The identified "protective" and "risk" factors, as well as the proposed classification system, represent helpful tools for clinical management and follow-up of patients with multiple hereditary exostoses; moreover, homogeneous cohorts of patients, useful for studies on the pathogenesis of multiple hereditary exostoses, have been identified.

During limb skeletogenesis the cartilaginous long bone anlagen and their growth plates become delimited by perichondrium with which they interact functionally. Yet, little is known about how, despite being so intimately associated with cartilage, perichondrium acquires and maintains its distinct phenotype and exerts its border function. Because perichondrium becomes deranged and interrupted by cartilaginous outgrowths in Hereditary Multiple Exostoses (HME), a pediatric disorder caused by EXT mutations and consequent heparan sulfate (HS) deficiency, we asked whether EXT genes and HS normally have roles in establishing its phenotype and function. Indeed, conditional Ext1 ablation in perichondrium and lateral chondrocytes flanking the epiphyseal region of mouse embryo long bone anlagen – a region encompassing the groove of Ranvier – caused ectopic cartilage formation. A similar response was observed when HS function was disrupted in long bone anlagen explants by genetic, pharmacological or enzymatic means, a response preceded by ectopic BMP signaling within perichondrium. These treatments also triggered excess chondrogenesis and cartilage nodule formation and overexpression of chondrogenic and matrix genes in limb bud mesenchymal cells in micromass culture. Interestingly, the treatments disrupted the peripheral definition and border of the cartilage nodules in such a way that many nodules overgrew and fused with each other into large amorphous cartilaginous masses. Interference with HS function reduced the physical association and interactions of BMP2 with HS and increased the cell responsiveness to endogenous and exogenous BMP proteins. In sum, Ext genes and HS are needed to establish and maintain perichondrium’s phenotype and border function, restrain pro-chondrogenic signaling proteins including BMPs, and restrict chondrogenesis. Alterations in these mechanisms may contribute to exostosis formation in HME, particularly at the expense of regions rich in progenitor cells including the groove of Ranvier.

Heparan sulfate (HS) is an essential component of cell surface and matrix-associated proteoglycans (HSPGs). Due to their sulfation patterns, the HS chains interact with numerous signaling proteins and regulate their distribution and activity on target cells. Many of these proteins, including bone morphogenetic protein family members, are expressed in the growth plate of developing skeletal elements, and several skeletal phenotypes are caused by mutations in HS-synthesizing and modifying enzymes. The disease we discuss here is Hereditary Multiple Exostoses (HME), a disorder caused by mutations in HS synthesizing enzymes EXT1 and EXT2, leading to HS deficiency. The exostoses are benign cartilaginous-bony outgrowths, form next to growth plates, can cause growth retardation and deformities, chronic pain and impaired motion, and progress to malignancy in 2-5% of patients. We describe recent advancements on HME pathogenesis and exostosis formation deriving from studies that have determined distribution, activities and roles of signaling proteins in wild type and HS-deficient cells and tissues. Aberrant distribution of signaling factors combined with aberrant responsiveness of target cells to those same factors appear to be a major culprit in exostosis formation. Insights from these studies suggest plausible and cogent ideas about how HME could be treated in the future.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths—called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Hereditary multiple exostoses is a pediatric skeletal disorder characterized by benign cartilaginous tumors called exostoses that form next to growing skeletal elements. Hereditary multiple exostoses patients carry heterozygous mutations in the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2, but studies suggest that EXT haploinsufficiency and ensuing partial HS deficiency are insufficient for exostosis formation. Searching for additional pathways, we analyzed presence and distribution of heparanase in human exostoses. Heparanase was readily detectable in most chondrocytes, particularly in cell clusters. In control growth plates from unaffected persons, however, heparanase was detectable only in hypertrophic zone. Treatment of mouse embryo limb mesenchymal micromass cultures with exogenous heparanase greatly stimulated chondrogenesis and bone morphogenetic protein signaling as revealed by Smad1/5/8 phosphorylation. It also stimulated cell migration and proliferation. Interfering with HS function both with the chemical antagonist Surfen or treatment with bacterial heparitinase up-regulated endogenous heparanase gene expression, suggesting a counterintuitive feedback mechanism that would result in further HS reduction and increased signaling. Thus, we tested a potent heparanase inhibitor (SST0001), which strongly inhibited chondrogenesis. Our data clearly indicate that heparanase is able to stimulate chondrogenesis, bone morphogenetic protein signaling, cell migration, and cell proliferation in chondrogenic cells. These properties may allow heparanase to play a role in exostosis genesis and pathogenesis, thus making it a conceivable therapeutic target in hereditary multiple exostoses.