Affiliations 1 Biomedical Sciences College, Shandong Medicinal Biotechnology Centre, Shandong First Medical University & Shandong Academy of Medical Sciences, Ji'nan, Shandong, China. 2 Department of Orthopedic Surgery, The First Affiliated Hospital of Shandong First Medical University, Ji'nan, Shandong, China. 3 Key Lab for Biotech-Drugs of National Health Commission, Ji'nan, Shandong, China. 4 Qingdao Academy of Chinese Medical Science, Shandong University of Traditional Chinese Medicine, Ji'nan, Shandong, China.
Hereditary Multiple Osteochondromas is a rare autosomal dominant skeletal disorder caused by heterozygous loss-of-function mutations in EXT1 or EXT2, which encode glycosyltransferases essential for heparan sulfate biosynthesis. Whether haploinsufficiency alone suffices or biallelic inactivation is required for osteochondroma formation remains a central unresolved question. In this study, we employed CRISPR/Cas9 combined with PiggyBac transposon technology to introduce a second pathogenic mutat...更多
Hereditary Multiple Osteochondromas is a rare autosomal dominant skeletal disorder caused by heterozygous loss-of-function mutations in EXT1 or EXT2, which encode glycosyltransferases essential for heparan sulfate biosynthesis. Whether haploinsufficiency alone suffices or biallelic inactivation is required for osteochondroma formation remains a central unresolved question. In this study, we employed CRISPR/Cas9 combined with PiggyBac transposon technology to introduce a second pathogenic mutation into patient-derived induced pluripotent stem cells carrying a heterozygous EXT1 c.1126C>T mutation. This approach enabled the generation of isogenic iPSC lines: wild-type , single-mutant , and double-mutant . These iPSCs were differentiated through induced mesenchymal stem cells into chondrocytes. Biallelic EXT1 mutation in DM cells led to significant upregulation of SOX9, COL2A1, and ACAN, elevated glycosaminoglycan levels, and markedly reduced HS, whereas SM cells remained indistinguishable from WT. Three-dimensional chondrogenic organoid cultures revealed that DM organoids were enlarged and structurally disorganized, partially recapitulating key histopathological features of osteochondromas. Transcriptomic analysis identified the Wnt signaling pathway as the most significantly enriched pathway among differentially expressed genes following EXT1 loss. Collectively, these findings provide direct human cellular evidence that complete EXT1 inactivation-not haploinsufficiency-drives aberrant chondrogenesis, likely through impaired sequestration of morphogen ligands, thereby supporting the Two-hit pathogenic model.收起
