Skip to main content

Advertisement

SpringerLink
Log in

Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the EXT1 gene

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at θ = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529_531delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  1. Solomon L (1961) Bone growth in diaphysial aclasis. J Bone Joint Surg Br 43:700–716

    PubMed  Google Scholar 

  2. Solomon L (1963) Hereditary multiple exostosis. J Bone Joint Surg Br 45:292–304

    Google Scholar 

  3. Hennekam RCM (1991) Hereditary multiple exostoses. J Med Genet 28:262–266

    Article  PubMed  CAS  Google Scholar 

  4. Schmale GA, Conrad EU III, Raskind WH (1994) The natural history of hereditary multiple exostoses. J Bone Joint Surg [Am] 76:986–992

    CAS  Google Scholar 

  5. Wicklund CL, Pauli RM, Johnston D, Hecht JT (1995) Natural history study of hereditary multiple exostoses. Am J Med Genet 55:43–46

    Article  PubMed  CAS  Google Scholar 

  6. Porter DE, Lonie L, Fraser M, Dobson-Stone C, Porter JR, Monaco AP, Simpson AH (2004) Severity of disease and risk of malignant change in hereditary multiple exostoses. A genotype-phenotype study. J Bone Joint Surg [Br] 86:1041–1046

    Article  CAS  Google Scholar 

  7. Ahn J, Ludecke HJ, Lindow S, Horton WA, Lee B, Wagner MJ, Horsthemke B, Wells DE (1995) Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1). Nat Genet 11:137–143

    Article  PubMed  CAS  Google Scholar 

  8. Wuyts W,Van Hul W, Wauters J, Nemtsova M, Reyniers E, Van Hul EV, De Boulle K, de Vries BB, Hendrickx J, Herrygers I, Bossuyt P, Balemans W, Fransen E, Vits L, Coucke P, Nowak NJ, Shows TB, Mallet L, van den Ouweland AM, McGaughran J, Halley DJ, Willems PJ (1996) Positional cloning of a gene involved in hereditary multiple exostoses. Hum Mol Genet 5:1547–1557

    Article  PubMed  CAS  Google Scholar 

  9. Le Merrer M, Legeai-Mallet L, Jeannin PM, Horsthemke B, Schinzel A, Plauchu H, Toutain A, Achard F, Munnich A, Maroteaux P (1994) A gene for hereditary multiple exostoses maps to chromosome 19p. Hum Mol Genet 3:717–722

    Article  PubMed  Google Scholar 

  10. Bartsch O, Wuyts W, Van Hul W, Hecht JT, Meinecke P, Hogue D, Werner W, Zabel B, Hinkel GK, Powell CM, Shaffer LG, Willems PJ (1996) Delineation of a contiguous gene syndrome with multiple exostoses, enlarged parietal foramina, craniofacial dysostosis, and mental retardation, caused by deletions in the short arm of chromosome 11. Am J Hum Genet 58:734–742

    PubMed  CAS  Google Scholar 

  11. Philippe C, Porter DE, Emerton ME, Wells DE, Simpson AH, Monaco AP (1997) Mutation screening of the EXT1 and EXT2 genes in patients with hereditary multiple exostoses. Am J Hum Genet 61:520–528

    Article  PubMed  CAS  Google Scholar 

  12. McCormick C, Leduc Y, Martindale D, Mattison K, Esford LE, Dyer AP, Tufaro F (1998) The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate. Nat Genet 19:158–161

    Article  PubMed  CAS  Google Scholar 

  13. 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

    Article  PubMed  CAS  Google Scholar 

  14. Salmivirta M, Lidholt K, Lindahl U (1996) Heparan sulfate: a piece of information. FASEB J 10:1270–1279

    PubMed  CAS  Google Scholar 

  15. Ornitz DM (2000) FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 22:108–112

    Article  PubMed  CAS  Google Scholar 

  16. Chen CL, Huang SS, Huang JS (2006) Cellular heparan sulfate negatively modulates transforming growth factor-β1 (TGF-β1) responsiveness in epithelial cells. J Biol Chem 281:11506–11514

    Article  PubMed  CAS  Google Scholar 

  17. Takada T, Katagiri T, Ifuku M, Morimura N, Kobayashi M, Hasegawa K, Ogamo A, Kamijo R (2003) Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins. J Biol Chem 278:43229–43235

    Article  PubMed  CAS  Google Scholar 

  18. Baeg GH, Lin X, Khare N, Baumgartner S, Perrimon N (2001) Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128:87–94

    PubMed  CAS  Google Scholar 

  19. Bellaiche Y, The I, Perrimon N (1998) Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. Nature 394:85–88

    Article  PubMed  CAS  Google Scholar 

  20. Han C, Belenkaya TY, Khodoun M, Tauchi M, Lin X, Lin X (2004) Distinct and collaborative roles of Drosophila EXT family proteins in morphogen signalling and gradient formation. Development 131:1563–1575

    Article  PubMed  CAS  Google Scholar 

  21. Stickens D, Zak BM, Rougier N, Esko JD, Werb Z (2005) Mice deficient in Ext2 lack heparan sulfate and develop exostoses. Development 132:5055–5068

    Article  PubMed  CAS  Google Scholar 

  22. Lin X, Wei G, Shi Z, Dryer L, Esko JD, Wells DE, Matzuk MM (2000) Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice. Dev Biol 224:299–311

    Article  PubMed  CAS  Google Scholar 

  23. Koziel L, Kunath M, Kelly OG, Vortkamp A (2004) Ext1-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification. Dev Cell 6:801–813

    Article  PubMed  CAS  Google Scholar 

  24. Bornemann DJ, Duncan JE, Staatz W, Selleck S, Warrior R (2004) Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways. Development 131:1927–1938

    Article  PubMed  CAS  Google Scholar 

  25. Dobson-Stone C, Cox RD, Lonie L, Southam L, Fraser M, Wise C, Bernier F, Hodgson S, Porter DE, Simpson AH, Monaco AP (2000) Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses. Eur J Hum Genet 8:24–32

    Article  PubMed  CAS  Google Scholar 

  26. Wuyts W, Van Hul W (2000) Molecular basis of multiple exostoses: mutations in the EXT1 and EXT2 genes. Hum Mutat 15:220–227

    Article  PubMed  CAS  Google Scholar 

  27. Signori E, Massi E, Matera MG, Poscente M, Gravina C, Falcone G, Rosa MA, Rinaldi M, Wuyts W, Seripa D, Dallapiccola B, Fazio VM (2007) A combined analytical approach reveals novel EXT1/2 gene mutations in a large cohort of Italian multiple osteochondromas patients. Genes Chromosomes Cancer 46:470–477

    Article  PubMed  CAS  Google Scholar 

  28. Wuyts W, Van Hul W, De Boulle K, Hendrickx J, Bakker E, Vanhoenacker F, Mollica F, Ludecke HJ, Sayli BS, Pazzaglia UE, Mortier G, Hamel B, Conrad EU, Matsushita M, Raskind WH, Willems PJ (1998) Mutations in the EXT1 and EXT2 genes in hereditary multiple exostoses. Am J Hum Genet 62:346–354

    Article  PubMed  CAS  Google Scholar 

  29. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215

    Article  PubMed  CAS  Google Scholar 

  30. Bouchlaka C, Abdelhak S, Amouri A, Ben Abid H, Hadiji S, Frikha M, Ben Othman T, Amri F, Ayadi H, Hachicha M, Rebaï A, Saad A, Dellagi K, The Tunisian Fanconi Anaemia Study Group (2003) Fanconi anaemia in Tunisia: high prevalence of group A and identification of new FANCA mutations. J Hum Genet 48:352–361

    Google Scholar 

  31. Lindner TH, Hoffmann K (2005) easyLINKAGE: a PERL script for easy and automated two-/multi-point linkage analyses. Bioinformatics 21:405–407

    Article  PubMed  CAS  Google Scholar 

  32. Wells DE, Hill A, Lin X, Ahn J, Brown N, Wagner MJ (1997) Identification of novel mutations in the human EXT1 tumor suppressor gene. Hum Genet 99:612–615

    Article  PubMed  CAS  Google Scholar 

  33. Hecht JT, Hogue D, Wang Y, Blanton SH, Wagner M, Strong LC, Raskind W, Hansen MF, Wells D (1997) Hereditary multiple exostoses (EXT): mutational studies of familial EXT1 cases and EXT-associated malignancies. Am J Hum Genet 60:80–86

    PubMed  CAS  Google Scholar 

  34. Seki H, Kubota T, Ikegawa S, Haga N, Fujioka F, Ohzeki S, Wakui K, Yoshikawa H, Takaoka K, Fukushima Y (2001) Mutation frequencies of EXT1 and EXT2 in 43 Japanese families with hereditary multiple exostoses. Am J Med Genet 99:59–62

    Article  PubMed  CAS  Google Scholar 

  35. Cheung PK, McCormick C, Crawford BE, Esko JD, Tufaro F, Duncan G (2001) Etiological point mutations in the hereditary multiple exostoses gene EXT1: a functional analysis of heparan sulfate polymerase activity. Am J Hum Genet 69:55–66

    Article  PubMed  CAS  Google Scholar 

  36. Raskind WH, Conrad EU 3rd, Matsushita M, Wijsman EM, Wells DE, Chapman N, Sandell LJ, Wagner M, Houck J (1998) Evaluation of locus heterogeneity and EXT1 mutations in 34 families with hereditary multiple exostoses. Hum Mutat 11:231–239

    Article  PubMed  CAS  Google Scholar 

  37. 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 Genet 38:430–434

    Article  PubMed  CAS  Google Scholar 

  38. Hall CR, Cole WG, Haynes R, Hecht JT (2002) Reevaluation of a genetic model for the development of exostosis in hereditary multiple exostosis. Am J Med Genet 112:1–5

    Article  PubMed  Google Scholar 

  39. Vink GR, White SJ, Gabelic S, Hogendoorn PC, Breuning MH, Bakker E (2005) Mutation screening of EXT1 and EXT2 by direct sequence analysis and MLPA in patients with multiple osteochondromas: splice site mutations and exonic deletions account for more than half of the mutations. Eur J Hum Genet 13:470–474

    Article  PubMed  CAS  Google Scholar 

  40. Pedrini E, De Luca A, Valente EM, Maini V, Capponcelli S, Mordenti M, Mingarelli R, Sangiorgi L, Dallapiccola B (2005) Novel EXT1 and EXT2 mutations identified by DHPLC in Italian patients with multiple osteochondromas. Hum Mutat 26:280

    Article  PubMed  Google Scholar 

  41. Wei G, Bai X, Gabb MM, Bame KJ, Koshy TI, Spear PG, Esko JD (2000) Location of the glucuronosyltransferase domain in the heparan sulfate copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants. J Biol chem 275:27733–27740

    PubMed  CAS  Google Scholar 

  42. McCormick C, Duncan G, Goutsos KT, Tufaro F (2000) 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

    Article  PubMed  CAS  Google Scholar 

  43. Legeai-Mallet L, Munnich A, Maroteaux P, Le Merrer M (1997) Incomplete penetrance and expressivity skewing in hereditary multiple exostoses. Clin Genet 52:12–16

    Article  PubMed  CAS  Google Scholar 

  44. Faiyaz-Ul-Haque M, Ahmad W, Zaidi SH, Hussain S, Haque S, Ahmad M, Cohn DH, Tsui LC (2004) Novel mutations in the EXT1 gene in two consanguineous families affected with multiple hereditary exostoses (familial osteochondromatosis). Clin Genet 66:144–151

    Article  PubMed  CAS  Google Scholar 

  45. Alvarez C, Tredwell S, De Vera M, Hayden M (2006) The genotype-phenotype correlation of hereditary multiple exostoses. Clin Genet 70:122–130

    Article  PubMed  CAS  Google Scholar 

  46. Jäger M, Westhoff B, Portier S, Leube B, Hardt K, Royer-Pokora B, Gossheger G, Krauspe R (2007) Clinical outcome and genotype in patients with hereditary multiple exostoses. J Orthop Res 25:1541–1551

    Article  PubMed  Google Scholar 

  47. Alvarez CM, De Vera MA, Heslip TR, Casey B (2007) Evaluation of the anatomic burden of patients with hereditary multiple exostoses. Clin Orthop Relat Res 462:73–79

    Article  PubMed  Google Scholar 

  48. Bovée JV, Cleton-Jansen AM, Wuyts W, Caethoven G, Taminiau AH, Bakker E, Van Hul W, Cornelisse CJ, Hogendoorn PC (1999) EXT-mutation analysis and loss of heterozygosity in sporadic and hereditary osteochondromas and secondary chondrosarcomas. Am J Hum Genet 65:689–698

    Article  PubMed  Google Scholar 

  49. Wuyts W, Radersma R, Storm K, Vits L (2005) An optimized DHPLC protocol for molecular testing of the EXT1 and EXT2 genes in hereditary multiple osteochondromas. Clin Genet 68:542–547

    Article  PubMed  CAS  Google Scholar 

  50. Ellis LA, Taylor CF, Taylor GR (2000) A comparison of fluorescent SSCP and denaturing HPLC for high throughput mutation scanning. Hum Mutat 15:556–564

    Article  PubMed  CAS  Google Scholar 

  51. Mogensen J, Bahl A, Kubo T, Elanko N, Taylor R, McKenna WJ (2003) Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy. J Med Genet 40:e59

    Article  PubMed  CAS  Google Scholar 

  52. Bouchlaka C, Maktouf C, Mahjoub B, Ayadi A, Sfar T, Sioud M, Gueddich N, Belhadjali Z, Rebaï A, Abdelhak S, Dellagi K (2007) Genetic heterogeneity of megaloblastic anemia type 1 in Tunisian patients. J Hum Genet 52:262–270

    Article  PubMed  Google Scholar 

  53. El Kares R, Barbouche MR, Elloumi-Zghal H, Bejaoui M, Chemli J, Mellouli F, Tebib N, Abdelmoula MS, Boukthir S, Fitouri Z, M’Rad S, Bouslama K, Touiri H, Abdelhak S, Dellagi MK (2006) Genetic and mutational heterogeneity of autosomal recessive chronic granulomatous disease in Tunisia. J Hum Genet 51:887–895

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank the members of the Tunisian families for their participation in this study. This work was supported by the Ministry of Higher Education and Scientific and Technological Research and the Ministry of Public Health of Tunisia. We would like to thank Mr. Adel Rdissi for the English revision.

Author information

Authors and Affiliations

  1. Department of Molecular Immuno-Oncology, Faculty of Medicine, University of Monastir, Monastir, Tunisia

    Sana Sfar, Wijden Mahfoudh & Lotfi Chouchane

  2. Department of Orthopedic, EPS Fattouma Bourguiba, Monastir, Tunisia

    Abderrazak Abid

  3. Research Unit on Molecular Investigation of Genetic Orphan Diseases, Institut Pasteur de Tunis, Tunis, Tunisia

    Houyem Ouragini, Farah Ouechtati & Sonia Abdelhak

  4. Department of Genetic Medicine, Weill Cornell Medical College in Qatar, P.O. Box 24144, Doha, Qatar

    Lotfi Chouchane

Authors
  1. Sana Sfar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abderrazak Abid
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wijden Mahfoudh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Houyem Ouragini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Farah Ouechtati
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sonia Abdelhak
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lotfi Chouchane
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lotfi Chouchane.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sfar, S., Abid, A., Mahfoudh, W. et al. Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the EXT1 gene. Mol Biol Rep 36, 661–667 (2009). https://doi.org/10.1007/s11033-008-9226-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-008-9226-3

Keywords

Search

Navigation