Human Genetics

, Volume 132, Issue 11, pp 1253–1264 | Cite as

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

  • Muhammad Farooq
  • Hiroyuki Nakai
  • Atsushi Fujimoto
  • Hiroki Fujikawa
  • Klaus Wilbrandt Kjaer
  • Shahid Mahmood Baig
  • Yutaka ShimomuraEmail author
Original Investigation


All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.


HEK293T Cell Brachydactyly Complete Mini Protease Inhibitor Cocktail GDF5 Gene Brachydactyly Type 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We acknowledge the family members involved in this study. We thank Drs. Satoshi Ishii (Tokyo University, Japan) and Junichi Miyazaki (Osaka University, Japan) for supplying pCXN2.1 vector. This study was supported in part by the Special Coordination Funds for Promoting Science and Technology, the Ministry of Education, Culture, Sports, Science and Technology, Japan (to Y.S.).

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

439_2013_1330_MOESM1_ESM.pdf (1.1 mb)
Supplementary material 1 (PDF 1079 kb)


  1. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201PubMedCrossRefGoogle Scholar
  2. Asai-Coakwell M, French CR, Ye M, Garcha K, Bigot K, Perera AG, Staehling-Hampton K, Mema SC, Chanda B, Mushegian A, Bamforth S, Doschak MR, Li G, Dobbs MB, Giampietro PF, Brooks BP, Vijayalakshmi P, Sauve Y, Abitbol M, Sundaresan P, van Heyningen V, Pourquie O, Underhill TM, Waskiewicz AJ, Lehmann OJ (2009) Incomplete penetrance and phenotypic variability characterize Gdf6-attributable oculo-skeletal phenotypes. Hum Mol Genet 18:1110–1121PubMedCrossRefGoogle Scholar
  3. Bakrania P, Efthymiou M, Klein JC, Salt A, Bunyan DJ, Wyatt A, Ponting CP, Martin A, Williams S, Lindley V, Gilmore J, Restori M, Robson AG, Neveu MM, Holder GE, Collin JR, Robinson DO, Farndon P, Johansen-Berg H, Gerrelli D, Ragge NK (2008) Mutations in BMP4 cause eye, brain, and digit developmental anomalies: overlap between the BMP4 and hedgehog signaling pathways. Am J Hum Genet 82:304–319PubMedCrossRefGoogle Scholar
  4. Bell J (1951) On hereditary digital anomalies, Part 1: On brachydactyly and symphalangism. In: The Treasury of Human Inheritance. Cambridge Univ. Press, CambridgeGoogle Scholar
  5. Buxton P, Edwards C, Archer CW, Francis-West P (2001) Growth/differentiation factor-5 (GDF-5) and skeletal development. J Bone Joint Surg Am 83-A(Suppl 1):S23–S30Google Scholar
  6. Chang SC, Hoang B, Thomas JT, Vukicevic S, Luyten FP, Ryba NJ, Kozak CA, Reddi AH, Moos M Jr (1994) Cartilage-derived morphogenetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development. J Biol Chem 269:28227–28234PubMedGoogle Scholar
  7. Dawson K, Seeman P, Sebald E, King L, Edwards M, Williams J 3rd, Mundlos S, Krakow D (2006) GDF5 is a second locus for multiple-synostosis syndrome. Am J Hum Genet 78:708–712PubMedCrossRefGoogle Scholar
  8. Di Pasquale E, Beck-Peccoz P, Persani L (2004) Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene. Am J Hum Genet 75:106–111PubMedCrossRefGoogle Scholar
  9. Drews F, Knobel S, Moser M, Muhlack KG, Mohren S, Stoll C, Bosio A, Gressner AM, Weiskirchen R (2008) Disruption of the latent transforming growth factor-beta binding protein-1 gene causes alteration in facial structure and influences TGF-beta bioavailability. Biochim Biophys Acta 1783:34–48PubMedCrossRefGoogle Scholar
  10. Everman DB, Bartels CF, Yang Y, Yanamandra N, Goodman FR, Mendoza-Londono JR, Savarirayan R, White SM, Graham JM Jr, Gale RP, Svarch E, Newman WG, Kleckers AR, Francomano CA, Govindaiah V, Singh L, Morrison S, Thomas JT, Warman ML (2002) The mutational spectrum of brachydactyly type C. Am J Med Genet 112:291–296PubMedCrossRefGoogle Scholar
  11. Faiyaz-Ul-Haque M, Ahmad W, Wahab A, Haque S, Azim AC, Zaidi SH, Teebi AS, Ahmad M, Cohn DH, Siddique T, Tsui LC (2002a) Frameshift mutation in the cartilage-derived morphogenetic protein 1 (CDMP1) gene and severe acromesomelic chondrodysplasia resembling Grebe-type chondrodysplasia. Am J Med Genet 111:31–37PubMedCrossRefGoogle Scholar
  12. Faiyaz-Ul-Haque M, Ahmad W, Zaidi SH, Haque S, Teebi AS, Ahmad M, Cohn DH, Tsui LC (2002b) Mutation in the cartilage-derived morphogenetic protein-1 (CDMP1) gene in a kindred affected with fibular hypoplasia and complex brachydactyly (DuPan syndrome). Clin Genet 61:454–458PubMedCrossRefGoogle Scholar
  13. Fitch N, Jequier S, Costom B (1979) Brachydactyly C, short stature, and hip dysplasia. Am J Med Genet 4:157–166PubMedCrossRefGoogle Scholar
  14. Francis-West PH, Abdelfattah A, Chen P, Allen C, Parish J, Ladher R, Allen S, MacPherson S, Luyten FP, Archer CW (1999) Mechanisms of GDF-5 action during skeletal development. Development 126:1305–1315PubMedGoogle Scholar
  15. Gray AM, Mason AJ (1990) Requirement for activin A and transforming growth factor–beta 1 pro-regions in homodimer assembly. Science 247:1328–1330PubMedCrossRefGoogle Scholar
  16. Harrison CA, Al-Musawi SL, Walton KL (2011) Prodomains regulate the synthesis, extracellular localisation and activity of TGF-beta superfamily ligands. Growth Factors 29:174–186PubMedCrossRefGoogle Scholar
  17. Hotten GC, Matsumoto T, Kimura M, Bechtold RF, Kron R, Ohara T, Tanaka H, Satoh Y, Okazaki M, Shirai T, Pan H, Kawai S, Pohl JS, Kudo A (1996) Recombinant human growth/differentiation factor 5 stimulates mesenchyme aggregation and chondrogenesis responsible for the skeletal development of limbs. Growth Factors 13:65–74PubMedCrossRefGoogle Scholar
  18. Janssens K, Vanhoenacker F, Bonduelle M, Verbruggen L, Van Maldergem L, Ralston S, Guanabens N, Migone N, Wientroub S, Divizia MT, Bergmann C, Bennett C, Simsek S, Melancon S, Cundy T, Van Hul W (2006) Camurati-Engelmann disease: review of the clinical, radiological, and molecular data of 24 families and implications for diagnosis and treatment. J Med Genet 43:1–11PubMedCrossRefGoogle Scholar
  19. Lorda-Diez CI, Montero JA, Garcia-Porrero JA, Hurle JM (2010) Tgfbeta2 and 3 are coexpressed with their extracellular regulator Ltbp1 in the early limb bud and modulate mesodermal outgrowth and BMP signaling in chicken embryos. BMC Dev Biol 10:69PubMedCrossRefGoogle Scholar
  20. Luyten FP (1997) Cartilage-derived morphogenetic protein-1. Int J Biochem Cell Biol 29:1241–1244PubMedCrossRefGoogle Scholar
  21. Merino R, Macias D, Ganan Y, Economides AN, Wang X, Wu Q, Stahl N, Sampath KT, Varona P, Hurle JM (1999) Expression and function of Gdf-5 during digit skeletogenesis in the embryonic chick leg bud. Dev Biol 206:33–45PubMedCrossRefGoogle Scholar
  22. Miyamoto Y, Mabuchi A, Shi D, Kubo T, Takatori Y, Saito S, Fujioka M, Sudo A, Uchida A, Yamamoto S, Ozaki K, Takigawa M, Tanaka T, Nakamura Y, Jiang Q, Ikegawa S (2007) A functional polymorphism in the 5′ UTR of GDF5 is associated with susceptibility to osteoarthritis. Nat Genet 39:529–533PubMedCrossRefGoogle Scholar
  23. Miyazono K, Hellman U, Wernstedt C, Heldin CH (1988) Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization. J Biol Chem 263:6407–6415PubMedGoogle Scholar
  24. Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199PubMedCrossRefGoogle Scholar
  25. Noguchi K, Ishii S, Shimizu T (2003) Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family. J Biol Chem 278:25600–25606PubMedCrossRefGoogle Scholar
  26. Olofsson A, Miyazono K, Kanzaki T, Colosetti P, Engstrom U, Heldin CH (1992) Transforming growth factor-beta 1, -beta 2, and -beta 3 secreted by a human glioblastoma cell line. Identification of small and different forms of large latent complexes. J Biol Chem 267:19482–19488PubMedGoogle Scholar
  27. Ploger F, Seemann P, Schmidt-von Kegler M, Lehmann K, Seidel J, Kjaer KW, Pohl J, Mundlos S (2008) Brachydactyly type A2 associated with a defect in proGDF5 processing. Hum Mol Genet 17:1222–1233PubMedCrossRefGoogle Scholar
  28. Polinkovsky A, Robin NH, Thomas JT, Irons M, Lynn A, Goodman FR, Reardon W, Kant SG, Brunner HG, van der Burgt I, Chitayat D, McGaughran J, Donnai D, Luyten FP, Warman ML (1997) Mutations in CDMP1 cause autosomal dominant brachydactyly type C. Nat Genet 17:18–19PubMedCrossRefGoogle Scholar
  29. Robertson I, Jensen S, Handford P (2011) TB domain proteins: evolutionary insights into the multifaceted roles of fibrillins and LTBPs. Biochem J 433:263–276PubMedCrossRefGoogle Scholar
  30. Saharinen J, Keski-Oja J (2000) Specific sequence motif of 8-Cys repeats of TGF-beta binding proteins, LTBPs, creates a hydrophobic interaction surface for binding of small latent TGF-beta. Mol Biol Cell 11:2691–2704PubMedCrossRefGoogle Scholar
  31. Saharinen J, Taipale J, Keski-Oja J (1996) Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1. EMBO J 15:245–253PubMedGoogle Scholar
  32. Saharinen J, Hyytiainen M, Taipale J, Keski-Oja J (1999) Latent transforming growth factor-beta binding proteins (LTBPs)–structural extracellular matrix proteins for targeting TGF-beta action. Cytokine Growth Factor Rev 10:99–117PubMedCrossRefGoogle Scholar
  33. Schwabe GC, Turkmen S, Leschik G, Palanduz S, Stover B, Goecke TO, Mundlos S (2004) Brachydactyly type C caused by a homozygous missense mutation in the prodomain of CDMP1. Am J Med Genet A 124A:356–363PubMedCrossRefGoogle Scholar
  34. Seemann P, Schwappacher R, Kjaer KW, Krakow D, Lehmann K, Dawson K, Stricker S, Pohl J, Ploger F, Staub E, Nickel J, Sebald W, Knaus P, Mundlos S (2005) Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2. J Clin Invest 115:2373–2381PubMedCrossRefGoogle Scholar
  35. Sengle G, Charbonneau NL, Ono RN, Sasaki T, Alvarez J, Keene DR, Bachinger HP, Sakai LY (2008) Targeting of bone morphogenetic protein growth factor complexes to fibrillin. J Biol Chem 283:13874–13888PubMedCrossRefGoogle Scholar
  36. Settle SH Jr, Rountree RB, Sinha A, Thacker A, Higgins K, Kingsley DM (2003) Multiple joint and skeletal patterning defects caused by single and double mutations in the mouse Gdf6 and Gdf5 genes. Dev Biol 254:116–130PubMedCrossRefGoogle Scholar
  37. Sha X, Yang L, Gentry LE (1991) Identification and analysis of discrete functional domains in the pro region of pre–pro-transforming growth factor beta 1. J Cell Biol 114:827–839PubMedCrossRefGoogle Scholar
  38. Shi M, Zhu J, Wang R, Chen X, Mi L, Walz T, Springer TA (2011) Latent TGF-beta structure and activation. Nature 474:343–349PubMedCrossRefGoogle Scholar
  39. Shimomura Y, Agalliu D, Vonica A, Luria V, Wajid M, Baumer A, Belli S, Petukhova L, Schinzel A, Brivanlou AH, Barres BA, Christiano AM (2010) APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex. Nature 464:1043–1047PubMedCrossRefGoogle Scholar
  40. Storm EE, Kingsley DM (1999) GDF5 coordinates bone and joint formation during digit development. Dev Biol 209:11–27PubMedCrossRefGoogle Scholar
  41. Suzuki S, Marazita ML, Cooper ME, Miwa N, Hing A, Jugessur A, Natsume N, Shimozato K, Ohbayashi N, Suzuki Y, Niimi T, Minami K, Yamamoto M, Altannamar TJ, Erkhembaatar T, Furukawa H, Daack-Hirsch S, L’Heureux J, Brandon CA, Weinberg SM, Neiswanger K, Deleyiannis FW, de Salamanca JE, Vieira AR, Lidral AC, Martin JF, Murray JC (2009) Mutations in BMP4 are associated with subepithelial, microform, and overt cleft lip. Am J Hum Genet 84:406–411PubMedCrossRefGoogle Scholar
  42. Taipale J, Koli K, Keski-Oja J (1992) Release of transforming growth factor-beta 1 from the pericellular matrix of cultured fibroblasts and fibrosarcoma cells by plasmin and thrombin. J Biol Chem 267:25378–25384PubMedGoogle Scholar
  43. Taipale J, Miyazono K, Heldin CH, Keski-Oja J (1994) Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein. J Cell Biol 124:171–181PubMedCrossRefGoogle Scholar
  44. Thomas JT, Lin K, Nandedkar M, Camargo M, Cervenka J, Luyten FP (1996) A human chondrodysplasia due to a mutation in a TGF-beta superfamily member. Nat Genet 12:315–317PubMedCrossRefGoogle Scholar
  45. Thomas JT, Kilpatrick MW, Lin K, Erlacher L, Lembessis P, Costa T, Tsipouras P, Luyten FP (1997) Disruption of human limb morphogenesis by a dominant negative mutation in CDMP1. Nat Genet 17:58–64PubMedCrossRefGoogle Scholar
  46. Thomas JT, Prakash D, Weih K, Moos M Jr (2006) CDMP1/GDF5 has specific processing requirements that restrict its action to joint surfaces. J Biol Chem 281:26725–26733PubMedCrossRefGoogle Scholar
  47. Vidricaire G, Denault JB, Leduc R (1993) Characterization of a secreted form of human furin endoprotease. Biochem Biophys Res Commun 195:1011–1018PubMedCrossRefGoogle Scholar
  48. Wakefield LM, Smith DM, Flanders KC, Sporn MB (1988) Latent transforming growth factor-beta from human platelets. A high molecular weight complex containing precursor sequences. J Biol Chem 263:7646–7654PubMedGoogle Scholar
  49. Wolfman NM, McPherron AC, Pappano WN, Davies MV, Song K, Tomkinson KN, Wright JF, Zhao L, Sebald SM, Greenspan DS, Lee SJ (2003) Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases. Proc Natl Acad Sci USA 100:15842–15846PubMedCrossRefGoogle Scholar
  50. Yang W, Cao L, Liu W, Jiang L, Sun M, Zhang D, Wang S, Lo WH, Luo Y, Zhang X (2008) Novel point mutations in GDF5 associated with two distinct limb malformations in Chinese: brachydactyly type C and proximal symphalangism. J Hum Genet 53:368–374PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Muhammad Farooq
    • 1
    • 2
  • Hiroyuki Nakai
    • 3
  • Atsushi Fujimoto
    • 1
  • Hiroki Fujikawa
    • 1
  • Klaus Wilbrandt Kjaer
    • 4
  • Shahid Mahmood Baig
    • 2
  • Yutaka Shimomura
    • 1
    Email author
  1. 1.Laboratory of Genetic Skin DiseasesNiigata University Graduate School of Medical and Dental SciencesNiigataJapan
  2. 2.Human Molecular Genetics Laboratory, Health Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)FaisalabadPakistan
  3. 3.Graduate School of Science and TechnologyNiigata UniversityNiigataJapan
  4. 4.Department of Cellular and Molecular Medicine, Faculty of Health SciencesUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations