1. Kevin B. Jones, MD Charles Searby, BS Gail Kurriger, BS James Martin, PhD Peter J. Roughley, PhD Jose A. Morcuende, MD, PhD Joseph A. Buckwalter, MD, MS Val C. Sheffield, MD, PhD Department of Orthopaedics and Rehabilitation University of Iowa Hospitals & Clinics Osteochondromagenesis: Loss of heterozygosity modeled via Cre-mediated inversion of the second exon of Ext1 in chondrocytes Connective Tissue Oncology Society London, UK Friday, 14 November 2008

2. Human EXT1: What is known clinically... Hereditary Multiple Exostosis Autosomal dominant inheritance Shortened long bones Multiple osteochondromas Homozygosity lethal

4. EXT1 & EXT2: Tumor Supressor Genes? Loss of heterozygosity in chondrocytes of cartilage cap of HME osteochondromas Both alleles somatically mutated in some solitary osteochondromas Raskind et al. 1995; Bovee et al. 1999; Hecht et al. 1995 and 1997.

6. Ext1 Knock-Out Mice by Lin et al. 2000 Mouse Ext1 has 99% Homology to Human EXT1 PROBLEMS: Homozygous Ext1-knock-out mice do not survive to birth Heterozygous Ext1-knock-out mice very rarely form osteochondromas Simlar with Ext2 knock-out mice, Sitckens et al. 2005.

7. Of Mice and Men

9. ~60 kg Human flesh ~30g Mouse flesh ~2000X the number of cells

10. Haploinsufficiency or Loss of Heterozygosity?

11. Methods

12. CRE loxP LoxP Cre Routine or cis orientation of loxP sites results in fragment excision

13. trans orientation of loxP sites results in reversible fragment inversion CRE PxolloxP CODING SEQUENCE ECNEUQES GNIDOC LoxP PxoL Cre CODING SEQUENCE ECNEUQES GNIDOC

14. You do the math... Cre-mediated inversion yields 40% reverse orientation per trans-floxed allele Homozygosity for trans-floxed alleles should yield 40% loss of total copies of functional gene across tissue –20% of cells will remain unaffected and fwd/fwd –20% of cells will end up fwd/fwd after inversion –40% of cells will end up fwd/rev after inversion –20% of cells will end up rev/rev after inversion

15. Targeting Construct Intron 1 loxP exon 2 loxP neo loxP intron 2 exon 3 Exon 2 contains two of the very few disease- causing missense mutations at highly conserved amino acid residues 339 and 340. Exon 2 inversion not only disrupts the sequence, but introduces a stop codon in the reading frame

16. Cre-Recombinase Driver Doxycycline-inducible Collagen type II promoter-Cre Doxycycline adminstered via maternal drinking water during second week of life Gover and Roughley et al., 2006

17. Results

18. PCR Testing with Multiple Primers Intron 1 loxP exon 2 loxP neo loxP intron 2 exon 3 All permutations of flipping were present in every cartilage containing tissue.

19. exon 2 neo reversed exon 2 neo reversed neo exon 2 excised neo excised neo reversed exon 2 excised neo reversed neo exon 2 excised neo

20. Effects of Cre-mediated inversion RT-PCR demonstrated the expected 40% reduction in Ext1 transcripts across homozygous tissue exposed to Cre

21. Phenotype: Osteochondromas?

22. Distal femur physis 6 week old Ext1 fl / fl with doxy-col2-Cre

24. Proximal tibia 6 week old Ext1 fl / fl with doxy- col2-Cre

26. Genotype _____________________________________________ fl/fl+doxy-col2a1-Cre12/12 fl/fl without Cre0/5 wt/fl+doxy-col2a1-Cre0/7 wt/fl without Cre0/1 wt/wt without Cre0/8 _______________________________ # of mice with osteochondromas/ # of mice over 6 weeks old

27. Comparison Traditional Knockout 50% tissue reduction in Ext1 alleles Very rare biallelic loss Osteochondromas very rare Trans-floxed Conditional Knockout 40% tissue reduction in Ext1 alleles 20% biallelic loss Osteochondromas rampant

28. Discussion Loss of heterozygosity is the mechanism of osteochondromagenesis in the setting of hereditary multiple exostoses

29. Hypothesis Disruption of both Ext1 alleles in a physeal chondrocyte is sufficient to form an osteochondroma

30. Alternate Hypothesis Disruption of both Ext1 alleles in some physeal chondrocytes is sufficient to derange signals and form osteochondromas

31. Hypothesis vs. Alternate Hypothesis Are osteochondromas clonal (at least consistent) for reverse orientation of exon 2? On-going work with laser capture micro-dissection and in situ hybridization

32. Conclusion Osteochondromageneis resulted from low- prevalence biallelic disruption of Ext1 in chondrocytes, modeling loss of heterozygosity with a unique twist on the standard conditional knock-out

33. Project Funding Support OREF Resident Research Award 2003 Department of Orthopaedics and Rehabilitation, University of Iowa Val C. Sheffield Genetics Laboratory Department of Orthopaedics and Rehabilitation University of Iowa Hospitals & Clinics

34. Kevin B. Jones, MD Charles Searby, BS Gail Kurriger, BS James Martin, PhD Peter J. Roughley, PhD Jose A. Morcuende, MD, PhD Joseph A. Buckwalter, MD, MS Val C. Sheffield, MD, PhD Department of Orthopaedics and Rehabilitation University of Iowa Hospitals & Clinics Osteochondromagenesis: Loss of heterozygosity modeled via Cre-mediated inversion of the second exon of Ext1 in chondrocytes Connective Tissue Oncology Society London, UK Friday, 14 November 2008