DLG2 variants in patients with pubertal disorders.

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 cosegregated 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 three 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.

Cartilage-Targeted IGF-1 Treatment to Promote Longitudinal Bone 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 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.

Effect of Antiandrogen, Aromatase Inhibitor, and Gonadotropin-releasing Hormone Analog on Adult Height in Familial Male Precocious Puberty.

OBJECTIVE: Antiandrogen, aromatase inhibitor, and gonadotropin-releasing hormone analog (GnRHa) treatment normalizes growth rate and bone maturation and increases predicted adult height (AH) in boys with familial male-limited precocious puberty (FMPP). To evaluate the effect of long-term antiandrogen, aromatase inhibitor, and GnRHa on AH, boys with FMPP who were treated were followed to AH. STUDY DESIGN: Twenty-eight boys with FMPP, referred to the National Institutes of Health, were started on antiandrogen and aromatase inhibitor at 4.9 ± 1.5 years of age; GnRHa was added at 6.9 ± 1.5 years of age. Treatment was discontinued at 12.2 ± 0.5 years of age (bone age, 14.4 ± 1.3). AH was assessed at 16.4 ± 1.3 years of age (bone age, 18.5 ± 0.6). RESULTS: AH (mean ± SD) for all treated subjects was 173.6 ± 6.8 cm (-0.4 ± 1.0 SD relative to adult US males). For 25 subjects with pretreatment predicted AH, AH significantly exceeded predicted AH at treatment onset (173.8 ± 6.9 vs 164.9 ± 10.7 cm; P < .001), but fell short of predicted AH at treatment discontinuation (177.3 ± 9.0 cm; P < .001). For 11 subjects with maternal or sporadic inheritance, the mean AH was 3.1 cm (0.4 SD score) below sex-adjusted midparental height (175.4 ± 5.8 vs 178.5 ± 3.1 cm [midparental height]; P = .10). For 16 subjects with affected and untreated fathers, AH was significantly greater than fathers' AH (172.8 ± 7.4 vs 168.8 ± 7.2 cm; P < .05). CONCLUSIONS: Long-term treatment with antiandrogen, aromatase inhibitor, and GnRHa in boys with FMPP results in AH modestly below sex-adjusted midparental height and within the range for adult males in the general population.

Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome.

Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes. Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth.

A set of imprinted genes required for normal body growth also promotes growth of rhabdomyosarcoma cells.

INTRODUCTION: 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. RESULTS: 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. DISCUSSION: 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.

Coordinated postnatal down-regulation of multiple growth-promoting genes: evidence for a genetic program limiting organ growth.

Children grow, but adults do not. The cessation of growth in multiple organs is the end result of a progressive decline in cell proliferation beginning in early life. The mechanisms responsible for this growth deceleration are largely unknown. Using expression microarray and real-time PCR, we identified a common program of gene expression in lung, kidney, and liver during growth deceleration in juvenile rats. Gene ontology analyses and siRNA-mediated knockdown in vitro indicated that many of the down-regulated genes are growth promoting. Down-regulated genes in the program showed declining histone H3K4 trimethylation with age, implicating underlying epigenetic mechanisms. To investigate the physiological processes driving the genetic program, a tryptophan-deficient diet was used to temporarily inhibit juvenile growth in newborn rats for 4 wk. Afterward, microarray analysis showed that the genetic program had been delayed, implying that it is driven by body growth itself rather than age. Taken together, the findings suggest that growth in early life induces progressive down-regulation of a large set of proliferation-stimulating genes, causing organ growth to slow and eventually cease.

Persistent Sox9 expression in hypertrophic chondrocytes suppresses transdifferentiation into osteoblasts.

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.

Catch-up growth after hypothyroidism is caused by delayed growth plate senescence.

Catch-up growth is defined as a linear growth rate greater than expected for age after a period of growth inhibition. We hypothesized that catch-up growth occurs because growth-inhibiting conditions conserve the limited proliferative capacity of growth plate chondrocytes, thus slowing the normal process of growth plate senescence. When the growth-inhibiting condition resolves, the growth plates are less senescent and therefore grow more rapidly than normal for age. To test this hypothesis, we administered propylthiouracil to newborn rats for 8 wk to induce hypothyroidism and then stopped the propylthiouracil to allow catch-up growth. In untreated controls, the growth plates underwent progressive, senescent changes in multiple functional and structural characteristics. We also identified genes that showed large changes in mRNA expression in growth plate and used these changes as molecular markers of senescence. In treated animals, after stopping propylthiouracil, these functional, structural, and molecular senescent changes were delayed, compared with controls. This delayed senescence included a delayed decline in longitudinal growth rate, resulting in catch-up growth. The findings demonstrate that growth inhibition due to hypothyroidism slows the developmental program of growth plate senescence, including the normal decline in the rate of longitudinal bone growth, thus accounting for catch-up growth.

Changes in cell-cycle kinetics responsible for limiting somatic growth in mice.

In mammals, the rate of somatic growth is rapid in early postnatal life but then slows with age, approaching zero as the animal approaches adult body size. To investigate the underlying changes in cell-cycle kinetics, [methyl-H]thymidine and 5'-bromo-2'deoxyuridine were used to double-label proliferating cells in 1-, 2-, and 3-wk-old mice for four weeks. Proliferation of renal tubular epithelial cells and hepatocytes decreased with age. The average cell-cycle time did not increase in liver and increased only 1.7 fold in kidney. The fraction of cells in S-phase that will divide again declined approximately 10 fold with age. Concurrently, average cell area increased approximately 2 fold. The findings suggest that somatic growth deceleration primarily results not from an increase in cell-cycle time but from a decrease in growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cells enlarge to a greater volume, perhaps because they are relieved of the size constraint imposed by cell division. In conclusion, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size.

Ezh2 Mutations Found in the Weaver Overgrowth Syndrome Cause a Partial Loss of H3K27 Histone Methyltransferase Activity.

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.

Wnt gene expression in the post-natal growth plate: regulation with chondrocyte differentiation.

Longitudinal growth of long bones occurs at the growth plate by endochondral ossification. In the embryonic mouse, this process is regulated by Wnt signaling. Little is known about which members of the Wnt family of secreted signaling proteins might be involved in the regulation of the postnatal growth plate. We used microdissection and real-time PCR to study mRNA expression of Wnt genes in the mouse growth plate. Of the 19 known members of the Wnt family, only six were expressed in postnatal growth plate. Of these, Wnts -2b, -4, and -10b signal through the canonical beta-catenin pathway and Wnts -5a, -5b, and -11 signal through the noncanonical calcium pathway. The spatial expression for these six Wnts was remarkably similar, showing low mRNA expression in the resting zone, increasing expression as the chondrocytes differentiated into the proliferative and prehypertrophic state and then (except Wnt-2b) decreasing expression as the chondrocytes underwent hypertrophic differentiation. This overall pattern is broadly consistent with previous studies of embryonic mouse growth cartilage suggesting that Wnt signaling modulates chondrocyte proliferation and hypertrophic differentiation. We also found that mRNA expression of these Wnt genes persisted at similar levels at 4 weeks, when longitudinal bone growth is waning. In conclusion, we have identified for the first time the specific Wnt genes that are expressed in the postnatal mammalian growth plate. The six identified Wnt genes showed a similar pattern of expression during chondrocyte differentiation, suggesting overlapping or interacting roles in postnatal endochondral bone formation.

Gradients in bone morphogenetic protein-related gene expression across the growth plate.

In the growth plate, stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone and then terminally differentiate into the non-dividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ) of 7-day-old male rats. Expression of approximately 29,000 genes was analyzed by microarray and selected genes verified by real-time PCR. The analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were upregulated in HZ compared with RZ and PZ (30-fold each, P < 0.01 and 0.001 respectively). In contrast, BMP signaling inhibitors were expressed early in the differentiation pathway; BMP-3 and gremlin were differentially expressed in RZ (100- and 80-fold, compared with PZ, P < 0.001 and 0.005 respectively) and growth differentiation factor (GDF)-10 in PZ (160-fold compared with HZ, P < 0.001). Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. Since BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes, these findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower RZ, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation. Thus, BMP signaling gradients may be a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in growth plate cartilage.

Depletion of resting zone chondrocytes during growth plate senescence.

With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2'-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate. Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation. Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle.

EZH1 and EZH2 promote skeletal growth by repressing inhibitors of chondrocyte proliferation and hypertrophy.

Histone methyltransferases EZH1 and EZH2 catalyse the trimethylation of histone H3 at lysine 27 (H3K27), which serves as an epigenetic signal for chromatin condensation and transcriptional repression. Genome-wide associated studies have implicated EZH2 in the control of height and mutations in EZH2 cause Weaver syndrome, which includes skeletal overgrowth. Here we show that the combined loss of Ezh1 and Ezh2 in chondrocytes severely impairs skeletal growth in mice. Both of the principal processes underlying growth plate chondrogenesis, chondrocyte proliferation and hypertrophy, are compromised. The decrease in chondrocyte proliferation is due in part to derepression of cyclin-dependent kinase inhibitors Ink4a/b, while ineffective chondrocyte hypertrophy is due to the suppression of IGF signalling by the increased expression of IGF-binding proteins. Collectively, our findings reveal a critical role for H3K27 methylation in the regulation of chondrocyte proliferation and hypertrophy in the growth plate, which are the central determinants of skeletal growth.

Growth plate senescence is associated with loss of DNA methylation.

The overall body size of vertebrates is primarily determined by longitudinal bone growth at the growth plate. With age, the growth plate undergoes programmed senescence, causing longitudinal bone growth to slow and eventually cease. Indirect evidence suggests that growth plate senescence occurs because stem-like cells in the growth plate resting zone have a finite proliferative capacity that is gradually exhausted. Similar limits on replication have been observed when many types of animal cells are placed in cell culture, an effect known as the Hayflick phenomenon. However, we found that the number of population doublings of rabbit resting zone chondrocytes in culture did not depend on the age of the animal from which the cells were harvested, suggesting that the mechanisms limiting replicative capacity of growth plate chondrocytes in vivo are distinct from those in vitro. We also observed that the level of DNA methylation in resting zone chondrocytes decreased with age in vivo. This loss of methylation appeared to occur specifically with the slow proliferation of resting zone chondrocytes in vivo and was not observed with the rapid proliferation of proliferative zone chondrocytes in vivo (i.e. the level of DNA methylation did not change from the resting zone to the hypertrophic zone), with proliferation of chondrocytes in vitro, or with growth of the liver in vivo. Thus, the overall level of DNA methylation decreases during growth plate senescence. This finding is consistent with the hypothesis that the mechanism limiting replication of growth plate chondrocytes in vivo involves loss of DNA methylation and, thus, loss of DNA methylation might be a fundamental biological mechanism that limits longitudinal bone growth in mammals, thereby determining the overall adult size of the organism.

Recovery from osteoporosis through skeletal growth: early bone mass acquisition has little effect on adult bone density.

It is often assumed that bone mineral accretion should be optimized throughout childhood to maximize peak bone mass. In contrast, we hypothesized that bone mineral acquisition early in life would have little or no effect on adult bone mass because many areas of the juvenile skeleton are replaced in toto through skeletal growth. To test this hypothesis, we induced osteoporosis by administering dexamethasone to 5-week-old rabbits for 5 weeks and then allowed them to recover for 16 weeks. Tibial bone mineral density (ash weight/volume) was decreased in the dexamethasone-treated animals at the end of treatment but recovered completely. Bone structure in the femur was assessed by histomorphometry. Trabecular and cortical bone in the distal metaphysis was made osteoporotic by dexamethasone, but was then replaced through endochondral bone formation and recovered. Periosteal bone formation rate in the diaphysis was decreased during dexamethasone treatment but afterwards rebounded above controls and normalized cortical width. Our data suggest that bone mineral acquisition early in life has little effect on adult bone density because the juvenile bone is largely replaced through growth. If this concept generalizes, then interventions to maximize peak bone mass should be directed at adolescents rather than young children.

The role of the resting zone in growth plate chondrogenesis.

In mammals, growth of long bones occurs at the growth plate, a cartilage structure that contains three principal layers: the resting, proliferative, and hypertrophic zones. The function of the resting zone is not well understood. We removed the proliferative and hypertrophic zones from the rabbit distal ulnar growth plate in vivo, leaving only the resting zone. Within 1 wk, a complete proliferative and hypertrophic zone often regenerated. Next, we manipulated growth plates in vivo to place resting zone cartilage ectopically alongside the proliferative columns. Ectopic resting zone cartilage induced a 90-degree shift in the orientation of nearby proliferative zone chondrocytes and seemed to inhibit their hypertrophic differentiation. Our findings suggest that resting zone cartilage makes important contributions to endochondral bone formation at the growth plate: 1) it contains stem-like cells that give rise to clones of proliferative chondrocytes; 2) it produces a growth plate-orienting factor, a morphogen, that directs the alignment of the proliferative clones into columns parallel to the long axis of the bone; and 3) it may also produce a morphogen that inhibits terminal differentiation of nearby proliferative zone chondrocytes and thus may be partially responsible for the organization of the growth plate into distinct zones of proliferation and hypertrophy.

Determinants of growth during gonadotropin-releasing hormone analog therapy for precocious puberty.

In children with precocious puberty (PP), treatment with GnRH analogs (GnRHa) often decreases height velocity below normal. Based on previous animal studies, we hypothesized that this impaired growth is due to excessive advancement in growth plate senescence induced by the prior estrogen exposure. This hypothesis predicts that the height velocity during treatment will be inversely related to the severity of prior estrogen exposure. We analyzed data from 100 girls (age, 5.8 +/- 2.1 yr; mean +/- SD) with central PP who were treated with GnRHa. During GnRHa therapy, height velocity was low for age (-1.6 +/- 1.7 SD score; mean +/- SD). The absolute height velocity correlated most strongly with the bone age (BA), which we used as a surrogate marker for growth plate senescence (r = -0.727, P < 0.001). The severity of the growth abnormality (height velocity SD score for age) correlated inversely with markers of the severity of prior estrogen exposure, including duration of PP (r = -0.375, P < 0.001), Tanner breast stage (r = -0.220, P < 0.05), and BA advancement (r = -0.283, P < 0.01). Stepwise regression confirmed that BA was the best independent predictor of growth during GnRHa therapy. The findings are consistent with our hypothesis that impaired growth during GnRHa therapy is due, at least in part, to premature growth plate senescence induced by the prior estrogen exposure.

In boys with abnormal developmental tempo, maturation of the skeleton and the hypothalamic-pituitary-gonadal axis remains synchronous.

The primary mechanism that initiates puberty is unknown. One possible clue is that pubertal maturation often parallels skeletal maturation. Conditions that delay skeletal maturation also tend to delay the onset of puberty, whereas conditions that accelerate skeletal maturation tend to hasten the onset of puberty. To examine this relationship, we studied boys with congenital adrenal hyperplasia (n = 13) and familial male-limited precocious puberty (n = 22), two conditions that accelerate maturational tempo, and boys with idiopathic short stature (n = 18) in which maturational tempo is sometimes delayed. In all three conditions, the onset of central puberty generally occurred at an abnormal chronological age but a normal bone age. Boys with the greatest skeletal advancement began central puberty at the earliest age, whereas boys with the greatest skeletal delay began puberty at the latest age. Furthermore, the magnitude of the skeletal advancement or delay matched the magnitude of the pubertal advancement or delay. This synchrony between skeletal maturation and hypothalamic-pituitary-gonadal axis maturation was observed among patients within each condition and also between conditions. In contrast, the maturation of the hypothalamic-pituitary-gonadal axis did not remain synchronous with other maturational processes including weight, height, or body mass index. We conclude that in boys with abnormal developmental tempo, maturation of the skeleton and the hypothalamic-pituitary-gonadal axis remains synchronous. This synchrony is consistent with the hypothesis that in boys, skeletal maturation influences hypothalamic-pituitary-gonadal axis maturation.

Mechanisms responsible for longitudinal growth of the cortex: coalescence of trabecular bone into cortical bone.

BACKGROUND: The purpose of the present study was to determine whether longitudinal growth of the cortex occurs through intramembranous bone formation involving the periosteum or through endochondral bone formation involving the growth plate and to explore the cellular and biochemical mechanisms responsible for this process. METHODS: Cortical bone formation was studied in the metaphyses of growing New Zealand White rabbits by means of (1) oxytetracycline labeling and fluorescence microscopy, (2) computer-assisted histomorphometry, (3) osteoblast culture and [(3) H]-thymidine incorporation in the presence of periosteum or periosteum-conditioned medium, and (4) surgical insertion of membranes between the periosteum and the underlying spongiosa. RESULTS: Within the metaphyseal cortex, oxytetracycline labeling produced fluorescent closed curves outlining enlarging trabeculae derived from coalescing endochondral trabecular bone. In this region of coalescing trabeculae close to the periosteum, osteoblast surface was increased compared with trabeculae farther from the periosteum (p < 0.001). The osteoclast surface did not differ. In vitro, osteoblast proliferation was increased in the presence of periosteum (p < 0.001) or periosteum-conditioned medium (p < 0.001). Surgical insertion of permeable or impermeable membranes between the periosteum and the spongiosa did not prevent cortex formation. CONCLUSIONS: These observations demonstrate that metaphyseal cortical bone is formed by coalescence of endochondral trabecular bone. This coalescence is associated with increased osteoblast surface in the peripheral spongiosa. The increased osteoblast surface could be due to inductive effects of periosteum; in the present study, periosteum stimulated osteoblast proliferation in vitro but was not required for metaphyseal cortical bone formation in vivo. CLINICAL RELEVANCE: Understanding metaphyseal cortical growth may help to elucidate the pathophysiology of osseous growth disorders in children.