Kevin B. Jones, MD Charles Searby, BS Gail Kurriger, BS James Martin, PhD Peter J. Roughley, PhD

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