title: Molecular analysis of two novel missense mutations in the GDF5 proregion that reduce protein activity and are associated with brachydactyly type C.
abstract: Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5(T201P) and proGDF5(L263P) revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFβ1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.
authors.affiliation: Berlin Brandenburg Center for Regenerative Therapies, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany; Berlin Brandenburg School for Regenerative Therapies, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Stange
authors.affiliation: Martin-Luther-University Halle-Wittenberg, Technical Biochemistry, 06120 Halle, Germany.
authors.forename: Tino
authors.initials: T
authors.lastname: Thieme
authors.affiliation: Martin-Luther-University Halle-Wittenberg, Technical Biochemistry, 06120 Halle, Germany.
authors.forename: Karen
authors.initials: K
authors.lastname: Hertel
authors.affiliation: Martin-Luther-University Halle-Wittenberg, Technical Biochemistry, 06120 Halle, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Kuhfahl
authors.affiliation: Department of Pediatrics I, Innsbruck Medical University, 6020 Innsbruck, Austria; Division of Human Genetics, Innsbruck Medical University, 6020 Innsbruck, Austria.
authors.forename: Andreas R
authors.initials: AR
authors.lastname: Janecke
authors.affiliation: Department of Plastic, Reconstructive and Aesthetic Surgery, Innsbruck Medical University, 6020 Innsbruck, Austria.
authors.forename: Hildegunde
authors.initials: H
authors.lastname: Piza-Katzer
authors.affiliation: Department of Clinical Genetics, Turku University Hospital, 20014 Turku, Finland.
authors.forename: Maila
authors.initials: M
authors.lastname: Penttinen
authors.affiliation: Department of Clinical Genetics, Turku University Hospital, 20014 Turku, Finland.
authors.forename: Marja
authors.initials: M
authors.lastname: Hietala
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
authors.forename: Katarina
authors.initials: K
authors.lastname: Dathe
authors.affiliation: Berlin Brandenburg Center for Regenerative Therapies, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany; Institute for Medical Genetics and Human Genetics, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany; Research Group Development and Disease, Max-Planck-Institut für Molekulare Genetik, 14195 Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Mundlos
authors.affiliation: Martin-Luther-University Halle-Wittenberg, Technical Biochemistry, 06120 Halle, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Schwarz
authors.affiliation: Berlin Brandenburg Center for Regenerative Therapies, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany; Berlin Brandenburg School for Regenerative Therapies, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany. Electronic address: petra.seemann@charite.de.
authors.forename: Petra
authors.initials: P
authors.lastname: Seemann
mesh_terms: D000595:Amino Acid Sequence; D001846:Bone Development / Q000235:genetics*; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000378:metabolism; D001842:Bone and Bones / Q000196:embryology; D059327:Brachydactyly / Q000235:genetics*; D002356:Cartilage / Q000196:embryology / Q000254:growth & development; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D020219:Chondrogenesis / Q000235:genetics*; D055428:Growth Differentiation Factor 5 / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D059785:Karyotype; D020125:Mutation, Missense; D016415:Sequence Alignment; D051785:Smad Proteins / Q000378:metabolism
chemical_list: C525893:GDF5 protein, human; D055428:Growth Differentiation Factor 5; D051785:Smad Proteins; C498211:BMPR1A protein, human; C498214:BMPR1B protein, human; D052005:Bone Morphogenetic Protein Receptors, Type I
keywords: BMP; chicken micromass; chondrogenesis; proprotein; skeletal diseases