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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.
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