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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.

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.

BackgroundMidkine (MDK), one of the heparin-binding growth factors, is highly expressed in multiple organs during embryogenesis. Plasma concentrations have been reported to be elevated in patients with a variety of malignancies, in adults with obesity, and in children with short stature, diabetes, and obesity. However, the concentrations in healthy children and their relationships to age, nutrition, and linear growth have not been well studied.Subjects and methodsPlasma MDK was measured by immunoassay in 222 healthy, normal-weight children (age 0-18 yrs, 101 boys), 206 healthy adults (age 18-91 yrs, 60 males), 61 children with BMI ≥ 95th percentile (age 4-18 yrs, 20 boys), 20 girls and young women with anorexia nervosa (age 14-23 yrs), and 75 children with idiopathic short stature (age 3-18 yrs, 42 boys). Body fat was evaluated by dual-energy X-ray absorptiometry (DXA) in a subset of subjects. The associations of MDK with age, sex, adiposity, race/ethnicity and stature were evaluated.ResultsIn healthy children, plasma MDK concentrations declined with age (r = -0.54, P < 0.001) with values highest in infants. The decline occurred primarily during the first year of life. Plasma MDK did not significantly differ between males and females or between race/ethnic groups. MDK concentrations were not correlated with BMI SDS, fat mass (kg) or percent total body fat, and no difference in MDK was found between children with anorexia nervosa, healthy weight and obesity. For children with idiopathic short stature, MDK concentrations did not differ significantly from normal height subjects, or according to height SDS or IGF-1 SDS.ConclusionsIn healthy children, plasma MDK concentrations declined with age and were not significantly associated with sex, adiposity, or stature-for-age. These findings provide useful reference data for studies of plasma MDK in children with malignancies and other pathological conditions.

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.