BMP2 Variants Research Articles

Monoallelic loss-of-function BMP2 variants result in BMP2-related skeletal dysplasia spectrum

PurposeBone morphogenic proteins (BMPs) regulate gene expression that is related to many critical developmental processes, including osteogenesis for which they are named. In addition, BMP2 is widely expressed in cells of mesenchymal origin, including bone, cartilage, skeletal and cardiac muscle, and adipose tissue. It also participates in neurodevelopment by inducing differentiation of neural stem cells. In humans, BMP2 variants result in a multiple congenital anomaly syndrome through a haploinsufficiency mechanism. We sought to expand the phenotypic spectrum and highlight phenotypes of patients harboring monoallelic missense variants in BMP2. MethodsWe used retrospective chart review to examine phenotypes from an international cohort of 18 individuals and compared these with published cases. Patient-derived missense variants were modeled in zebrafish to examine their effect on the ability of bmp2b to promote embryonic ventralization. ResultsThe presented cases recapitulated existing descriptions of BMP2-related disorders, including craniofacial, cardiac, and skeletal anomalies and exhibit a wide phenotypic spectrum. We also identified patients with neural tube defects, structural brain anomalies, and endocrinopathies. Missense variants modeled in zebrafish resulted in loss of protein function. ConclusionWe use this expansion of reported phenotypes to suggest multidisciplinary medical monitoring and management of patients with BMP2-related skeletal dysplasia spectrum.

Genetic variants in bone morphogenetic proteins signaling pathway might be involved in palatal rugae phenotype in humans

This study investigated, if genetic variants in BMP2, BMP4 and SMAD6 are associated with variations in the palatal rugae pattern in humans. Dental casts and genomic DNA from 75 patients were evaluated. Each patient was classified as follows: total amount of rugae; bilateral symmetry in the amount, length and shape of the palatal rugae; presence of secondary or fragmentary palatal rugae; presence of unifications; predominant shape; and predominant direction of the palatal rugae. The genetic variants in BMP2 (rs1005464 and rs235768), BMP4 (rs17563) and SMAD6 (rs2119261 and rs3934908) were genotyped. Genotype distribution was compared between palatal rugae patterns using the chi-square test (alpha = 0.05). The allele A was associated with the presence of secondary or fragmentary rugae for rs1005464 (OR = 2.5, 95%CI 1.1–6.3; p = 0.014). Secondary or fragmentary rugae were associated with the G allele in rs17563 (OR = 2.1, 95%CI 1.1–3.9; p = 0.017). rs17563 was also associated with rugae unification (p = 0.017 in the additive model). The predominant shape (wavy) was associated with rs2119261 (p = 0.023 in the additive model). The left–right symmetry of the length of primary rugae was associated with rs3934908 in the recessive model (OR = 3.6, 95%CI 1.2–11.7; p = 0.025). In conclusion, genetic variants in the BMP pathway impacted on palatal rugae pattern.

Optimized expression and purification of a soluble BMP2 variant based on in-silico design

Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of l-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.

BMP-2 Variants in Breast Epithelial to Mesenchymal Transition and Microcalcifications Origin.

This study aims to investigate the possible different roles of the BMP-2 variants, cytoplasmic and nuclear variant, in both epithelial to mesenchymal transition and in microcalcifications origin in human breast cancers. To this end, the in situ expression of cytoplasmic and nuclear BMP-2 was associated with the expression of the main epithelial to mesenchymal transition biomarkers (e-cadherin and vimentin) and molecules involved in bone metabolisms (RUNX2, RANKL, SDF-1) by immunohistochemistry. In addition, the expression of cytoplasmic and nuclear BMP-2 was associated with the presence of microcalcifications. Our data showed a significant association among the number of cytoplasmic BMP-2-positive cells and the number of both vimentin (positive association) and e-cadherin (negative association) positive breast cells. Conversely, no associations were found concerning the nuclear BMP-2-positive breast cells. Surprisingly, the opposite result was obtained by analyzing the variants of BMP-2 and both the expression of RANKL and SDF-1 and the presence of microcalcifications. Specifically, the presence of microcalcifications was related to the expression of nuclear BMP-2 variant rather than the cytoplasmic one, as well as a strong association between the number of nuclear BMP-2 and the expression of the main breast osteoblast-like cells (BOLCs) biomarkers. To further corroborate these data, an in vitro experiment for demonstrating the co-expression of nBMP-2 and RANKL or vimentin or SDF-1 in breast cancer cells that acquire the capability to produce microcalcifications was developed. These investigations confirmed the association between the nBMP-2 expression and both RANKL and SDF-1. The data supports the idea that whilst cytoplasmic BMP-2 can be involved in epithelial to mesenchymal transition phenomenon, the nuclear variant is related to the essential mechanisms for the formation of breast microcalcifications. In conclusion, from these experimental and translational perspectives, the complexity of BMP-2 signaling will require a detailed understanding of the involvement of specific BMP-2 variants in breast cancers.

Robinow Syndrome and Brachydactyly: An Interplay of High-Throughput Sequencing and Deep Phenotyping in a Kindred

We report a family with a spectrum of short stature, craniofacial dysmorphism, and digital anomalies in a father and 2 daughters, with the youngest (proband) displaying a severe phenotype. Clinically, autosomal dominant Robinow syndrome (ADRS) was diagnosed. Whole-exome sequencing identified a heterozygous pathogenic BMP2 variant in the father and his daughters. The phenotype of short stature, facial dysmorphism, and skeletal anomalies with or without cardiac anomalies related to BMP2 haploinsufficiency has some facial and digital resemblance to ADRS. Although this variant segregated in the affected members, it failed to explain the severe phenotype of the proband. A reanalysis of the girl's raw data confirmed 2 disorders: a de novo likely pathogenic DVL1 variant implicated in ADRS and the familial BMP2 variant. A close interplay of high-throughput sequencing and deep phenotyping unraveled the complexities of the blended phenotype in the proband.

BMP2 variants in the risk of ankylosing spondylitis.

The purpose of the study was to explore the genetic effects of bone morphogenetic protein (BMP2) polymorphisms on the susceptibility to ankylosing spondylitis (AS) in Chinese Han population. The case-control study included 120 AS cases and 110 healthy controls. Hardy-Weinberg equilibrium test was performed in control group. BMP2 rs235768 and rs3178250 polymorphisms were analyzed by polymerase chain reaction and direct sequencing. Additionally, the χ2 test was used to estimate association strength between BMP2 genetic polymorphisms and AS susceptibility, and the results were assessed via odds ratio (OR) with the corresponding 95% confidence interval (95%CI). Results adjustment was performed using logistic regression analysis. AA, AT, TT genotype and A, T allele frequencies of BMP2 rs235768 polymorphism presented no significant differences between case and control groups (P > .05 for all). TC genotype of rs3178250 polymorphism showed significantly higher in case group than that in control group (P = .048). After adjusting, TC genotype was a risk factor for AS (OR = 2.095;95%CI = 1.086-4.038;P = .027). BMP2 rs3178250 polymorphism may increase individual susceptibility to AS in Chinese Han population.

S5A-07 SESSION 5A: GENERALITIES IN CRANIOSYNOSTOSIS – PART II SMAD6 GENOTYPE PREDICTS NEURODEVELOPMENT IN NON-SYNDROMIC CRANIOSYNOSTOSIS

Introduction: De novo or rare transmitted mutations in the SMAD6 gene interact with common BMP2 variants to affect 7% of non-syndromic midline synostosis (NSC). In addition to cranial abnormalities and brain deformation, neurodevelopmental outcomes may be influenced by genetics. This study aimed to determine the cognitive sequelae of SMAD6 NSC in relation to non-SMAD6 NSC. Methods: Midline NSC patients ≥6 years of age, with SMAD6 mutations (identified in a national genetic study) and age/race/gender/synostosis type/operation matched non-SMAD6 NSC controls were recruited. All patients completed a double-blinded neurodevelopmental battery (Wechsler Fundamentals, Wechsler Abbreviated Scale of Intelligence, Beery-Buktenica Developmental Test) and parents/guardians completed behavioral surveys (Behavior Rating Inventory of Executive Function and Behavior Rating System for Children). Twenty demographic/patient factors, known to influence cognition, were collected. Results: Twenty subjects participated including all ten known SMAD6 patients nationwide (mean age at testing 10.1 years; 1 female; 8 metopic, 2 sagittal; 9 treated with cranial vault remodeling [CVR], 1 strip) and ten matched controls (mean age 9.8 years; 1 female; 8 metopic, 2 sagittal; 9 CVR, 1 strip). There was no significant difference between all 20 demographics. In head to head comparison, SMAD6 cases scored significantly worse on numerical operations (p=0.012), performance IQ (PIQ; p=0.004), full IQ (FIQ, p=0.007), and motor coordination (p=0.007). Individual correlations revealed age at testing, age at surgery, parental education, and household income significantly correlated with cognition. Multiple regressions were performed to control for these 4 variables. SMAD6 patients still performed worse on numerical operations(p=0.046), PIQ(p=0.018), FIQ(p=0.010), motor coordination(p=0.043). On behavioral surveys, SMAD6 scored worse on inhibition(p=0.003), behavior regulation(p=0.032), hyperactivity(p=0.007), aggression(p=0.008), conduct(p=0.029), social skills (p=0.039), and communication(p=0.018). Post-hoc power analysis yielded power between 84.4%>92.4%. Conclusion: This prospective double blind comparison revealed that genetic influences of NSC may play a role in functional neuropsychiatric development. Midline synostosis with SMAD6 mutations led to poorer non-adjusted and adjusted performance on mathematics, performance IQ, full IQ and motor coordination compared to non-SMAD6 NSC. SMAD6 patients had additional behavioral deficits in inhibition, hyperactivity, aggression, conduct, social skills, and communication. Moving forward, genetic testing may serve as a critical advocate for early adjunctive neuropsychiatric therapy in patients with NSC.

Evidence of distinct RELN and TGFB1 genetic associations in familial and non-familial otosclerosis in a British population

Otosclerosis is a common form of hearing loss which typically presents in young adults. The disease has a familial, monogenic form and a non-familial form with a more complex aetiology. A previous genome wide association study identified evidence that variants within RELN are associated with the condition. Other genes in which an association has been reported include BMP2, COL1A1, FGF2, PPP2R5B and TGFB1. However, follow up studies have often failed to replicate initial positive results. The aim of this study was to establish if an association exists between eight single nucleotide polymorphisms (SNPs) in these six previously implicated genes and otosclerosis in a British case–control cohort (n = 748). Evidence of an association between rs1800472 in TGFB1 and otosclerosis was found (p = 0.034), this association was strongest amongst non-familial cases (p = 0.011). No evidence of an association was detected with variants in COL1A1, FGF2, BMP2, and PPP2R5B. No association between variation in RELN and otosclerosis was observed in the whole cohort. However, a significant association (p = 0.0057) was detected between one RELN SNP (rs39399) and otosclerosis in familial patients. Additionally, we identify expression of one RELN transcript in 51 of 81 human stapes tested, clarifying previous conflicting data as to whether RELN is expressed in the affected tissue. Our findings strengthen the association of TGFB1 (rs1800472) with otosclerosis and support a relationship between RELN and familial otosclerosis only, which may explain previous variable replications.

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry.

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

A variant associated with sagittal nonsyndromic craniosynostosis alters the regulatory function of a non-coding element.

Craniosynostosis presents either as a nonsyndromic congenital anomaly or as a finding in nearly 200 genetic syndromes. Our previous genome-wide association study of sagittal nonsyndromic craniosynostosis identified associations with variants downstream from BMP2 and intronic in BBS9. Because no coding variants in BMP2 were identified, we hypothesized that conserved non-coding regulatory elements may alter BMP2 expression. In order to identify and characterize noncoding regulatory elements near BMP2, two conserved noncoding regions near the associated region on chromosome 20 were tested for regulatory activity with a Renilla luciferase assay. For a 711 base pair noncoding fragment encompassing the most strongly associated variant, rs1884302, the luciferase assay showed that the risk allele (C) of rs1884302 drives higher expression of the reporter than the common allele (T). When this same DNA fragment was tested in zebrafish transgenesis studies, a strikingly different expression pattern of the green fluorescent reporter was observed depending on whether the transgenic fish had the risk (C) or the common (T) allele at rs1884302. The in vitro results suggest that altered BMP2 regulatory function at rs1884302 may contribute to the etiology of sagittal nonsyndromic craniosynostosis. The in vivo results indicate that differences in regulatory activity depend on the presence of a C or T allele at rs1884302.

Utilizing BMP-2 muteins for treatment of multiple myeloma.

Multiple myeloma (MM) represents a haematological cancer characterized by the pathological hyper proliferation of antibody-producing B-lymphocytes. Patients typically suffer from kidney malfunction and skeletal disorders. In the context of MM, the transforming growth factor β (TGFβ) member Activin A was recently identified as a promoter of both accompanying symptoms. Because studies have shown that bone morphogenetic protein (BMP)-2-mediated activities are counteracted by Activin A, we analysed whether BMP2, which also binds to the Activin A receptors ActRII and ActRIIB but activates the alternative SMAD-1/5/8 pathway, can be used to antagonize Activin A activities, such as in the context of MM. Therefore three BMP2 derivatives were generated with modified binding activities for the type II (ActRIIB) and/or type I receptor (BMPRIA) showing either increased or decreased BMP2 activity. In the context of MM these BMP2 muteins show two functionalities since they act as a) an anti-proliferative/apoptotic agent against neoplastic B-cells, b) as a bone-formation promoting growth factor. The molecular basis of both activities was shown in two different cellular models to clearly rely on the properties of the investigated BMP2 muteins to compete for the binding of Activin A to the Activin type II receptors. The experimental outcome suggests new therapeutic strategies using BMP2 variants in the treatment of MM-related pathologies.

The BMP2 variant L51P restores the osteogenic differentiation of human mesenchymal stromal cells in the presence of intervertebral disc cells.

Spinal fusion is hampered by the presence of remaining intervertebral disc (IVD) tissue and leads to spinal non-union. While the exact mechanism remains unknown, we hypothesise that factors preventing disc ossification, such as antagonists of the bone morphogenetic proteins (BMP), could be responsible for this process. The objective of this study was to investigate spinal non-union using an in vitro human model with a focus on the BMP signalling components and to identify factors contributing to the incomplete and delayed ossification. Human bone marrow-derived mesenchymal stromal cells (MSC) were cocultured with IVD cells in the presence of L51P, a BMP2 variant with osteoinductive potential. The ossification of MSC was evaluated by quantitative reverse transcription polymerase chain reaction (qPCR), alkaline phosphatase (ALP) activity and alizarin red staining. Endogenous expression of major BMP antagonists, namely Gremlin (GREM1), Noggin (NOG) and Chordin (CHRD) was detected in IVD-derived cells, with abundance in nucleus pulposus cells. Osteogenesis of MSC was hindered by IVD cells as shown by reduced alizarin red staining, ALP activity and qPCR. L51P, added to the cocultures, restored mineralisation, blocking the activity of the BMP antagonists secreted by IVD cells. It is possible that the BMP antagonists secreted by IVD cells are responsible for spinal non-unions. The inhibition of BMP antagonists with L51P may result in an efficient and more physiological osteoinduction rather than delivery of exogenous osteogenic factors. Therefore, L51P might represent an attractive therapeutic candidate for bone healing.

Comprehensive genetic exploration of selective tooth agenesis of mandibular incisors by exome sequencing

Tooth agenesis is described as the absence of one or more teeth. It is caused by a failure in tooth development and is one of the most common human developmental anomalies. We herein report genomic analyses of selective mandibular incisor agenesis (SMIA) using exome sequencing. Two Japanese families with SMIA were subjected to exome sequencing, and family with sequence similarity 65 member A (FAM65), nuclear factor of activated T-cells 3 (NFATC3) and cadherin-related 23 gene (CDH23) were detected. In the follow-up study, 51 Japanese and 32 Korean sporadic patients with SMIA were subjected to exome analyses, and 18 reported variants in PAX9, AXIN2, EDA, EDAR, WNT10A, BMP2 and GREM2 and 27 variants of FAM65, NFATC3 and CDH23 were found in 38 patients. Our comprehensive genetic study of SMIA will pave the way for a full understanding of the genetic etiology of SMIA and provide targets for treatment.

Site-Directed Immobilization of BMP-2: Two Approaches for the Production of Innovative Osteoinductive Scaffolds.

The regenerative potential of bone is strongly impaired in pathological conditions, such as nonunion fractures. To support bone regeneration various scaffolds have been developed in the past, which have been functionalized with osteogenic growth factors such as bone morphogenetic proteins (BMPs). However, most of them required supra-physiological levels of these proteins leading to burst releases, thereby causing severe side effects. Site-specific, covalent coupling of BMP2 to implant materials might be an optimal strategy in order to overcome these problems. Therefore, we created a BMP-2 variant (BMP2-K3Plk) containing a noncanonical amino acid (propargyl-l-lysine) substitution introduced by genetic code expansion that allows for site-specific and covalent immobilization onto polymeric scaffold materials. To directly compare different coupling strategies, we also produced a BMP2 variant containing an additional cysteine residue (BMP2-A2C) allowing covalent coupling by thioether formation. The BMP2-K3Plk mutant was coupled to functionalized beads by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) either directly or via a short biotin-PEG linker both with high specificity. After exposing the BMP-coated beads to C2C12 cells, ALP expression appeared locally restricted in close proximity to these beads, showing that both coupled BMP2 variants trigger cell differentiation. The advantage of our approach over non-site-directed immobilization techniques is the ability to produce fully defined osteogenic surfaces, allowing for lower BMP2 loads and concomitant higher bioactivities, for example, due to controlled orientation toward BMP2 receptors. Such products might provide superior bone healing capabilities with potential safety advantages as of homogeneous product outcome.

Genetic Variants in the Bone Morphogenic Protein Gene Family Modify the Association between Residential Exposure to Traffic and Peripheral Arterial Disease.

There is a growing literature indicating that genetic variants modify many of the associations between environmental exposures and clinical outcomes, potentially by increasing susceptibility to these exposures. However, genome-scale investigations of these interactions have been rarely performed particularly in the case of air pollution exposures. We performed race-stratified genome-wide gene-environment interaction association studies on European-American (EA, N = 1623) and African-American (AA, N = 554) cohorts to investigate the joint influence of common single nucleotide polymorphisms (SNPs) and residential exposure to traffic (“traffic exposure”)—a recognized vascular disease risk factor—on peripheral arterial disease (PAD). Traffic exposure was estimated via the distance from the primary residence to the nearest major roadway, defined as the nearest limited access highways or major arterial. The rs755249-traffic exposure interaction was associated with PAD at a genome-wide significant level (P = 2.29x10-8) in European-Americans. Rs755249 is located in the 3’ untranslated region of BMP8A, a member of the bone morphogenic protein (BMP) gene family. Further investigation revealed several variants in BMP genes associated with PAD via an interaction with traffic exposure in both the EA and AA cohorts; this included interactions with non-synonymous variants in BMP2, which is regulated by air pollution exposure. The BMP family of genes is linked to vascular growth and calcification and is a novel gene family for the study of PAD pathophysiology. Further investigation of BMP8A using the Genotype Tissue Expression Database revealed multiple variants with nominally significant (P < 0.05) interaction P-values in our EA cohort were significant BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10-6) and rs1180341 (tibial artery, eQTL P = 5.3x10-6). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes.

The BMP2-variant L51P Enhances the Osteogenic Differentiation of Human Mesenchymal Stromal Cells in the Presence of Intervertebral Disc Cells

Introduction Discectomy and spinal fusion represents the gold standard treatment for spinal surgery to relieve pain. Fusion can be hindered, however, for yet unknown reasons that lead to non-fusions with pseudo-arthrosis. We previously showed that the intervertebral disc (IVD)-derived cells hinder the ossification process of human bone marrow-derived stromal cells (hMSC).1 Within this study, we hypothesized that BMP-antagonists secreted by IVD cells are the factors responsible for such inhibition and this can be reversed by addition of L51P. L51P is an engineered BMP2 variant that has been recently demonstrated to be a generic antagonist of a variety of BMP-inhibitors that control osteoinduction of bone.2,3 Material and Methods The experimental work was ethically approved and written consent of patients was obtained. hMSCs, primary nucleus pulposus (NPC) and annulus fibrosus cells (AFC) ( N = 6) were obtained from patients undergoing spinal surgery, isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4M/mL) and separated by culture inserts from MSCs in a co-culture (CC)-set-up. The allogenic CCs were paired in 11 repeated experiments. MSCs were kept in 1: control medium (±alginate beads), 2: osteogenic medium+NPC (±100ng/mL L51P) and 3: osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related markers and of BMP antagonists such as noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family were quantified with qPCR, and histological staining for calcium deposition and Alkaline Phosphatase (ALP) assay were performed. The endogenous expression of the BMP-antagonists in IVD cells (passage 1) was evaluated by immunohistochemistry. Results Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC and AFC. However, L51P added to CCs of MSCs with either NPC or AFC induced mineralization by blocking the activity of the IVD cell's secreted BMP-antagonists. It was noted that L51P caused a general reduction in ALP activity in all experimental groups. ALP activity was significantly up-regulated in positive control, CCNPC, and in CCAFC+L51P relative to negative controls, suggesting osteogenesis in these groups. CCNPC+L51P was significantly different from positive control but not CCNPC+L51P and CCAFC+L51P suggesting a reduction of the ALP activity. For the relative gene expression of potential BMP inhibitors (i.e., chordin, gremlin, noggin and TWSG-1), RNA and protein level using qPCR and immunohistochemistry confirmed expression of these BMP-antagonists inside the cells. Conclusion Alizarin red staining revealed an inhibition of the osteogenic differentiation of MSCs in CC with NPC or AFC. L51P could rescue osteogenesis of MSCs in the presence of NPC and AFC. Addition of L51P caused a general reduction in the ALP activity after 21 days of culture. L51P could thus be an attractive therapeutic treatment for spinal fusion, where it could enhance bone formation in the presence of NP and AF tissue. Acknowledgments This project was funded by two projects of the Swiss National Science Foundation grant number #IZK0Z3_154384 and #310030_153411. Eva Roth assisted in the biochemical assays and Hans-Jörg Sebald, provided the BMP2 variant L51P. References Chan SCW, Benneker LM, Heini P, Gantenbein B. An in vitro investigation into bone inhibition in non-unions caused by intervertebral disc cells. In: Proceedings of Biospine 5; Berlin, Germany, 2015 Albers CE, Hofstetter W, Sebald HJ, Sebald W, Siebenrock KA, Klenke FM. L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin. Bone 2012;51(3):401–406 Khattab HM, Ono M, Sonoyama W, et al. The BMP2 antagonist inhibitor L51P enhances the osteogenic potential of BMP2 by simultaneous and delayed synergism. Bone 2014;69:165–173

Hereditary hemochromatosis type 1 phenotype modifiers in Italian patients. The controversial role of variants in HAMP, BMP2, FTL and SLC40A1 genes.

Hereditary hemochromatosis (HH) is a heterogeneous disorder of iron metabolism. The most common form of the disease is Classic or type 1 HH, mainly caused by a biallelic missense p.Cys282Tyr (c.845G>A) mutation in the HFE gene. However, the penetrance of p.Cys282Tyr/p.Cys282Tyr genotype is incomplete in terms of both biochemical and clinical expressivity. Lack of penetrance is thought to be caused by several genetic and environmental factors. Recently, a lot of evidences on HH genetic modifiers were produced, often without conclusive results. We investigated 6 polymorphisms (rs10421768 in HAMP gene, rs235756 in BMP2 gene, rs2230267 in FTL gene, rs1439816 in SLC40A1 gene, rs41295942 in TFR2 gene and rs2111833 in TMPRSS6 gene) with uncertain function in order to further evaluate their role in an independent cohort of 109 HH type 1 patients. Our results make it likely the role of rs10421768, rs235756, rs2230267 and rs1439816 polymorphisms, respectively in HAMP, BMP2, FTL and SLC40A1 genes in HH expressivity. In addition, previous and our findings support a hypothetical multifactorial model of HH, characterized by a principal gene (HFE in HH type 1) and minor genetic and environmental factors that still have to be fully elucidated.

Rare Variants in BMP2 and BMP4 Found in Otosclerosis Patients Reduce Smad Signaling

Genetic variation in BMP2 and BMP4 found in otosclerosis patients result in altered Smad signaling. Otosclerosis is a common form of adult-onset conductive hearing loss resulting from abnormal bone remodeling of the bony labyrinth that surrounds the inner ear. Both genetic and environmental factors are implicated in the disease, yet very little is known about its pathogenesis. The evidence for a genetic component has been established through family-based linkage and population-based association studies. Previously, members of the TGF-β superfamily of genes have been associated with otosclerosis. Sequencing of BMP2 and BMP4 coding regions was performed to identify common and rare variation in German otosclerosis patients compared with controls. Functional analyses of rare variation in the patient cohort were conducted by exposing an osteosarcoma cell line to conditioned media containing either wild type or variant forms of BMP2 or BMP4 and analyzing Smad1/5/8 phosphorylation. Although no significant association with common variation in these 2 genes was detected, there were 8 singleton variants identified in the German population. Of the 4 coding variants found solely in otosclerosis patients, two--BMP4(N150K) and BMP2(K357-R396del)--were found to decrease Smad1/5/8 signaling. Rare variants in BMP2 and BMP4 are not a major genetic component in the otosclerosis population. However, those with functional affect showed decreased Smad signaling. Further analysis of Smad signaling molecules should be performed to determine if these pathways in combination are a major contributor to otosclerosis, which could lead to additional treatment options for otosclerosis patients.

Identification of mutations in bone morphogenetic protein 2, 4 and 7 in congenital heart disease patients from Indian population

Background Congenital heart disease (CHD) is the most common congenital defect, affecting 1% of all livebirths. Initial cardiac development and the subsequent maturation of the heart are temporally and spatially regulated by several Bone Morphogenetic Proteins (BMPs). BMP signaling plays an important role in the specification and patterning of the early embryo. Animal model studies have established a strong link between BMPs and cardiac development. At least 6 BMPs (BMP2, BMP4, BMP5, BMP6, BMP7 & BMP10) are expressed in the heart where they have both independent and redundant functions. BMP2, 4 & 7 are expressed in the AtrioVentricular cushion and Out Flow Tract (OFT) and helps in their formation. However due to functional redundancy of BMP ligands, relatively little is known about the role of individual BMPs. The association of BMPs with CHD has not been studied so far in human subjects. Here we report the genetic variants in BMP2 (NM_001200.2), BMP4 (NM_001202.3) and BMP7 (NM_001719.2) in individuals with CHD.

Clinical and genetic analysis of a nonsyndromic oligodontia in a child.

The etiology of tooth agenesis may be related to several factors, among them, the genetic alterations that play a fundamental role in the development of this dental anomaly, so that knowledge about it helps the clinician to have a greater understanding of their patients. Thus, the aim of this study was to report the case of a nonsyndromic child, with tooth agenesis of one premolar, three first permanent molars, and all second permanent molars. In addition, a genetic research between polymorphic variants in genes MMP3 and BMP2 was performed in order to observe the association between changes in these genes and congenital tooth absences. For this purpose, DNA from child was extracted and polymorphisms were investigated. It was clinically and radiographically observed that this was a case of oligodontia, in which the authors suggested an association between the polymorphisms found and tooth agenesis diagnosed in that child.