200700 (Faiyaz-Ul-

**/homozygous**

*121delG* prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 (Polinkovsky

*158delT* prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 (Everman *et* 

*158insC* prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 (Everman *et* 

*206insG* prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 (Polinkovsky

*493delC* prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 (Galjaard *et* 

M173V prodomain *gdf5* gene homozygous Brachydactyly type C # 113100 (Schwabe *et* 

S204R prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 (Everman *et* 

*759delG* prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 (Polinkovsky

*811ins23* prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 Everman, D.

*830delT* prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 Everman, D.

R301X prodomain *gdf5* gene heterozygous Brachydactyly type C # 113100 (Polinkovsky

R399C mature domain heterozygous Brachydactyly type A1 # 112500 (Byrnes *et al.*,

C400Y/*del1144G* mature domain/ compound Chondrodysplasia, # 200700 (Thomas *et* 

homozygous Chondrodysplasia, Grebe type

compound heterozygous

rs143383 5´UTR *gdf5* gene heterozygous Osteoarthritis

rs143384 5´UTR *gdf5* gene heterozygous Osteoarthritis

2250ct 3´UTR *gdf5* gene heterozygous Osteoarthritis

*206insG* prodomain *gdf5* gene homozygous Chondrodysplasia,

*297insC* prodomain *gdf5* gene homozygous Chondrodysplasia,

1114insGAGT prodomain *gdf5* gene homozygous Chondrodysplasia,

R378Q/P436T prodomain *gdf5* gene;

R380Q prodomain *gdf5* gene;

C400Y mature domain; no

C400Y mature domain; no

processing site / mature domain

processing site

processing/secretion

processing/secretion

**mutation location hetero-**

Two SNPs in the 5' untranslated regions (UTR) of *GDF5*, rs143383 and further downstream rs143384, share both a T-to-C transition in the *GDF5* core promoter. Functional studies using RNA extracted from the articular cartilage of OA patients harboring the SNP rs143383 revealed a significant, up to 27% reduced expression level of the osteoarthritis-associated Tallele relative to the C-allele, a phenomenon termed differential allelic expression (DAE) (Southam *et al.*, 2007). This allelic expression imbalance of *GDF5* could be extended to other soft tissues of the whole synovial joint, emphasizing that the single nucleotide

**Figure 8.** Localization of *GDF5* mutations. Arrowheads indicate the location of all currently known mutations linked to human skeletal malformation diseases affecting the limb. The specific inherited disease caused by each mutation is displayed in the legend underneath.

A GDF-5 monomer consists of an N-terminal signal peptide domain (black box), a prodomain (dark grey box) and the C-terminal mature part (light grey box) containing six highly conserved cysteine residues forming the cystine knot motif, whereas the seventh cysteine connects two monomers via an intermolecular disulfide bond. Italic type indicates nucleotide nomenclature; normal type represents single amino acid nomenclature. For references see Table 1.

Table 1 and Fig. 8).

**3.4. GDF-5: A key molecule in joint development and maintenance** 

Besides Noggin, the *GDF5* gene has been identified as a mutational hotspot in skeletal malformation diseases. To date, 14 missense mutations as well as a multitude of frameshift mutations have been identified in the translated region of the *GDF5* gene. Furthermore single nucleotide polymorphisms (SNPs) in the 5' and 3' untranslated region of the *GDF5* gene, three of which could be linked to enhanced susceptibility of developing osteoarthritis (OA), suggest that tempero-spatially highly defined gene expression of GDF-5 is required throughout life and is not limited to limb and joint development during embryogenesis (see

Two SNPs in the 5' untranslated regions (UTR) of *GDF5*, rs143383 and further downstream rs143384, share both a T-to-C transition in the *GDF5* core promoter. Functional studies using RNA extracted from the articular cartilage of OA patients harboring the SNP rs143383 revealed a significant, up to 27% reduced expression level of the osteoarthritis-associated Tallele relative to the C-allele, a phenomenon termed differential allelic expression (DAE) (Southam *et al.*, 2007). This allelic expression imbalance of *GDF5* could be extended to other soft tissues of the whole synovial joint, emphasizing that the single nucleotide

**Figure 8.** Localization of *GDF5* mutations. Arrowheads indicate the location of all currently known mutations linked to human skeletal malformation diseases affecting the limb. The specific inherited

A GDF-5 monomer consists of an N-terminal signal peptide domain (black box), a prodomain (dark grey box) and the C-terminal mature part (light grey box) containing six highly conserved cysteine residues forming the cystine knot motif, whereas the seventh cysteine connects two monomers via an intermolecular disulfide bond. Italic type indicates nucleotide nomenclature; normal type represents

disease caused by each mutation is displayed in the legend underneath.

single amino acid nomenclature. For references see Table 1.



Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 33

a butterfly shaped assembly with the monomeric subunits adopting an architecture resembling a left hand (Sebald *et al.*, 2004). The dimer interface is formed by the palm of the hand, two two-stranded β-sheets resembling two fingers emanate from the cystine knot containing palm. Mutagenesis was used to determine the receptor binding epitopes (Kirsch *et al.*, 2000). The BMP type I receptors bind to the so-called wrist epitope, the type II receptors bind to the so-called knuckle epitope (Kirsch *et al.*, 2000). The location of these receptor binding epitopes were then confirmed by structure analyses of various BMP ligand-receptor complexes (Kirsch *et al.*, 2000, Greenwald *et al.*, 2003, Allendorph *et al.*, 2006,

Homozygous non-sense or frame-shift mutations in the pro- or mature part of *GDF5* will result in a complete knockout of *GDF5*. However, also heterozygous non-sense and frameshift mutations in *GDF5* will severely lower the level of intact protein; assuming equal transcriptional and translational efficiency from both alleles by statistics only 25% of the protein produced will be intact due to its dimeric nature. Hence the complete knockout or partial knockdown of *GDF5* achieved by this type of mutation leads to rather severe skeletal malformation phenotypes such as brachydactyly type C (BDC), symphalangism (SYM1) or multiple synostosis syndrome (SYNS1). One potentially underappreciated possibility is also the formation of nonfunctional heterodimeric ligands if a cell produces more than one TGFβ factor at a time and thus a possible influence of non-sense GDF-5 mutations onto other BMP signals. It is a known fact that in Drosophila the BMP-2 and BMP-7 orthologs Dpp and Screw can form heterodimers with unique functions required for proper development of certain tissues (Shimmi *et al.*, 2005, O'Connor *et al.*, 2006), however in vertebrates existence of such BMP heterodimers has only been postulated or recombinant proteins have been used in the analysis, but existence of such heterodimers has not really been proven *in vivo* (Schmid *et al.*, 2000, Butler & Dodd, 2003) thus a potential "cross"-influence of non-

Of the 14 missense mutations known in the *GDF5* gene four are located within the pro-part of the GDF-5 protein. Whereas for the TGF-βs the pro-part fulfills an important regulatory role, termed latency, its role for the BMP and GDF subgroup of the TGF-β superfamily is much less clear. Latency was discovered for TGF-β1 in 1984 showing that TGF-β proteins are secreted as large protein complexes that require activation for TGF-β signaling (Lawrence *et al.*, 1984). It is known today that upon secretion the pro-part of TGF-βs is cleaved in the Golgi apparatus by furin proteases at a site between the pro- and mature part containing a consensus RXXR motif (other proteases might substitute for furin proteases but providing for TGF-β proteins with different N-termini) (Dubois *et al.*, 1995). The pro-part also called latency-associated peptide (LAP) however is still non-covalently attached thereby interfering with TGF-β signaling. Activation corresponding to release of the mature part from this intermediate latent complex is achieved either by physicochemical changes in the environment, e.g. acidification or by further proteolysis. Proteins specifically binding LAP have been identified (Miyazono *et al.*, 1988), these latent TGF-β binding proteins (LTBP) interact with the extracellular matrix and play an important role in the TGF-β activation process (for review see (Annes *et al.*, 2003)). For BMPs a process identical to latency as

functional GDF-5 mutations on other BMPs can only be hypothesized.

Weber *et al.*, 2007, Kotzsch *et al.*, 2009).

**Table 1.** Table of all known mutations in *GDF5* gene linked to skeletal malformation diseases affecting the limb. Mutations depicted in red represent single nuclear polymorphisms (SNPs) located in 5´ or 3´ regulatory regions of *GDF5* gene. Shown in black are mutations situated in the prodomain, whereas mutations in the mature part are represented in blue. Frameshift mutations are highlighted in italics, non-sense mutations are underlined.

polymorphism is not restricted to cartilage (Egli *et al.*, 2009). In addition, recent analysis showed that expression of GDF-5 could be further modulated epigenetically as both Calleles of the SNPs rs143383 and rs143384 form CpG sites thereby explaining the intra- and inter-individual variations observed (Reynard *et al.*, 2011). A third SNP influencing GDF-5 expression, 2250ct, is found in the 3' UTR of *GDF5*. It acts independently from the 5' SNP rs143383 and can similarly reduce protein expression levels by 20-25% (Egli *et al.*, 2009). The independent reduction in expression by these SNPs can be additive thereby showing that even moderate imbalances in the allelic expression levels of *GDF5* can result in severe disturbances in synovial joint maintenance. This idea is further emphasized by the identification of a duplication in the 3´ UTR of the *BMP2* gene including a distant enhancer of BMP2 expression in BDA2 patients. The phenotype described by Dathe *et al.* resembles those caused by specific mutations in the *GDF5* or the *BMPR1B* gene (Dathe *et al.*, 2009). As BMP-2 is expressed in regions surrounding future joints as well as in the joint interzone during the development of interphalangeal joints in close proximity to GDF-5 expression, one could hypothesize that by either increasing BMP-2 levels due to the duplication of an enhancer or by decreasing the GDF-5 expression due to regulatory SNPs as described above, the fine-tuned balance between signals from different BMPs may be severely disturbed.

#### **3.5. Proper folding and processing of pro-GDF-5 is essential for GDF-5 signaling**

Like other ligands of the TGF-β superfamily GDF-5 is expressed and secreted as a dimeric pro-protein consisting of a large (354aa per monomer) pro-part and a smaller (120aa per monomer) mature part at the C-terminus. The C-terminal mature part harbors the characteristic motif present in all TGF-β ligands comprising of seven (BMPs, GDFs) highly conserved cysteine residues (Activins, TGF-βs have two further Cys residues at the Nterminus of the mature part) of which six form the so-called cystine knot. The seventh cysteine residue is involved in an intermolecular disulfide bond thereby stabilizing the (usually homo-)dimeric ligand assembly. The dimeric mature part of TGF-β ligand exhibits a butterfly shaped assembly with the monomeric subunits adopting an architecture resembling a left hand (Sebald *et al.*, 2004). The dimer interface is formed by the palm of the hand, two two-stranded β-sheets resembling two fingers emanate from the cystine knot containing palm. Mutagenesis was used to determine the receptor binding epitopes (Kirsch *et al.*, 2000). The BMP type I receptors bind to the so-called wrist epitope, the type II receptors bind to the so-called knuckle epitope (Kirsch *et al.*, 2000). The location of these receptor binding epitopes were then confirmed by structure analyses of various BMP ligand-receptor complexes (Kirsch *et al.*, 2000, Greenwald *et al.*, 2003, Allendorph *et al.*, 2006, Weber *et al.*, 2007, Kotzsch *et al.*, 2009).

32 Mutations in Human Genetic Disease

non-sense mutations are underlined.

mW408R (hW414R)

mW408R (hW414R) prodomain; no processing/secretion

mature domain; location in type I receptor binding site

mature domain; location in type I receptor binding site

C429R mature domain homozygous Chondrodysplasia,

heterozygous Grebe type *al.*, 1997)

heterozygous Brachypodism (Masuya *et* 

homozygous severe Brachypodism, Osteoarthritis

**Table 1.** Table of all known mutations in *GDF5* gene linked to skeletal malformation diseases affecting the limb. Mutations depicted in red represent single nuclear polymorphisms (SNPs) located in 5´ or 3´ regulatory regions of *GDF5* gene. Shown in black are mutations situated in the prodomain, whereas mutations in the mature part are represented in blue. Frameshift mutations are highlighted in italics,

polymorphism is not restricted to cartilage (Egli *et al.*, 2009). In addition, recent analysis showed that expression of GDF-5 could be further modulated epigenetically as both Calleles of the SNPs rs143383 and rs143384 form CpG sites thereby explaining the intra- and inter-individual variations observed (Reynard *et al.*, 2011). A third SNP influencing GDF-5 expression, 2250ct, is found in the 3' UTR of *GDF5*. It acts independently from the 5' SNP rs143383 and can similarly reduce protein expression levels by 20-25% (Egli *et al.*, 2009). The independent reduction in expression by these SNPs can be additive thereby showing that even moderate imbalances in the allelic expression levels of *GDF5* can result in severe disturbances in synovial joint maintenance. This idea is further emphasized by the identification of a duplication in the 3´ UTR of the *BMP2* gene including a distant enhancer of BMP2 expression in BDA2 patients. The phenotype described by Dathe *et al.* resembles those caused by specific mutations in the *GDF5* or the *BMPR1B* gene (Dathe *et al.*, 2009). As BMP-2 is expressed in regions surrounding future joints as well as in the joint interzone during the development of interphalangeal joints in close proximity to GDF-5 expression, one could hypothesize that by either increasing BMP-2 levels due to the duplication of an enhancer or by decreasing the GDF-5 expression due to regulatory SNPs as described above, the fine-tuned balance between signals from different BMPs may be severely disturbed.

**3.5. Proper folding and processing of pro-GDF-5 is essential for GDF-5 signaling** 

Like other ligands of the TGF-β superfamily GDF-5 is expressed and secreted as a dimeric pro-protein consisting of a large (354aa per monomer) pro-part and a smaller (120aa per monomer) mature part at the C-terminus. The C-terminal mature part harbors the characteristic motif present in all TGF-β ligands comprising of seven (BMPs, GDFs) highly conserved cysteine residues (Activins, TGF-βs have two further Cys residues at the Nterminus of the mature part) of which six form the so-called cystine knot. The seventh cysteine residue is involved in an intermolecular disulfide bond thereby stabilizing the (usually homo-)dimeric ligand assembly. The dimeric mature part of TGF-β ligand exhibits

Grebe type

*al.*, 2007)

(Masuya *et al.*, 2007)

Haque *et al.*, 2008)
