Osteochondroma Formation: Haploinsufficiency or Two Hits?
CMA Reijnders, CJF Waaijer, A Hamilton, E Buddingh, PDS Dijkstra, SJ Ham, E Bakker, K Szuhai, M Karperien, PCW Hogendoorn, SE Stringer, JVMG Bovee. Leiden University Medical Center, Leiden, Netherlands; University of Manchester, Manchester, United Kingdom; OLVG, Amsterdam, Netherlands; University of Twente, Enschede, Netherlands
Background: Multiple osteochondromas (MO) is an autosomal dominant disorder caused by germline mutations in EXT1 and/or EXT2, whereas solitary osteochondroma is a non-hereditary lesion. EXT is involved in heparan sulfate biosynthesis. We investigated the controversial issue whether osteochondromas arise via the classical two-hit model for tumor suppressor genes or via haploinsufficiency.
Design: An in vitro 3D chondrogenic pellet model was used to compare heterozygous mesenchymal stem cells (MSCs)(EXTwt/-) of MO patients with normal MSCs and the corresponding tumor specimens (presumed EXT-/-). EXT mutations and mRNA expression levels were assessed. HS chain length and structure of normal and heterozygous MSCs in monolayer culture was determined. Immunohistochemistry was performed on heparan sulfate (HS), heparan sulfate proteoglycans (HSPGs)(SDC2-4, perlecan, CD44v3), and HS dependent signaling pathways: TGFbeta/BMP (phosphosmad-1, phosphosmad-2, PAI-1), Wnt (beta-catenin) and PTHLH (PTHR1, bcl2).
Results: Germline EXT1 and EXT2 mutations were present in MO patients (6/8). We demonstrated a second hit in the EXT genes in 5 out of 8 osteochondromas (both solitary and hereditary). MSCs with a heterozygous EXT mutation are identical to wildtype MSCs with regard to HS chain length and structure, in vitro chondrogenesis and the expression of EXT and EXT downstream signaling molecules.
Conclusions: In conclusion, since I) a heterozygous EXT mutation does not affect chondrogenesis, heparan sulfate and downstream signaling pathways and II) we show a second hit in the majority of osteochondromas our results refute the haploinsufficiency theory and strongly support the two-hit model for osteochondroma formation.
Category: Bone & Soft Tissue
Tuesday, March 23, 2010 1:45 PM
Platform Session: Section E, Tuesday Afternoon