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 juvenile bones. Consequently, mutations in genes that regulate growth plate chondrogenesis cause abnormal bone growth in children. Depending on the severity and nature of the 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 who are brought to medical attention for linear growth disorders, clinical evaluation and laboratory evaluation fail to identify the underlying etiology. To discover new genetic causes of childhood growth disorders, we are using powerful genetic approaches including SNP arrays to detect large 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. This analysis has led to our identification of heterozygous mutations in ACAN causing autosomal dominant short stature with advanced bone age and premature osteoarthritis. Recently, we have participated in an international collaboration to identify more patients with this condition and thereby elucidate the phenotypic spectrum of the disorder We have also explored the epigenetic regulation of skeletal growth at the growth plate. Histone methyltransferases EZH1 and EZH2 catalyze the trimethylation of histone H3 at lysine 27 (H3K27), which serves as an epigenetic signal for chromatin condensation and transcriptional repression. In humans, heterozygous mutations in EZH2 cause Weaver syndrome, which includes marked skeletal overgrowth. In addition, the EZH2 gene lies in a locus associated with human height variation by genome-wide association studies, providing further evidence that EZH2 plays an important role in regulating skeletal growth. Because longitudinal bone growth results from chondrogenesis at the growth plate, we explored the role of Ezh1 and 2 in this process. In mice, neither cartilage-specific knockout of Ezh2 nor generalized knockout of Ezh1 affected skeletal growth, but the combined losses of both histone methyltransferases in cartilage diminished H3K27 trimethylation and severely impaired skeletal growth. Both of the principal process underlying growth plate chondrogenesis, chondrocyte proliferation and hypertrophy, were compromised. The decrease in chondrocyte proliferation is due in part to derepression of cyclin dependent kinase inhibitors Ink4a/b, while the ineffective chondrocyte hypertrophy is due to suppression of IGF signaling by 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. We are currently studying the missense mutations in EZH2 that cause Weaver syndrome to understand the molecular pathophysiology of the disorder. Our research has also focused on the role of bone morphogenetic proteins in the regulation of the growth plate and articular cartilage. 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. 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 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. We found evidence that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton.
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