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Osteochondromagenesis: Loss of heterozygosity modeled via Cre-mediated inversion of the second exon of Ext1 in chondrocytes Department of Orthopaedics and Rehabilitation University of Iowa Hospitals & Clinics 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 Connective Tissue Oncology Society London, UK Friday, 14 November 2008 Human EXT1: What is known clinically. . . Hereditary Multiple Exostosis • • • • Autosomal dominant inheritance Shortened long bones Multiple osteochondromas Homozygosity lethal 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. 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. Of Mice and Men Of Mice and Men ~60 kg Human flesh ~30g Mouse flesh ~2000X the number of cells Haploinsufficiency or Loss of Heterozygosity? Methods loxP LoxP loxP LoxP CRE Cre Routine or cis orientation of loxP sites results in fragment excision trans orientation of loxP sites results in reversible fragment inversion LoxP Pxol PxoL loxP CODING SEQUENCE CODING SEQUENCE CRE Cre ECNEUQES GNIDOC ECNEUQES GNIDOC 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 Targeting Construct Intron 1 loxP exon 2 loxP neo loxP intron 2 • Exon 2 contains two of the very few diseasecausing 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 exon 3 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 Results 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. excised neo exon 2 exon 2 excised neo neo reversed neo 2 exon excised neo reversed exon 2 reversed exon 2 neo excised neo reversed neo reversed exon 2 Effects of Cre-mediated inversion RT-PCR demonstrated the expected 40% reduction in Ext1 transcripts across homozygous tissue exposed to Cre Phenotype: Osteochondromas? Distal femur physis 6 week old Ext1 fl/fl with doxy-col2-Cre Proximal tibia 6 week old Ext1 fl/fl with doxycol2-Cre Genotype # of mice with osteochondromas/ # of mice over 6 weeks old _____________________________________________ fl/fl+doxy-col2a1-Cre 12/12 fl/fl without Cre 0/5 wt/fl+doxy-col2a1-Cre 0/7 wt/fl without Cre 0/1 wt/wt without Cre 0/8 _______________________________ Comparison Traditional Knockout Trans-floxed Conditional Knockout • 50% tissue reduction in Ext1 alleles • Very rare biallelic loss • 40% tissue reduction in Ext1 alleles • 20% biallelic loss • Osteochondromas very rare • Osteochondromas rampant Discussion Loss of heterozygosity is the mechanism of osteochondromagenesis in the setting of hereditary multiple exostoses Hypothesis Disruption of both Ext1 alleles in a physeal chondrocyte is sufficient to form an osteochondroma Alternate Hypothesis Disruption of both Ext1 alleles in some physeal chondrocytes is sufficient to derange signals and form osteochondromas 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 Conclusion Osteochondromageneis resulted from lowprevalence biallelic disruption of Ext1 in chondrocytes, modeling loss of heterozygosity with a unique twist on the standard conditional knock-out 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 Osteochondromagenesis: Loss of heterozygosity modeled via Cre-mediated inversion of the second exon of Ext1 in chondrocytes Department of Orthopaedics and Rehabilitation University of Iowa Hospitals & Clinics 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 Connective Tissue Oncology Society London, UK Friday, 14 November 2008
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