Skeletal Phenotype Research Articles

Carrying both COL1A2 and FBN2 gene heterozygous mutations results in a severe skeletal clinical phenotype: an affected family

BackgroundOsteogenesis imperfecta (OI) is the most common monogenic disease of the skeletal system and is usually caused by mutations in the COL1A1 or COL1A2 genes. Congenital contractural arachnodactyly syndrome (CCA) is an autosomal dominant hereditary disease of connective tissue. To date, the FBN2 gene is the only gene reported to cause CCA. Researchers found that COL1A2 and FBN2 are both involved in the extracellular matrix organization pathway. These findings suggest that these two genes play an important role in a similar mechanism and may trigger a synergistic effect.MethodsTrio-whole-exome sequencing (Trio-WES) was performed to analyse the underlying genetic cause of a proband with OI in a Chinese family. Sanger sequencing was used to validate the mutations in 3 members of the family with OI with varying degrees of severity of skeletal abnormalities and the members with no clinical signs.ResultA c.3304G > C mutation in the COL1A2 gene (p.Gly1102Arg) and a novel c.4108G > T mutation in the FBN2 gene (p.Glu1370*) were detected in the proband, an affected member of the family. The affected individuals with both mutations present a more severe phenotype, while affected individuals present a milder phenotype if only the mutation in COL1A2 is detected (c.3304G > C). The unaffected individual in this family did not have any mutations in the COL1A2 gene or FBN2 gene.ConclusionOur study is the first clinical report to indicate that patients carrying concomitant mutations in both the COL1A2 and FBN2 genes may present with more severe skeletal abnormalities. Furthermore, our study suggests the possibility of synergistic effects between the COL1A2 and FBN2 genes.

Bioinspired Nanovesicles Convert the Skeletal Endothelium-Associated Secretory Phenotype to Treat Osteoporosis.

Recently, bone marrow endothelial cells (BMECs) were found to play an important role in regulating bone homeostasis. However, few studies utilized BMECs to treat bone metabolic diseases including osteoporosis. Here, we reported bioinspired nanovesicles (BNVs) prepared from human induced pluripotent stem cells-derived endothelial cells under hypoxia culture through an extrusion approach. Abundant membrane C-X-C motif chemokine receptor 4 conferred these BNVs bone-targeting ability and the endothelial homology facilitated the BMEC tropism. Due to their unique endogenous miRNA cargos, these BNVs re-educated BMECs to secret cytokines favoring osteogenesis and anti-inflammation. Owing to the conversion of secretory phenotype, the osteogenic differentiation of bone mesenchymal stem cells was facilitated, and the M1-macrophage-dominant pro-inflammatory microenvironment was ameliorated in osteoporotic bones. Taken together, this study proposed BMEC-targeting nanovesicles treating osteoporosis via converting the skeletal endothelium-associated secretory phenotype.

Clinical, biochemical and genetic characteristics of MOGS-CDG: a rare congenital disorder of glycosylation

PurposeTo summarise the clinical, molecular and biochemical phenotype of mannosyl-oligosaccharide glucosidase-related congenital disorders of glycosylation (MOGS-CDG), which presents with variable clinical manifestations, and to analyse which clinical biochemical assay consistently...

A Baseline for Skeletal Investigations in Medaka (Oryzias latipes): The Effects of Rearing Density on the Postcranial Phenotype.

Oryzias latipes is increasingly used as a model in biomedical skeletal research. The standard approach is to generate genetic variants with particular skeletal phenotypes which resemble skeletal diseases in humans. The proper diagnosis of skeletal variation is key for this type of research. However, even laboratory rearing conditions can alter skeletal phenotypes. The subject of this study is the link between skeletal phenotypes and rearing conditions. Thus, wildtype medaka were reared from hatching to an early juvenile stage at low (LD: 5 individuals/L), medium (MD: 15 individuals/L), and high (HD: 45 individuals/L) densities. The objectives of the study are: (I) provide a comprehensive overview of the postcranial skeletal elements in medaka; (II) evaluate the effects of rearing density on specific meristic counts and on the variability in type and incidence of skeletal anomalies; (III) define the best laboratory settings to obtain a skeletal reference for a sound evaluation of future experimental conditions; (IV) contribute to elucidating the structural and cellular changes related to the onset of skeletal anomalies. The results from this study reveal that rearing densities greater than 5 medaka/L reduce the animals’ growth. This reduction is related to decreased mineralization of dermal (fin rays) and perichondral (fin supporting elements) bone. Furthermore, high density increases anomalies affecting the caudal fin endoskeleton and dermal rays, and the preural vertebral centra. A series of static observations on Alizarin red S whole mount-stained preural fusions provide insights into the etiology of centra fusion. The fusion of preural centra involves the ectopic formation of bony bridges over the intact intervertebral ligament. An apparent consequence is the degradation of the intervertebral ligaments and the remodeling and reshaping of the fused vertebral centra into a biconoid-shaped centrum. From this study it can be concluded that it is paramount to take into account the rearing conditions, natural variability, skeletal phenotypic plasticity, and the genetic background along with species-specific peculiarities when screening for skeletal phenotypes of mutant or wildtype medaka.

Octanoic Acid-Enrichment Diet Improves Endurance Capacity and Reprograms Mitochondrial Biogenesis in Skeletal Muscle of Mice.

Background: Medium Chain Fatty Acids (MCFAs) are a dietary supplement that exhibit interesting properties, due to their smaller molecular size. The acute consumption of MCFAs is expected to enhance exercise performance. However, the short-term effects of MCFAs on endurance performance remains poorly understood. The aim of our study is to evaluate the octanoic acid (C8)-rich diet effect on endurance capacity, and to explore their molecular and cellular effects. Methods: C57BL/6J mice were fed with a chow diet (Control group) or an octanoic acid-rich diet (C8 diet) for 6 weeks. Spontaneous activity, submaximal and maximal exercise tests were carried out to characterize the exercise capacities of the mice. Beta-oxidation and mitochondrial biogenesis pathways were explored in skeletal muscle by RT-qPCR, Western Blot (Quadriceps) and histochemical staining (Gastrocnemius). Results: Mice fed with a C8-rich diet presented a higher spontaneous activity (p < 0.05) and endurance capacities (p < 0.05) than the control, but no effect on maximal effort was observed. They also presented changes in the skeletal muscle metabolic phenotype, with a higher number of the oxidative fibers, rich in mitochondria. At the molecular level, the C8-diet induced an AMPK activation (p < 0.05), associated with a significant increase in PGC1a and CS gene expression and protein levels. Conclusion: Our study provided evidence that C8-enrichment as a food supplementation improves endurance capacities and activates mitochondrial biogenesis pathways leading to higher skeletal muscle oxidative capacities.

Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton.

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (Tfr1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin, and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of Tfr1 and Tfr1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of Tfr1 in osteoclast lineage cells in vivo and in vitro, we crossed Tfrc (encoding Tfr1)-floxed mice with Lyz2 (LysM)-Cre and Cathepsin K (Ctsk)-Cre mice to generate Tfrc conditional knockout mice in myeloid osteoclast precursors (Tfr1ΔLysM) or differentiated osteoclasts (Tfr1ΔCtsk), respectively. Skeletal phenotyping by µCT and histology unveiled a significant increase in trabecular bone mass with normal osteoclast number in long bones of 10-week-old young and 6-month-old adult female but not male Tfr1ΔLysM mice. Although high trabecular bone volume in long bones was observed in both male and female Tfr1ΔCtsk mice, this phenotype was more pronounced in female knockout mice. Consistent with this gender-dependent phenomena, estrogen deficiency induced by ovariectomy decreased trabecular bone mass in Tfr1ΔLysM mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts in vitro by attenuating mitochondrial respiration and activation of the Src-Rac1-WAVE regulatory complex axis, respectively, leading to decreased bone resorption with little impact on osteoclast differentiation. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling in a gender-dependent manner.

EPLINβ Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINβ. In this study, we investigated the role of EPLINβ in osteoblasts using EPLINβ-deficient (EPLINβGT/GT) mice. The skeletal phenotype of EPLINβGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINβGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINβGT/GT mice, suggesting aberrant cell adhesion. In EPLINβGT/GT osteoblasts, α- and β-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINβ knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINβ knockdown cells, suggesting an important role for EPLINβ in osteoblast formation. In conclusion, we propose that EPLINβ is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

Lack of CCR3 leads to a skeletal phenotype only in male mice

We recently showed that adult male mice that lacked the C–C-chemokine receptor 3 (CCR3) exhibited disturbed bone remodeling, which resulted in a cortical bone phenotype of thin femoral cortical bone. However, it remains unknown whether this phenotype would be present during bone modeling, or it affects female mice. Here, we analyzed juvenile and adolescent CCR3-deficient mice to determine when bone modeling was affected in the absence of CCR3 signaling. To investigate whether the CCR3 bone phenotype was sex-related, we analyzed both young female and male mice, and adult females.Micro-computed tomography (μCT) and histomorphometric analyses in adolescent CCR3-deficient male mice revealed reduced cortical bone volume and thickness, and an increase in periosteal mineralization. Interestingly, no skeletal phenotype was observed in adolescent or adult female CCR3-deficient mice. Among juvenile CCR3-deficient mice, neither males nor females showed a skeletal phenotype, which indicated that bone modeling was not affected by the CCR3 deficiency.In summary, adolescent and adult male mice that lacked CCR3 receptors exhibited a cortical phenotype that was not present in female mice, probably due to an estrogen protective mechanism. Based on these and our previous results, we suggest that the importance of CCR3 in cortical bone turnover is related to sex hormones. Because only a few molecules are known to control cortical bone turnover, our novel finding that CCR3 regulated cortical bone thickness only in males suggested that CCR3 is a novel target for controlling cortical bone morphology in male individuals, and perhaps, in post-menopausal women.

Mouse models of type 1 diabetes and their use in skeletal research.

In this review, we describe the three primary mouse models of insulin-deficiency diabetes that have been used to study the effects of type 1 diabetes (T1D) on skeletal outcomes. These models include streptozotocin (chemically)-induced diabetes, autoimmune-mediated diabetes (the nonobese diabetes mouse), and a mutation in the insulin gene (the Akita mouse). We then describe the skeletal findings and/or skeletal phenotypes that have been delineated using these models. Humans with T1D have decreased bone mineral density and an increased risk for fragility fracture. Mouse models of insulin-deficiency diabetes (hereafter denoted as T1D) in many ways recapitulate these skeletal deficits. Utilizing techniques of microcomputed tomography, bone histomorphometry, biomechanical testing and fracture modeling, bone biomarker analysis, and Raman spectroscopy, mouse models of T1D have demonstrated abnormalities in bone mineralization, bone microarchitecture, osteoblast function, abnormal bone turnover, and diminished biomechanical properties of bone. Mouse models have provided significant insights into the underlying mechanisms involved in the abnormalities of bone observed in T1D in humans. These translational models have provided targets and pathways that may be modifiable to prevent skeletal complications of T1D.

GIRK3 deletion facilitates kappa opioid signaling in chondrocytes, delays vascularization and promotes bone lengthening in mice.

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K+ (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2-/-, Girk3-/- and Girk2/3-/- mice. Bones from 12-week-old Girk2-/- mice were normal in length, but femurs and tibiae from Girk3-/- and Girk2/3-/- mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3-/- mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3-/- chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3-/- mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

Polygenic Models Partially Predict Muscle Size and Strength but Not Low Muscle Mass in Older Women.

Background: Heritability explains 45-82% of muscle mass and strength variation, yet polygenic models for muscle phenotypes in older women are scarce. Therefore, the objective of the present study was to (1) assess if total genotype predisposition score (GPSTOTAL) for a set of polymorphisms differed between older women with low and high muscle mass, and (2) utilise a data-driven GPS (GPSDD) to predict the variance in muscle size and strength-related phenotypes. Methods: In three-hundred 60- to 91-year-old Caucasian women (70.7 ± 5.7 years), skeletal muscle mass, biceps brachii thickness, vastus lateralis anatomical cross-sectional area (VLACSA), hand grip strength (HGS), and elbow flexion (MVCEF) and knee extension (MVCKE) maximum voluntary contraction were measured. Participants were classified as having low muscle mass if the skeletal muscle index (SMI) < 6.76 kg/m2 or relative skeletal muscle mass (%SMMr) < 22.1%. Genotyping was completed for 24 single-nucleotide polymorphisms (SNPs). GPSTOTAL was calculated from 23 SNPs and compared between the low and high muscle mass groups. A GPSDD was performed to identify the association of SNPs with other skeletal muscle phenotypes. Results: There was no significant difference in GPSTOTAL between low and high muscle mass groups, irrespective of classification based on SMI or %SMMr. The GPSDD model, using 23 selected SNPs, revealed that 13 SNPs were associated with at least one skeletal muscle phenotype: HIF1A rs11549465 was associated with four phenotypes and, in descending number of phenotype associations, ACE rs4341 with three; PTK2 rs7460 and CNTFR rs2070802 with two; and MTHFR rs17421511, ACVR1B rs10783485, CNTF rs1800169, MTHFR rs1801131, MTHFR rs1537516, TRHR rs7832552, MSTN rs1805086, COL1A1 rs1800012, and FTO rs9939609 with one phenotype. The GPSDD with age included as a predictor variable explained 1.7% variance of biceps brachii thickness, 12.5% of VLACSA, 19.0% of HGS, 8.2% of MVCEF, and 9.6% of MVCKE. Conclusions: In older women, GPSTOTAL did not differ between low and high muscle mass groups. However, GPSDD was associated with muscle size and strength phenotypes. Further advancement of polygenic models to understand skeletal muscle function during ageing might become useful in targeting interventions towards older adults most likely to lose physical independence.

A Mild Case of Autosomal Recessive Osteopetrosis Masquerading as the Dominant Form Involving Homozygous Deep Intronic Variations in the CLCN7 Gene

Osteopetrosis is a heterogeneous group of rare hereditary diseases characterized by increased bone mass of poor quality. Autosomal-dominant osteopetrosis type II (ADOII) is most often caused by mutation of the CLCN7 gene leading to impaired bone resorption. Autosomal recessive osteopetrosis (ARO) is a more severe form and is frequently accompanied by additional morbidities. We report an adult male presenting with classical clinical and radiological features of ADOII. Genetic analyses showed no amino-acid-converting mutation in CLCN7 but an apparent haploinsufficiency and suppression of CLCN7 mRNA levels in peripheral blood mononuclear cells. Next generation sequencing revealed low-frequency intronic homozygous variations in CLCN7, suggesting recessive inheritance. In silico analysis of an intronic duplication c.595-120_595-86dup revealed additional binding sites for Serine- and Arginine-rich Splicing Factors (SRSF), which is predicted to impair CLCN7 expression. Quantitative backscattered electron imaging and histomorphometric analyses revealed bone tissue and material abnormalities. Giant osteoclasts were present and additionally to lamellar bone, and abundant woven bone and mineralized cartilage were observed, together with increased frequency and thickness of cement lines. Bone mineralization density distribution (BMDD) analysis revealed markedly increased average mineral content of the dense bone (CaMean T-score + 10.1) and frequency of bone with highest mineral content (CaHigh T-score + 19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except for an unusually circular shape. Together, our findings suggest that the reduced expression of CLCN7 mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture. While the skeletal phenotype and medical history were suggestive of ADOII, genetic analysis revealed that this is a possible mild case of ARO due to deep intronic mutation.

Tissue-specific isoform expression of GNE gene in human tissues.

Mutations in the sialic acid biosynthesis enzyme GNE lead to a late-onset, debilitating neuromuscular disorder, GNE myopathy, characterized by progressive skeletal muscle weakness. The mechanisms responsible for skeletal muscle specificity, late-onset, and disease progression are unknown. Our main aim is to understand the reason for skeletal muscle-specific phenotype. To answer this question, we have analyzed the expression profile of the GNE gene and its multiple mRNA variants in different human tissues. A combinatorial approach encompassing bioinformatics tools and molecular biology techniques was used. NCBI, Ensembl, and GTEx were used for data mining. The expression analysis of GNE and its variants was performed with cDNA tissue panel using PCR and targeted RNA-seq. Among nine different GNE isoforms reported in this study, transcript variants 1, X1, and X2 were not tissue specific. Transcript variants 1, 6, X1, and X2, were found in skeletal muscles suggesting their possible role in GNE myopathy. In the current study, we present new data about GNE expression patterns in human tissues. Our results suggest that there may be a link between tissue-specific pathology and isoform pattern in skeletal muscles, which could provide clues for the development of new treatment strategies for GNE myopathy.

Trabecular Bone Structural Variation of the Human Distal Tibia, Talus, and Calcaneus

Based on the principles of bone functional adaptation, bone structure has been interpreted as reflective, at least in part, of the mechanical loading environment experienced by an individual during their lifetime. However, the extent to which reduced skeletal robusticity in modern humans over the last 10,000 years is the result of reduced activity and increasing reliance on agricultural subsistence strategies, rather than genetic or environmental factors known to influence skeletal phenotype, is not well understood. The objective of this study is to investigate how bone structure varies between human groups with distinct subsistence strategies, and thus different levels of physical activity and mechanical loading. Here we compare trabecular bone structure in the distal tibia, talus, and calcaneus of three archaeological groups from central and southern Illinois with varying levels of mobility from relatively low mobility mixed strategy agriculturalists to relatively high mobility hunter‐gatherers. High‐resolution CT image data of the distal tibia, talus, and calcaneus was used to quantify trabecular bone microarchitecture variables including bone volume fraction and degree of anisotropy. Bone structural variables are compared between subsistence groups using registered point clouds and Bayesian multilevel modeling to test whether bone structure differs between groups with different inferred mobility and activity levels, but similar population history and environmental context. Results indicate that variation in trabecular bone structure for the distal tibia, talus, and calcaneus support the hypothesis that more highly mobile subsistence groups have greater trabecular robusticity. Investigating the relationship between bone structure variation and differences in activity and mobility is important for understanding the role of behavioral, ecological, and biological factors in determining human skeletal phenotype.

Dose dependent effects of green tea extracts in the skeletal development of a Down syndrome mouse model

Down syndrome (DS) is a congenital disorder caused by trisomy of chromosome 21. The genetic imbalance results in intellectual disability, but also alters the development of different body systems, including the cranial and postcranial skeleton. To normalize altered development and to rescue skeletal phenotypes, green tea extracts enriched in EGCG (GTE‐EGCG) have been proposed for therapy. In this study, we longitudinally followed the skeletal postnatal development in a mouse model for DS, Ts65Dn, and assessed the effects of both a high dose and a low dose of pre‐ and postnatal GTE‐EGCG treatment. We administered a green tea extract containing 45% EGCG at doses of 100 or 30 mg/kg/day to pregnant Ts65Dn females, starting at embryonic day 9 and continued until adulthood. We longitudinally scanned wild‐type and Ts65Dn mice at postnatal days 3, 14 and 29 using in vivo micro‐computed tomography (µCT).Quantitative morphometric analyses showed that TS mice have a significantly different skull and mandibular shape than WT mice, as well as reduced bone mineral density (BMD) of the premaxilla and humerus bones that were detected already after birth. Moreover, Ts65Dn mice presented shorter faces, humerus and scapula, with significant differences appearing later in development, at the juvenile stage. Treatment with GTE‐EGCG generally deviated both WT and TS mice from normal growth trajectories, exacerbating some morphological anomalies, such as skull brachycephaly and reduced bone dimensions and mineral density. However, the effects of pre‐ and postnatal GTE‐EGCG administration were dose‐dependent, and low dose GTE‐EGCG treatment presented rescuing effects in facial shape. Our results underscore the complex nature of the DS phenotype and the need of further research to characterize the diverse effects of GTE‐EGCG treatment on different systems and structures and to fine‐tune dose and timing to achieve optimal safety and therapeutic potential.

Hoxa5 Activity Across the Lateral Somitic Frontier Regulates Development of the Mouse Sternum.

The skeletal system derives from multiple embryonic sources whose derivatives must develop in coordination to produce an integrated whole. In particular, interactions across the lateral somitic frontier, where derivatives of the somites and lateral plate mesoderm come into contact, are important for proper development. Many questions remain about genetic control of this coordination, and embryological information is incomplete for some structures that incorporate the frontier, including the sternum. Hox genes act in both tissues as regulators of skeletal pattern. Here, we used conditional deletion to characterize the tissue-specific contributions of Hoxa5 to skeletal patterning. We found that most aspects of the Hoxa5 skeletal phenotype are attributable to its activity in one or the other tissue, indicating largely additive roles. However, multiple roles are identified at the junction of the T1 ribs and the anterior portion of the sternum, or presternum. The embryology of the presternum has not been well described in mouse. We present a model for presternum development, and show that it arises from multiple, paired LPM-derived primordia. We show evidence that HOXA5 expression marks the embryonic precursor of a recently identified lateral presternum structure that is variably present in therians.

Lfng and Dll3 cooperate to modulate protein interactions in cis and coordinate oscillatory Notch pathway activation in the segmentation clock

In mammalian development, oscillatory activation of Notch signaling is required for segmentation clock function during somitogenesis. Notch activity oscillations are synchronized between neighboring cells in the presomitic mesoderm (PSM) and have a period that matches the rate of somite formation. Normal clock function requires cyclic expression of the Lunatic fringe (LFNG) glycosyltransferase, as well as expression of the inhibitory Notch ligand Delta-like 3 (DLL3). How these factors coordinate Notch activation in the clock is not well understood. Recent evidence suggests that LFNG can act in a signal-sending cell to influence Notch activity in the clock, raising the possibility that in this context, glycosylation of Notch pathway proteins by LFNG may affect ligand activity. Here we dissect the genetic interactions of Lfng and Dll3 specifically in the segmentation clock and observe distinctions in the skeletal and clock phenotypes of mutant embryos showing that paradoxically, loss of Dll3 is associated with strong reductions in Notch activity in the caudal PSM. The patterns of Notch activity in the PSM suggest that the loss of Dll3 is epistatic to the loss of Lfng in the segmentation clock, and we present direct evidence for the modification of several DLL1 and DLL3 EGF-repeats by LFNG. We further demonstrate that DLL3 expression in cells co-expressing DLL1 and NOTCH1 can potentiate a cell's signal-sending activity and that this effect is modulated by LFNG, suggesting a mechanism for coordinated regulation of oscillatory Notch activation in the clock by glycosylation and cis-inhibition.

Genetic Deletion of Menin in Mouse Mesenchymal Stem Cells: An Experimental and Computational Analysis.

ABSTRACTLoss‐of‐function mutations in the MEN1 tumor‐suppressor gene cause the multiple endocrine neoplasia type 1 syndrome. Menin, the MEN1 gene product, is expressed in many tissues, including bone, where its function remains elusive. We conditionally inactivated menin in mesenchymal stem cells (MSCs) using paired‐related homeobox 1 (Prx1)‐Cre and compared resultant skeletal phenotypes of Prx1‐Cre;Men1 f/f menin‐knockout mice (KO) and wild‐type controls using in vivo and in vitro experimental approaches and mechanics simulation. Dual‐energy X‐ray absorptiometry demonstrated significantly reduced bone mineral density, and 3‐dimensional micro‐CT imaging revealed a decrease in trabecular bone volume, altered trabecular structure, and an increase in trabecular separation in KO mice at 6 and 9 months of age. Numbers of osteoblasts were unaltered, and dynamic histomorphometry demonstrated unaltered bone formation; however, osteoclast number and activity and receptor activator of NF‐κB ligand/osteoprotegerin (RANKL/OPG) mRNA profiles were increased, supporting increased osteoclastogenesis and bone resorption. In vitro, proliferative capabilities of bone marrow stem cells and differentiation of osteoblasts and mineralization were unaltered; however, osteoclast generation was increased. Gross femur geometrical alterations observed included significant reductions in length and in mid‐metaphyseal cross‐sectional area. Atomic force microscopy demonstrated significant decreases in elasticity of both cortical and trabecular bone at the nanoscale, whereas three‐point bending tests demonstrated a 30% reduction in bone stiffness; finite element analysis showed morphological changes of the femur microgeometry and a significantly diminished femur flexural rigidity. The biomechanical results demonstrated the detrimental outcome of the accelerated osteoclastic bone resorption. Our studies have a twofold implication; first, MEN1 deletion from MSCs can negatively regulate bone mass and bone biomechanics, and second, the experimental and computational biomechanical analyses employed in the present study should be applicable for improved phenotypic characterization of murine bone. Furthermore, our findings of critical menin function in bone may underpin the more severe skeletal phenotype found in hyperparathyroidism associated with loss‐of‐function of the MEN1 gene. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Delineation of dual molecular diagnosis in patients with skeletal deformity

BackgroundSkeletal deformity is characterized by an abnormal anatomical structure of bone and cartilage. In our previous studies, we have found that a substantial proportion of patients with skeletal deformity could be explained by monogenic disorders. More recently, complex phenotypes caused by more than one genetic defect (i.e., dual molecular diagnosis) have also been reported in skeletal deformities and may complicate the diagnostic odyssey of patients. In this study, we report the molecular and phenotypic characteristics of patients with dual molecular diagnosis and variable skeletal deformities.ResultsFrom 1108 patients who underwent exome sequencing, we identified eight probands with dual molecular diagnosis and variable skeletal deformities. All eight patients had dual diagnosis consisting of two autosomal dominant diseases. A total of 16 variants in 12 genes were identified, 5 of which were of de novo origin. Patients with dual molecular diagnosis presented blended phenotypes of two genetic diseases. Mendelian disorders occurred more than once include Osteogenesis Imperfecta Type I (COL1A1, MIM:166200), Neurofibromatosis, Type I (NF1, MIM:162200) and Marfan Syndrome (FBN1, MIM:154700).ConclusionsThis study demonstrated the complicated skeletal phenotypes associated with dual molecular diagnosis. Exome sequencing represents a powerful tool to detect such complex conditions.

Novel Small Molecule Fibroblast Growth Factor 23 Inhibitors Increase Serum Phosphate and Improve Skeletal Abnormalities in Hyp Mice.

Excess fibroblast growth factor (FGF) 23 causes hereditary hypophosphatemic rickets, such as X-linked hypophosphatemia (XLH) and tumor-induced osteomalacia (TIO). A small molecule that specifically binds to FGF23 to prevent activation of the fibroblast growth factor receptor/α-Klotho complex has potential advantages over the currently approved systemically administered FGF23 blocking antibody. Using structure-based drug design, we previously identified ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine) as a small molecule antagonist for FGF23. Additional structure-activity studies developed a series of ZINC13407541 analogs with enhanced drug-like properties. In this study, we tested in a preclinical Hyp mouse homolog of XLH a direct connect analog [(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] (8n), which exhibited the greatest stability in microsomal assays, and [(E)-2-((E)-4-methylstyryl)benzaldehyde oxime] (13a), which exhibited increased in vitro potency. Using cryo-electron microscopy structure and computational docking, we identified a key binding residue (Q156) of the FGF23 antagonists, ZINC13407541, and its analogs (8n and 13a) in the N-terminal domain of FGF23 protein. Site-directed mutagenesis and bimolecular fluorescence complementation-fluorescence resonance energy transfer assay confirmed the binding site of these three antagonists. We found that pharmacological inhibition of FGF23 with either of these compounds blocked FGF23 signaling and increased serum phosphate and 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations in Hyp mice. Long-term parenteral treatment with 8n or 13a also enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate in Hyp mice. The more potent 13a compound had greater therapeutic effects in Hyp mice. Further optimization of these FGF23 inhibitors may lead to versatile drugs to treat excess FGF23-mediated disorders. SIGNIFICANCE STATEMENT: This study used structure-based drug design and medicinal chemistry approaches to identify and optimize small molecules with different stability and potency, which antagonize excessive actions of fibroblast growth factor 23 (FGF23) in hereditary hypophosphatemic rickets. The findings confirmed that these antagonists bind to the N-terminus of FGF23 to inhibit its binding to and activation of the fibroblast growth factor receptors/α-Klotho signaling complex. Administration of these lead compounds improved phosphate homeostasis and abnormal skeletal phenotypes in a preclinical Hyp mouse model.