Regulation Of Childhood Growth

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

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Abstract

Children grow taller because their bones grow longer. This bone elongation results from chondrogenesis in the growth plate, a thin layer of cartilage found near the ends of children’s bones. Growth plate chondrogenesis is controlled by multiple endocrine, paracrine, extracellular matrix-related, and intracellular regulatory systems. Our group has helped elucidate growth plate regulation by FGFs, BMPs, C-type natriuretic peptide, retinoids, WNTs, PTHrP/IHH, IGFs, estrogens, glucocorticoids, transcription factors such as SOX9, microRNAs, and epigenetic mechanisms. Variants in genes that regulate growth plate chondrogenesis cause abnormal bone growth in children, often resulting in short or tall stature. In some of these disorders, the bones are malformed, in which case the condition is termed a skeletal dysplasia. Some genetic disorders affect other tissues, in addition to the growth plate cartilage, producing a more complex genetic syndrome. To discover new genetic causes of skeletal growth disorders, we have studied families with inherited growth disorders using exome sequencing to detect causative genetic variants. With this approach, we have previously helped elucidate the roles of ACAN, QRICH1, BRF1, and CYP26A1/C1 in disorders of human growth. We also discovered that neomorphic variants in SP7 cause a high-turnover bone disorder. We recently discovered that variants in SPIN4, an epigenetic reader, cause an overgrowth syndrome with X-linked inheritance. We then explored the molecular mechanisms responsible for this condition. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro. Recently we have extended our investigations into the molecular mechanisms by which SPIN4 causes overgrowth, finding evidence that SPIN4 acts locally to expand the population of growth plate chondroprogenitor cells in the resting zone. We also recently performed exome sequencing in three unrelated families with short stature, distinctive facial appearances, and neurodevelopmental abnormalities. In these families, we identified variants in WASHC3, a component of the WASH complex. In the first family, we found evidence that a de novo dominant missense variant impaired WASHC3 participation in the WASH complex, altered PTH1R endosomal trafficking, diminished PTH1R signaling, and affected growth plate chondrocyte hypertrophic differentiation, providing a likely explanation for the short stature. Knockdown of other WASH complex components also diminished PTH1R signaling. In the second and third family, a homozygous variant in the start codon markedly reduced WASHC3 protein expression. In combination with prior studies of WASH complex proteins, our findings strongly suggest that the WASH complex is required for normal skeletal growth and that, consequently, genetic abnormalities impairing the function of the WASH complex cause short stature as well as distinctive facies and variable neurodevelopmental abnormalities. We have also continued to develop new treatment approaches to skeletal growth disorders by targeting therapeutic molecules to growth plate cartilage. We have focused particularly on targeting insulin-like growth factgor-1 (IGF-1) to the growth plate. Recombinant human IGF-1 is currently used to treat severe primary IGF-1 deficiency, but this treatment requires twice-daily injection, often does not fully correct the growth deficit, and has important adverse effects. We therefore sought to target IGF-1 to growth plate cartilage by generating fusion proteins combining IGF-1 with single-chain human antibody fragments that target matrilin-3, a cartilage matrix protein. We have shown evidence that this cartilage-targeting IGF-1 fusion protein promotes growth plate function without increasing kidney cell proliferation. Furthermore, we found that the fusion protein showed significantly reduced hypoglycemic effect compared to IGF-1 itself. To gain mechanistic insights into the role of matrilin-3 targeting, we assessed the ability of the fusion protein to activate AKT signaling in vitro and found that it caused a prolonged increase in AKT signaling compared to IGF-1 and that this effect was dependent on matrilin-3. Taken together, our findings provide further evidence that cartilage-targeted therapy could provide new pharmacological approaches for the treatment of childhood growth disorders, such as GH-insensitivity syndrome.