Decreased EXT expression and intracellular accumulation of heparan sulphate proteoglycan
in osteochondromas and peripheral chondrosarcomas
L Hameetman 1, G David 2, A Yavas 1, SJ White 3, AHM Taminiau 4, A-M Cleton-Jansen 1, PCW
Hogendoorn 1, JVMG Bovée 1 *
1Department of Pathology, Leiden University Medical Centre, Leiden, The Netherlands
2Centre for Human Genetics, University of Leuven, Leuven, Belgium
3Centre for Human and Clinical Genetics, Leiden University Medical Centre, Leiden, The Netherlands
4Department of Orthopaedic Surgery, Leiden University Medical Centre, Leiden, The Netherlands
Funded by:
Dutch Cancer Society; Grant Number: RUL2002-2738
Abstract
Mutational inactivation of EXT1 or EXT2 is the cause of hereditary multiple osteochondromas. These
genes function in heparan sulphate proteoglycan (HSPG) biosynthesis in the Golgi apparatus. Loss of
heterozygosity of the EXT1 locus at 8q24 is frequently found in solitary osteochondromas, whereas
somatic mutations are rarely found. We investigated the expression of EXT1 and EXT2 (quantitative
RT-PCR) and of different HSPGs (immunohistochemistry) in solitary and hereditary osteochondromas
and in cases with malignant progression to secondary peripheral chondrosarcoma, in relation to
possible mutations and promoter methylation. The mutation status of patients with multiple
osteochondromas correlated with decreased EXT1 or EXT2 expression found in their resected
tumours. We could not show somatic point mutations or promoter hypermethylation in 17 solitary
tumours; however, EXT1 expression was decreased in 15 cases, whereas EXT2 was not. Intracellular
accumulation of syndecan-2 and heparan sulphate-bearing isoforms of CD44 (CD44v3) was found in
most tumours, which concentrated in the Golgi apparatus as shown by confocal microscopy. This
contrasted with the extracellular expression found in normal growth plates. In conclusion, mutational
inactivation of either EXT1 or EXT2 leads to loss of mRNA expression of the corresponding gene. We
hypothesize that loss of EXT expression disrupts the function of the EXT1/2 complex in HSPG
biosynthesis, resulting in the intracellular accumulation of HSPG core proteins that we found in these
tumours.
Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons,
Ltd.
in osteochondromas and peripheral chondrosarcomas
L Hameetman 1, G David 2, A Yavas 1, SJ White 3, AHM Taminiau 4, A-M Cleton-Jansen 1, PCW
Hogendoorn 1, JVMG Bovée 1 *
1Department of Pathology, Leiden University Medical Centre, Leiden, The Netherlands
2Centre for Human Genetics, University of Leuven, Leuven, Belgium
3Centre for Human and Clinical Genetics, Leiden University Medical Centre, Leiden, The Netherlands
4Department of Orthopaedic Surgery, Leiden University Medical Centre, Leiden, The Netherlands
Funded by:
Dutch Cancer Society; Grant Number: RUL2002-2738
Abstract
Mutational inactivation of EXT1 or EXT2 is the cause of hereditary multiple osteochondromas. These
genes function in heparan sulphate proteoglycan (HSPG) biosynthesis in the Golgi apparatus. Loss of
heterozygosity of the EXT1 locus at 8q24 is frequently found in solitary osteochondromas, whereas
somatic mutations are rarely found. We investigated the expression of EXT1 and EXT2 (quantitative
RT-PCR) and of different HSPGs (immunohistochemistry) in solitary and hereditary osteochondromas
and in cases with malignant progression to secondary peripheral chondrosarcoma, in relation to
possible mutations and promoter methylation. The mutation status of patients with multiple
osteochondromas correlated with decreased EXT1 or EXT2 expression found in their resected
tumours. We could not show somatic point mutations or promoter hypermethylation in 17 solitary
tumours; however, EXT1 expression was decreased in 15 cases, whereas EXT2 was not. Intracellular
accumulation of syndecan-2 and heparan sulphate-bearing isoforms of CD44 (CD44v3) was found in
most tumours, which concentrated in the Golgi apparatus as shown by confocal microscopy. This
contrasted with the extracellular expression found in normal growth plates. In conclusion, mutational
inactivation of either EXT1 or EXT2 leads to loss of mRNA expression of the corresponding gene. We
hypothesize that loss of EXT expression disrupts the function of the EXT1/2 complex in HSPG
biosynthesis, resulting in the intracellular accumulation of HSPG core proteins that we found in these
tumours.
Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons,
Ltd.
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Judith V.M.G. Bovée M.D., Ph.D.
Research authored by Dr. Bovée
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List of Publications via PubMed
(NIH National Library of Medicine)
List of Publications via PubMed
(NIH National Library of Medicine)
The role of EXT1 in nonhereditary osteochondroma: identification of homozygous deletions.
Hameetman L, Szuhai K, Yavas A, Knijnenburg J, van Duin M, van Dekken H, Taminiau AH,
Cleton-Jansen AM, Bovée JV, Hogendoorn PC.
Department of Pathology, Leiden University Medical Center, PO Box 9600 L1-Q, 2300 RC Leiden, The
Netherlands.
J Natl Cancer Inst. 2007 Mar 7;99(5):396-406.
BACKGROUND: Multiple osteochondromas is a hereditary syndrome that is characterized by the
formation of cartilage-capped bony neoplasms (osteochondromas), for which exostosis (multiple)-1
(EXT1) has been identified as a causative gene. However, 85% of all osteochondromas present as
solitary (nonhereditary) lesions in which somatic mutations in EXT1 are extremely rare, but loss of
heterozygosity and clonal rearrangement of 8q24 (the chromosomal locus of EXT1) are common. We
examined whether EXT1 might act as a classical tumor suppressor gene for nonhereditary
osteochondromas. METHODS: Eight nonhereditary osteochondromas were subjected to
high-resolution array-based comparative genomic hybridization (array-CGH) analysis for
chromosome 8q. The array-CGH results were validated by subjecting tumor DNA to multiple
ligation-dependent probe amplification (MLPA) analysis for EXT1. EXT1 locus-specific fluorescent in
situ hybridization (FISH) was performed on nuclei isolated from the three tissue components of
osteochondroma (cartilage cap, perichondrium, bony stalk) to examine which parts of the tumor are
of clonal origin. RESULTS: Array-CGH analysis of tumor DNA revealed that all eight
osteochondromas had a large deletion of 8q; five tumors had an additional small deletion of the
other allele of 8q that contained the EXT1 gene. MLPA analysis of tumor DNA confirmed these
findings and identified two additional deletions that were smaller than the limit of resolution of
array-CGH. FISH analysis of the cartilage cap, perichondrium, and bony stalk showed that these
homozygous EXT1 deletions were present only in the cartilage cap of osteochondroma.
CONCLUSION: EXT1 functions as a classical tumor suppressor gene in the cartilage cap of
nonhereditary osteochondromas.
Hameetman L, Szuhai K, Yavas A, Knijnenburg J, van Duin M, van Dekken H, Taminiau AH,
Cleton-Jansen AM, Bovée JV, Hogendoorn PC.
Department of Pathology, Leiden University Medical Center, PO Box 9600 L1-Q, 2300 RC Leiden, The
Netherlands.
J Natl Cancer Inst. 2007 Mar 7;99(5):396-406.
BACKGROUND: Multiple osteochondromas is a hereditary syndrome that is characterized by the
formation of cartilage-capped bony neoplasms (osteochondromas), for which exostosis (multiple)-1
(EXT1) has been identified as a causative gene. However, 85% of all osteochondromas present as
solitary (nonhereditary) lesions in which somatic mutations in EXT1 are extremely rare, but loss of
heterozygosity and clonal rearrangement of 8q24 (the chromosomal locus of EXT1) are common. We
examined whether EXT1 might act as a classical tumor suppressor gene for nonhereditary
osteochondromas. METHODS: Eight nonhereditary osteochondromas were subjected to
high-resolution array-based comparative genomic hybridization (array-CGH) analysis for
chromosome 8q. The array-CGH results were validated by subjecting tumor DNA to multiple
ligation-dependent probe amplification (MLPA) analysis for EXT1. EXT1 locus-specific fluorescent in
situ hybridization (FISH) was performed on nuclei isolated from the three tissue components of
osteochondroma (cartilage cap, perichondrium, bony stalk) to examine which parts of the tumor are
of clonal origin. RESULTS: Array-CGH analysis of tumor DNA revealed that all eight
osteochondromas had a large deletion of 8q; five tumors had an additional small deletion of the
other allele of 8q that contained the EXT1 gene. MLPA analysis of tumor DNA confirmed these
findings and identified two additional deletions that were smaller than the limit of resolution of
array-CGH. FISH analysis of the cartilage cap, perichondrium, and bony stalk showed that these
homozygous EXT1 deletions were present only in the cartilage cap of osteochondroma.
CONCLUSION: EXT1 functions as a classical tumor suppressor gene in the cartilage cap of
nonhereditary osteochondromas.
Multiple Osteochondromas: Clinicopathological and Genetic Spectrum and Suggestions for
Clinical Management
Hereditary Cancer Clin Pract 2004; 2(4);161-173
authors: Liesbeth Hameetman, Liesbeth Hameetman, Judith V.M.G. Bovée, Antonie H.M. Taminiau,
Antonie H.M. Taminiau, Herman M. Kroon, Herman M. Kroon, Pancras C.W. Hogendoorn,
Pancras C.W. Hogendoorn
Multiple Osteochondromas is an autosomal dominant disorder characterised by the presence of
multiple osteochondromas and a variety of orthopaedic deformities. Two genes causative of Multiple
Osteochondromas, Exostosin-1 (EXT1) and Exostosin-2 (EXT2), have been identified, which act as
tumour suppressor genes. Osteochondroma can progress towards its malignant counterpart,
secondary peripheral chondrosarcoma and therefore adequate follow-up of Multiple Osteochondroma
patients is important in order to detect malignant transformation early.
This review summarizes the considerable recent basic scientific and clinical understanding resulting in
a multi-step genetic model for peripheral cartilaginous tumorigenesis. This enabled us to suggest
guidelines for clinical management of Multiple Osteochondroma patients. When a patient is suspected
to have Multiple Osteochondroma, the radiologic documentation, histology and patient history have
to be carefully reviewed, preferably by experts and if indicated for Multiple Osteochondromas,
peripheral blood of the patient can be screened for germline mutations in either EXT1 or EXT2. After
the Multiple Osteochondroma diagnosis is established and all tumours are identified, a regular
follow-up including plain radiographs and base-line bone scan are recommended
Clinical Management
Hereditary Cancer Clin Pract 2004; 2(4);161-173
authors: Liesbeth Hameetman, Liesbeth Hameetman, Judith V.M.G. Bovée, Antonie H.M. Taminiau,
Antonie H.M. Taminiau, Herman M. Kroon, Herman M. Kroon, Pancras C.W. Hogendoorn,
Pancras C.W. Hogendoorn
Multiple Osteochondromas is an autosomal dominant disorder characterised by the presence of
multiple osteochondromas and a variety of orthopaedic deformities. Two genes causative of Multiple
Osteochondromas, Exostosin-1 (EXT1) and Exostosin-2 (EXT2), have been identified, which act as
tumour suppressor genes. Osteochondroma can progress towards its malignant counterpart,
secondary peripheral chondrosarcoma and therefore adequate follow-up of Multiple Osteochondroma
patients is important in order to detect malignant transformation early.
This review summarizes the considerable recent basic scientific and clinical understanding resulting in
a multi-step genetic model for peripheral cartilaginous tumorigenesis. This enabled us to suggest
guidelines for clinical management of Multiple Osteochondroma patients. When a patient is suspected
to have Multiple Osteochondroma, the radiologic documentation, histology and patient history have
to be carefully reviewed, preferably by experts and if indicated for Multiple Osteochondromas,
peripheral blood of the patient can be screened for germline mutations in either EXT1 or EXT2. After
the Multiple Osteochondroma diagnosis is established and all tumours are identified, a regular
follow-up including plain radiographs and base-line bone scan are recommended
Dr. Bovée is one of the team members of EuroBoNet pathologist specialised in the pathology of bone
and soft tissue tumours. She is part of the bone tumour research group. The main emphasis of her
research is focused on enchondromas, osteochondromas and chondrosarcomas.
and soft tissue tumours. She is part of the bone tumour research group. The main emphasis of her
research is focused on enchondromas, osteochondromas and chondrosarcomas.
Press Release 2 / 13 / 08
Multiple osteochondromas
Judith V.M.G. Bovée
Orphanet Journal of Rare Diseases 2008, 3:3 (13 February 2008)
To Read this publication Click Here
Multiple osteochondromas
Judith V.M.G. Bovée
Orphanet Journal of Rare Diseases 2008, 3:3 (13 February 2008)
To Read this publication Click Here
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By the Health On the Net Foundation. Click here to verify.# HON Conduct 282463 and is linked on the NIH
National Library of Medicine, Directory of Health Organizations (SIS) website, as well as the link for Patient
Information on The Diseases Database a cross-referenced index of human disease, and the Intute: health & life
sciences a free online service providing access to the very best Web resources for education and research
located in the UK
The MHE Research Foundation is proud to be working with the EuroBoNeT consortium, a European Commission
granted Network of Excellence for studying the pathology and genetics of bone tumors.
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