Dr. Wuyts serves on the Scientific and Medical Advisory Board of the MHE Research Foundation
Research authored by Dr. Wuyts
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List of Publications via PubMed
(NIH National Library of Medicine)
Research authored by Dr. Wuyts
Click the tab and a window will appear.
List of Publications via PubMed
(NIH National Library of Medicine)
Current Research project being conducted by Dr. Wuyts, along with the help of the MHE
Research Foundation and its MHE Research Registry.
Research Foundation and its MHE Research Registry.
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Mutation detection strategies for molecular screening in patients with MHE.
Abstract 2005 MHE Conference
Wim Wuyts PH.D.
Department of Medical Genetics University of Antwerp, Belgium.
Hereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by the presence of bony outgrowths
(exostoses) on the long bones. HME is a genetically heterogeneous condition and at present two causal genes have been
identified: EXT1 on chromosome 8q23-q24 and EXT2 on chromosome 11p11.2. A third locus, EXT3 on chromosome 19p has
been suggested but is controversial.
Since the identification of the HME causing genes, molecular analysis of HME patients has been optimized to increase sensitivity
of the testing and reduce the cost. Recently, we further optimized the mutation screening protocol for both EXT1 and EXT2.
For all coding exons DHPLC conditions were optimized and validated in a large set of HME patients with a known EXT1 or EXT2
mutation. All mutations could be detected under at least 1 DHPLC condition, providing a robust and sensitive alternative for
labor extensive and more expensive sequencing analysis.
However, approximately 15 to 20% of HME patients does not show a mutation after extended sequence analysis of EXT1 and
EXT2. We therefore expanded the screening protocol with FISH, MLPA and RNA analysis. This enabled us to detect (partial)
EXT1 or EXT2 deletions in approximately 30%of EXT1/EXT2 mutation negative patients. At least one patient with two copies of
both EXT genes showed loss of one EXT1 allele on the RNA level, but the underlying cause is still under investigation.
The various mutation detection strategies will be discussed as well as the mutation spectrum observed in HME patients.
Abstract 2005 MHE Conference
Wim Wuyts PH.D.
Department of Medical Genetics University of Antwerp, Belgium.
Hereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by the presence of bony outgrowths
(exostoses) on the long bones. HME is a genetically heterogeneous condition and at present two causal genes have been
identified: EXT1 on chromosome 8q23-q24 and EXT2 on chromosome 11p11.2. A third locus, EXT3 on chromosome 19p has
been suggested but is controversial.
Since the identification of the HME causing genes, molecular analysis of HME patients has been optimized to increase sensitivity
of the testing and reduce the cost. Recently, we further optimized the mutation screening protocol for both EXT1 and EXT2.
For all coding exons DHPLC conditions were optimized and validated in a large set of HME patients with a known EXT1 or EXT2
mutation. All mutations could be detected under at least 1 DHPLC condition, providing a robust and sensitive alternative for
labor extensive and more expensive sequencing analysis.
However, approximately 15 to 20% of HME patients does not show a mutation after extended sequence analysis of EXT1 and
EXT2. We therefore expanded the screening protocol with FISH, MLPA and RNA analysis. This enabled us to detect (partial)
EXT1 or EXT2 deletions in approximately 30%of EXT1/EXT2 mutation negative patients. At least one patient with two copies of
both EXT genes showed loss of one EXT1 allele on the RNA level, but the underlying cause is still under investigation.
The various mutation detection strategies will be discussed as well as the mutation spectrum observed in HME patients.
1/ 1/ 07 Abstract information concerning this research project.
Clinical and molecular study of factors implicated in Multiple Osteochondroma
(MHE / MO / HME).
This project aims the identification and study of genes involved in the disorder Multiple Osteochondroma (MO) / Multiple
Hereditary Exostoses (MHE). MHE / MO / HME is a hereditary bone disorder characterized by the presence of bony outgrowths
(osteochondromas / exostoses) on the long bones. MHE / MO / HME patients suffer from pain caused by the pressure of the
osteochondromas / exostoses on neighboring tissues, organs or nerves. Often MHE / MO / HME patients also show skeletal
deformities. However, great clinical variability is observed between the various patients, even within one family.
It has been shown that mutations in one of two genes, EXT1 or EXT2, are responsible for the majority of MHE / MO / HME
cases. However, the exact mechanism leading to the development of osteochondromas is still not fully understood.
This project concentrates on the molecular aspects of MHE / MO / HME. Tumor and blood samples of MHE / MO / HME patients
are collected and studied to see how they differ from samples from healthy individuals. This may provide valuable information
on the mechanisms of osteochondroma / exostoses development and may give us more insight in factors leading to the clinical
variability.
Clinical and molecular study of factors implicated in Multiple Osteochondroma
(MHE / MO / HME).
This project aims the identification and study of genes involved in the disorder Multiple Osteochondroma (MO) / Multiple
Hereditary Exostoses (MHE). MHE / MO / HME is a hereditary bone disorder characterized by the presence of bony outgrowths
(osteochondromas / exostoses) on the long bones. MHE / MO / HME patients suffer from pain caused by the pressure of the
osteochondromas / exostoses on neighboring tissues, organs or nerves. Often MHE / MO / HME patients also show skeletal
deformities. However, great clinical variability is observed between the various patients, even within one family.
It has been shown that mutations in one of two genes, EXT1 or EXT2, are responsible for the majority of MHE / MO / HME
cases. However, the exact mechanism leading to the development of osteochondromas is still not fully understood.
This project concentrates on the molecular aspects of MHE / MO / HME. Tumor and blood samples of MHE / MO / HME patients
are collected and studied to see how they differ from samples from healthy individuals. This may provide valuable information
on the mechanisms of osteochondroma / exostoses development and may give us more insight in factors leading to the clinical
variability.
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| Wim Wuyts, Ph.D., research |
Press Release 1 / 19 / 08
Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing
Analysis, Fluorescence in Situ Hybridization, and a New Multiplex Ligation-Dependent Probe Amplification Probe Set
in Patients with Multiple Osteochondromas
Ivy Jennes*, Mark M. Entius{dagger}, Els Van Hul*, Alessandro Parra{ddagger}, Luca Sangiorgi {ddagger} and Wim Wuyts*
To Read this publication Click Here
Both Luca Sangiorgi, M.D., Ph.D. and Wim Wuyts, Ph.D. are members of our Scientific & Medical Advisory Board and our
foundation helped support this research
Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing
Analysis, Fluorescence in Situ Hybridization, and a New Multiplex Ligation-Dependent Probe Amplification Probe Set
in Patients with Multiple Osteochondromas
Ivy Jennes*, Mark M. Entius{dagger}, Els Van Hul*, Alessandro Parra{ddagger}, Luca Sangiorgi {ddagger} and Wim Wuyts*
To Read this publication Click Here
Both Luca Sangiorgi, M.D., Ph.D. and Wim Wuyts, Ph.D. are members of our Scientific & Medical Advisory Board and our
foundation helped support this research
Questions to the Scientific & Medical Advisory Board,
Please use the contact Scientific & Medical Advisory Board Tab or you may email directly
AdvisoryBoard@mheresearchfoundation.org
Please use the contact Scientific & Medical Advisory Board Tab or you may email directly
AdvisoryBoard@mheresearchfoundation.org
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Have a Question or Comments for the Board of Directors,
Please use the contact Board of Directors Tab or General enquiries can be emailed to
Boardofdirectors@mheresearchfoundation.org
Please use the contact Board of Directors Tab or General enquiries can be emailed to
Boardofdirectors@mheresearchfoundation.org
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2009 Conference abstract
Genetics of Multiple Osteochondromas; Overview of the Current Status
Wim Wuyts, PhD
Department of Medical Genetics, University of Antwerp, Belgium.
e-mail: wim.wuyts@ua.ac.be
Multiple osteochondromas (MO; Hereditary multiple exostoses-HME) is an autosomal dominant bone disorder characterized by
the presence of osteochondromas (exostoses) on the long bones. This condition is genetic heterogeneous with at present two
proteins known to be mutated in MO patients: Extosin 1 encoded by the EXT1 gene and Extosin 2 encoded by the EXT2 gene.
Both EXT genes belong to a larger gene family of EXT-EXT-like (EXTL) genes which encode glycosyltransferases involved in the
adhesion and/or polymerization of heparin sulphate (HS) chains at HS proteoglycans (HSPGs). EXT1 is located at
8q24.11-q24.13 and comprises 11 exons, while EXT2 is located at 11p12-p11 and contains 16 exons.
To date, an EXT1 or EXT2 mutation is detected in 70-95% of affected individuals and various mutation detection protocols have
been developed to screen both EXT genes. Mutation data gathered during the last 15 years is available and can be consulted at
the online Multiple Osteochondromas Mutation Database (Modb; http://medgen.ua.ac.be/LOVD/home.ph). Currently, the MOdb
lists over 900 variant entries of which more than 550 are unique. It provides a global overview of mutation distribution,
frequency and nature of the identified mutation.
A small percentage of patients does not show a mutation with standard mutation detection techniques including DHPLC, direct
sequencing and MLPA. These are in general sporadic patients and extended analysis has shown that at least a fraction of these
patients may have mosaic mutations.
There is great variation in the phenotypic manifestation of MO in various patients. It has been suggested that part of the
interfamilial variation is due to the EXT mutation, with EXT1 patients showing more severe clinical spectrum than EXT2 patients.
However, as also intrafamilial variation is observed also other factors may play a role and the identification of such other genomic
variants is an ongoing challenge.
Genetics of Multiple Osteochondromas; Overview of the Current Status
Wim Wuyts, PhD
Department of Medical Genetics, University of Antwerp, Belgium.
e-mail: wim.wuyts@ua.ac.be
Multiple osteochondromas (MO; Hereditary multiple exostoses-HME) is an autosomal dominant bone disorder characterized by
the presence of osteochondromas (exostoses) on the long bones. This condition is genetic heterogeneous with at present two
proteins known to be mutated in MO patients: Extosin 1 encoded by the EXT1 gene and Extosin 2 encoded by the EXT2 gene.
Both EXT genes belong to a larger gene family of EXT-EXT-like (EXTL) genes which encode glycosyltransferases involved in the
adhesion and/or polymerization of heparin sulphate (HS) chains at HS proteoglycans (HSPGs). EXT1 is located at
8q24.11-q24.13 and comprises 11 exons, while EXT2 is located at 11p12-p11 and contains 16 exons.
To date, an EXT1 or EXT2 mutation is detected in 70-95% of affected individuals and various mutation detection protocols have
been developed to screen both EXT genes. Mutation data gathered during the last 15 years is available and can be consulted at
the online Multiple Osteochondromas Mutation Database (Modb; http://medgen.ua.ac.be/LOVD/home.ph). Currently, the MOdb
lists over 900 variant entries of which more than 550 are unique. It provides a global overview of mutation distribution,
frequency and nature of the identified mutation.
A small percentage of patients does not show a mutation with standard mutation detection techniques including DHPLC, direct
sequencing and MLPA. These are in general sporadic patients and extended analysis has shown that at least a fraction of these
patients may have mosaic mutations.
There is great variation in the phenotypic manifestation of MO in various patients. It has been suggested that part of the
interfamilial variation is due to the EXT mutation, with EXT1 patients showing more severe clinical spectrum than EXT2 patients.
However, as also intrafamilial variation is observed also other factors may play a role and the identification of such other genomic
variants is an ongoing challenge.
2009 Conference abstract
Characterisation of the Promoter Region of the Human Exostosin-1 Gene
Ivy Jennes1, Ines Cilissen1, Monia Zuntini2, Luca Sangiorgi2 and Wim Wuyts1
1Department of Medical Genetics, University and University Hospital of Antwerp, Belgium. 2Department of Medical Genetics,
Rizzoli Orthopaedic Institute, Bologna, Italy.
e-mail: ivy.jennes@ua.ac.be.
Mutations in Exostosin-1 (EXT1) or Exostosin-2 (EXT2) cause multiple osteochondromas (MO). EXT1 and EXT2 are both
tumour suppressor genes that encode proteins that function as glycosyltransferases in the biosynthesis of heparan sulphate
(HS). At present, very little is known about the regulation of the EXT genes. Mutations in the EXT1 gene are responsible for 47-
64% of the MO families. To elucidate the transcriptional regulation of EXT1, we isolated and characterized the EXT1 promoter
region.
Theoretical analysis of the 10 kb upstream of the EXT1 start codon was performed with promoter prediction programs TSSG,
TSSW, FPROM, BDGP, Promoter 2.0 Prediction Server and Web Promoter Scan. These programs showed presence of a CpG
island containing CG and CAAT boxes but no TATA box, which is characteristic for a housekeeping gene. Two potential
functional promoter regions were identified, located respectively ~2.650 bp and ~900 bp upstream of the start codon.
To confirm the correct promoter region experimentally, overlapping PCR fragments in the 10 kb putative EXT1 promoter region
were generated and cloned in the pGL4.72 Luciferase Reporter Vector. After transfection in Human Embryonic Kidney cells,
promoter activity was determined by performing luciferase assays, which located the actual core promoter within the 560 bp
fragment containing the predicted promoter sequence at ~-900 bp. Further fine mapping located the minimal core promoter
within in a fragment of 350 kb.
Subsequently, the promoter region was analysed for protein binding capacities with transcription binding prediction programs
AliBaBa2, Cister, TFsearch, TFSiteScan and TESS. This analysis revealed various putative transcription factor binding sites.
Additional analysis revealed the presence of two polymorphic G/C SNP’s in which the presence of a cytosine destroys the binding
site of a predicted transcription factor. Subsequently, new luciferase assays were designed to test whether these SNP’s might
indeed influence the EXT1 promoter activity. They revealed a promoter activity that was up to 37% lower in CC genotypes
compared to GG genotypes. Consequently, we identified both SNP’s as primary modifiers that might explain part of the clinical
variation observed in MO patients.
To test this hypothesis, both SNP’s were characterised in 269 MO patients from 2 populations (148 patients from mixed ethnic
origin from the Antwerp research group and 121 patients from Italian origin from the Bologna research group). The clinical
features evaluated included the presence of deformities and complications, patients’ stature and the number of osteochondroma
and osteochondroma sites. First of all, significant association was observed between the frequency of a cytosine in the first SNP
location and short stature in patients (≤P25) (p<0,05). Secondly, the presence of a cytosine in the first SNP location in trans
with the EXT1 mutation showed significant association with the presence of deformities (p<0,05). Finally, analysis of the first
SNP location in EXT2 associated patients revealed association with the presence of deformities (p<0,02), short stature in
patients (≤P25) (p<0,02) and the number of osteochondroma sites (p<0,05). These observations are the first indication for a
genotype-phenotype association for promoter SNP’s in EXT1.
Characterisation of the Promoter Region of the Human Exostosin-1 Gene
Ivy Jennes1, Ines Cilissen1, Monia Zuntini2, Luca Sangiorgi2 and Wim Wuyts1
1Department of Medical Genetics, University and University Hospital of Antwerp, Belgium. 2Department of Medical Genetics,
Rizzoli Orthopaedic Institute, Bologna, Italy.
e-mail: ivy.jennes@ua.ac.be.
Mutations in Exostosin-1 (EXT1) or Exostosin-2 (EXT2) cause multiple osteochondromas (MO). EXT1 and EXT2 are both
tumour suppressor genes that encode proteins that function as glycosyltransferases in the biosynthesis of heparan sulphate
(HS). At present, very little is known about the regulation of the EXT genes. Mutations in the EXT1 gene are responsible for 47-
64% of the MO families. To elucidate the transcriptional regulation of EXT1, we isolated and characterized the EXT1 promoter
region.
Theoretical analysis of the 10 kb upstream of the EXT1 start codon was performed with promoter prediction programs TSSG,
TSSW, FPROM, BDGP, Promoter 2.0 Prediction Server and Web Promoter Scan. These programs showed presence of a CpG
island containing CG and CAAT boxes but no TATA box, which is characteristic for a housekeeping gene. Two potential
functional promoter regions were identified, located respectively ~2.650 bp and ~900 bp upstream of the start codon.
To confirm the correct promoter region experimentally, overlapping PCR fragments in the 10 kb putative EXT1 promoter region
were generated and cloned in the pGL4.72 Luciferase Reporter Vector. After transfection in Human Embryonic Kidney cells,
promoter activity was determined by performing luciferase assays, which located the actual core promoter within the 560 bp
fragment containing the predicted promoter sequence at ~-900 bp. Further fine mapping located the minimal core promoter
within in a fragment of 350 kb.
Subsequently, the promoter region was analysed for protein binding capacities with transcription binding prediction programs
AliBaBa2, Cister, TFsearch, TFSiteScan and TESS. This analysis revealed various putative transcription factor binding sites.
Additional analysis revealed the presence of two polymorphic G/C SNP’s in which the presence of a cytosine destroys the binding
site of a predicted transcription factor. Subsequently, new luciferase assays were designed to test whether these SNP’s might
indeed influence the EXT1 promoter activity. They revealed a promoter activity that was up to 37% lower in CC genotypes
compared to GG genotypes. Consequently, we identified both SNP’s as primary modifiers that might explain part of the clinical
variation observed in MO patients.
To test this hypothesis, both SNP’s were characterised in 269 MO patients from 2 populations (148 patients from mixed ethnic
origin from the Antwerp research group and 121 patients from Italian origin from the Bologna research group). The clinical
features evaluated included the presence of deformities and complications, patients’ stature and the number of osteochondroma
and osteochondroma sites. First of all, significant association was observed between the frequency of a cytosine in the first SNP
location and short stature in patients (≤P25) (p<0,05). Secondly, the presence of a cytosine in the first SNP location in trans
with the EXT1 mutation showed significant association with the presence of deformities (p<0,05). Finally, analysis of the first
SNP location in EXT2 associated patients revealed association with the presence of deformities (p<0,02), short stature in
patients (≤P25) (p<0,02) and the number of osteochondroma sites (p<0,05). These observations are the first indication for a
genotype-phenotype association for promoter SNP’s in EXT1.
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