMP) activity showed that SOSTTG had ∼2-fold less MMP activation than WT or Sost-/- , and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra-articular administration of recombinant Sost protein, immediately post-injury, also significantly decreased MMP activity levels relative to PBS-treated controls, and Sost activation in response to injury was TNFα and NF-κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Joint injury causes post-traumatic osteoarthritis (PTOA). About ∼50% of patients rupturing their anterior cruciate ligament (ACL) will develop PTOA within 1-2 decades of the injury, yet the mechanisms responsible for the development of PTOA after joint injury are not well understood. In this study, we examined whole joint gene expression by RNA sequencing (RNAseq) at 1 day, 1-, 6-, and 12 weeks post injury, in a non-invasive tibial compression (TC) overload mouse model of PTOA that mimics ACL rupture in humans. We identified 1446 genes differentially regulated between injured and contralateral joints. This includes known regulators of osteoarthritis such as MMP3, FN1, and COMP, and several new genes including Suco, Sorcs2, and Medag. We also identified 18 long noncoding RNAs that are differentially expressed in the injured joints. By comparing our data to gene expression data generated using the surgical destabilization of the medial meniscus (DMM) PTOA model, we identified several common genes and shared mechanisms. Our study highlights several differences between these two models and suggests that the TC model may be a more rapidly progressing model of PTOA. This study provides the first account of gene expression changes associated with PTOA development and progression in a TC model. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 35:474-485, 2017.

Wnt3a is a major regulator of bone metabolism however, very few of its target genes are known in bone. Wnt3a preferentially signals through transmembrane receptors Frizzled and co-receptors Lrp5/6 to activate the canonical signaling pathway. Previous studies have shown that the canonical Wnt co-receptors Lrp5 and Lrp6 also play an essential role in normal postnatal bone homeostasis, yet, very little is known about specific contributions by these co-receptors in Wnt3a-dependent signaling. We used high-throughput sequencing technology to identify target genes regulated by Wnt3a in osteoblasts and to elucidate the role of Lrp5 and Lrp6 in mediating Wnt3a signaling. Our study identified 782 genes regulated by Wnt3a in primary calvarial osteoblasts. Wnt3a up-regulated the expression of several key regulators of osteoblast proliferation/ early stages of differentiation while inhibiting genes expressed in later stages of osteoblastogenesis. We also found that Lrp6 is the key mediator of Wnt3a signaling in osteoblasts and Lrp5 played a less significant role in mediating Wnt3a signaling.

Loss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 (Sostdc1(-/-)) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1(-/-) cortical bone measurements revealed larger bones with higher BMD, suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1(-/-) mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1, and that Sostdc1(-/-) 5day calluses harbor >2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1-positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21days post fracture. These data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1(-/-) mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum.

Type 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21days post-fracture, and examined bone quality and callus outcomes at 21days and 42days post-fracture (11 and 14weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ+SostAb mice, also reversed the lower mineralization seen in STZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls. Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by activin A, an effect countered by inhibition of anti-activin A via monoclonal antibody treatment. Hence, we surmised that anti-ACVR1 antibodies that block activation of ACVR1 by ligands should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated anti-ACVR1 monoclonal antibodies that block ACVR1's activation by its ligands. Surprisingly, in vivo, these anti-ACVR1 antibodies stimulated HO and activated signaling of FOP-mutant ACVR1. This property was restricted to FOPmutant ACVR1 and resulted from anti-ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild-type ACVR1 was inhibited by anti-ACVR1 antibodies. These results uncover an additional property of FOP-mutant ACVR1 and indicate that anti-ACVR1 antibodies should not be considered as therapeutics for FOP., Introduction Fibrodysplasia ossificans progressiva (FOP) (OMIM #135100) is a rare, autosomal dominant disorder characterized by congenital skeletal dysplasias and progressive and cumulative heterotopic ossification (HO) of skeletal muscles, tendons, ligaments, [...]

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.

Glaucoma is a leading cause of blindness, afflicting more than 60 million people worldwide. Increased intraocular pressure (IOP) due to impaired aqueous humor drainage is a major risk factor for the development of glaucoma. Here, we demonstrated that genetic disruption of the angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway results in high IOP, buphthalmos, and classic features of glaucoma, including retinal ganglion degeneration and vision loss. Eyes from mice with induced deletion of Angptl and Angpt2 (A1A2[Flox.sup.WB] mice) lacked drainage pathways in the corneal limbus, including Schlemm's canal and lymphatic capillaries, which share expression of the PROX1, VEGFR3, and FOXC family of transcription factors. VEGFR3 and FOXCs have been linked to lymphatic disorders in patients, and FOXC1 has been linked to glaucoma. In contrast to blood endothelium, in which ANGPT2 is an antagonist of ANGPT1, we have shown that both ligands cooperate to regulate TIE2 in the lymphatic network of the eye. While A1A2[Flox.sup.WB] mice developed high IOP and glaucoma, expression of ANGPT1 or ANGPT2 alone was sufficient for ocular drainage. Furthermore, we demonstrated that loss of FOXC2 from lymphatics results in TIE2 downregulation, suggesting a mechanism for ocular defects in patients with FOXC mutations. These data reveal a pathogenetic and molecular basis for glaucoma and demonstrate the importance of angiopoietin ligand cooperation in the lymphatic endothelium., Introduction The angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway is a major regulator of vascular development, and altered expression of ANGPT ligands or activity of the TIE2 receptor is linked to a variety [...]

The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization-associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in [Col4a3.sup.[-/-]] mice, which model Alport syndrome. Ablation of Usag1 in [Col4a3.sup.[-/-]]mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in [Col4a3.sup.[-/-]] mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome., Introduction The renal glomerular basement membrane (GBM) contributes importantly to maintenance of the structural integrity of the glomerular capillaries (1), (2). Type IV collagen is the major component of the [...]