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 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. This analysis has led to our identification of heterozygous mutations in ACAN causing autosomal dominant short stature with advanced bone age and premature osteoarthritis. ACAN encodes the proteoglycan aggrecan which is an important component of cartilage extracellular matrix. We have recently shown that ACAN mutations can also present as nonfamilial short stature and can present as short stature without accelerated skeletal maturation. Our genetic studies of children with growth disorders has also led to the identification of biallelic mutations in BRF1 in children with growth failure, central nervous system abnormalities, and facial anomalies. The findings confirm a previous report that biallelic mutations in BRF1 cause cerebellar-facial-dental syndrome. Our findings also help define the growth phenotype, indicating that the linear growth failure can become clinically evident before the neurological abnormalities and that a severely delayed bone age may serve as a diagnostic clue. We also studied a subject with tall stature, advanced bone age, and mild dysmorphic features. Exome sequencing revealed a de novo missense mutation in EZH2. EZH2 encodes a histone methyltransferase that methylates histone 3 at lysine 27 (H3K27). This finding was finding was consistent with previous reports that heterozygous missense mutations in EZH2 cause Weaver syndrome, which is characterized by tall stature, advanced bone age, characteristic facies, and variable intellectual disability. The molecular pathogenesis of this disorder is poorly understood. We previously showed that EZH2 plays a critical role in the regulation of chondrocyte proliferation and hypertrophy in the growth plate, which are the central determinants of skeletal growth. To determine whether the EZH2 mutations found in Weaver syndrome cause a gain of function or a loss of function, EZH2 with the mutation found in our patient was expressed in mouse growth plate chondrocytes. The mutant protein showed decreased histone methyltransferase activity compared to wild-type protein. The EZH2 mutation found in our subject was then introduced into mice using CRISPR/Cas9. Heterozygotes showed mild overgrowth, recapitulating the Weaver overgrowth phenotype. Both homozygous and heterozygous embryos showed decreased H3K27 methylation. The findings demonstrate that EZH2 mutations found in Weaver syndrome cause a partial loss of function. For normal bone growth to occur, cells in the growth plate must differentiate from proliferative zone (PZ) chondrocytes to hypertrophic zone (HZ) chondrocytes. The important role of microRNAs (miRNAs) in growth plate chondrocyte differentiation was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. We sought to identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes. First, we microdissected individual growth plate zones and performed miRNA profiling using a solution hybridization method and also miRNA-seq to identify miRNAs that are preferentially expressed in PZ compared to HZ. We found that some of these preferentially expressed miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) serve to promote proliferation and inhibit hypertrophic differentiation. We also found evidence that the observed differential expression of mir-374-5p, mir-379-5p and mir-503-5p between PZ and HZ is induced by the normal concentration gradient across the growth plate of parathyroid hormone-related protein (PTHrP). 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 PZ and HZ. In PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.
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