Clinical features of the affected family members with mutations in <i>GDF5</i>.

<p>The features are coded using terms from the Human Phenotype Ontology <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003846#pgen.1003846-Robinson1" target="_blank">[47]</a>. + present; − absent. <i>GDF5</i> mutations are presented with either features of brachydactyly (GDF5 p.R399C) or features of synostosis (GDF5 p.E491K) or a combination of multiple synostosis with additional brachydactyly (GDF5 p.W414R).</p

GDF5^W414R is positioned within the NOG and BMPR1A/B binding interface of the GDF5 dimer.

<p><b>A</b>: 3D presentation of the human GDF5 homodimer (PDB 1waq). The topology of the GDF5 monomer comprises two ß-sheets forming the fingers as well as the four-turn α-helix with the preceding pre-helix loop. The mutations are highlighted in pink (GDF5^W414R), violet (GDF5^R399C) and orange (GDF5^E491K). The image of the GDF5 structure was visualized using PyMol (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>). <b>B</b>: Protein sequence alignment of human, mouse and chicken GDF5 comprising the seven cysteine residues (bold) of the mature domain. Numbering is referred to the pro-protein sequence. Amino acids predicted to form the NOG binding interface are depicted as framed white boxes and based on the BMP7:NOG complex (PDB 1m4u). Residues predicted to be involved in BMPR1A binding are shown as grey boxes and refer to the BMP2:BMPR1A structure (PDB 1rew). Black boxes mark amino acids that bind to BMPR1B (PDB 3evs). Arrows indicate the mutated sites for GDF5^W414R, GDF5^R399C and GDF5^E491K. Note that GDF5^W414R and GDF5^E491K are located within the NOG binding site. Moreover, all three mutations interfere with the BMP type I receptor (BMPR1A and BMPR1B) binding interface.</p

Binding affinities of GDF5^W414R to immobilized receptor ectodomains.

<p>GDF5^W414R shows altered NOG and BMP receptor type I binding affinities in the Biacore assay.</p><p>Mean values from two experiments using at least six different analyte concentrations are shown.</p

GDF5^W414R shows impaired Bmpr1a signaling in a SBE-Luciferase reporter gene assay.

<p>NIH/3T3 cells were transfected with the BMP type I receptors, <i>Bmpr1a</i> or <i>Bmpr1b,</i> as well as with wild type <i>GDF5</i> and the GDF5 variants <i>GDF5^W414R</i>, <i>GDF5^R399C</i> and <i>GDF5^E491K</i>. As reporter, the SMAD binding element (SBE) was used and firely luciferase was normalized against TK-Renilla luciferase. <b>A</b>: No Bmp type I receptor was co-expressed which resulted in a weak SBE reporter activation for wild type GDF5 and GDF5^E491K, whereas in case of GDF5^W414R and GDF5^R399C signaling activity was absent. <b>B</b>: <i>Bmpr1a</i> co-expression increased the signaling activity of wild type GDF5 and GDF5^E491K; however, GDF5^W414R and GDF5^R399C were not able to induce reporter gene expression. <b>C</b>: Co-expression of <i>Bmpr1b</i> further increased the signaling activity of wild type GDF5 and GDF5^E491K compared to co-expression with <i>Bmpr1a</i>. In case of GDF5^W414R and GDF5^R399C, <i>Bmpr1b</i> co-expression rescued their signaling activity. The means of triplicate measurements are shown, error bars indicate standard deviation and a represent experiment is shown. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; **p≤0.01). Significances are related to the respective wild type GDF5 value.</p

<i>Gdf5</i>, <i>Nog</i> and <i>Bmpr1b</i> are co-expressed during murine limb development.

<p>Mouse embryo<i>s</i> with the C57BL/6 genetic background at embryonic stages 11.5 (A–H), 12.5 (A′–H′) and 13.5 (A″–H″) were labeled with probes of <i>Gdf5</i> (A and E), <i>Nog</i> (B and F), <i>Bmpr1a</i> (C and G) or <i>Bmpr1b</i> (D and H) and signals are shown in red. Representatively, two sections of the coronal dorsal axis (A–D) and the autopod transversal axis (E–H) are depicted. The signal for <i>Gdf5</i> strongly co-localizes with the <i>Nog</i> and <i>Bmpr1b</i> expression pattern, whereas <i>Bmpr1a</i> expression is in direct proximity in the surrounding epithelium and underlying mesenchyme.</p

GDF5^W414R is resistant towards inhibition by NOG in chicken micromass cultures.

<p>Chicken micromass cells were infected with RCASBP(A) containing the coding sequence (cds) of either wild type <i>GDF5</i> or the GDF5 variants <i>GDF5^W414R</i>, <i>GDF5^R399C</i> or <i>GDF5^E491K</i>. RCASBP(B) contained the cds of <i>NOG</i> and was used for co-transfection. Chicken micromass cultures and quantification of Alcian blue incorporation at 595 nm into the extracellular matrix (ECM) are shown for day 5. In the chicken micromass system, wild type GDF5 strongly induced chondrogenesis compared to the untransfected control. Chondrogenic differentiation was completely blocked in both, the control and wild type GDF5 cultures, when <i>NOG</i> is co-transfected. A similar pattern was observed for GDF5^R399C. Contrary, GDF5^W414R and GDF5^E491K exhibited insensitivity towards the antagonist. Values represent the mean of triplicates and error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; ***p≤0.001).</p