Kauz Matsumoto, M.D., Ph.D., research
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2009 Conference abstract
Stochastic Conditional Knockout of Ext1 Reveals an Unexpected Relationship between Biallelic Inactivation of the
Gene and the Development of Multiple Exostoses

Kazu Matsumoto1, Fumitoshi Irie1, Susan Mackem2, and Yu Yamaguchi1
1Sanford Children's Health Research Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla,
CA 92037, USA. 2Laboratory of Pathology, National Cancer Institute, Bethesda, MD, 20892, USA.


Individuals with MHE carry heterozygous loss-of-function mutations of Ext1 or Ext2, which together encode an enzyme
essential for heparan sulfate synthesis. Despite the unambiguous identification of causative genes, there are a number of
enigmatic issues and unanswered questions surrounding MHE. Among them, three questions are of particular interest: (i)
whether osteochondroma in MHE is a true neoplasm or a developmental defect; (ii) whether loss of heterozygosity is the
underlying genetic mechanism of MHE; and (iii) why Ext1+/– mutant mice, which faithfully mimic the genotype of human MHE,
are resistant to osteochondroma formation, especially in long bones.

To test the hypothesis that biallelic inactivation of Ext1 occurring in a small fraction of chondrocytes is the pathogenic
mechanism of MHE, we employed a method of stochastic inactivation of loxP-flanked Ext1 alleles (Ext1flox) using a tamoxifen-
dependent Cre transgene driven by the Col2a1 promoter (Col2-CreERT). We originally intended to control the level of
recombination using different doses of tamoxifen. Unexpectedly, Col2-CreERT;Ext1flox/flox mice developed multiple
osteochondromas and other MHE-like bone deformities without tamoxifen treatment. We found that the non-induced Col2-
CreERT transgene drives stochastic recombination in a small fraction of chondrocytes (~5% in long bones). (Col2-CreERT;
Ext1flox/flox mice that are raised without tamoxifen treatment are designated as Ext1-SKO [stochastic knockout] mice.) The
penetrance of the long bone exostosis phenotype in Ext1-SKO mice was 100%, whereas bowing deformity and subluxation of
the radius and scoliosis were observed in 92% and 58% of Ext1-SKO mice, respectively. In contrast, neither heterozygous
Ext1-SKO mice (i.e., Col2-CreERT;Ext1flox/+) or Prx1-Cre;Ext1flox/+ mice developed these phenotypes at all, supporting the
requirement for biallelic inactivation. Surprisingly, osteochondromas (cartilage cap region) developed in Ext1-SKO mice are not
clonal growths of Ext1-null chondrocytes, but mixtures of Ext1-null and wild-type chondrocytes at highly variable ratios. This
heterogeneous nature of osteochondroma might be a part of the reason why previous studies on loss of heterozygosity have
not generated an unequivocal conclusion. Our results indicate that, although biallelic inactivation of Ext1 is required for its
initiation, chondrocytes comprising osteochondroma are not clonal, and therefore osteochondroma is not considered to be a
neoplasm in its strictest sense. Our results also suggest that Ext1-null chondrocytes exert unexpectedly potent cell non-
autonomous effects on the behavior of wild-type chondrocytes. This mouse model provides novel insight not only into the
genetic mechanism of MHE but also how heparan sulfate controls tissue development.
Photo's taken during the
Third International MHE Research Conference

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