ORIG INAL ART ICLE

The molecular and cellular basis of exostosis formation inhereditary multiple exostoses

Meirav Trebicz-Geffen*, Dror Robinson*,�, Zoharia Evron*, Tova Glaser*, Mati Fridkin�, Yehuda

Kollander§, Israel Vlodavsky–, Neta Ilan–, Kit Fong Law**, Kathryn S.E. Cheah**, Danny Chan**,

Haim Werner* and Zvi Nevo*

*Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel

Aviv, Israel,�Department of Orthopedics and Traumatology, Golda-Hasharon Campus, Petach-Tiqua, Israel,

�Department of

Organic Chemistry, The Weizman Institute of Science, Rehovot, Israel,§Department of Orthopedic Oncology, Tel Aviv Sourasky

Medical Center, Tel Aviv, Israel,–Cancer and Vascular Biology Research Center, Rappaport Faculty of Medicine, Technion, Haifa,

Israel and **Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong

INTERNATIONAL

JOURNAL OF

EXPERIMENTAL

PATHOLOGY

Received for publication:

29 January 2008

Accepted for publication:

2 March 2008

Correspondence:

Zvi Nevo

Department of Human Molecular

Genetics and Biochemistry

Sackler School of Medicine

Tel-Aviv University

Ramat-Aviv

Tel Aviv 69978

Israel

Tel.: 972 3 6409319

Fax: 972 3 6406087

E-mail: zvinevo@post.tau.ac.il

Summary

The different clinical entities of osteochondromas, hereditary multiple exostoses (HME)

and non-familial solitary exostosis, are known to express localized exostoses in their joint

metaphyseal cartilage. In the current study biopsies of osteochondromas patients were

screened with respect to a number of cellular and molecular parameters. Specifically, car-

tilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10

articular cartilages of control subjects were collected and cell cultures were established.

Results obtained showed that one of the two HME samples that underwent DNA

sequencing analysis (HME-1) had a novel mutation for an early stop codon, which led to

an aberrant protein, migrating at a lower molecular weight position. The EXT-1 mRNA

and protein levels in chondrocyte cultures derived from all nine HME patients were ele-

vated, compared with solitary exostosis patients or control subjects. Furthermore, cell

cultures of HME patients had significantly decreased pericellular heparan sulphate (HS)

in comparison with cultures of solitary exostosis patients or control subjects. Immuno-

histochemical staining of tissue sections and Western blotting of cell cultures derived

from HME patients revealed higher levels of heparanase compared with solitary exostosis

patients and of control subjects. Further investigations are needed to determine whether

the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS,

increased degradation or a combination of both. In conclusion, it appears that due to a

mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to

the anatomical deformations with exostoses formation. Hence, elevation of HS content in

the pericellular regions should be a potential molecular target for correction.

Keywords

chondrocytes, EXT1, heparan sulphate, heparanase, hereditary multiple exostoses,

solitary exostosis

Int. J. Exp. Path. (2008)

doi: 10.1111/j.1365-2613.2008.00589.x

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd 1

Hereditary multiple exostoses (HME), also termed osteo-

chondromas, is the most prevalent genetic skeletal dysplasia

in orthopaedic surgery. HME is characterized by the forma-

tion of cartilage-capped bone projections (exostoses) local-

ized mainly to the juxta-ephipyseal region of long bones

(Solomon 1963). These bony protuberances appear shortly

after birth and continue to develop throughout childhood

and into puberty. The protuberances seem to develop at the

expense of the linear bone growth, thus leading to short

stature, a common clinical feature of HME patients

(Solomon 1963).

In approximately two-thirds of HME-afflicted individuals,

other skeletal complications have also been reported, includ-

ing malignant complications (Solomon 1963; Carmel &

Cramer 1968; Vinstein & Franken 1971; Schmale et al.

1994; Wicklund et al. 1995; Wuyts et al. 1996). Exostosis

development can also occur as a solitary lesion, with a prev-

alence of 1–2% in the general population. Solitary exostosis,

however, is believed to constitute a separate non-familial

clinical entity, and it is a typical hallmark of the Langer-

Giedion syndrome or Trevor disease. Figure 1 demonstrates

X-rays of patients afflicted with either HME or solitary

exostosis. In HME patients the exostoses appear in several

joints and in multiple sites in the same joint, while in soli-

tary only one exostosis emerges. Furthermore, while the for-

mat of the exostosis in solitary resembles a mushroom

shape, the shapes of the exostosis in HME patients are less

defined.

Hereditary multiple exostoses is an autosomal dominant

disorder associated with mutations in the exostosin (EXTs)

gene family. The HME patient population has been found to

be linked to three major genetic loci: EXT1, which maps to

chromosome 8q24.1 (Cook et al. 1993; Ahn et al. 1995),

EXT2, which maps to 11p13 (Potocki & Shaffer 1996; Stic-

kens et al. 1996; Wuyts et al. 1996), and EXT3, which is

located on the short arm of chromosome 19, although its

exact position has not yet been defined (Le Merrer et al.

1994). The majority of HME patients are believed to bear

nonsense or frameshift mutations in the EXT1 (60–70%) or

EXT2 (30–40%) genes, leading to the synthesis of truncated

forms of the EXT proteins (Wuyts & Van Hul 2000; Fran-

cannet et al. 2001). Further analysis revealed that EXT1 is a

Type II transmembrane glycoprotein, localized in the endo-

plasmic reticulum and Golgi apparatus, a distribution that is

characteristic of glycosyltransferases (Lin et al. 1998;

McCormick et al. 1998). Of importance, EXT1 and EXT2

have been identified as tumour suppressor genes. Thus, loss

of heterozygosity at these loci was detected in HME patients

whose benign tumours transformed into chondrosarcomas

(Hecht et al. 1995).

Although it has been known for years that HME is linked

to molecular defects in EXT loci, it was only recently shown

that the proteins encoded by these genes are related with

heparan sulphate (HS) biosynthesis. HS proteoglycans

(HSPGs) are ubiquitous macromolecules which were shown

to be associated with cell membrane surfaces (pericellular)

and with extracellular matrix (ECM) constituents (Vlodav-

sky et al. 1999; Vlodavsky & Friedmann 2001). The HS

sugar chain synthesis is initiated at a specific serine residue

of the core protein, followed by the formation of a tri-

saccharide (xylose-galactose-galactose) termed linkage

region, linking the core protein and the sugar side chain

polymers (Fritz et al. 1994; Lind et al. 1998; McCormick

et al. 1998, 2000a,b; Kitagawa et al. 1999). Chain elonga-

tion then takes place by the alternating addition of GlcA

and GlcNAc from the corresponding nucleotide sugars

(UDP-GlcA and UDP-GlcNAc respectively).

The ability of HS to interact with different attachment

sites on cell plasma membranes, including fibroblast growth

factor (FGF) receptors, as well as with ECM macromole-

cules such as collagen, laminin and fibronectin, indicates

that this proteoglycan is essential for activating growth fac-

tors, in the self-assembly of supramolecular aggregates, and

in the insolubility of ECM components, as well as in cell

adhesion and locomotion processes (Vlodavsky et al. 1999;

Vlodavsky & Friedmann 2001). The endoglycosidase hepa-

ranase is the predominant enzyme responsible for the degra-

dation of HS. Therefore, heparanase plays a crucial role in

fundamental biological processes associated with ECM

remodelling, cell migration, angiogenesis and cancer metas-

tasis (Edovitsky et al. 2004).

The present study was designed to provide a molecular

analysis of the HME-associated EXT1 gene, including DNA

sequence analysis, and mRNA and protein measurements. In

addition, novel information is provided regarding the peri-

cellular glycosaminoglycan profile and heparanase content of

cartilaginous tissue sections and chondrocyte cultures

derived from HME, solitary exostosis patients and control

subjects. These later parameters are associated with

anatomical deformation.

Materials and methods

Collection of biopsies

Biopsies of HME and solitary exostosis patients and of con-

trol subjects were collected from discarded tissues obtained

in the operation room. Helsinki approvals were obtained

from the relevant hospital committees and from the Tel Aviv

University Committee. The study included nine HME (HME

2 M. Trebicz-Geffen et al.

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

1–9) and 10 solitary exostosis (Solitary 1–10) patients. The

samples derived from control subjects were collected from

normal articular cartilage of subjects undergoing total hip or

total knee replacements.

Tissue processing

Fresh tissue samples of the two types of exostoses and of

control subjects were used for the establishment of cell cul-

tures as well as for the immunohistochemical procedures

and tissue sections. Paraffin was removed with xylol and the

samples were rehydrated in serial alcohol dilutions. Each

sample was stained with Mayer’s haematoxylin eosin, Mas-

son’s trichrome and alcian blue (pH of 1.0 and 2.5), using

routine staining techniques.

Cell cultures

Cell cultures were established as described previously (Rob-

inson et al. 1994). Monolayer cultures derived from biopsies

had a heterogeneous appearance, exhibiting both a fibro-

blast-like morphology as well as typical polygonal chondro-

cytic features. The cultures were grown in MEM medium

(Biological Industries, Bet Haemek, Israel) containing 1%

glutamine, antibiotics and 10% foetal calf serum (Biological

Industries). Cultures were seeded at high density to either

maintain or regain the chondrogenic phenotype, judged mor-

phologically and by biochemical parameters, of the carti-

laginous characteristic ECM composition. Cultures were

subcultured twice and P2 cultures were used in experiments.

Histological and immunohistochemical procedures

Tissue samples were processed and stained as described pre-

viously (Robinson et al. 1990). Immunohistochemical stain-

ing was performed in tissue sections using anti-EXT1

(dilution 1:100) and anti-heparanase (dilution 1:50) anti-

bodies. The protocols used in this study have been described

previously (Robinson et al. 1994).

Generation of an anti-EXT1-specific antibody

To generate an EXT1 antibody a synthetic peptide was syn-

thesized corresponding to residues Val733–Leu746 of the

mouse EXT1 protein sequence (NCBI, Nucleotide Database,

Locus NM_000127, GI: 4557570) with an additional

cysteine residue at the N terminus, and conjugated to malei-

mide-activated KLH (Pierce, Rockford, IL, USA). The immu-

nogenic sample at each injection time point was 200 lg of

the synthetic peptide ⁄ rabbit, together with complete or

incomplete Freund’s adjuvant (Sigma-Aldrich, Saint Louis,

MO, USA). The mixture was subcutaneously injected into

New Zealand white rabbits at 3-week intervals. Ten days

after the sixth booster, sera were collected and total IgG was

precipitated with saturated ammonium sulphate (Harlow &

Lane 1988). Antibody specificity was confirmed by ELISA

and by immunohistochemistry (Kobayashi et al. 2000). The

antibody-specific signal was abolished by introducing an

excess of the synthetic antigenic peptide. An ELISA assay

was used to assess the specificity of the EXT1 antibody.

Ninety-six-well plates were coated overnight with the fol-

lowing peptides: peptide 1, Val733–Leu746 of the mouse

EXT1 sequence (positive control); peptide 2, the reverse

sequence of the Val733–Leu746 peptide (negative control);

and peptide 3, the random tripeptide Ser-Gly-Gly (negative

control). The EXT1 antibody was assayed at dilutions rang-

ing from 1:3 to 1:531,441. The mouse EXT1 sequence was

found to have �95% homology to the human sequence. Spe-

cific binding of the antibody was detected to peptide number

1, but not to peptides 2 and 3, at dilutions below 1:1000.

Immunoblotting analysis

Cells were harvested by mechanical scraping, washed and

collected with phosphate-buffered saline (PBS) containing

5 mm EDTA. The cells were centrifuged at 600 g for

10 min, and the pellet resuspended in a lysis buffer contain-

ing 1% Triton X-100, 150 mm NaCl, 2 mm EDTA, 2 mm

EGTA, and protease and phosphatase inhibitors. The lysates

were spun down at maximum speed in a microfuge. Proteins

in the Triton X-100-soluble supernatant were resolved by

SDS-PAGE, transferred onto nitrocellulose membranes and

immunoblotted (Towbin et al. 1979) with the rabbit EXT1

polyclonal antibody. Bound antibodies were detected using

an enhanced chemiluminescence detection system (Super-

Signal; Pierce).

EXT1 mutation screening

DNA samples were isolated from cultured chondrocytes of

HME patients (HME 1 and 2), solitary exostoses patients

(Solitary1–4), and from normal subjects using the High Pure

PCR template preparation kit (Roche Molecular Biochemi-

cals, Indianapolis, IN, USA). PCR amplifications of the

entire EXT1 coding sequence were performed for each

sample, using primers and conditions as previously described

(Wuyts et al. 1998). Mutational analysis was performed by

conformation sensitive gel electrophoresis (CSGE) compared

to a normal control. PCR products with conformational

changes were purified using a PCR purification kit (Roche)

Hereditary multiple exostoses 3

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

and then sequenced. Sequencing results were blasted against

the corresponding EXT1 exon sequences (gi: 51511724),

and against the human genome database sequence in Ensem-

ble (Wuyts et al. 1998).

RNA isolation and RT-PCR analysis of EXT1 mRNA

Total RNA was isolated from explant-derived cultures from

HME, solitary exostosis patients and controls using TRI-

REAGENT (MRC, Cincinnati, OH, USA). For the synthesis

of cDNA, 5 lg of total RNA was reverse transcribed with

an oligo(dT)12–18 primer (Promega, Madison, WI, USA) and

SuperScript II reverse transcriptase (Gibco BRL�, Paisley,

UK). The cDNA products were used directly as templates

for PCR amplification. PCR was performed in a final volume

of 25 ll containing 10· PCR buffer [1x = 20 mm Tris-HCl

(pH 8.4), 50 mm KCl], 1.5 mm MgCl2, 1.25 U Taq DNA

Polymerase (Gibco BRL�), 0.1 mm dNTPs (Gibco BRL�),

10 lm each of the primers and 10 ng ⁄ ll template cDNA.

The primers used for EXT1 human cDNA were: forward:

5¢-TGCAAGCATGGCAAAGACTGGCAAAAG-3¢; reverse:

5¢-AGAAGCTTCAACACTGGCTGGGACTG-3¢. The expec-

ted size of the EXT1 mRNA was 598 bp and of the EXT1

DNA 1098 bp. Human glyceraldehyde-3-phosphate dehy-

drogenase (GAPDH) mRNA was used as an internal control.

Primers used were: forward: 5¢-CCACCCATGGCAAATTC-

CATGGCA-3¢; reverse: 5¢-TCAAGACGGCAGGTCAGGTC-

CACC-3¢. The expected size of the GAPDH mRNA was

610 bp. PCR was performed for 25 cycles: 94 �C for 1 min,

72 �C for 4 min and 72 �C for 10 min. The PCR products

were resolved by 1.5% agarose gel electrophoresis and

visualized by ethidium bromide staining (Sigma-Aldrich).

Isolation and quantitative analysis of the

glycosaminoglycan (GAG) profile in cultured cells

The rate of synthesis and GAG content in cultured cells

were measured by incubating the cells in 2 ml medium sup-

plemented with 10 lCi ⁄ ml of radioactive sodium sulphate

[35S Carrier free] (Amersham, Buckinghamshire, UK). The

radioactive sulphate precursor is incorporated into GAG

molecules. Cells were incubated for 24 h and the GAG

molecules were isolated according to the protocol described

previously (Nevo & Dorfman 1972; Nevo et al. 1972).

Briefly, cells and medium were boiled to denature the

proteins as pretreatment to protein digestion with papain,

followed by exhaustive dialysis against distilled water. The

GAG molecules were precipitated with 0.5–1% cetyl pyridi-

nium chloride (CPC) in the presence of 1 mg of chondroitin

sulphate, as a carrier. The release of the GAGs from the

complex GAG-CPC was achieved using 2 m CaCl2, and

recovery of the soluble GAG molecules was achieved by

precipitation in an ethanol-ether solution (2:1 v ⁄ v). An

aliquot was taken for radioactive reading and the rest of the

samples were used for GAG analyses using different GAG

degrading enzymes. The GAGs were dried and dissolved

in a small aliquot of distilled water. Chondroitin

sulphate digestion was performed by chondroitinase ABC

(0.1 U ⁄ sample ⁄ 200 ll) (Sigma) in 0.01 m Tris-acetate buffer

(pH 8.0) for 48 h at 37 �C. HS digestion was performed by

heparanase (0.1 U ⁄ sample ⁄ 200 ll) (Sigma) incubation in

0.1 m sodium acetate (pH 7.0) for 48 h at 37 �C (Nevo &

Dorfman 1972; Nevo et al. 1972).

Statistical analysis

To evaluate the statistical significance of the experiments we

used unpaired Student’s t-test. Differences were considered

as significant at P < 0.05.

Results

Mutational analyses of the EXT1 gene in HME and

solitary exostosis patients

The EXT1 gene has 11 exons, with exon 1 being the largest

with 1735 bp. Exon 1 was amplified using four sets of prim-

ers to produce short overlapping fragments covering the cod-

ing region and the splice junctions (Wuyts et al. 1998).

Exons 2–11 were amplified using primer pairs that flank the

coding region (Wuyts et al. 1998). CSGE analysis was per-

formed for all PCR products from the patients and the pat-

tern was compared to the equivalent products from a

normal control. PCR products with conformational changes

were subjected to DNA sequencing. Results of sequencing

analysis showed a heterozygous 1 bp deletion at nt 418 of

the cDNA sequence in one of the HME patients (HME-1)

(Figure 2a). In the mutant allele, the deletion would predict

a frameshift in codon usage. The predicted protein product

is expected to contain a 17-aa novel peptide extending from

aa 157 until a termination codon at aa 173 (Figure 2b). In

the HME-2 patient, a silent mutation was found (exon 6 C

to T, and exon 9 G to A, data not shown). No mutations

were detected in Solitary 1–4.

EXT1 mRNA measurements in HME, solitary exostosis

and control patients’ cultured cells

EXT1 mRNA levels were determined in cell cultures

derived from HME and solitary exostosis explants using a

4 M. Trebicz-Geffen et al.

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

(a) (b) (c) (d)

Figure 1 X-ray appearance of different

exostoses. (a) Solitary exostosis in a

femur. (b) Multiple exostoses in the

knee joint, antero-posterior view. (c)

Multiple exostoses in the knee joint,

lateral view. (d) Multiple exostoses in

the upper hip and trochanter (arrows

indicate the exostoses sites).

(a)

Normalallele

Mutantallele

MATG GCC AAA AAA CGC TAT TTC ATC CTG CTC GCTTCA TCT TGT CTC GCCGGCCAG

Q A K K R Y F I L L S A G S C L A 1854

36108

54162

72216

90270

108324

126378

144

162486

180540

432

LCTT TTT TAT TTC GGA GGC CAG TTT AGG TCGGCA AGC CAC AGC CGGAGGTTG

F F G G Q F R A S R S S RLCTT TTT TAT TTC GGA GGC TTG CAG TTT AGG TCGGCA AGC CAC AGC CGGAGGTTG

L F Y F G G L Q F R

R

A S R S H S R

R

X P F P A LDR

AGA

TGG CGC TTC CCG GAC GCT CTG CGC CCC TTC GIT TGG GAT CAA TTG GAACCTCCC

GAA CAC AGC GGT AGG AAT GGC TTG CAC CCCCAC CCG GAT CAT TTC AGTGAAR K H S G R

F V P X D Q L E

N G

G

L H H P

P

S P D H P

S

S

N

M

R S

S

S H

KXY K K C C F F T L

I

I

VDAAC GAT TCC AGC GTG CAC ATT TCC CCC CGG CAG CGA GAT GCC AAC TCCAAGGAG

R

R

Q K R D

D

A N SP

AGC TAC AAA GGC AAG AAG TGC CGC ATG GAG TCC TTC TTCGAT ACC CTTTGCATC

G PVC YNX F K V Q Q G K KX Y KTGC AAA AAC GGC TTC AAA GTC TAC GTA TAC CCA CAA AAA GAGGGG AAACAGAAG

ATC GAA AGT TAC CAA AAC ATT CTA GCG GCC ATC GCT CCA TCTGGT ACAGAGGCCS AL AKA Y Q N I K P G S TI I A

CCT ACC CCA GCC AGG CGT GCC TCT TTG TCC TGA TGG ATA TAGCTT ACAGTCCGGP SS LTR A R R · V I L · TP

K T S C H L S M C T I P K C R VD·

A X

GAG AGT TGT CAC CTC AGT ATG TGC ACA ATT TGA CCA AAG AGATGC GTCGATACC

594

216648

234702

252 756

270810

288864

918

320961

306

198

ACA

TCCS

L

P

P

S

T

G

T

CTT

CCA

CCC

TCA

ACC

GGG

ATT

TGTC

L

P

F

P

Q

T

I

CTG

CCA

TTT

CCA

CAG

ACG

CTC

GGAG

T

A

L

S

G

L

L

ACT

GCA

CTA

TCC

GGA

TTG

GCT

ACAT

T

S

Q

L

·

C

A

ACA

TCA

AGG

CTC

TAG

TGC

GTG

ATGM

P

V

I

L

D

S

V

CCG

GTA

ATC

CTC

GAT

TCC

ACA

GTAV

R

L

I

S

Q

S

T

AGG

CTG

ATC

TCA

CAG

TCA

GAG

GGAG

T

K

P

G

T

P

R

ACG

AAA

CCA

GGA

ACA

CCA

ACA

ATCI

X

T

G

S

P

P

T

TGG

ACT

GGA

AGT

CCA

CCT

ACA

ATTI

G

S

Q

T

G

A

T

GGT

TCC

CAG

ACA

GGA

GCA

CCG

TAA·

L

D

E

C

X

S

P

TTG

GAC

GAG

TGC

ATG

AGC

ATG

TTAL

S

T

R

Y

Y

A

X

TCG

ACT

AGA

TAT

TAT

GCA

I

A

L

G

S

I

K

R

AGA

ATT

GCC

TTG

GGG

TCA

ATC

AAG

A

TATY

R

X

G

R

T

T

AGG

ATG

GGT

AGG

ACG

ACT

ATTI

R

F

F

G

S

G

CGA

TTT

TTT

GGA

TCC

GGC

CCGP

C

L

·

R

I

K

TGC

CTA

TGA

AGA

ATA

AAA

GCAA

X

F

S

G

T

S

TGG

TTC

AGT

GGT

ACG

AGC

AGT

TTTF

T

P

G

X

P

M

S

ACA

CCA

GGG

TGG

CCT

ATG

AGG

ACTT

G

K

S

T

·

R

R

GGC

AAG

TCT

ACA

TGA

AGG

(b)

A A A AA AA A A A A A A A A A A A A AA AAT T T T T T T T T T T T T T T TN

N N N N NN N N N N N N N N N N N N N N N N NNNNN NNNN

N N N N N N N N N N N NNCG G G G G G G G G G GGGG GGGC C10

100 110 120 130 140 150 160 170 180 190

20 30 7040 50 60 80 90C C C C C C C C C C C C C C C CCCCG

A A A A A A A A A A A A A A A A A A A A A A A A A A A A AA AT T T T T T T T T T TG G G G G G G G G G G GG GGGC C C C C C C C CC

Figure 2 Sequencing analysis of EXT1

exon 1b in an HME patient. (a) DNA

sequencing analysis shows a heterozy-

gous 1 bp deletion at nt 418 in exon

1b. The red arrow indicates the position

of the deleted G. Beyond this point,

two overlapping sequences are

observed: one normal sequence and one

with 1bp shift, indicating that the

deletion is a heterozygous event. (b) In

the mutant allele, the deletion results in

a frameshift in codon usage. The pre-

dicted protein product contains a 17-aa

novel peptide from aa 157 (highlighted

in red) until a termination codon at aa

173.

3

2.5

OD

ELISA for the specificity of EXT1 Ab

EXT1 reverse

EXT1

L-seryl-glycyl-glycyl

2

1.5

1

0.5

0

1:3

1:9

1:27

1:81

1:24

31:

729

1:21

87

1:65

61

1:19

,683

1:59

,049

1:17

7,14

7

1:53

1,44

1

Figure 4 ELISA analysis of EXT1 antibody specificity. Ninety-

six-well plates were covered with coating buffer overnight with

three different peptides at 1 and 10 mg ⁄ ml concentrations: (1)

synthetic peptide of mouse EXT1 residues Val733–Leu746; (2)

reverse peptide sequence of EXT1 Val733–Leu746; and (3) con-

trol tripeptide (Ser-gly-gly). The EXT1 antibody dilutions ran-

ged from 1:3 to 1:531,441. The secondary antibody was goat

anti-rabbit (1:70000). O-phenylenediamine dihydrate (OPD)

was used for the colour reaction. The intensity of the colour

reaction was determined by spectrophotometric reading at

450 nm.

EXTl

GAPDH

RT

Normal

(a)

(b)

+ – + – + –

Expression of EXT1 mRNA in normal

EX

T1

/ GA

PDH

Normal HME Solitary exostosis

P-value < 0.05 chondrocytes

chondrocytes compared to solitary and HME

* 1.5

1

0.5

0

*

HME Solitary

610 bp

598 bp

exostoses chondrocytes

Figure 3 EXT1 mRNA levels in HME and solitary exostosis

patients. (a) EXT1 mRNA levels were measured by RT-PCR in

cultured cells of HME and solitary exostosis patients, and

control subjects. (b) Densitometric analysis of EXT1 mRNA

levels. Statistical analysis was performed using Student’s t-test

comparing both HME and solitary exostosis patients with

normals. *Significantly different vs. controls (P < 0.05).

Hereditary multiple exostoses 5

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

semi-quantitative RT-PCR protocol. As shown in

Figure 3(a), EXT1 mRNA levels in HME patients were

higher than in solitary exostosis patients and controls. The

levels of GAPDH mRNA remained unchanged in all sam-

ples. Figure 3(b) shows the results of densitometric scanning

of the EXT1 mRNA bands normalized to the corresponding

GAPDH mRNA bands. Similar results were obtained using

Northern blot analyses and real-time PCR (data not

shown).

Western blot analyses of EXT1 protein in cell culture

homogenates

Western blot analysis was performed using a specific home-

made EXT1 antibody. The biological activity of the anti-

body was determined by ELISA, which showed that the

EXT1 antibody displayed high specificity against an EXT1

peptide (Figure 4). In addition, the EXT1 protein was

stained specifically as an epitope in the ossifying region near

the tide mark of the articular cartilage (Figure 5). Further

corroboration of the antibody specificity was provided by a

competition-linked immunohistochemical staining procedure.

In this assay, a synthetic EXT1 peptide was incubated for

1 h with the EXT1 antibody in order to block the free bind-

ing sites for EXT1. As shown in Figure 6(a), a dose-depen-

dent decrease in the staining signal of EXT1 was seen in

the presence of the EXT1 peptide while a positive signal

(a) (b) Articular cartilage

Ossifying region

Human femurEXT1 antibody 1:100

Human femurPre-immune serum 1:100

Figure 5 Immunohistochemical staining of EXT1. Normal

human femur paraffin sections were incubated with pre-immune

(a) and immune (b) EXT1 antiserum (1:100). Secondary

antibody was goat anti-rabbit (1:70,000). Staining is seen in the

ossification region of the articular cartilage (b).

X40

10 mg/mlαEXTl With peptide

αEXTl W/O peptide

X40

Figure 6 Immunohistochemical staining with aEXT1 in the

presence of a synthetic EXT1 peptide. Normal human femur

(articular cartilage) paraffin sections were incubated with

pre-immune and immune EXT1 antiserum (1:100), with or

without a synthetic peptide for EXT1 from Val733–Leu746 at a

concentration of 10 mg ⁄ ml.

Normal

Normal

Densitometric values of ααEXT1(b)

(a)

vs Pre-immune serum

Pre-immune serum 1:500 αEXT1 1:500

32.5 *

EXT1

Pre-immuneserum

*

21.5

10.5

0

HME Solitaryexostosischondrocytes

Arb

itra

ry u

nits

HME Solitaryexostosischondrocytes

Normal HME Solitaryexostosis

80 kDa

P-value <0.05

chondrocytes

Figure 7 Western blot analyses of EXT1. (a) EXT1 protein

levels were measured in HME, solitary exostosis and normal

chondrocyte cultures and compared with pre-immune serum.

The cells were scraped from the plates mechanically, lysed, and

100 lg total protein was analysed using SDS-PAGE. (b)

Densitometric analysis of EXT1 protein levels. Bars denote

mean ± SD of five independent cultures of each clinical entity.

Statistical analysis was performed using Student’s t-test.

*Significantly different vs. control (P < 0.05).

Pre-immune serum 1:500 ααEXT1 1:500

HME Solitary exostosis

Normalchondrocytes

Normal HME

80 kDa

Solitary exostosis chondrocytes

Figure 8 Pattern of expression of EXT1 of the HME-1 patient.

EXT1 expression in the HME-1 patient (bearing an early stop

codon mutation) was analysed using Western blot analysis, in

comparison with samples of solitary exostosis and normal

chondrocyte cultures, using both specific and pre-immune

serum. The cells were scraped off the plates mechanically, and

then the lysates (100 lg) were separated on SDS-PAGE.

6 M. Trebicz-Geffen et al.

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

concentrated mainly in the ossifying region of the articular

cartilage (Figure 6b).

Western blot analyses of cultured cells from HME, soli-

tary exostosis and controls revealed that EXT1 is expressed

at higher levels in HME than in solitary exostosis patients

or controls (Figure 7). Furthermore, in one of the HME

patients (HME-1) the EXT1 protein was seen in Western

blots as two bands, with one of the bands migrating at a

lower position (Figure 8).

Biochemical analyses of the GAG profiles in cultured

cells

The total content of GAG molecules measured in the

pericellular and intracellular fractions was similar in all

the cultures tested. The lowest levels of chondroitin

sulphate were measured in the pericellular and intracellu-

lar fractions of the solitary exostosis cultures, while the

highest levels of chondroitin sulphate were seen in the

HME cultures. Control cultures were intermediate in this

respect (Figure 9). The HS content in intracellular and

pericellular domains were the lowest in HME cultured

cells and highest in the solitary exostosis cultures, with

intermediate levels in control cultures (Figure 9). GAG

profiles in the extracellular fractions of the cultures reflect

in general the GAG profile detected in the parallel intra-

cellular and pericellular cultures. Hence, there are distinct

and significant differences in the contents of chondroitin

sulphate and HS found in the fractions of the two clinical

entities.

140

120

100

80

* *

*

Heparinase Chondroitinase treated

Normal ch. HME Solitary exo.

Residual GAG- Residual GAG- P-value <0.05

P-value <0.05CS content HS content

Intracellular & Pericellular

Per

cent

age

of t

otal

GA

G

Per

cent

age

of t

otal

GA

G

Medium

Residual GAG- Residual GAG- CS content HS content

Normal ch. HME Solitary exo.

treated Untreated

Heparinase Chondroitinase treated treated

Untreated

60

40

20

0

120

100

80

60

40

20

0

Figure 9 GAG profiles in the different cultures, separating the extracellular domains from the intracellular and pericellular GAG

fractions. The experiment was conducted in parallel on 4 different patients of each entity, and each experiment was carried out in

tetraplicates. The GAG contents postchondroitinase and postheparanase digestion were compared with untreated GAG samples. A

value of 100% was given to the cpm counts of untreated normal chondrocytes, HME and solitary exostoses, and compared with the

enzymatically digested residues. Statistical analysis was performed using Student’s t-test, comparing HME and solitary exostosis to

normal. *Significantly different vs. controls (P < 0.05).

(a) (b)

(d)(c)

X40 X40

X100X100Figure 10 Immunohistochemical

staining of heparanase. Immunohisto-

chemical staining with an heparanase

antibody was performed on normal

human articular cartilage (a) and HME

patients (b–d), using standard staining

procedures.

Hereditary multiple exostoses 7

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

As shown in Figure 10, the HME sections exhibited inten-

sely positive staining for heparanase in the cartilage regions

and also in the bone marrow (data not shown). The sections

from the controls did not stain for heparanase. Finally, we

checked the protein levels of heparanase by Western blot-

ting, and found that they were higher in HME patients than

in controls and solitary exostosis patients (Figure 11).

Discussion

Although the entire scope of function of the EXT protein is

not yet fully known, all EXT gene products exhibit glycosyl-

transferase activity related to the proteoglycan HS biosyn-

thesis pathway (Lind et al. 1998; Kitagawa et al. 1999; Kim

et al. 2001). HS along with other PGs-GAGs, including

chondroitin sulphate and others, function physiologically at

the cell surface and in the ECM in a variety of processes,

including activation of growth factors, receptor signalling,

cellular proliferation and differentiation (McCormick et al.

1998; Selleck 2000). Although GAGs represent simple poly-

mers of repeating disaccharide units attached to a core pro-

tein via a linkage region, the polymers exist in discrete

structural forms, generated by varying patterns of epimeriza-

tion, glycosyltransferring and sulphation (Selleck 2000). This

structural diversity in GAG chains might account for the

functional diversity and specificity of tissue-derived proteo-

glycans (San Antonio et al. 1994; Chintala et al. 1995; Sel-

leck 2000). As patients bearing mutations in the EXT1 and

EXT2 genes are at higher risk of developing chondrosarco-

ma or osteosarcoma, it was suggested that the malignant

transformation is caused by a double hit in the oligosaccha-

ride chains of HS and chondroitin sulphate (the two defec-

tive glycosyltransferases). According to the literature 60–

70% of HME patients bear mutations in the EXT1 gene

belonging either to the nonsense or frameshift mutations

(Wuyts & Van Hul 2000; Francannet et al. 2001). These

findings were confirmed in our current study where in one

of two HME patients who underwent DNA sequencing

analysis a novel stop codon mutation was detected.

We then determined the EXT1 mRNA and protein levels

in cell cultures and tissue sections. Our results show that

EXT1 mRNA levels are higher in all HME-derived cultures

than in those derived from solitary exostosis or normal con-

trols. These results were corroborated using Northern blot

and real-time PCR. Western blot analyses of EXT1 protein

showed that, similar to EXT1 mRNA, the protein levels in

HME patients are higher than in solitary exostosis or con-

trols. Elevated EXT1 mRNA levels in cells derived from

HME patients might reflect an attempt to compensate for

the defective EXT1 protein.

The findings of the current study support the notion that

the clinical entities of HME and of solitary exostosis differ

from each other not only in the clinical symptoms but also in

most major genetic and biochemical aspects, i.e. the genetic-

molecular profile of the EXT genes, as well as the analytical

profile of the pericellular and extracellular PG contents. It is

important to emphasize that there is considerable diversity in

the population of HME patients in respect of the EXT1

mutations, as was evident from the DNA sequencing analyses

in our subjects. These analyses showed that one HME patient

had a silent mutation, while the other patient was found to

have an early stop codon mutation, that most probably led

to an aberrant protein. Indeed, it was found that the patient

with the early stop codon mutation had two EXT1 bands on

Western blots. One band showed the expected MW, similar

to the solitary exostosis patients and controls, while the other

band displayed a lower MW.

Importantly, scanning densitometry of Western blots

showed that EXT1 protein levels tended to be higher in the

HME patients compared with solitary exostosis patients and

controls. However, because of the heterogeneity of the HME

patients, this difference did not reach statistical significance.

Larger numbers of patients need to be studied to confirm

these findings. The population of HME patients demon-

strated autosomal dominant inheritance and heterozygosity

in the EXT1 alleles. Clinical relevance has not yet been

Normalchondrocytes

10 *

(a)

(b)

P-value < 0.05

86420

Densitometric levels of Heparanse protein

Normalchondrocytes

HMEArb

itra

ry u

nits

Solitaryexostosis

Solitaryexostosis

65 kDaHeparanase

Tubulin 50 kDa

HME

Figure 11 Protein levels of heparanase in lysates of cultured

cells. (a) Protein levels of heparanase were measured in HME,

solitary exostosis and normal cultured cells. The cells were

scraped off the plates mechanically with buffer that causes com-

plete lysis of the cells; lysates were run on SDS-PAGE and blot-

ted with anti-heparanase. Blots were then re-probed with anti-

tubulin. (b) Densitometric averages of heparanase levels normal-

ized to tubulin values (n = 5). Statistical analysis was performed

using Student’s t-test. *Significantly different vs. controls

(P < 0.05).

8 M. Trebicz-Geffen et al.

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

correlated with the degree of gene penetration, i.e. ‘silent’

vs. early stop codon mutations. Furthermore, patients differ

in the severity of the clinical features, i.e. number, size and

distribution of exostoses, as well as in malignant transforma-

tion. Of notice, malignant transformation can be affected by

other genes, such as EXT2, which is also involved in HS

chain elongation and might create a hetero-oligomer in vivo

with EXT1 in the Golgi apparatus (Kobayashi et al. 2000;

McCormick et al. 2000a).

Recent studies have shown that clones of proliferating

chondrocytes without functional EXT1 (or EXT2) expres-

sion fail to produce HS (Hecht et al. 2005; Stickens et al.

2005). Lack of HS at the cell surface disrupts FGF signalling

and Indian hedgehog diffusion, leading to focal over-prolif-

eration and adjacent bone collar deficiency and osteoporosis

respectively (Lemos et al. 2005). A combination of these

effects was proposed to contribute to osteochondroma path-

ogenesis.

Heparan sulphate chains are associated with many pro-

teins both pericellularly and extracellularly, and are involved

in a number of normal and pathological processes such as

morphogenesis, inflammation, autoimmunity, tumour metas-

tasis and angiogenesis (Kjellen & Lindahl 1991; Vlodavsky

et al. 1992, 1994-1995; Iozzo 1998; Bernfield et al. 1999).

The involvement of EXTs in the synthesis of HS chains led

us to measure HS levels in cells derived from HME patients.

Analysis of the pericellular levels of HS showed that there is

indeed a decrease in HS chains content in HME patients

compared with solitary exostosis and controls (Hecht et al.

2005; Stickens et al. 2005). Furthermore, chondroitin sul-

phate content was highest in cells derived from HME

patients in the intracellular and pericellular domains, as well

as in the extracellular GAG fraction. These findings corrobo-

rate previous reports on GAG biosynthesis with diminished

amount of HS chains in HME-derived cells (Hecht et al.

2005; Stickens et al. 2005). Taken together, these results

suggest that most probably the mutation in the EXT1 gene,

coding for a defective HS glycosyltransferase enzyme, is

responsible for the deficit in HS chains (McCormick et al.

2000a; Hecht et al. 2005; Stickens et al. 2005). It appears

that in HME patients the deficiency in HS polymerase might

not be reflected in total GAG molecules due to the compen-

sation of the glycosyltransferase of chondroitin sulphate,

resulting in almost normal content of total GAG molecules.

Alternatively, a low content of HS might also result from

over-expression of the degrading enzyme heparanase. This

possibility was examined by immunohistochemical staining

with anti-heparanse on histological sections of cartilaginous

tissues of HME and solitary exostosis patients and control

subjects. Heparanase is a glycosylated endo-d-glucouroni-

dase which is capable of cleaving HS at specific intra-chain

sites and which is involved in the catabolism of HS glycos-

aminoglycan chains (Parish et al. 1987, 1999; Vlodavsky

et al. 1992, 1994-1995; Edovitsky et al. 2004). Heparanase

precursors bind to the cell surface, most likely to HS, and

are then converted to the highly active 50 kDa form (Oosta

et al. 1982; Hoogewerf et al. 1995; Freeman & Parish 1998;

Vlodavsky et al. 2003).

Our study shows that heparanase has an important role in

the development of HME. Heparanase expression was

higher in paraffin block sections of HME patients compared

with controls. In addition, protein levels of heparanase in

Western blots of cell homogenates were higher in HME than

in solitary exostosis patients or controls. Therefore, we can

conclude that there may be more than one explanation for

the low levels of HS in HME patients, i.e. reduced synthesis

and increased degradation. Hence, the clinical diversity and

the degree of the ailment severity among HME patients

might be explained by the combined effects of molecular

failures of HS glycosyltransferases and ⁄ or heparanase. Both

of these findings are directing the search and efforts towards

clinical treatment of HME patients by finding ways to sup-

port and elevate the content of HS.

Acknowledgements

This work was conducted by Meirav Trebicz-Geffen as par-

tial fulfilment of the requirements for a PhD degree at the

Sackler School of Medicine, Tel Aviv University, Tel Aviv,

Israel. This work was supported partially by grant 1267 ⁄ 04

from the Israel Science Foundation and grant

N_HKU011 ⁄ 00 from the Hong Kong Research Council.

References

Ahn J., Ludecke H.J., Lindow S. et al. (1995) Cloning of the

putative tumour suppressor gene for hereditary multiple

exostoses (EXT1). Nat. Genet. 11, 137–143.

Bernfield M., Gotte M., Park P.W. et al. (1999) Functions of

cell surface heparan sulfate proteoglycans. Annu. Rev.

Biochem. 68, 729–777.

Carmel P.W. & Cramer F.J. (1968) Cervical cord compression

due to exostosis in a patient with hereditary multiple exosto-

ses. Case report. J. Neurosurg. 28, 500–503.

Chintala S.K., Miller R.R., McDevitt C.A. (1995) Role of hepa-

ran sulfate in the terminal differentiation of growth plate

chondrocytes. Arch. Biochem. Biophys. 316, 227–234.

Cook A., Raskind W., Blanton S.H. et al. (1993) Genetic

heterogeneity in families with hereditary multiple exostoses.

Am. J. Hum. Genet. 53, 71–79.

Hereditary multiple exostoses 9

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

Edovitsky E., Elkin M., Zcharia E., Peretz T., Vlodavsky I.

(2004) Heparanase gene silencing, tumor invasiveness,

angiogenesis, and metastasis. J. Natl Cancer Inst. 96, 1219–

1230.

Francannet C., Cohen-Tanugi A., Le Merrer M., Munnich A.,

Bonaventure J., Legeai-Mallet L. (2001) Genotype-phenotype

correlation in hereditary multiple exostoses. J. Med. Gene 38,

430–434.

Freeman C. & Parish C.R. (1998) Human platelet heparanase:

purification, characterization and catalytic activity. Biochem.

J. 330 (Pt 3), 1341–1350.

Fritz T.A., Gabb M.M., Wei G., Esko J.D. (1994) Two N-acety-

lglucosaminyltransferases catalyze the biosynthesis of heparan

sulfate. J. Biol. Chem. 269, 28809–28814.

Harlow E. & Lane D. (1988) Antibodies: A Laboratory Man-

ual, pp. 298–299. Cold Spring Harbor, NY: Cold Spring Har-

bor Laboratory Press.

Hecht J.T., Hogue D., Strong L.C., Hansen M.F., Blanton S.H.,

Wagner M. (1995) Hereditary multiple exostosis and chon-

drosarcoma: linkage to chromosome II and loss of heterozy-

gosity for EXT-linked markers on chromosomes II and 8.

Am. J. Hum. Genet. 56, 1125–1131.

Hecht J.T., Hayes E., Haynes R. et al. (2005) Differentiation-

induced loss of heparan sulfate in human exostosis derived

chondrocytes. Differentiation 73, 212–221.

Hoogewerf A.J., Leone J.W., Reardon I.M. (1995) CXC chemo-

kines connective tissue activating peptide-III and neutrophil

activating peptide-2 are heparin ⁄ heparan sulfate-degrading

enzymes. J. Biol. Chem. 270, 3268–3277.

Iozzo R.V. (1998) Review. Matrix proteoglycans: from molecu-

lar design to cellular function. Annu. Rev. Biochem. 67, 609–

652.

Kim B.T., Kitagawa H., Tamura J. et al. (2001) Human tumor

suppressor EXT gene family members EXTL1 and EXTL3

encode alpha 1,4-N-acetylglucosaminyltransferases that likely

are involved in heparan sulfate ⁄ heparin biosynthesis. Proc.

Natl Acad. Sci. USA 98, 7176–7181.

Kitagawa H., Shimakawa H., Sugahara K. (1999) The tumor

suppressor EXT-like gene EXTL2 encodes an alpha1, 4-N-

acetylhexosaminyltransferase that transfers N-acetylgalactos-

amine and N-acetylglucosamine to the common glycosamino-

glycan-protein linkage region. The key enzyme for the chain

initiation of heparan sulfate. J. Biol. Chem. 274, 13933–13937.

Kjellen L. & Lindahl U. (1991) Proteoglycans: structures and

interactions. Annu. Rev. Biochem. 60, 443–475.

Kobayashi S., Morimoto K., Shimizu T., Takahashi M., Kurosa-

wa H., Shirasawa T. (2000) Association of EXT1 and EXT2,

hereditary multiple exostoses gene products, in Golgi appara-

tus. Biochem. Biophys. Res. Commun. 268, 860–867.

Le Merrer M., Legeai-Mallet L., Jeannin P.M. et al. (1994) A

gene for hereditary multiple exostoses maps to chromosome

19p. Hum. Mol. Genet. 3, 717–722.

Lemos M.C., Kotanko P., Christie P.T. et al. (2005) A novel

EXT1 splice site mutation in a kindred with hereditary multi-

ple exostosis and osteoporosis. J. Clin. Endocrinol. Metab.

90, 5386–5392.

Lin X., Gan L., Klein W.H., Wells D. (1998) Expression and

functional analysis of mouse EXT1, a homolog of the human

multiple exostoses type 1 gene. Biochem. Biophys. Res. Com-

mun. 248, 738–743.

Lind T., Tufaro F., McCormick C., Lindahl U., Lidholt K.

(1998) The putative tumor suppressors EXT1 and EXT2 are

glycosyltransferases required for the biosynthesis of heparan

sulfate. J. Biol. Chem. 273, 26265–26268.

McCormick C., Leduc Y., Martindale D. et al. (1998) The

putative tumor suppressor EXT1 alters the expression of

cell-surface heparan sulfate. Nat. Genet. 19, 158–161.

McCormick C., Duncan G., Goutsos K.T., Tufaro F. (2000a)

The putative tumor suppressors EXT1 and EXT2 form a

stable complex that accumulates in the Golgi apparatus and

catalyzes the synthesis of heparan sulfate. Proc. Natl Acad.

Sci. USA 97, 668–673.

McCormick C., Duncan G., Tufaro F. (2000b) Herpes simplex

virus: discovering the link between heparan sulphate and

hereditary bone tumors. Rev. Med. Virol. 10, 373–384.

Nevo Z. & Dorfman A. (1972) Stimulation of chondromuco-

protein synthesis in chondrocytes by extracellular chondro-

mucoprotein. Proc. Natl Acad. Sci. USA 69, 2069–2072.

Nevo Z., Horwitz A.L., Dorfmann A. (1972) Synthesis of chon-

dromucoprotein by chondrocytes in suspension culture. Dev.

Biol. 28, 219–228.

Oosta G.M., Favreau L.V., Beeler D.L., Rosenberg R.D. (1982)

Purification and properties of human platelet heparitinase. J.

Biol. Chem. 257, 11249–11255.

Parish C.R., Coombe D.R., Jakobsen K.B., Bennett F.A., Under-

wood P.A. (1987) Evidence that sulphated polysaccharides

inhibit tumor metastasis by blocking tumor-cell-derived hepa-

ranases. Int. J. Cancer 40, 511–518.

Parish C.R., Freeman C., Brown K.J., Francis D.J., Cowden

W.B. (1999) Identification of sulfated oligosaccharide-based

inhibitors of tumor growth and metastasis using novel in vitro

assays for angiogenesis and heparanase activity. Cancer Res.

59, 3433–3441.

Potocki L. & Shaffer L.G. (1996) Interstitial deletion of

11(p11.2p12): a newly described contiguous gene deletion

syndrome involving the gene for hereditary multiple exostoses

(EXT2). Am. J. Med. Genet. 62, 319–325.

Robinson D., Halperin N., Nevo Z. (1990) Regenerating hyaline

cartilage in articular defects of old chickens using implants of

embryonal chick chondrocytes embedded in a new natural

delivery substance. Calcif. Tissue Int. 46, 246–253.

Robinson D., Tieder M., Halperin N., Burshtein D., Nevo Z.

(1994) Maffucci’s syndrome – the result of neural abnormali-

ties? Evidence of mitogenic meurotrasmitters present in

10 M. Trebicz-Geffen et al.

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology

enchondromas and soft tissue hemangiomas. Cancer 7, 949–

957.

San Antonio J.D., Karnovsky M.J., Gay S., Sanderson R.D.,

Lander A.D. (1994) Interactions of syndecan-1 and heparin

with human collagens. Glycobiology 4, 327–332.

Schmale G.A., Conrad E.U. 3rd, Raskind W.H. (1994) The nat-

ural history of hereditary multiple exostoses. J. Bone Joint

Surg. Am. 76, 986–992.

Selleck S.B. (2000) Proteoglycans and pattern formation: sugar

biochemistry meets developmental genetics. Trends Genet. 16,

206–212.

Solomon L. (1963) Hereditary multiple exostosis. J. Bone Joint

Surg. 45B, 292–304.

Stickens D., Clines G., Burbee D. et al. (1996) The EXT2

multiple exostoses gene defines a family of putative tumour

suppressor genes. Nat. Genet. 14, 25–32.

Stickens D., Zak B.M., Rougier N., Esko J.D., Werb Z. (2005)

Mice deficient in EXT2 lack heparan sulfate and develop

exostoses. Development 132, 5055–5068.

Towbin H., Staehelin T., Gordon J. (1979) Electrophoretic

transfer of proteins from polyacrylamide gels to nitrocellulose

sheets: procedure and some applications. Proc. Natl Acad.

Sci. USA 76, 4350–4354.

Vinstein A.L. & Franken E.A. Jr (1971) Hereditary multiple

exostoses. Report of a case with spinal cord compression.

Am. J. Roentgenol. Radium Ther. Nucl. Med. 112, 405–407.

Vlodavsky I. & Friedmann Y. (2001) Review. Molecular prop-

erties and involvement of heparanase in cancer metastasis and

angiogenesis. J. Clin. Invest. 108, 341–347.

Vlodavsky I., Eldor A., Haimovitz-Friedman A. et al. (1992)

Expression of heparanase by platelets and circulating cells of

the immune system: possible involvement in diapedesis and

extravasation. Invasion Metastasis 12, 112–127.

Vlodavsky I., Mohsen M., Lider O. et al. (1994-1995) Inhibi-

tion of tumor metastasis by heparanase inhibiting species of

heparin. Invasion Metastasis 14, 290–302.

Vlodavsky I., Friedmann Y., Elkin M. et al. (1999) Mammalian

heparanase: gene cloning, expression and function in tumor

progression and metastasis. Nat. Med. 5, 793–802.

Vlodavsky I., Zcharia E., Goldshmidt O. et al. (2003) Involve-

ment of heparanase in tumor progression and normal differ-

entiation. Pathophysiol. Haemost. Thromb. 33 (Suppl. 1) ,

59–61.

Wicklund C.L., Pauli R.M., Johnston D., Hecht J.T. (1995)

Natural history study of hereditary multiple exostoses. Am. J.

Med. Genet. 55, 43–46.

Wuyts W. & Van Hul W. (2000) Molecular basis of multiple

exostoses: mutations in the EXT1 and EXT2 genes. Hum.

Mutat. 15, 220–227.

Wuyts W., Van Hul W., Wauters J. et al. (1996) Positional

cloning of a gene involved in hereditary multiple exostoses.

Hum. Mol. Genet. 5, 1547–1557.

Wuyts W., Van Hul W., De Boulle K. et al. (1998) Mutations

in the EXT1 and EXT2 genes in hereditary multiple exo-

stoses. Am. J. Hum. Genet. 62, 346–354.

Hereditary multiple exostoses 11

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, International Journal of Experimental Pathology