Children grow taller because their bones grow longer. This bone elongation occurs at the growth plate, a thin layer of cartilage found near the ends of children's bones. Consequently, mutations in genes that regulate growth plate chondrogenesis cause abnormal bone growth in children. Depending on the specific genetic abnormality, the clinical phenotype can range from chondrodysplasias with short, malformed bones, to severe, often disproportionate, short stature, to mild proportionate short stature. If the genetic defect affects tissues other than the growth plate cartilage, the child may present with a more complex syndrome that includes other clinical abnormalities. For many children with growth disorders, the etiology remains unknown. To discover new genetic causes of childhood growth disorders, we are using powerful genetic approaches including SNP arrays to detect deletions, duplications, mosaicism, and uniparental disomy combined with exome sequencing to detect single nucleotide variants and small insertions/deletions in coding regions and splice sites. Recently, this analysis has led us to find that mutations in a gene called QRICH1 impairs growth at the growth plates, causing short stature. We studied a child 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 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, a step that is critical for bone elongation. 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. We have also sought to improve the treatment of children with growth disorders. 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 sought to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. For this purpose, we developed cartilage-targeting single-chain human antibody fragments and then created fusion proteins of these antibody fragments, combined with insulin-like growth factor I (IGF-1), an endocrine/paracrine factor that positively regulates chondrogenesis. These fusion proteins retained both cartilage binding and IGF-1 biological activity and were able to stimulate bone growth in an organ culture system. Using a growth hormone-deficient mouse model, we found evidence that subcutaneous injections of these fusion proteins had greater on-target efficacy at the growth plate and less off-target effect than IGF-1 alone. Our findings provide proof-of-principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders. Our group also studies the fundamental mechanisms governing skeletal growth. Recently, we focused on why 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. We found 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 found evidence that the molecular mechanisms that underlie the differential aging between different bones involve modulation of critical paracrine regulatory pathways, including Igf, Bmp, and Wnt signaling. Taken together, the findings reveal that the striking disparities in lengths of different bones, which characterize normal mammalian skeletal proportions, is achieved in part by modulating the progression of growth plate senescence. We also have explored the transdifferentiation of growth plate chondrocytes into 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. We found 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.
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