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Purpose Impaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions. Methods Exome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH. Results We identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that co-segregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in 3 unrelated families with IHH. Conclusion The findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion.

The resting zone houses a group of slowly proliferating ‘reserve’ chondrocytes and has long been speculated to serve as the stem cell niche of the postnatal growth plate. But are these resting chondrocytes bona fide stem cells? Recent technological advances in lineage tracing and next-generation sequencing have finally allowed researchers to answer this question. Several recent studies have also shed light into the signaling pathways and molecular mechanisms involved in the maintenance of resting chondrocytes, thus providing us with important new insights into the role of the resting zone in the paracrine and endocrine regulation of childhood bone growth.

The ability to identify, isolate, and study pure populations of cells is critical for understanding normal physiology in organs and tissues, which involves spatial regulation of signaling pathways and interactions between cells with different functions, expression profiles, and lineages. Here, we focus on assessing the growth plate cartilage, composed of multiple functionally and histologically distinct zones, to investigate temporally and spatially dependent gene expression differences. In this chapter, we describe the method of laser capture microdissection to isolate chondrocytes from different zones of differentiation in the mouse growth plate cartilage for RNA isolation, and subsequent downstream applications, such as RNA-sequencing and quantitative real-time PCR. We also provide an assessment of different factors contributing to the integrity of the isolated RNA, such as staining methods and procedures in RNA isolation.

The ability to identify, isolate, and study pure populations of cells is critical for understanding normal physiology in organs and tissues, which involves spatial regulation of signaling pathways and interactions between cells with different functions, expression profiles, and lineages. Here, we focus on assessing the growth plate cartilage, composed of multiple functionally and histologically distinct zones, to investigate temporally and spatially dependent gene expression differences. In this chapter, we describe the method of laser capture microdissection to isolate chondrocytes from different zones of differentiation in the mouse growth plate cartilage for RNA isolation, and subsequent downstream applications, such as RNA-sequencing and quantitative real-time PCR. We also provide an assessment of different factors contributing to the integrity of the isolated RNA, such as staining methods and procedures in RNA isolation.

Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy, particularly in severe, non-GH-deficient conditions such as chondrodysplasias, and potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we developed a cartilage-targeting single-chain human antibody fragment (CaAb) aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. To this end, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor 1 (IGF-1), an endocrine and/or paracrine factor that positively regulates chondrogenesis. These CaAb-IGF-1 fusion proteins retained both cartilage binding and IGF-1 biological activity, and they were able to stimulate bone growth in an organ culture system. Using a GH-deficient (lit) mouse model, we found that subcutaneous injections of these CaAb-IGF-1 fusion proteins increased overall growth plate height without increasing proliferation in kidney cortical cells, suggesting on-target efficacy at the growth plate and less off-target effect on the kidney than IGF-1 alone. Alternate-day injections of these fusion proteins, unlike IGF-1 alone, were sufficient to produce a therapeutic effect. Our findings provide proof of principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders.

Growth plate chondrocytes undergo sequential differentiation to form the resting zone, the proliferative zone (PZ), and the hypertrophic zone (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and miRNA sequencing. Thirty-four miRNAs were differentially expressed between the PZ and the HZ, and we hypothesized that some of the miRNAs that are preferentially expressed in the PZ may promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in the PZ is driven by the parathyroid hormone–related protein (PTHrP) concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a parathyroid hormone (PTH)/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p, and mir-503-5p between the PZ and the HZ. In the PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In the HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.

Context Weaver syndrome is characterized by tall stature, advanced bone age, characteristic facies, and variable intellectual disability. It is caused by heterozygous mutations in enhancer of zeste homolog 2 (EZH2), a histone methyltransferase responsible for histone H3 at lysine 27 (H3K27) trimethylation. However, no early truncating mutations have been identified, suggesting that null mutations do not cause Weaver syndrome. Objective To test alternative hypotheses that EZH2 variants found in Weaver syndrome cause either a gain of function or a partial loss of function. Design Exome sequencing was performed in a boy with tall stature, advanced bone age, and mild dysmorphic features. Mutant or wild-type EZH2 protein was expressed in mouse growth plate chondrocytes with or without endogenous EZH2, and enzymatic activity was measured. A mouse model was generated, and histone methylation was assessed in heterozygous and homozygous embryos. Results A de novo missense EZH2 mutation [c.1876G>A (p.Val626Met)] was identified in the proband. When expressed in growth plate chondrocytes, the mutant protein showed decreased histone methyltransferase activity. A mouse model carrying this EZH2 mutation was generated using CRISPR/Cas9. Homozygotes showed perinatal lethality, whereas heterozygotes were viable, fertile, and showed mild overgrowth. Both homozygous and heterozygous embryos showed decreased H3K27 methylation. Conclusion We generated a mouse model with the same mutation as our patient, found that it recapitulates the Weaver overgrowth phenotype, and demonstrated that EZH2 mutations found in Weaver syndrome cause a partial loss of function.

In many children with short stature, the genetic etiology remains unknown. Short stature due to defects in trafficking of membrane-associated proteins, such as receptors, has not been reported. A mother and two daughters presented with disproportionate short stature and Noonan-like facies. Exome sequencing, confirmed by Sanger sequencing, identified a de novo dominant mutation (L69F) in CCDC53. CCDC53 normally binds to WASH1 within a protein complex termed the WASH complex. To study the effect of the mutation on this interaction, CCDC53 was first ablated in HEK293 cells (CCDC53KO-HEK) by CRISPER-Cas9 and then wild-type (WT) or mutant (mut) CCDC53 or empty vector (EV) was expressed in these cells. The identified mutation impaired CCDC53 interaction with WASH1 as assessed by co-immunoprecipitation. CCDC53 and the WASH complex are involved in trafficking of cell-surface proteins that have undergone endocytosis, including G-coupled protein receptors that contain a PDZ motif, such as PTH1R. We therefore hypothesized that the mutation would affect PTH1R endocytic trafficking. CCDC53KO-HEK cells were transfected with vectors to express PTH1R and either EV, WT or mut CCDC53 and treated with PTH, after which endosomes were extracted and PTH1R was detected by western blot. Compared to WT, EV and mut CCDC53 showed decreased PTH1R localization in endosomes. In this system, the mutation also decreased generation of cAMP by extracellular PTH (P=0.001 vs WT), indicating that the mutation interferes with PTH1R signal transduction. PTH1R acts as the receptor for PTHrP and prevents premature hypertrophy of growth plate chondrocytes. We therefore hypothesized that the CCDC53 mutation would allow premature hypertrophy of growth plate chondrocytes. Primary rat epiphyseal chondrocytes were transfected with siRNA against Ccdc53 and with vectors to express either WT or mut Ccdc53. Compared to WT, mut Ccdc53 led to increased expression of hypertrophy-associated genes: Col10a1 (P=0.001), Ihh (P=0.031), and Mmp13 (P=0.003), consistent with the hypothesized effect on hypertrophic differentiation. In conclusion, a mutation in CCDC53 was identified in a family with disproportionate short stature and Noonan-like facies. The mutation interfered with the ability of CCDC53 to participate in the WASH complex, affecting endosomal localization of PTH1R, PTH1R signal transduction, and chondrocyte hypertrophy. Because the timing of growth plate chondrocyte hypertrophy is critical for linear growth, the findings provide a potential explanation for the short stature in this disorder. More generally, the findings also indicate that endocytic trafficking of PTH1R by the WASH complex plays an important role in the regulation of human linear growth.

Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy particularly in severe, non-GH deficient conditions such as chondrodysplasias, and has potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we previously developed a cartilage-targeting single-chain human antibody fragment (CaAb) (1), aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. In the current study, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor-I (IGF-1), an endocrine/paracrine factor which positively regulates chondrogenesis. We first used ELISA to assess the ability of the CaAb-IGF1 fusion proteins to bind tissue lysates from different mouse organs and found that the fusion proteins bound to cartilage with high tissue specificity. We also found that these fusion proteins retain potent IGF-1 biological activity, as demonstrated by their ability to stimulate AKT and ERK phosphorylation in cell culture and stimulate metatarsal bone growth in organ culture. To assess in vivo efficacy, we administered daily subcutaneous injections to growth hormone-deficient (lit) mice for 5 days. In this model, twice-daily IGF-1 increased growth plate height but daily IGF-1 did not. In contrast, daily equimolar injections of the targeted fusion proteins did increase growth plate height whereas a fusion protein lacking the cartilage-targeting domain did not (P

Nutritional excess of vitamin A, a precursor for retinoic acid (RA), causes premature epiphyseal fusion, craniosynostosis, and light-dependent retinopathy. Similarly, homozygous loss-of-function mutations in CYP26B1, one of the major RA-metabolizing enzymes, cause advanced bone age, premature epiphyseal fusion, and craniosynostosis. In this paper, a patient with markedly accelerated skeletal and dental development, retinal scarring, and autism-spectrum disease is presented and the role of retinoic acid in longitudinal bone growth and skeletal maturation is reviewed. Genetic studies were carried out using SNP array and exome sequencing. RA isomers were measured in the patient, family members, and in 18 age-matched healthy children using high-performance liquid chromatography coupled to tandem mass spectrometry. A genomic SNP array identified a novel 8.3 megabase microdeletion on chromosome 10q23.2–23.33. The 79 deleted genes included CYP26A1 and C1 , both major RA-metabolizing enzymes. Exome sequencing did not detect any variants that were predicted to be deleterious in the remaining alleles of these genes or other known retinoic acid-metabolizing enzymes. The patient exhibited elevated plasma total RA (16.5 vs. 12.6±1.5 nM, mean±SD, subject vs. controls) and 13- cis RA (10.7 nM vs. 6.1±1.1). The findings support the hypothesis that elevated RA concentrations accelerate bone and dental maturation in humans. CYP26A1 and C1 haploinsufficiency may contribute to the elevated retinoic acid concentrations and clinical findings of the patient, although this phenotype has not been reported in other patients with similar deletions, suggesting that other unknown genetic or environmental factors may also contribute.

Longitudinal bone growth is driven by endochondral ossification, a process in which cartilage tissue is generated by growth plate chondrocytes and then remodeled into bone by osteoblasts. In the postnatal growth plate, as hypertrophic chondrocytes approach the chondro-osseous junction, they may undergo apoptosis, or directly transdifferentiate into osteoblasts. The molecular mechanisms governing this switch in cell lineage are poorly understood. Here we show that the physiological downregulation of Sox9 in hypertrophic chondrocyte is associated with upregulation of osteoblast-associated genes (such as Mmp13, Cola1, Ibsp) in hypertrophic chondrocytes, before they enter the metaphyseal bone. In transgenic mice that continued to express Sox9 in all cells derived from the chondrocytic lineage, upregulation of these osteoblast-associated genes in the hypertrophic zone failed to occur. Furthermore, lineage tracing experiments showed that, in transgenic mice expressing Sox9, the number of chondrocytes transdifferentiating into osteoblasts was markedly reduced. Collectively, our findings suggest that Sox9 downregulation in hypertrophic chondrocytes promotes expression of osteoblast-associated genes in hypertrophic chondrocytes and promotes the subsequent transdifferentiation of these cells into osteoblasts.

In many children with short stature, the etiology of the decreased linear growth remains unknown. We sought to identify the underlying genetic etiology in a patient with short stature, irregular growth plates of the proximal phalanges, developmental delay, and mildly dysmorphic facial features. Exome sequencing identified a de novo, heterozygous, nonsense mutation (c.1606C> T:p.R536X) in QRICH1. In vitro studies confirmed that the mutation impaired expression of the QRICH1 protein. siRNA-mediated knockdown of Qrich1 in primary mouse epiphyseal chondrocytes caused downregulation of gene expression associated with hypertrophic differentiation. We then identified an unrelated individual with another heterozygous de novo nonsense mutation in QRICH1 who had a similar phenotype. A recently published study identified QRICH1 mutations in three patients with developmental delay, one of whom had short stature. Our findings indicate that QRICH1 mutations cause not only developmental delay but also a chondrodysplasia characterized by diminished linear growth and abnormal growth plate morphology due to impaired growth plate chondrocyte hypertrophic differentiation.

Recent basic studies have yielded important new insights into the molecular mechanisms that regulate growth locally. Simultaneously, clinical studies have identified new molecular defects that cause growth failure and overgrowth, and genome-wide association studies have elucidated the genetic basis for normal human height variation.The Hippo pathway has emerged as one of the major mechanisms controlling organ size. In addition, an extensive genetic program has been described that allows rapid body growth in the fetus and infant but then causes growth to slow with age in multiple tissues. In human genome-wide association studies, hundreds of loci associated with adult stature have been identified; many appear to involve genes that function locally in the growth plate. Clinical genetic studies have identified a new genetic abnormality, microduplication of Xq26.3, that is responsible for growth hormone excess, and a gene, DNMT3A, in which mutations cause an overgrowth syndrome through epigenetic mechanisms.These recent advances in our understanding of somatic growth not only provide insight into childhood growth disorders but also have broader medical applications because disruption of these regulatory systems contributes to oncogenesis.

Mechanical stimulation is crucial to bone growth and triggers osteogenic differentiation through a process involving Rho and protein kinase A. We previously cloned a gene (AKAP13, aka BRX) encoding a protein kinase A-anchoring protein in the N-terminus, a guanine nucleotide-exchange factor for RhoA in the mid-section, coupled to a carboxyl region that binds to estrogen and glucocorticoid nuclear receptors. Because of the critical role of Rho, estrogen, and glucocorticoids in bone remodeling, we examined the multifunctional role of Akap13. Akap13 was expressed in bone, and mice haploinsufficient for Akap13 (Akap13+/–) displayed reduced bone mineral density, reduced bone volume/total volume, and trabecular number, and increased trabecular spacing; resembling the changes observed in osteoporotic bone. Consistent with the osteoporotic phenotype, Colony forming unit-fibroblast numbers were diminished in Akap13+/– mice, as were osteoblast numbers and extracellular matrix production when compared to control littermates. Transcripts of Runx2, an essential transcription factor for the osteogenic lineage, and alkaline phosphatase (Alp), an indicator of osteogenic commitment, were both reduced in femora of Akap13+/– mice. Knockdown of Akap13 reduced levels of Runx2 and Alp transcripts in immortalized bone marrow stem cells. These findings suggest that Akap13 haploinsufficient mice have a deficiency in early osteogenesis with a corresponding reduction in osteoblast number, but no impairment of mature osteoblast activity. © 2015 American Society for Bone and Mineral Research.

Bones at different anatomical locations vary dramatically in size. For example, human femurs are 20-fold longer than the phalanges in the fingers and toes. The mechanisms responsible for these size differences are poorly understood. Bone elongation occurs at the growth plates and advances rapidly in early life but then progressively slows due to a developmental program termed “growth plate senescence.” This developmental program includes declines in cell proliferation and hypertrophy, depletion of cells in all growth plate zones, and extensive underlying changes in the expression of growth-regulating genes. Here, we show evidence that these functional, structural, and molecular senescent changes occur earlier in the growth plates of smaller bones (metacarpals, phalanges) than in the growth plates of larger bones (femurs, tibias) and that this differential aging contributes to the disparities in bone length. We also show evidence that the molecular mechanisms that underlie the differential aging between different bones involve modulation of critical paracrine regulatory pathways, including insulin-like growth factor (Igf), bone morphogenetic protein (Bmp), and Wingless and Int-1 (Wnt) signaling. Taken together, the findings reveal that the striking disparities in the lengths of different bones, which characterize normal mammalian skeletal proportions, is achieved in part by modulating the progression of growth plate senescence., Author summary The various bones found in human extremities vary dramatically in size. For example, the femur in the thigh is approximately 20 times longer than the phalanges of the toes. The mechanisms that regulate how much a particular bone grows to attain the skeletal proportions of each animal remain mostly unknown. In this study, we sought to uncover these mechanisms and found that this scaling in skeletal size is achieved in part by modulating the rate of aging of growth plates in different bones. Bone elongation occurs at the cartilaginous growth plates and proceeds rapidly in early life but slows and eventually stops due to a developmental program termed “growth plate senescence,” which is used to describe the gradual decline in growth plate function with age. We discovered that this developmental program is more advanced in shorter bones than in longer bones and that this differential aging contributes to the disparities in bone growth. We show that the molecular mechanisms that underlie this differential aging between bones involve differential expression of specific local signaling pathways, which regulate cell proliferation and differentiation in the growth plate. Taken together, we conclude that the striking disparities in the lengths of different bones—characteristic of normal mammalian skeletal proportions—are achieved in part by modulating the progression of aging in the growth plates.

Many children with idiopathic short stature have a delayed bone age. Idiopathic short stature with advanced bone age is far less common.The aim was to identify underlying genetic causes of short stature with advanced bone age.We used whole-exome sequencing to study three families with autosomal-dominant short stature, advanced bone age, and premature growth cessation.Affected individuals presented with short stature [adult heights -2.3 to -4.2 standard deviation scores (SDS)] with histories of early growth cessation or childhood short stature (height SDS -1.9 to -3.5 SDS), advancement of bone age, and normal endocrine evaluations. Whole-exome sequencing identified novel heterozygous variants in ACAN, which encodes aggrecan, a proteoglycan in the extracellular matrix of growth plate and other cartilaginous tissues. The variants were present in all affected, but in no unaffected, family members. In Family 1, a novel frameshift mutation in exon 3 (c.272delA) was identified, which is predicted to cause early truncation of the aggrecan protein. In Family 2, a base-pair substitution was found in a highly conserved location within a splice donor site (c.2026+1GA), which is also likely to alter the amino acid sequence of a large portion of the protein. In Family 3, a missense variant (c.7064TC) in exon 14 affects a highly conserved residue (L2355P) and is strongly predicted to perturb protein function.Our study demonstrates that heterozygous mutations in ACAN can cause a mild skeletal dysplasia, which presents clinically as short stature with advanced bone age. The accelerating effect on skeletal maturation has not previously been noted in the few prior reports of human ACAN mutations. Our findings thus expand the spectrum of ACAN defects and provide a new molecular genetic etiology for the unusual child who presents with short stature and accelerated skeletal maturation.

Body size varies enormously among mammalian species. In small mammals, body growth is typically suppressed rapidly, within weeks, whereas in large mammals, growth is suppressed slowly, over years, allowing for a greater adult size. We recently reported evidence that body growth suppression in rodents is caused in part by a juvenile genetic program that occurs in multiple tissues simultaneously and involves the downregulation of a large set of growth-promoting genes. We hypothesized that this genetic program is conserved in large mammals but that its time course is evolutionarily modulated such that it plays out more slowly, allowing for more prolonged growth. Consistent with this hypothesis, using expression microarray analysis, we identified a set of genes that are downregulated with age in both juvenile sheep kidney and lung. This overlapping gene set was enriched for genes involved in cell proliferation and growth and showed striking similarity to a set of genes downregulated with age in multiple organs of the juvenile mouse and rat, indicating that the multiorgan juvenile genetic program previously described in rodents has been conserved in the 80 million years since sheep and rodents diverged in evolution. Using microarray and real-time PCR, we found that the pace of this program was most rapid in mice, more gradual in rats, and most gradual in sheep. These findings support the hypothesis that a growth-regulating genetic program is conserved among mammalian species but that its pace is modulated to allow more prolonged growth and therefore greater adult body size in larger mammals.

RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1f/f Sos2−/− mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.

Insulin-like growth factor 2 (IGF2) is an important fetal growth factor. Its expression is dramatically down-regulated in multiple organs after birth but is frequently up-regulated in cancers. The mechanisms that drive down-regulation of IGF2 in postnatal tissues or the up-regulation in malignancy are unclear. We found evidence that E2F transcription factor 3 (E2F3) drives these changes in expression. E2f3 mRNA expression, protein expression, and binding to the Igf2 promoter all decreased with age postnatally in multiple mouse organs. In late juvenile hepatocytes, restoration of high E2f3 expression restored high Igf2 expression, indicating a causal relationship, but this induction did not occur in fetal hepatocytes, which already have high E2f3 and Igf2 expression. Transient expression of E2f3 in both HEK293 cells and in late juvenile hepatocytes were able to activate reporter constructs containing the mouse Igf2 promoter P2, which includes consensus E2F-binding sites. In humans, microarray data revealed declines in E2F3 and IGF2 expression with age similar to the mouse. In addition, E2F3-overexpressing human prostate and bladder cancers showed increased IGF2 expression, and levels of E2F3 and IGF2 mRNA in these cancers were positively correlated. Taken together, the findings suggest that down-regulation of E2f3 with age helps drive the dramatic decline in Igf2 expression in postnatal organs, and E2F3 overexpression in human cancers induces IGF2 overexpression.

As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders.

Regulation of body growth remains a fascinating and unresolved biological mystery. One key component of body growth is skeletal and longitudinal bone growth. Children grow taller because their bones grew longer, and the predominant driver of longitudinal bone growth is a cartilaginous structure found near the ends of long bone named the growth plate. Numerous recent studies have started to unveil the importance of microRNAs in regulation of growth plate functions, therefore contributing to regulation of linear growth. In addition to longitudinal growth, other organs in our body need to increase in size and cell number as we grow, and the regulation of organ growth involves both systemic factors like hormones; and other intrinsic mechanisms, which we are just beginning to understand. This review aims to summarize some recent important findings on how microRNAs are involved in both of these processes: the regulation of longitudinal bone growth, and the regulation of organs and overall body growth.

Microcephalic primordial dwarfism (MPD) is a rare, severe form of human growth failure in which growth restriction is evident in utero and continues into postnatal life. Single causative gene defects have been identified in a number of patients with MPD, and all involve genes fundamental to cellular processes including centrosome functions.The objective of the study was to find the genetic etiology of a novel presentation of MPD.The design of the study was whole-exome sequencing performed on two affected sisters in a single family. Molecular and functional studies of a candidate gene were performed using patient-derived primary fibroblasts and a zebrafish morpholino oligonucleotides knockdown model.Two sisters presented with a novel subtype of MPD, including severe intellectual disabilities.NIN, encoding Ninein, a centrosomal protein critically involved in asymmetric cell division, was identified as a candidate gene, and functional impacts in fibroblasts and zebrafish were studied.From 34,606 genomic variants, two very rare missense variants in NIN were identified. Both probands were compound heterozygotes. In the zebrafish, ninein knockdown led to specific and novel defects in the specification and morphogenesis of the anterior neuroectoderm, resulting in a deformity of the developing cranium with a small, squared skull highly reminiscent of the human phenotype.We identified a novel clinical subtype of MPD in two sisters who have rare variants in NIN. We show, for the first time, that reduction of ninein function in the developing zebrafish leads to specific deficiencies of brain and skull development, offering a developmental basis for the myriad phenotypes in our patients.

Previous meta-analysis of genome-wide association (GWA) studies has identified 180 loci that influence adult height. However, each GWA locus typically comprises a set of contiguous genes, only one of which presumably modulates height. We reasoned that many of the causative genes within these loci influence height because they are expressed in and function in the growth plate, a cartilaginous structure that causes bone elongation and thus determines stature. Therefore, we used expression microarray studies of mouse and rat growth plate, human disease databases and a mouse knockout phenotype database to identify genes within the GWAS loci that are likely required for normal growth plate function. Each of these approaches identified significantly more genes within the GWA height loci than at random genomic locations (P < 0.0001 each), supporting the validity of the approach. The combined analysis strongly implicates 78 genes in growth plate function, including multiple genes that participate in PTHrP-IHH, BMP and CNP signaling, and many genes that have not previously been implicated in the growth plate. Thus, this analysis reveals a large number of novel genes that regulate human growth plate chondrogenesis and thereby contribute to the normal variations in human adult height. The analytic approach developed for this study may be applied to GWA studies for other common polygenic traits and diseases, thus providing a new general strategy to identify causative genes within GWA loci and to translate genetic associations into mechanistic biological insights.

In many normal tissues, proliferation rates decline postnatally, causing somatic growth to slow. Previous evidence suggests that this decline is due, in part, to decline in the expression of growth-promoting imprinted genes including Mest, Plagl1, Peg3, Dlk1, and Igf2. Embryonal cancers are composed of cells that maintain embryonic characteristics and proliferate rapidly in childhood. We hypothesized that the abnormal persistent rapid proliferation in embryonal cancers occurs in part because of abnormal persistent high expression of growth-promoting imprinted genes.Analysis of microarray data showed elevated expression of MEST, PLAGL1, PEG3, DLK1, and IGF2 in various embryonal cancers, especially rhabdomyosarcoma, as compared to nonembryonal cancers and normal tissues. Similarly, mRNA expression, assessed by real-time PCR, of MEST, PEG3, and IGF2 in rhabdomyosarcoma cell lines was increased as compared to nonembryonal cancer cell lines. Furthermore, siRNA-mediated knockdown of MEST, PLAGL1, PEG3, and IGF2 expression inhibited proliferation in Rh30 rhabdomyosarcoma cells.These findings suggest that the normal postnatal downregulation of growth-promoting imprinted genes fails to occur in some embryonal cancers, particularly rhabdomyosarcoma, and contributes to the persistent rapid proliferation of rhabdomyosarcoma cells and, more generally, that failure of the mechanisms responsible for normal somatic growth deceleration can promote tumorigenesis.

Autophagy is a normal intracellular mechanism for recycling unnecessary or dysfunctional cellular components and proteins, thus preserving energy and alleviating stress to promote cellular survival. Numerous recent studies have demonstrated the importance of autophagy in normal cartilage maintenance and implicated deregulation of autophagy in the pathogenesis of osteoarthritis (OA), which remains as the most common chronic joint disease and the leading cause of disability worldwide.

In mammals, proliferation is rapid in many tissues during early postnatal life, causing rapid somatic growth. This robust proliferation is then suppressed as the animal approaches adult size, bringing many tissues to a quiescent state where proliferation occurs only as needed to replace dying cells. Recent evidence suggests that the mechanism responsible for this decline in proliferation involves a multi-organ genetic program. We hypothesized that this genetic program continues to progress into later adult life, eventually suppressing proliferation to levels below those needed for tissue renewal, thus contributing to aging. We therefore used expression microarray to compare the temporal changes in gene expression that occur in adult mouse organs during aging to those occurring as juvenile proliferation slows. We found that many of the changes in gene expression that occur during the aging process originate during the period of juvenile growth deceleration. Bioinformatic analyses of the genes that show persistent decline in expression throughout postnatal life indicated that cell-cycle-related genes are strongly over-represented. Thus, the findings support the hypothesis that the genetic program that slows juvenile growth to limit body size persists into adulthood and thus may eventually hamper tissue maintenance and repair, contributing to the aging process.

The involvement of caspase-3 and its failure in the induction of DNA fragmentation during erythropoiesis were investigated with TF-1 cells. During erythroid differentiation, caspase-3 activation and cleavage of caspase-3 substrates such as ICAD (inhibitor of caspase-activated DNase) were detected without concomitant phosphatidyl-serine (PS) externalization and DNA fragmentation. These observations are in contrast to our understanding that DNA is degraded by CAD (caspase-activated DNase) when ICAD is cleaved by caspase-3. Our study demonstrates that CAD is downregulated at the mRNA and protein level during the erythroid differentiation in TF-1 cells. This provides a mechanism for the first time how cells avoid DNA fragmentation with activated caspase-3.

Many genetic disorders, including chondrodysplasias, and acquired disorders impair growth plate function, resulting in short and sometimes malformed bones. There are multiple endocrine and paracrine factors that promote chondrogenesis at the growth plate, which could potentially be used to treat these disorders. Targeting these growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing undesirable effects on non-target tissues. Using yeast display technology, we selected single-chain variable antibody fragments that bound to human and mouse matrilin-3, an extracellular matrix protein specifically expressed in cartilage tissue. The ability of the selected antibody fragments to bind matrilin-3 and to bind cartilage tissue in vitro and in vivo was assessed by ELISA and immunohistochemistry. We identified antibody fragments that bound matrilin-3 with high affinity and also bound with high tissue specificity to cartilage homogenates and to cartilage structures in mouse embryo sections. When injected intravenously in mice, the antibody fragments specifically homed to cartilage. Yeast display successfully selected antibody fragments that are able to target cartilage tissue in vivo. Coupling these antibodies to chondrogenic endocrine and paracrine signaling molecules has the potential to open up new pharmacological approaches to treat childhood skeletal growth disorders.

Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers-the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton.

Background/Aims: In addition to genome-wide association studies (GWAS), height-associated genes may be uncovered by studying individuals with extreme short or tall stature. Methods: Genome-wide analysis for copy number variants (CNVs), using single nucleotide polymorphism (SNP) arrays, was performed in 49 index cases born small for gestational age with persistent short stature. Segregation analysis was performed, and genes in CNVs were compared with information from GWAS, gene expression in rodents' growth plates, and published information. Results: CNVs were detected in 13 cases. In 5 children a known cause of short stature was found: UPD7, UPD14, a duplication of the SHOX enhancer region, an IGF1R deletion, and a 22q11.21 deletion. In the remaining 8 cases, potential pathogenic CNVs were detected, either de novo (n = 1), segregating (n = 2), or not segregating with short stature (n = 5). Bioinformatic analysis of the de novo and segregating CNVs suggested that HOXD4, AGPS, PDE11A, OSBPL6, PRKRA and PLEKHA3, and possibly DGKB and TNFRSF11B are potential candidate genes. A SERPINA7 or NRK defect may be associated with an X-linked form of short stature. Conclusion: SNP arrays detected 5 known causes of short stature with prenatal onset and suggested several potential candidate genes.

In juvenile mammals, the epiphyses of long bones grow by chondrogenesis within the articular cartilage. A better understanding of the molecular mechanisms that regulate the growth of articular cartilage may give insight into the antecedents of joint disease, such as osteoarthritis.We used laser capture microdissection to isolate chondrocytes from the superficial, middle, and deep zones of growing tibial articular cartilage in the 1-wk-old mouse and then investigated expression patterns by microarray. To identify molecular markers for each zone of the growing articular cartilage, we found genes showing zone-specific expression and confirmed by real-time PCR and in situ hybridization.Bioinformatic analyses implicated ephrin receptor signaling, Wnt signaling, and bone morphogenetic protein signaling in the spatial regulation of chondrocyte differentiation during growth. Molecular markers were identified for superficial (e.g., Cilp, Prg4), middle (Cxcl14, Tnn), and deep zones (Sfrp5, Frzb). Comparison between juvenile articular and growth plate cartilage revealed that the superficial-to-deep zone transition showed similarity with the hypertrophic-to-resting zone transition.Laser capture microdissection combined with microarray analysis identified novel signaling pathways that are spatially regulated in growing mouse articular cartilage and revealed similarities between the molecular architecture of the growing articular cartilage and that of growth plate cartilage.

For most bones, elongation is driven primarily by chondrogenesis at the growth plates. This process results from chondrocyte proliferation, hypertrophy, and extracellular matrix secretion, and it is carefully orchestrated by complex networks of local paracrine factors and modulated by endocrine factors. We review here recent advances in the understanding of growth plate physiology. These advances include new approaches to study expression patterns of large numbers of genes in the growth plate, using microdissection followed by microarray. This approach has been combined with genome-wide association studies to provide insights into the regulation of the human growth plate. We also review recent studies elucidating the roles of bone morphogenetic proteins, fibroblast growth factors, C-type natriuretic peptide, and suppressor of cytokine signaling in the local regulation of growth plate chondrogenesis and longitudinal bone growth.

Articular and growth plate cartilage are discrete tissues but arise from a common cartilaginous condensation and have comparable spatial architectures consisting of distinct layers of chondrocytes. To investigate similarities and differences between articular and growth plate cartilage and to explore transcriptional changes that occur during the onset of their divergence, we performed manual microdissection of 10-day-old rat proximal tibias, microarray analysis, bioinformatics, and real-time PCR to compare gene expression profiles in individual cartilage layers. We found that many genes that were spatially upregulated in the intermediate/deep zone of articular cartilage were also spatially upregulated in the resting zone of growth plate cartilage (overlap greater than expected by chance, P

Item does not contain fulltext Height is a highly heritable and classic polygenic trait. Recent genome-wide association studies (GWAS) have revealed that at least 180 genetic variants influence adult height. However, these variants explain only about 10% of the phenotypic variation in height. Genetic analysis of short individuals can lead to the discovery of novel rare gene defects with a large effect on growth. In an effort to identify novel genes associated with short stature, genome-wide analysis for copy number variants (CNVs), using single-nucleotide polymorphism arrays, in 162 patients (149 families) with short stature was performed. Segregation analysis was performed if possible, and genes in CNVs were compared with information from GWAS, gene expression in rodents' growth plates and published information. CNVs were detected in 40 families. In six families, a known cause of short stature was found (SHOX deletion or duplication, IGF1R deletion), in two combined with a de novo potentially pathogenic CNV. Thirty-three families had one or more potentially pathogenic CNVs (n=40). In 24 of these families, segregation analysis could be performed, identifying three de novo CNVs and nine CNVs segregating with short stature. Four were located near loci associated with height in GWAS (ADAMTS17, TULP4, PRKG2/BMP3 and PAPPA). Besides six CNVs known to be causative for short stature, 40 CNVs with possible pathogenicity were identified. Segregation studies and bioinformatics analysis suggested various potential candidate genes.

Mechanical stimulation is crucial to bone growth and triggers osteogenic differentiation through a process involving Rho and protein kinase A. We previously cloned a gene (AKAP13, aka BRX) encoding a protein kinase A-anchoring protein in the N-terminus, a guanine nucleotide-exchange factor for RhoA in the mid-section, coupled to a carboxyl region that binds to estrogen and glucocorticoid nuclear receptors. Because of the critical role of Rho, estrogen, and glucocorticoids in bone remodeling, we examined the multifunctional role of Akap13. Akap13 was expressed in bone, and mice haploinsufficient for Akap13 (Akap13(+/-)) displayed reduced bone mineral density, reduced bone volume/total volume, and trabecular number, and increased trabecular spacing; resembling the changes observed in osteoporotic bone. Consistent with the osteoporotic phenotype, Colony forming unit-fibroblast numbers were diminished in Akap13(+/-) mice, as were osteoblast numbers and extracellular matrix production when compared to control littermates. Transcripts of Runx2, an essential transcription factor for the osteogenic lineage, and alkaline phosphatase (Alp), an indicator of osteogenic commitment, were both reduced in femora of Akap13(+/-) mice. Knockdown of Akap13 reduced levels of Runx2 and Alp transcripts in immortalized bone marrow stem cells. These findings suggest that Akap13 haploinsufficient mice have a deficiency in early osteogenesis with a corresponding reduction in osteoblast number, but no impairment of mature osteoblast activity.

Proteoglycans are a component of the extracellular matrix and are critical for cellular and tissue function. Mutations in proteoglycan components and enzymes involved in proteoglycan synthesis have been implicated in several growth disorders, with common features including short stature and skeletal dysplasia. For example, mutations in B4GALT7, a gene whose protein product catalyzes proteoglycan synthesis, have been associated with the rare progeroid variant of Ehlers-Danlos syndrome. Here, we conducted exome sequencing in a patient with a previously undiagnosed growth disorder and identified compound heterozygous mutations in B4GALT7. This patient is just the fourth individual with genetically confirmed progeroid variant of Ehlers-Danlos syndrome. The mutations include a previously characterized c.808C>T p.Arg270Cys substitution, and a novel c.122T>C p.Leu41Pro substitution. We demonstrate that the novel mutation caused decreased levels of the enzyme, supporting the pathogenicity of the mutation. Our report identifies a novel mutation in B4GALT7 causing the progeroid variant of Ehlers-Danlos syndrome and contributes an extensive phenotypic characterization of a patient with the syndrome. We also reviewed the previous literature in addition to the present patient, and conclude that the key features associated with B4GALT7 deficiency are short stature, developmental anomalies of the forearm bones and elbow, and bowing of the extremities, in addition to the classic features of Ehlers-Danlos syndrome. This report helps define the phenotype of the progeroid variant of Ehlers-Danlos syndrome and furthers our understanding of the effect of proteoglycan defects in growth disorders.

During early postnatal life, extensive changes in gene expression occur concomitantly in multiple major organs, indicating the existence of a common core developmental genetic program. This program includes hundreds of growth-promoting genes that are downregulated with age in liver, kidney, lung, and heart, and there is evidence that this component of the program drives the widespread decline in cell proliferation that occurs in juvenile life, as organs approach adult sizes. To investigate epigenetic changes that might orchestrate this program, we performed chromatin immunoprecipitation-promoter tiling array to assess temporal changes in histone H3K4 and H3K27 trimethylation (me3) at promoter regions throughout the genome in kidney and lung, comparing 1- to 4-wk-old mice. We found extensive genome-wide shifts in H3K4me3 and H3K27me3 occurring with age in both kidney and lung. The number of genes with concordant changes in the two organs was far greater than expected by chance. Temporal changes in H3K4me3 showed a strong, positive association with changes in gene expression, assessed by microarray, whereas changes in H3K27me3 showed a negative association. Gene ontology analysis indicated that shifts in specific histone methylation marks were associated with specific developmental functions. Of particular interest, genes with decreases in H3K4me3 with age in both organs were strongly implicated in cell cycle and cell proliferation functions. Taken together, the findings suggest that the common core developmental program of gene expression which occurs in multiple organs during juvenile life is associated with a common core developmental program of histone methylation. In particular, declining H3K4me3 is strongly associated with gene downregulation and occurs in the promoter regions of many growth-regulating genes, suggesting that this change in histone methylation may contribute to the component of the genetic program that drives juvenile body growth deceleration.

As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders.

In embryonic growth cartilage, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP) participate in a negative feedback loop that regulates chondrocyte differentiation. Postnatally, this region undergoes major structural and functional changes. To explore the organization of the Ihh–PTHrP system in postnatal growth plate, we microdissected growth plates of 7-day-old rats into their constituent zones and assessed expression of genes participating in the Ihh–PTHrP feedback loop.Ihh, Patched 1, Smoothened,Gli1, Gli2, Gli3, andPthr1were expressed in regions analogous to the expression domains in embryonic growth cartilage. However, PTHrP was expressed in resting zone cartilage, a site that differs from the embryonic source, the periarticular cells. We then used mice in whichlacZhas replaced coding sequences ofGli1and thus serves as a marker for active hedgehog signaling. At 1, 4, 8, and 12 weeks of age,lacZexpression was detected in a pattern analogous to that of embryonic cartilage. The findings support the hypothesis that the embryonic Ihh–PTHrP feedback loop is maintained in the postnatal growth plate except that the source of PTHrP has shifted to a more proximal location in the resting zone.