Hereditary multiple exostoses (HME) is a genetically heterogeneous human disease characterized by the development of bony outgrowths near the ends of long bones. HME results from mutations in EXT1 and EXT2,genes that encode glycosyltransferases that synthesize heparan sulfate chains. To study the relationship of the disease to mutations in these genes, we generated Ext2-null mice by gene targeting. Homozygous mutant embryos developed normally until embryonic day 6.0, when they became growth arrested and failed to gastrulate, pointing to the early essential role for heparan sulfate in developing embryos. Heterozygotes had a normal lifespan and were fertile; however, analysis of their skeletons showed that about one-third of the animals formed one or more ectopic bone growths (exostoses). Significantly, all of the mice showed multiple abnormalities in cartilage differentiation, including disorganization of chondrocytes in long bones and premature hypertrophy in costochondral cartilage. These changes were not attributable to a defect in hedgehog signaling, suggesting that they arise from deficiencies in other heparan sulfate-dependent pathways. The finding that haploinsufficiency triggers abnormal cartilage differentiation gives insight into the complex molecular mechanisms underlying the development of exostoses.

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Multiple Hereditary Exostoses (MHE) syndrome is caused by haploinsufficiency in Golgi-associated heparan sulfate polymerases EXT1 or EXT2 and is characterized by formation of exostoses next to growing long bones and other skeletal elements. Recent mouse studies have indicated that formation of stereotypic exostoses requires a complete loss of Ext expression, suggesting that a similar local loss of EXT function may underlie exostosis formation in patients. To further test this possibility and gain greater insights into pathogenic mechanisms, we created heterozygous Ext1+/− and compound Ext1+/−/Ext2+/− mice. Like Ext2+/− mice described previously (Stickens et al. Development 132:5055), Ext1+/− mice displayed rib-associated exostosis-like outgrowths only. However, compound heterozygous mice had nearly twice as many outgrowths and, more importantly, displayed stereotypic growth plate-like exostoses along their long bones. Ext1+/−Ext2+/− exostoses contained very low levels of immuno-detectable heparan sulfate, and Ext1+/−Ext2+/− chondrocytes, endothelial cells and fibroblasts in vitro produced shortened heparan sulfate chains compared to controls and responded less vigorously to exogenous factors such as FGF-18. We also found that rib outgrowths formed in Ext1f/+Col2Cre and Ext1f/+Dermo1Cre mice, suggesting that ectopic skeletal tissue can be induced by conditional Ext ablation in local chondrogenic and/or perichondrial cells. The study indicates that formation of stereotypic exostoses requires a significant, but not complete, loss of Ext expression and that exostosis incidence and phenotype are intimately sensitive to, and inversely related to, Ext expression. The data also indicate that the nature and organization of ectopic tissue may be influenced by site-specific anatomical cues and mechanisms.