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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 - PowerPoint PPT Presentation
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Kevin B. Jones, MDCharles Searby, BSGail Kurriger, BS
James Martin, PhDPeter J. Roughley, PhD
Jose A. Morcuende, MD, PhDJoseph A. Buckwalter, MD, MS
Val C. Sheffield, MD, PhD
Department ofOrthopaedics and RehabilitationUniversity of IowaHospitals & 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
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
~30gMouse flesh
~2000X the number of cells
Haploinsufficiency
or
Loss of Heterozygosity?
Methods
CRE
loxP loxPLoxP LoxP
Cre
Routine or cis orientation of loxP sites results in fragment excision
trans orientation of loxP sites results in reversible fragment inversion
CRE
Pxol loxP
CODING SEQUENCE
ECNEUQES GNIDOC
LoxP PxoL
Cre
CODING SEQUENCE
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 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
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.
exon 2 neo
reversed exon 2 neo
reversed reversed neo exon 2
excised neo
excised neo
reversed exon 2
exon 2
excised neo
reversed neo exon 2
excised neo
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 physis6 week old Ext1 fl/fl
with doxy-col2-Cre
Proximal tibia6 week old Ext1 fl/fl
with doxy-col2-Cre
Genotype_____________________________________________
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
_______________________________
# of mice with osteochondromas/ # of mice over 6 weeks old
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
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 low-prevalence 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 ofOrthopaedics and RehabilitationUniversity of IowaHospitals & Clinics
Kevin B. Jones, MDCharles Searby, BSGail Kurriger, BS
James Martin, PhDPeter J. Roughley, PhD
Jose A. Morcuende, MD, PhDJoseph A. Buckwalter, MD, MS
Val C. Sheffield, MD, PhD
Department ofOrthopaedics and RehabilitationUniversity of IowaHospitals & 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