Growth Plate Chondrocytes Research Articles

Loss of Sprouty Produces a Ciliopathic Skeletal Phenotype in Mice Through Upregulation of Hedgehog Signaling.

The Sprouty family is a highly conserved group of intracellular modulators of receptor tyrosine kinase (RTK)-signaling pathways, which have been recently linked to primary cilia. Disruptions in the structure and function of primary cilia cause inherited disorders called ciliopathies. We aimed to evaluate Sprouty2 and Sprouty4 gene-dependent alterations of ciliary structure and to focus on the determination of its association with Hedgehog signaling defects in chondrocytes. Analysis of the transgenic mice phenotype with Sprouty2 and Sprouty4 deficiency revealed several defects, including improper endochondral bone formation and digit patterning, or craniofacial and dental abnormalities. Moreover, reduced bone thickness and trabecular bone mass, skull deformities, or chondroma-like lesions were revealed. All these pathologies might be attributed to ciliopathies. Elongation of the ciliary axonemes in embryonic and postnatal growth plate chondrocytes was observed in Sprouty2-/- and Sprouty2+/- /Sprouty4-/- mutants compared with corresponding littermate controls. Also, cilia-dependent Hedgehog signaling was upregulated in Sprouty2/4 mutant animals. Ptch1 and Ihh expression were upregulated in the autopodium and the proximal tibia of Sprouty2-/- /Sprouty4-/- mutants. Increased levels of the GLI3 repressor (GLI3R) form were detected in Sprouty2/4 mutant primary fibroblast embryonic cell cultures and tissues. These findings demonstrate that mouse lines deficient in Sprouty proteins manifest phenotypic features resembling ciliopathic phenotypes in multiple aspects and may serve as valuable models to study the association between overactivation of RTK and dysfunction of primary cilia during skeletogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Reconstitution of the host holobiont in germ-free born male rats acutely increases bone growth and affects marrow cellular content.

Integration of microbiota in a host begins at birth and progresses during adolescence, forming a multidirectional system of physiological interactions. Here, we present an instantaneous effect of natural, bacterial gut colonization on the acceleration of longitudinal and radial bone growth in germ-free born, 7-wk-old male rats. Changes in bone mass and structure were analyzed after 10 days following the onset of colonization through cohousing with conventional rats and revealed unprecedented acceleration of bone accrual in cortical and trabecular compartments, increased bone tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. In addition, the number of small in size adipocytes increased, whereas the number of megakaryocytes decreased, in the bone marrow of conventionalized germ-free rats indicating that not only bone mass but also bone marrow environment is under control of gut microbiota signaling. The changes in bone status paralleled with a positive shift in microbiota composition toward short-chain fatty acids (SCFA)-producing microbes and a considerable increase in cecal SCFA concentrations, specifically butyrate. Furthermore, reconstitution of the host holobiont increased hepatic expression of IGF-1 and its circulating levels. Elevated serum levels of 25-hydroxy vitamin D and alkaline phosphatase pointed toward an active process of bone formation. The acute stimulatory effect on bone growth occurred independently of body mass increase. Overall, the presented model of conventionalized germ-free rats could be used to study microbiota-based therapeutics for combatting dysbiosis-related bone disorders.

Correction of a knock-in mouse model of acrodysostosis with gene therapy using a rAAV9-CAG-human PRKAR1A vector.

Acrodysostosis is a rare skeletal dysplasia caused by loss-of-function mutations in the regulatory subunit of protein kinase A (PRKAR1A). In a knock-in mouse model (PRKAR1Awt/mut) expressing one copy of the recurrent R368X mutation, we tested the effects of a rAAV9-CAG-human PRKR1A (hPRKAR1A) vector intravenously administered at 4 weeks of age. Caudal vertebrae and tibial diaphyses contained 0.52 ± 0.7 and 0.13 ± 0.3 vector genome per cell (VGC), respectively, at 10 weeks of age and 0.22 ± 0.04 and 0.020 ± 0.04 at 16 weeks while renal cortex contained 0.57 ± 0.14 and 0.26 ± 0.05 VGC. Vector-mediated hPRKAR1A expression was found in growth plate chondrocytes, osteoclasts, osteoblasts, and kidney tubular cells. Chondrocyte architecture was restored in the growth plates. Body length, tail length, and body weight were improved in vector treated PRKAR1Awt/mut mice, not the bone length of their limbs. These results provide one of the few proofs for gene therapy efficacy in a mouse model of chondrodysplasia. In addition, the increased urinary cAMP of PRKAR1Awt/mut mice was corrected almost to normal. In conclusion, gene therapy with hPRKAR1A improved skeletal growth and kidney dysfunction, the hallmarks of acrodysostosis in R368X mutated mice and humans.

The hypertrophic chondrocyte: To be or not to be.

Hypertrophic chondrocytes are the master regulators of endochondral ossification; however, their ultimate cell fates cells remain largely elusive due to their transient nature. Historically, hypertrophic chondrocytes have been considered as the terminal state of growth plate chondrocytes, which are destined to meet their inevitable demise at the primary spongiosa. Chondrocyte hypertrophy is accompanied by increased organelle synthesis and rapid intracellular water uptake, which serve as the major drivers of longitudinal bone growth. This process is delicately regulated by major signaling pathways and their target genes, including growth hormone (GH), insulin growth factor-1 (IGF-1), indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic proteins (BMPs), sex determining region Y-box 9 (Sox9), runt-related transcription factors (Runx) and fibroblast growth factor receptors (FGFRs). Hypertrophic chondrocytes orchestrate endochondral ossification by regulating osteogenic-angiogenic and osteogenic-osteoclastic coupling through the production of vascular endothelial growth factor (VEGF), receptor activator of nuclear factor kappa-B ligand (RANKL) and matrix metallopeptidases-9/13 (MMP-9/13). Hypertrophic chondrocytes also indirectly regulate resorption of the cartilaginous extracellular matrix, by controlling formation of a special subtype of osteoclasts termed "chondroclasts". Notably, hypertrophic chondrocytes may possess innate potential for plasticity, reentering the cell cycle and differentiating into osteoblasts and other types of mesenchymal cells in the marrow space. We may be able to harness this unique plasticity for therapeutic purposes, for a variety of skeletal abnormalities and injuries. In this review, we discuss the morphological and molecular properties of hypertrophic chondrocytes, which carry out important functions during skeletal growth and regeneration.

Fractions of Shen-Sui-Tong-Zhi Formula Enhance Osteogenesis Via Activation of β-Catenin Signaling in Growth Plate Chondrocytes.

Background: Shen-sui-tong-zhi formula (SSTZF) has been used to treat osteoporosis for decades and shows excellent clinical efficacy. This article aims to explore the optimal anti-osteoporotic ingredient and its precise mechanisms in mice models. Methods: In this study, we first screened the optimal anti-osteoporosis fraction of SSTZF extract in vivo, and then further explored the mechanism of its effects both in vivo and in vitro. Ten-week-old female C57BL/6J mice were administrated with each fraction of SSTZF. At 10 weeks after ovariectomy (OVX), femurs were collected for tissue analyses, including histology, micro-CT, biomechanical tests, and immunohistochemistry for ALP, FABP4, and β-catenin. Additionally, we also evaluated the mRNA expression level of ALP and FABP4 and the protein expression level of β-catenin after being treated with SSTZF extract in C3H10T1/2 cells. Moreover, we investigated the anti-osteoporosis effect of SSTZF extract on mice with β-catenin conditional knockout in growth plate chondrocytes (β-catenin Gli1ER mice) through μCT, histology, and immunohistochemistry analyzes. Results: At 10 weeks after treatment, osteoporosis-like phenotype were significantly ameliorated in SSTZF n-butanol extract (SSTZF-NB) group mice, as indicated by increased trabecular bone area and ALP content, and decreased lipid droplet area and FABP4 content. No such improvements were observed after being treated with other extracts, demonstrating that SSTZF-NB is the optimal anti-osteoporosis fraction. Additionally, the elevated β-catenin was revealed in both OVX mice and C3H10T1/2 cells with SSTZF-NB administered. Furthermore, a significant osteoporosis-like phenotype was observed in β-catenin Gli1ER mice as expected. However, SSTZF-NB failed to rescue the deterioration in β-catenin Gli1ER mice, no significant re-upregulated ALP and downregulated FABP4 were observed after being treated with SSTZF-NB, demonstrating that SSTZF-NB prevents bone loss mainly via β-catenin signaling. Conclusion: SSTZF-NB enhances osteogenesis mainly via activation of β-catenin signaling in growth plate chondrocytes. SSTZF-NB is the optimal anti-osteoporosis fraction of SSTZF and it can be considered a salutary alternative therapeutic option for osteoporosis.

Piezo1 inactivation in chondrocytes impairs trabecular bone formation

In brief: Chondrocyte-specific ablation of the mechano-sensory Piezo1 results in substantially impaired formation of secondary spongiosa during endochondral bone formation. The study explores this unexpected finding and show that mechano-sensing in growth plate chondrocytes directly regulates the formation of bone trabeculae in the secondary spongiosa.

Readdressing Short Stature in India: The "Long and the Short" of it.

Readdressing Short Stature in India: The "Long and the Short" of it.

Manganese deficiency induces avian tibial dyschondroplasia by inhibiting chondrocyte proliferation and differentiation

Manganese (Mn) is an essential trace element for bone growth, and its deficiency has been shown to increase the incidence of leg abnormalities in fast-growing broilers, such as tibial dyschondroplasia (TD). Proliferation and differentiation of growth plate chondrocyte are critical for tibia development, but their roles in Mn deficiency-induced TD remains to be elucidated. Thirty 1-day-old Arbor Acres chicks were randomly divided into two groups and fed with control diet (60 mg Mn/kg diet) and Mn-deficiency diet (22 mg Mn/kg diet) for 42 days, respectively. Mn deficiency-induced TD model was successfully established and samples from proximal tibia metaphysis and growth plate were collected for assays. Pathological observation showed that Mn deficiency induced morphological abnormality and irregular arrangement of chondrocytes in proliferative and hypertrophic zone of tibial growth plate. Also, Mn deficiency decreased mRNA and protein expression levels of type II collagen and type X collagen in tibial growth plate, indicating the impairment of proliferating and hypertrophic chondrocytes. Moreover, down-regulated gene expression levels of Sox9, Tgf-β, Ihh, Runx2, Mef2c and Bmp-2 were shown in tibial growth plate of Mn-deficiency group, demonstrating that Mn deficiency inhibited the transcription levels of key regulators to disrupt chondrocyte proliferation and differentiation. Collectively, these findings confirmed that Mn deficiency affected the proliferation and differentiation of chondrocytes in tibial growth plate via inhibiting related regulatory factors, leading to TD in broilers.

Deletion of Glut1 in early postnatal cartilage reprograms chondrocytes toward enhanced glutamine oxidation

Glucose metabolism is fundamental for the functions of all tissues, including cartilage. Despite the emerging evidence related to glucose metabolism in the regulation of prenatal cartilage development, little is known about the role of glucose metabolism and its biochemical basis in postnatal cartilage growth and homeostasis. We show here that genetic deletion of the glucose transporter Glut1 in postnatal cartilage impairs cell proliferation and matrix production in growth plate (GPs) but paradoxically increases cartilage remnants in the metaphysis, resulting in shortening of long bones. On the other hand, articular cartilage (AC) with Glut1 deficiency presents diminished cellularity and loss of proteoglycans, which ultimately progress to cartilage fibrosis. Moreover, predisposition to Glut1 deficiency severely exacerbates injury-induced osteoarthritis. Regardless of the disparities in glucose metabolism between GP and AC chondrocytes under normal conditions, both types of chondrocytes demonstrate metabolic plasticity to enhance glutamine utilization and oxidation in the absence of glucose availability. However, uncontrolled glutamine flux causes collagen overmodification, thus affecting extracellular matrix remodeling in both cartilage compartments. These results uncover the pivotal and distinct roles of Glut1-mediated glucose metabolism in two of the postnatal cartilage compartments and link some cartilage abnormalities to altered glucose/glutamine metabolism.

G-Protein-Coupled Estrogen Receptor-1 Positively Regulates the Growth Plate Chondrocyte Proliferation in Female Pubertal Mice.

Estrogen enhances long bone longitudinal growth during early puberty. Growth plate chondrocytes are the main cells that contribute to long bone elongation. The role of G-protein-coupled estrogen receptor-1 (GPER-1) in regulating growth plate chondrocyte function remains unclear. In the present study, we generated chondrocyte-specific GPER-1 knockout (CKO) mice to investigate the effect of GPER-1 in growth plate chondrocytes. In control mice, GPER-1 was highly expressed in the growth plates of 4- and 8-week-old mice, with a gradual decline through 12 to 16 weeks. In CKO mice, the GPER-1 expression in growth plate chondrocytes was significantly lower than that in the control mice (80% decrease). The CKO mice also showed a decrease in body length (crown–rump length), body weight, and the length of tibias and femurs at 8 weeks. More importantly, the cell number and thickness of the proliferative zone of the growth plate, as well as the thickness of primary spongiosa and length of metaphysis plus diaphysis in tibias of CKO mice, were significantly decreased compared with those of the control mice. Furthermore, there was also a considerable reduction in the number of proliferating cell nuclear antigens and Ki67-stained proliferating chondrocytes in the tibia growth plate in the CKO mice. The chondrocyte proliferation mediated by GPER-1 was further demonstrated via treatment with a GPER-1 antagonist in cultured epiphyseal cartilage. This study demonstrates that GPER-1 positively regulates chondrocyte proliferation at the growth plate during early puberty and contributes to the longitudinal growth of long bones.

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E2), testosterone, and dihydrotestosterone (DHT). Mechanistically, E2 and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E2 signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E2 enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E2 treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E2 and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E2 and DHT membrane-associated PKC activity in costochondral cartilage cells.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

Changes in the avascular area of the meniscus using mesenchymal stem cells and growth plate chondrocytes in a pig model.

Menisci are wedge-shaped cartilage discs that are divided into two parts: the avascular and vascular regions. They are formed by fibrocartilage tissue, which contains round cartilage-like cells and extracellular matrix. Meniscus injury in animals is a common orthopedic problem, but data on the natural healing process mainly deals with the vascular zone. The healing processes in the avascular zone of the meniscus are significantly limited. Thus, this study aimed to evaluate autologous growth plate chondrocytes' impact on the healing process of a damaged meniscus in the avascular zone based on a growing animal model. The study group consisted of 10 pigs at about three months of age. From each animal, chondrocytes from the iliac growth plate and from concentrated bone marrow were taken. Knee joints were divided into right (R) and left (L). The medial meniscus of the R knee joint was treated with a hyaluronic acid based scaffold incubated with bone marrow cells from marrow aspirates (nCHON). The medial meniscus of the L knee joint was treated with a hyaluronic acid based scaffold incubated with bone marrow cells from marrow aspirates supplemented with immature chondrocytes isolated from growth plates (wCHON). The meniscus was damaged in the avascular zone in both knee joints. Followingly, the damaged part of the meniscus was filled with a scaffold with cells from the concentrated bone marrow and from growth plate chondrocytes. In the control group, a scaffold with concentrated bone marrow cells was used. After three months the animals were euthanized and preparations (microscopic slides) were made from the meniscus' damaged part. A qualitative and quantitative analysis have been prepared. The wCHON group in comparison with the nCHON group showed a statistically significantly higher number of fusiform cells on the surface of the graft as well as better healing of the graft. In addition, the degree of vascularization was higher in specimens from the wCHON group than in the nCHON group. The results of our research on immature pig knees revealed that mesenchymal stem cell and growth plate chondrocytes could be treated as the cell source for meniscus reconstruction, and growth plate chondrocytes enhance healing processes in the avascular zone of the injured meniscus.

Snail/HDAC1/2 mediate skeletal growth retardation in fetuses caused by prenatal nicotine exposure

Previous findings have confirmed that prenatal nicotine exposure (PNE) leads to retarded cartilage development in the fetal growth plate. It is characterized by insufficient matrix synthesis and decreased expression of matrix phenotype genes aggrecan (ACAN) and Col2A1 in the fetal growth plate chondrocytes; however, the specific molecular mechanism is yet unclear. This study intends to clarify the specific molecular mechanism of fetal osteochondral retardation caused by PNE through animal and cellular experiments. The present study demonstrated that in male offspring of the PNE group (the pregnant rats were subcutaneously administered nicotine 1.0 mg/kg twice per day (2.0 mg/kg.d) at GD11–20), the cartilage matrix of the fetal growth plate was lightly stained, the collagen was reduced, and expression of the matrix phenotype genes, ACAN and Col2A1, was significantly decreased. It was further found that PNE decreased histone acetylation (H3K9/H3K14) levels in the ACAN and Col2A1 promoter regions. Moreover, the expression of Snail and HDAC1/2 was increased in the PNE group. in vitro, the nicotine treatment at different concentrations elevated the expression of Snail/HDAC1/2 while decreasing the H3K9/H3K14 levels in the ACAN and Col2A1 promoter regions. Snail-siRNA transfection partially abolished the nicotine-induced increase in HDAC1/2 expression and decreased the histone acetylation levels in the ACAN and Col2A1 promoter regions. Trichostatin A (TSA) treatment partially reversed the nicotine-induced changes in downstream parameters. In summary, PNE-induced decreased cartilage matrix synthesis in the fetal growth plate of male offspring is effectuated by Snail/HDAC1/2-mediated decreased H3K9/H3K14 levels in the ACAN and Col2A1 promoter regions.

Statistical image analysis and escort histograms in characterization of articular cartilage repair in a skeleton animal model.

Statistical image analysis of an ensemble of digital images of histological samples is performed as an auxiliary investigation a result of the recently proposed method of articular cartilage repair utilizing growth plate chondrocytes in a skeleton animal model. A fixed–shift model of maximal likelihood estimates of image histograms applied for monochromatic (grayscale) images or their RGB components confirms the statistically significant effect of the previously proposed medical treatment. The type of staining used to prepare images of histological samples is related to the visibility of the effectiveness of medical treatment. Hellinger distance of escort distributions for maximal likelihood estimates of image histograms of medically treated and control samples is investigated to identify grayscale (or RGB) intensities responsible for statistically significant difference of the estimates. A difference of Shannon entropy quantifying informational content of the histograms allows one to identify staining and image colors which are most suitable to visualize cluster formation typical for articular cartilage repair processes.

DDIT3/CHOP promotes autophagy in chondrocytes via SIRT1-AKT pathway

Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.

Specific MicroRNAs Found in Extracellular Matrix Vesicles Regulate Proliferation and Differentiation in Growth Plate Chondrocytes.

Matrix vesicles (MVs) are extracellular organelles produced by growth plate cartilage cells in a zone-specific manner. MVs are similar in size to exosomes, but they are tethered to the extracellular matrix (ECM) via integrins. Originally associated with matrix calcification, studies now show that they contain matrix processing enzymes and microRNA that are specific to their zone of maturation. MVs produced by costochondral cartilage resting zone (RC) chondrocytes are enriched in microRNA 503 whereas those produced by growth zone (GC) chondrocytes are enriched in microRNA 122. MVs are packaged by chondrocytes under hormonal and factor regulation and release of their contents into the ECM is also under hormonal control, suggesting that their microRNA might have a regulatory role in growth plate proliferation and maturation. To test this, we selected a subset of these enriched microRNAs and transfected synthetic mimics back into RC and GC cells. Transfecting growth plate chondrocytes with select microRNA produced a broad range of phenotypic responses indicating that MV-based microRNAs are involved in the regulation of these cells. Specifically, microRNA 122 drives both RC and GC cells toward a proliferative phenotype, stabilizes the matrix and inhibits differentiation whereas microRNA 22 exerts control over regulatory factor production. This study demonstrates the strong regulatory capability possessed by unique MV enriched microRNAs on growth plate chondrocytes and their potential for use as therapeutic agents.

Growth Plate Genes Are Key Regulators of Growth: Lessons Learned From Children of Consanguineous Families From Kurdistan, Iraq

Introduction: The genetic basis of human growth regulation has only been partly elucidated thus far. Therefore, finding causative genes in patients with short stature help in understanding precise pathophysiological mechanisms, establishing genotype-phenotype relationships and optimizing treatment. In order to extend our knowledge about the genes involved in short stature, we studied a unique cohort of consanguineous families with children with short stature from Sulaymani in Kurdistan, Iraq. Patients and Methods: Fifty-five consanguineous families, with children shorter than -2.3SDS at the time of examination (median height -3.3SDS, range -2.3 to -15SDS), who were examined at the endocrine outpatient clinic of the Department of Pediatrics, Sulaymani University College of Medicine between January 2018 and February 2020, were included in the study. In families with more than one child with short stature, the shorter sibling was selected as the proband (median age 8 years, range 1 - 15 years). Three probands were subsequently excluded due to the diagnosis of Turner’s syndrome and Edward’s syndrome Consent was obtained from all families and probands’ DNA was analyzed by Whole Exome Sequencing (WES) methods. The data were processed by a bioinformatic pipeline and detected variants were filtered using variant analysis software. Selected potentially pathogenic variants were confirmed using Sanger sequencing methods and evaluated by the American College of Medical Genetics (ACMG) standards and guidelines. Results: A monogenic cause of short stature, which explained the patient phenotype, was elucidated in 13 of 26 families who were analyzed thus far. Seven families had multiple affected children making a total of 22 patients with a positive genetic diagnosis. Pathogenic or likely pathogenic variants (according to the ACMG standards) were found in genes involved in the GH-IGF-1 axis (GHR), in the extracellular matrix of the growth plate (COL1A2, MMP13, LTBP3, and ADAMTS17), in the regulation of chondrocytes (NPR2 and CTSK), transporter coding genes (SLC34A3), and other genes (PTCH1, GALNS, DNACJ21, ZSWIM6, GNPTG). Among them, there are 9 novel variants and 10 homozygous variants including variants in genes causing syndromic short stature. Unexpectedly, we successfully identified three cases of autosomal dominant short stature (variants in genes NPR2, COL1A2, PTCH1) as well. The remaining probands from 26 families are still being analyzed. Conclusion: With the help of NGS methods, we have successfully elucidated the genetic cause of short stature in nearly 50% of patients who were analyzed thus far. These results further strength the concept that genes affecting the growth plate (chondrocytes and the extracellular matrix) play a crucial role in growth regulation. Acknowledgements: The study was co-funded by grants AZV 18-07-00283 and GAUK 340420.

Specificity for the Epiphyseal Round Cell Layer is Significantly Associated With Height GWAS​

Human height is a model polygenic trait with thousands of height-related SNPs identified in GWAS to date. An important determinant of height is the proliferation and hypertrophy of growth plate chondrocytes during childhood long bone elongation. Connecting the expression of specific genes that affect skeletal biology to associated variants in GWAS remains a difficult challenge. To connect the genetics of height and growth plate gene expression, we studied the relationship between gene expression in the murine growth plate and common-variant associations from GWAS of height. To obtain gene expression data from the growth plate, we dissected three layers of murine tibial growth plates, extracted RNA from each layer, and measured expression using the Affymetrix GeneChip 430 3.0. For each gene, we derived a specificity score for each growth plate layer, and SNP-level p-values from a published GWAS of height (N~700000) were combined into gene-level p-values using MAGMA. We then used MAGMA to test for association between specificity of expression for each growth plate layer and the GWAS gene level p-values for height. We found that specificity for the round cell layer is significantly associated with height GWAS p-values (p = 8.5x10-9). This association remains when we condition on each of the other cell layers and on membership in a set of genes from OMIM that cause skeletal growth disorders (3.3x10-8 < p < 4.1x10-6). We replicated this result in a RNA-seq dataset of maturing chondrocytes sampled at three time points during development in vitro (days 3, 5, and 10): we found that z-scores for expression in the earliest two days of development are significantly associated with gene-level p-values from height GWAS (pDay3 = 1.2x10-21 and pDay5 = 2.0x10-20) and that this association remains after conditioning on the other timepoints and on the OMIM gene set (3.1x10-20 < pDay3 < 8.3x10-5; 3.7x10-19 < pDay5 < 0.002). We then performed pathway analysis of genes that are both highly specific to the round layer and highly significant in GWAS using Enrichr. Together, our results suggest that genes expressed in early chondrocyte development (the round cell layer) are particularly relevant to the contribution of growth plate-expressed genes to height. This conclusion both sheds light on the regulation of human skeletal growth and also helps prioritize relevant genes implicated from the height GWAS in skeletal biology.

Gut Microbiota Accelerates Bone Growth and Marrow Adiposity of the Adolescent Gnotobiotic Rat

Skeletal growth and bone homeostasis are significant holobiome components strongly tied to the gut microbiota. We aimed to explore the requirement of gut microbiome for skeletal maturation in an adolescent organism. For our approach, we co-housed young (7-week-old) germ-free (GF) rats with conventional rats of the same age, sex (male), and strain (Sprague Dawley) for 10 days to naturally conventionalize GF (GFC) rats with the gut microbiota by coprophagy. Bone morphometry by micro-CT scanning and histological staining revealed accelerated longitudinal and radial bone growth in GFC rats when compared to GF rats. Changes in bone mass and structure for GFC rats encompassed an increase of bone accrual in cortical and trabecular compartments, greater tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. Analysis of bone marrow at the tibia midshaft depicted more adipocyte number, greater adipocyte density, and altered cell size distribution to favor small adipocytes, but reduced megakaryocytes, in GFC rats. Interestingly, putative effector genes responsible for bone mass accumulation were found to be comparable between GF and GFC rats, which shifted our focus onto systemic host and microbial regulatory factors. Assessment of serum markers indicated that the active bone formation could be due to elevated levels of 25-hydroxy vitamin D and alkaline phosphatase. Importantly, 16S ribosomal RNA gene sequencing demonstrated that the prominent bacterial colonizers were Clostridiaceae and Clostridium members, which are potent short chain fatty acid (SCFA) producers. 1H-NMR metabolomics in the cecal contents showed a dramatic increase of SCFA, specifically butyrate, following bacterial colonization. In relation with the literature, our observation of increased hepatic insulin-like growth factor-1 (IGF-1) expression and systemic IGF-1 levels suggest that butyrate promotes bone growth through the somatotropic axis. Collectively, our study demonstrates that gut microbiota can deliver signals to the skeleton for accelerating bone expansion and accumulating marrow fat.