Missense Mutations in GDF5 Signaling Molecular Mechanisms Behind Skeletal Malformation

ASR Joint Regen Review

Rheumatoid Arthritis Holistic Treatments

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Tina V. Hellmann, Joachim Nickel and Thomas D. Mueller

Additional information is available at the end of the chapter http://dx.doi.org/10.5772/35195

1. Introduction

Members of the large transforming growth factor P (TGF-P) superfamily of secreted growth factors initiate cellular signal transduction via binding to and oligomerization of two different types of membrane bound serine/threonine kinase receptors termed type I and type II (Carcamo et al., 1994, ten Dijke et al., 1996, Massague, 2000). They execute important functions in early (e.g. gastrulation) as well as in later stages (e.g. patterning) of embryonal development, but are also essential for regulation of tissue homeostasis and repair in the adult organism (Rosen & Thies, 1992, Kingsley, 1994, Hogan, 1996, Reddi, 1998, Massague, 2000). A characteristic feature of this protein family is the high degree of promiscuity in the ligand-receptor interaction (for review see (Sebald et al., 2004, Nickel et al., 2009)). This is exemplified by the numeral discrepancy of a likewise large number of ligands - more than 30 ligands are known in mammals to date - and a comparably small number of receptors available for binding and signaling (Miyazawa et al., 2002). Only 12 receptors exist in the TGF-P superfamily of which seven belong to the type I and five to the type II receptor subclass (Newfeld et al., 1999). This implies that a given receptor typically binds more than one TGF-P member, but we usually see that even a particular TGF-P ligand binds more than one receptor of either subtype (for review see (Sebald et al., 2004, Nickel et al., 2009)). Noteworthy, another seemingly reduction in the signaling output is due to the fact that principally only two primary pathways are activated by all TGF-P members (Hoodless et al., 1996, Nakao et al., 1997). After ligand-dependent oligomerization of the single transmembrane receptors, the intracellular kinase domain of the type II receptor activates the type I receptor kinase domain by transphosphorylation of a type I receptor exclusive membrane-proximal glycine/serine-rich region, termed GS-box (Shi & Massague, 2003). This phosphorylation unleashes the binding site for a group of transcription factors called SMADs whose naming derives from their homology to Drosophila's mothers against decapentaplegic (MAD) and the C. elegans protein Sma (Derynck et al., 1996). Dependent on the nature of the type I receptor present in the TGF-P ligand-receptor signaling complex R-SMAD proteins (for receptor-regulated SMADs) either belonging to the so-called SMAD1/5/8 or the SMAD2/3 family become phosphorylated. Subsequently, the so activated SMAD1/5/8 or SMAD2/3 proteins form heteromeric SMAD complexes comprising one R-SMAD of either of the aforementioned subfamilies and the common mediator SMAD protein SMAD4. This heteromeric SMAD complex then translocates into the nucleus where it regulates gene transcription by functioning as a transcription or co-transcription factor (see Fig. 1) (Heldin et al., 1997, Miyazono, 2000, Massague et al., 2005).

Figure 1. Signal transduction of BMPs and GDFs. Signal transduction is initiated by binding of the dimeric ligand to two types of transmembrane serine-/threonine kinase receptors termed type I and type II. Upon ligand binding the receptor chains oligomerize and the type II receptor transphosphorylates the type I receptor at the so-called GS-box thereby activating the kinase domain. Consequently, intracellular

Figure 1. Signal transduction of BMPs and GDFs. Signal transduction is initiated by binding of the dimeric ligand to two types of transmembrane serine-/threonine kinase receptors termed type I and type II. Upon ligand binding the receptor chains oligomerize and the type II receptor transphosphorylates the type I receptor at the so-called GS-box thereby activating the kinase domain. Consequently, intracellular downstream signaling components termed receptor-regulated SMADs (R-SMADs) are activated by phosphorylation. These R-SMADs then oligomerize with the common mediator SMAD (co-SMAD), SMAD4, translocate into the nucleus and in concert with other transcriptional modulators regulate target gene transcription. Regulation of this signaling pathway can occur at multiple levels as indicated. Thus, extracellular signaling modulators (e.g. Noggin, Follistatin) can bind to BMP/GDF ligands thereby preventing the interaction with their signaling receptors. On the membrane level coreceptors like ROR2 or members of the repulsive guidance molecule (RGM) family are thought to interact with the receptors and/or the ligands thereby amplifying the BMP/GDF signal. On the contrary, the pseudoreceptor BAMBI is an inhibitor of BMP as well as Activin signaling. The extracellular domain resembles the ligand binding interface of the type I receptors, while an intracellular kinase domain is lacking. The inhibitory function of the pseudoreceptor is potentially due to the formation of complexes with type I and/or type II receptors, thereby interfering with regular signal transduction. Amongst others, signal transduction can also be modulated intracellularly by the so-called inhibitory SMADs (I-SMADs), SMAD6 and SMAD7, where the I-SMADs compete with activated R-SMADs for interaction with SMAD4.

1.1. The multitude of biological functions of TGF-P members is established by a highly complex regulatory "cross-reactive" signaling network

Analysis of the patterning function of TGF-P members showed that they act as classical morphogens, i.e. the factors form a concentration gradient across the developing tissue and a specific cellular response is triggered dependent on the morphogen concentration (for review see (Wu & Hill, 2009)). A precise morphogenic function of an individual ligand can therefore only be explained in that either distinct tempero- and/or spatial distribution patterns of this ligand and its respective receptor(s) exist, which provide for specific signals at individual sites of action or in that the signaling event is tightly controlled by additional regulatory mechanisms. In the past years various studies identified a multitude of different components modulating the signal transduction of TGF-P members either outside the cell through secreted antagonists/modulator proteins (Ueno et al., 1987, Smith & Harland, 1992, Francois et al., 1994, Merino et al., 1999b, Shimmi & O'Connor, 2003), at the cell surface level via activating coreceptors or deactivating pseudoreceptors or extracellular matrix components (Lopez-Casillas et al., 1993, Onichtchouk et al., 1999, Gray et al., 2002, Wiater & Vale, 2003, Babitt et al., 2005, Samad et al., 2005, Lin et al., 2007), or in the cell interior through proteins interacting with the receptors, SMAD components or via influencing receptor turnover or degradation (see Fig. 1) (Zhu et al., 1999, Wotton & Massague, 2001, Chen et al., 2006). The majority of these modulating mechanisms again involve proteins, which themselves exhibit promiscuous binding to several partners, thus resulting in a highly complex regulatory "cross-reactive" network. It thus seems logical that attempts or incidents, which in vitro seem to manipulate individual interactions by a defined mechanism, will in vivo inevitably lead to a massive intervention in an interweaved signaling network with established equilibrium of cross-interacting partners.

1.2. What can be learned from individual gene deletions?

Due to the morphogen's inherent coupling of ligand concentration and signaling activity it is therefore expected, that mutations causing an alteration in signaling capacities become visible in a broad variety of different phenotypes. Consistently, a vast number of mutations could be correlated with inherited diseases (see OMIM database). Although often a clear correlation between mutation and phenotype can be drawn, in most of the cases the molecular mechanism translating the individual mutation into the corresponding phenotype remains unclear. An alternative strategy to identify functions of individual signaling components in the above-described signaling network is to eliminate their signaling input or function by null mutations. In the past decades a large number of knockout mice have been generated (TGF-P ligands, receptors, modulator proteins, etc.) and the loss of individual or combinations of genes of the TGF-P signaling network were analyzed in detail in hetero- as well as in homozygous situations (Zhao, 2003). Surprisingly, given the importance of TGF-P members for embryonic development and organogenesis, deletion of some genes of this superfamily did not result in prominent phenotypes (e.g. BMP-6) indicating that others can maximally compensate for a loss of these signaling components. On the other extreme some individual gene deletion resulted in embryonic lethality (e.g. BMP-2 or BMPR-IA) indicating that these components might occupy invariable key signaling positions, but thereby also impeding a detailed elucidation of gene function during development. In these situations, gene function was often further analyzed using conditional knockout mice to overcome lethality or to allow a cell- or tissue-specific deletion of the target gene to study the gene function in a more restricted environment. For some of the genes investigated it could be demonstrated, that a multitude of biological functions are strongly connected to the presence of one gene product in a strict temporal and spatial manner. For instance, it could be demonstrated for the receptor BMPR-IA that this receptor is essential for the formation of mesoderm during embryogenesis, (Mishina et al., 1995) but also for the differentiation and proliferation in postnatal hair follicles (Andl et al., 2004). However, these examples should emphasize the main problem of identifying individual relations between the factors and their biological function in such regulatory signaling networks. For the analysis of such mutation/function relations it is essential that a particular mutation translates into a visible phenotype and that this mutation does not result in embryonic lethality.

2. The role of GDFs in limb development

Astonishingly, within the complex machinery of TGF-P signaling only a few components seem to fulfill these criteria and for those a collection of mutations have been identified in the past years. One of these genes encodes for growth and differentiation factor 5 (GDF-5), which - like the other members of the TGF-P superfamily - binds as secreted signaling molecule to a defined subset of type I and type II receptors and initiates the activation of downstream signaling cascades. The biological role of GDF-5 in vivo became first apparent from the genetic analysis of the brachypodism mice (bp) (Storm et al., 1994), which also finally led to the discovery of GDF-5, -6 and -7. In brachypodism mice length and number of bones in the limbs are altered, but the axial skeleton does not seem to be affected (Gruneberg & Lee, 1973). It has already been suggested in the early 1980's that the bp mutation very likely disrupts a signaling event, which naturally leads to mesenchyme aggregation and chondrogenesis in the limb (Owens & Solursh, 1982). Initially three independent bp mutations have been described, which were all mapped to the GDF5 locus on chromosome 2 all resulting in a frame-shift of the open reading frame and thus basically representing GDF5 null mutations (Storm et al., 1994). As a result of the bp mutations several long bones show reduced length and the first two phalanges in the digits II-V are replaced by a single bony element in all four extremities (Gruneberg & Lee, 1973). It is important to note that despite GDF5 mRNA expression was reported to occur in a variety of non-skeletal tissues, e.g. the uterus, placenta, brain, heart, lung, kidney, etc., bp mice are fertile and do neither show behavioral abnormalities nor do they exhibit any morphological changes outside a few defined limb elements.

Figure 2. Schematic representation of the skeletal elements of a human limb and autopod.

A) Skeletal elements of a human limb. The stylopod gives rise to the humerus, the most proximal element of the limb skeleton, followed by the bony elements of radius and ulna, which derive from the zeugopod. Most distally, the autopod forms the bones of the hand.

B) Representation of the bony elements of the human autopod subdivided into the bones of the wrist (carpals), palm (metacarpals) and digits (phalanges).

Figure 2. Schematic representation of the skeletal elements of a human limb and autopod.

A) Skeletal elements of a human limb. The stylopod gives rise to the humerus, the most proximal element of the limb skeleton, followed by the bony elements of radius and ulna, which derive from the zeugopod. Most distally, the autopod forms the bones of the hand.

B) Representation of the bony elements of the human autopod subdivided into the bones of the wrist (carpals), palm (metacarpals) and digits (phalanges).

The elements of the vertebrate limb originate from mesenchymal cells that first condense and subsequently initiate a differentiation program leading to the production of cartilage and bones in a highly defined fashion. These skeletal elements develop from single condensations in a proximal-to-distal sequence, which first grow and then branch and segment starting with the condensation forming the humerus at 10.5 days post coitus (dpc) (Wanek et al., 1989, Storm & Kingsley, 1996, Francis-West et al., 1999). The humerus aggregate then branches distally at 11.5 dpc thereby forming the condensations for the radius and the ulna (for nomenclature see Fig. 2). The digits develop as continuous structures called digital rays, which lengthen distally during further outgrowth. In order to build regular hands or feet the rays will then (13.5 - 15.5 dpc) be further separated in a sequential segmentation process to form the metacarpals and the phalanges. In mice GDF5 mRNA is first detectable in the developing forelimb at 11.5 dpc in the proximal and distal region that will later form the shoulder and the elbow (Storm & Kingsley, 1996, FrancisWest et al., 1999). At 12.5 dpc GDF-5 is additionally expressed within the developing digital ray at a site that likely forms the future joint between the metacarpals and proximal phalanges. One day later at 13.5 dpc GDF5 mRNA is expressed in the developing rows of carpals and in an additional stripe across the digital rays, with the sites coinciding with developing joints in the wrist and the first interphalangeal joint (Storm & Kingsley, 1996). At 14.5 dpc the segmentation process seems completed, an additional stripe of GDF-5 expression separates the developing intermediate and distal phalanges and now all elements of a mice forelimb are defined and undergo chondrogenesis (Fig. 3) (Storm & Kingsley, 1996).


11.5 dpc

12.5 dpc

13.5 dpc

14.5 dpc

Figure 3. Expression pattern of BMP2, GDF5, BMPR1A and BMPR1B in the developing mouse fore limb.

Whole-mount in situ hybridization of BMP2, GDF5 and their receptors BMPR1A and BMPR1B in a mouse fore limb at different embryonic stages. GDF5 expression marks the developing cellular condensations. At 11.5 dpc GDF5 is expressed in regions later forming shoulder and elbow. At 12.5 dpc GDF5 is additionally visible in the future joints between the metacarpals and proximal phalanges. Later it is expressed in a stripe of the digital ray corresponding to the future interphalangeal joints separating the proximal from the intermediate (13.5 dpc) and the intermediate from the distal phalanges (14.5 dpc). BMP2 expression is seen in the apical ectodermal ridge, the underlying mesenchyme and at the posterior side of the limb at 11.5 dpc. One day later, BMP2 expression is mainly restricted to the interdigital mesenchyme as well as to the posterior wrist forming region, the wrist and the distal joints of radius and ulna. At 13.5 dpc BMP2 expression can be localized to a region surrounding the cartilage condensations of the dorsal tendons, whereas at 14.5 dpc it is mainly found around the regions of future interphalangeal joints. BMPR1A shows a more or less uniform expression throughout the whole developing mouse limb at all stages depicted above. In contrast, BMPR1B expression at 11.5 dpc is restricted to developing condensations of the digit anlagen. Later, at 13.5 dpc 14.5 dpc, BMPR1B expression can be found in regions of the future interphalangeal joints. Reprinted from The American Journal of Human Genetics (2009) 84, 483-492, K. Dathe et al., "Duplications involving a conserved regulatory element downstream of BMP2 are associated with Brachydactyly type A2", Copyright 2011, with permission from Elsevier.

The full process of joint formation occurs in three steps: First, special regions with high cell densities so-called interzones are formed corresponding to the stripes across the developing cartilage elements. Second, apoptosis leads to the removal of cells in the center of this interzone. Together with changes in the extracellular matrix on neighboring cells this creates a three-layered structure characteristic for the developing joint. Third, at both extremes of the interzone differentiation of the articular cartilage takes place leading to a fluid-filled gap between the (now segmented) skeletal elements (Haines, 1947, Mitrovic, 1978, Craig et al., 1987). The above observations highlight GDF-5 as one of the earliest markers for joint formation, whose mRNA can be detected in the developing joint 24 to 36h prior to visible morphological changes in the interzone and its expression continues for 2 to 3 days (for details see Fig. 4). The reduction of the number of phalanges in the brachypodism mouse, which is basically a GDF5 knockout mouse, is likely due to a failure in the segmentation in the digital rays (Storm et al., 1994). In bp mice limb-bud development as well as the condensations for the initial digital rays seem normal, but during segmentation of the digital rays during 12.5 to 14.5 dpc the formation of an interzone leading to the separation of proximal and intermediate phalanges is absent in bp mice. However, as GDF-5 is expressed in all synovial joints in wildtype mice and not just in the first interphalangeal joints of digits II to V it seems apparent that GDF-5 cannot be the sole factor for the formation of all joints in the whole limb (Storm & Kingsley, 1996). Without knowing the nature and molecular functions of GDF-5 Hinchliffe and Johnson in 1980 already suggested that the brachypodism phenotype might be caused by the disruption of a pattern (of various factors) that determines the location of joints in the limb (Hinchliffe & Johnson, 1980). As GDF-5 shares between 80 and 86% amino acid sequence identity in its C-terminal mature part with GDF-6 and GDF-7 and the latter factors are also expressed during limb development it seemed logical to assume that these factors might compensate for the loss of GDF5 in the brachypodism mutations (Storm & Kingsley, 1996). This hypothesis whether the two GDF-5 family members GDF-6 and GDF-7 can either substitute in case of a loss of GDF5 or act in a synergistic manner was again tested by generating knockout animal models.

Both genes GDF6 and GDF7 are expressed in and around the developing joint (Hattersley et al., 1995, Wolfman et al., 1995), furthermore the mRNA expression pattern does not strictly overlap with that of GDF5 (Wolfman et al., 1997). Strong mRNA levels of GDF6 can be observed in elbow and the carpal joints as well as the perimeter of the digital ray, whereas GDF7 expression is restricted to the proximal interphalangeal joint (Settle et al., 2003). Indeed, studies on GDF6 knockout mice show fusions in joints different from those seen in the brachypodism mice - in GDF6-/- mice fusions of specific bones in the wrist and ankle correlate with the strongest GDF6 expression in wildtype mice - possibly suggesting that a particular member of the GDF-5/6/7 family might be responsible for the formation of a subset of joints in the limb system (Settle et al., 2003). Expression analysis using other joint markers such as GDF5 (Storm & Kingsley, 1996), PTHRP (Parathyroid hormone-related protein, (Lanske et al., 1996, Vortkamp et al., 1996)) or DELTAEF1 (a zinc-finger homeobox transcription factor, (Takagi et al., 1998)) shows that the earliest stages of joint formation also occur in the absence of GDF6 expression, but similar to the brachypodism mutations these morphological changes do not proceed and thus segmentation of these skeletal elements is

Figure 4. Schematic representation of limb bud outgrowth and determination of digit identities. A-C) Limb bud outgrowth. During limb bud initiation morphogen gradients determine the three main axes of the limb: proximo-distal, antero-posterior and dorso-ventral. Development of these gradients is under control of specific signaling centers such as the apical ectodermal ridge (AER) providing a proximo-distal gradient, the zone of polarizing activity (ZPA) producing an anterior-posterior gradient and the dorsal and ventral ectoderm establishing a dorso-ventral signal, thereby generating a morphogenic field inheriting the information for skeletal pattern formation (for review see Tickle, 2003 & 2006; Zeller, 2009). Skeletal elements of the vertebrate limb originate from mesenchymal cells that condense to form the cartilage anlagen, which develop in a proximo-to-distal manner starting with the condensation forming the humerus at 10.5 dpc. The humerus aggregate then branches distally at 11.5 dpc thereby forming the condensations of radius and ulna. The digits develop as continuous structures termed digital rays, which lengthen distally during further outgrowth. In order to build regular hands the rays will then (13.5 - 15.5 dpc) be further separated in a sequential segmentation process to form the metacarpals and the phalanges. D) Formation of the initial condensation in the human autopod. Distal mesenchymal cells under control of fibroblast growth factors (FGFs) derived from the AER and ectodermal Wnts (eWnts) remain in an undifferentiated, proliferative state. As cells escape from AER signaling they start to differentiate into prechondrogenic cells and later into chondrocytes, whereas chondrogenesis is negatively regulated by eWnt/P-catenin signaling. Mesodermally derived BMPs as well as GDF-5 positively influence differentiation by signaling via type I receptors BMPR-IA and BMPR-IB expressed in the chondrogenic precursor cells. E) Elongation and segmentation of the digit condensations. Directed outgrowth of the condensations is achieved by BMP signaling in a region termed phalanx-forming region (PFR). This process is negatively regulated by eWnt signaling. Within the condensation pre-hypertrophic chondrocytes arise expressing Ihh, which positively influences PFR located BMP signaling. At the side of the future joint locally acting Wnt signals derived from the surrounding mesenchyme induce the differentiation of chondroprogenitor cells into flatened interzone cells expressing GDF-5. This process is encouraged by Ihh signaling from pre-hypertrophic condrocytes. Furthermore, GDF-5 and Ihh positively influence proliferation of columnar chondrocytes. F-G) Cavitation of the joint and growth of the digit. Ihh induces parathyroid hormone-related peptide (PTHrP) expressed in proliferative columnar chondrocytes underneath the future joint. PTHrP itself is a negative regulator of Ihh expression, thereby forming a negative feedback loop with Ihh. Interzone cells express BMP-2, which has a role in regulating apoptosis of these cells, thereby forming the joint cavity. The establishment of the so-called growth plate initiates further growth of the digit. This region is composed of zones of progressively differentiated chondrocytes: proliferating, columnar chondrocytes, followed by pre-hypertrophic chondrocytes expressing Ihh and finally hypertrophic chondrocytes eventually undergoing apoptosis thereby giving rise to the formation of the bone marrow cavity (BMC).

halted (Settle et al., 2003). In contrast to GDF5'/' mice, which had fusions restricted to synovial joint, GDF6'1' mutants also showed defects in the cartilage and ligament structures of the middle ear and the coronal suture (a non-synovial joint) in the skull (Settle et al., 2003). Analysis of the GDF5/GDF6 double knockout mouse showed additional skeletal defects with many bones being strongly reduced in length or even being absent. As these defects are not observed in either one of the single knockout mice and are also observed in synovial joints outside the limbs it suggests that GDF-5 and GDF-6 act synergistically during the formation of specific joints (Settle et al., 2003).

For GDF-7 function the effects in GDF7'1' mice are subtler and no changes in the skeletal patterning have been observed (Settle et al., 2001). The phenotypes described comprise abnormal vesicle development in male mice (Settle et al., 2001), smaller cross-sectional diameter of various long bones (Maloul et al., 2006) and minor differences in tendon and ligament structures (Mikic et al., 2006). A possible explanation for the very mild phenotype seen in GDF7-/- mice might be due to the upregulation of GDF5 and GDF6 mRNA expression above levels seen in wildtype mice leading to a partial compensation in the absence of GDF7 (Mikic et al., 2006). The above-described effects seen upon single or double deletion of GDF members indeed underline that GDF-5 alone, despite its patterning structure throughout the skeleton, does not induce the joint forming process in all joints of the developing limb. Moreover, it rather acts only on specific joints or might address additional ones throughout the limb in combination with GDF-6 or other factors (possibly in varying ratios) giving rise to the hypothesis that additional morphogens, e.g. members of the BMP superfamily, contribute to joint formation in vivo.

This idea that GDF-5 possibly acts via a defined combination with other factors to induce and maintain joint formation is supported by overexpression studies applying either locally ectopically GDF-5 protein (Storm & Kingsley, 1999) or by expressing GDF-5 systemically via retroviral transfection (Francis-West et al., 1999). Interestingly, implantation of agarose beads soaked with recombinant GDF-5 into the limbs of chicken embryos did not lead to the development of additional ectopic joints. Instead, GDF-5 stimulated cartilage growth of existing cartilage, which - dependent on the location of the implantation - could even interfere with joint development (Storm & Kingsley, 1999). Studies using developing limbs of mice show similar results, implanting recombinant GDF-5 in hind limbs at 12.5 or 13.5 dpc showed that GDF-5 stimulated growth of currently present cartilage cells whereas the interdigital mesenchyme did not respond to GDF-5 treatment after 12.5 dpc. This different response of both cell types could also be seen when different cartilage differentiation markers such as Collagen2 and Indian hedgehog (IHH) were analyzed with both markers being induced upon GDF-5 treatment in the existing cartilage but not in the interdigital mesenchymal cells (Storm & Kingsley, 1999). This suggests that the different cells present in the developing joints lose their GDF-5 responsiveness at different times. GDF-5 can thus be considered as a pro-chondrogenic factor that acts in a stage-dependent manner and is required but not sufficient for joint formation.

3. Disorders in limb development

A group of skeletal malformation diseases observed in humans, i.e. brachydactyly, symphalangism and chondrodysplasia, exhibits similar limb deforming phenotypes as observed in brachypodism mice suggesting that similar mechanisms and factors are affected in humans (for review see (Temtamy & Aglan, 2008, Mundlos, 2009)). All phenotypes describe skeletal malformations of extremities - especially of the phalanges - caused by abnormalities in cartilage development. Typically all the brachydactyly-causing mutations affect the formation of synovial joints due to a deregulation of chondrocyte proliferation and/or differentiation. The classification of the different diseases has initially been done by examining the skeletal malformation phenotype (Bell, 1951). Genetic analyses later revealed disease-causing mutations not only in GDF-5, but also in other TGF-P ligands, receptors or modulator proteins as well as in other differentiation factors. Nowadays the different brachydactyly phenotypes are classified into eight different forms (BDA1-3, BDB1-2, BDC, BDD, BDE), which show clear differences regarding affected phalanges (see Fig. 5).

Of those the brachydactylies BDA1, BDD and BDE are caused by genes that are seemingly unrelated to the TGF-P/BMP signaling pathway. In BDA1, which is characterized by shortened intermediate digits in all phalanges, inactivating mutations in the gene encoding for the secreted morphogen of the Hedgehog family Indian hedgehog (IHH) seem to be the molecular cause (Gao et al., 2001, Liu et al., 2006). Indian hedgehog is regulating chondrocyte proliferation and is also required for ossification of endochondral bones (St-Jacques et al., 1999, Karp et al., 2000). The skeletal malformation phenotype resembles that of the IHH-/-knockout mice (St-Jacques et al., 1999) and suggested that binding to the receptor Patched (PTCH) and its subsequent activation is impaired in patients suffering from BDA1. Modelling of a potential receptor interaction of IHH on the basis of the crystal structure of Sonic hedgehog bound to the hedgehog antagonist HHIP indicates that the four missense mutations at position Gly95, Asp100, Glu131 and Thr154 inactivate IHH via two different mechanisms (Bosanac et al., 2009). The mutations of Gly95, Asp100 or Glu131 disrupt the conserved calcium coordination site present in hedgehog proteins, which was shown to be



Figure 5. Clinical features of non-syndromic brachydactylies. In the top row, schematic representations of human hands depict specific phalanges and interdigital tissue affected in each skeletal malformation disease. Typical clinical features of hands are shown in the middle, corresponding X-rays underneath. Reprinted from Clinical Genetics (2009) 76, 123-136, S. Mundlos, "The brachydactylies: a molecular disease family", Copyright 2011, with permission from John Wiley and Sons.

required for high-affinity receptor binding (McLellan et al., 2006, Gao et al., 2009, Guo et al., 2010). For the fourth mutation - T154I - identified recently no clear mechanistic explanation can be given, however based on the IHH 3D model Thr154 is located in close proximity to the other BDA1-associated missense mutations (Liu et al., 2006) and thus possibly also interferes with receptor binding. Although neither IHH nor its receptors directly bind to TGF-P signaling components, BMP and IHH signals interact at various stages to regulate chondrocyte development. First of all, it has been shown that treatment of limb explants with the BMP antagonist Noggin leads to a decreased expression of IHH message (Minina et al., 2001). Later Seki and Hata found that the IHH gene is a direct target of the BMP/SMAD signaling pathway due to the fact that GC-rich boxes in the promoter region of IHH confer binding of SMAD4 (Seki & Hata, 2004). This allows an upregulation of IHH expression in response to BMP signals. In the GDF-5 implantation experiments performed by Storm and Kingsley the GDF-5 dependent increase in the IHH mRNA message was used as a marker for chondrocyte differentiation (Storm & Kingsley, 1999). Secondly, there also seems to be a positive feedback loop as in chicken ectopic expression of IHH leads to an increased expression of BMP-2 and BMP-4 and similar results could be obtained in mice using transgenic animals in which the IHH gene expression is driven by a COL2 promoter (Pathi et al., 1999, Minina et al., 2001). However, the effects of the deactivating IHH mutations in BDA1 are not exclusively transmitted via its direct regulatory roles on the BMP signaling pathway, besides the above described feedback loop between IHH and BMP pathways, both factors also exhibit independent functions in chondrocyte development (Minina et al., 2001).

The brachydactylies BDD and BDE are characterized by a shortened distal phalanx in finger I and shortened metacarpals in fingers I to V, respectively. In both diseases mutations in the HOXD13 gene seem to be the molecular cause (Caronia et al., 2003, Johnson et al., 2003). HOXD proteins represent homeobox transcription factors and disruption of the 5' HOXD genes HOXD11, HOXD12, and HOXD13 in mice have shown that these transcription factors exhibit important position-specific functions during limb development (Davis & Capecchi, 1996, Villavicencio-Lorini et al., 2010). Two of three mutations described, I314L and Q371R seem to disrupt binding of the HOXD transcription factor to its target DNA site as deduced from structural modeling of the protein:DNA complex (Johnson et al., 2003, Zhao et al., 2007). Although the amino acid replacement is rather conservative, the leucine sidechain seems to introduce a steric hindrance to a neighboring pyrimidine base of the bound target DNA possibly altering the specificity for DNAs containing either a thymine or a cytosine in this sequence. For the second mutation, serine 308 to cysteine, it is difficult to deduce a molecular mechanism explaining the skeletal phenotype. Serine 308 located in the homeobox domain of HOXD13 is not in contact with the DNA and placed in a less conserved region, thus misfolding of the HOXD13 protein due to the different sidechain size and polarity of the introduced cysteine residue might explain the altered HOXD13 function. The effect of both mutations on DNA binding was however confirmed experimentally by electrophoretic mobility shift assays (EMSA) (Johnson et al., 2003). Similar to BDA1 a direct regulatory or physical interaction of HOXD proteins and members of the TGF-P/BMP pathway is not apparent and thus it seems unclear at first sight whether the skeletal malformation phenotype of the HOXD13 mutants results from an independent parallel disturbed signaling pathway involved in limb development or whether HOXD13 might be an upstream or downstream target of the TGF-P/BMP signaling cascade. Suzuki et al. have found that both HOXA13 and HOXD13 transcription factors can enhance transcription of the BMP4 promoter and may thus increase BMP expression (Suzuki et al., 2003). Recently the group of Stefan Mundlos investigated the effect of the HOXD11, -12, -13 and HOXA13 genes on joint formation in mice and discovered that HOXD13 can directly bind and regulate the RUNX2 promoter, whose activation is crucial for formation of cortical bone (Villavicencio-Lorini et al., 2010). Studies using mice with defective HOXA13 revealed that upon loss of HOXA13 function mRNA expression for GDF5 is downregulated, whereas mRNA for BMP2 is upregulated (Perez et al., 2010). As HOXA and HOXD proteins might form regulatory complexes, BDE initiating mutations in HOXD13 may thus act via altering a defined concentration balance between GDF-5 and BMP-2 in the developing joint.

3.1. Disrupted GDF-5 signaling correlates with impaired joint formation

The other brachydactyly forms are caused by mutations in either GDF5, or other BMP genes, BMP receptors or modulator proteins thereby highlighting the central regulatory role of the GDF/BMP signals for proper joint formation. Mutations in the GDF5 gene are found in brachydactylies of the type BDA1, BDA2 and BDC, but also in symphalangism and multiple synostosis syndrome phenotypes as well as in chondrodysplasias of the Grebe, Hunter-Thompson and DuPan type, which are more severe skeletal malformation diseases possibly due to the fact that in the latter syndromes the mutations in GDF5 are homozygous or compound heterozygous (see Table 1). Mutations in the BMP type I receptor BMPR-IB as well as a duplication of an about 6kb element in the 3' regulatory untranslated domain of the BMP2 gene also lead to brachydactyly of the type BDA2 (Lehmann et al., 2003, Lehmann et al., 2006, Dathe et al., 2009). Mutations in the orphan tyrosine receptor kinase ROR2, which might possibly act as a GDF-5 specific coreceptor thereby influencing receptor activation of this TGF-P member, lead to brachydactyly of the type BDB1 (Oldridge et al., 2000, Schwabe et al., 2000). Amino acid exchanges in the BMP modulator protein Noggin are observed in patients suffering from brachydactyly type B2 (BDB2) (Lehmann et al., 2007). As there is a wealth of structural and functional data available for almost all of the above-mentioned factors a more in-depth analysis can be performed to analyze the molecular mechanism behind these disease-causing mutations.

3.2. Mutations interfering with BMPR-IB kinase activity and signaling

So far three mutations in the BMP type I receptor BMPR-IB could be correlated with brachydactyly BDA2. In the BMP/GDF signaling pathway three type I receptors, BMPR-IA (Alk3), BMPR-IB (Alk6) and ActR-I (Alk2) can be addressed by the different ligands for binding and signaling (Sebald et al., 2004). In vitro interaction analyses show that GDF-5 can bind only to BMPR-IA and BMPR-IB with affinities in the nano-molar range (Nickel et al., 2005), whereas it shows no measureable interaction with the type I receptor ActR-I (Heinecke et al., 2009). These and other in vitro studies also showed that GDF-5 interacts preferentially with BMPR-IB exhibiting a 10 to 15-fold higher affinity for BMPR-IB than for BMPR-IA (Nickel et al., 2005, Heinecke et al., 2009). Furthermore, performing a more in vivo-like radioligand binding assay in order to analyze the interaction of radiolabeled GDF-5 via chemical crosslinking to cells that were either transfected with the different type I and type II receptors or endogenously express BMP receptors, an exclusive binding of GDF-5 to BMPR-IB could be detected (Nishitoh et al., 1996). Despite this rather strong binding specificity of GDF-5 to BMPR-IB on whole cells measuring transcriptional activation in mink lung cells transfected with different combinations of BMP type I and type II receptors showed that GDF-5 can activate SMAD signaling via BMPR-IB and BMPR-IA with almost identical efficiency (Nishitoh et al., 1996). However, BMPR-IA cannot substitute for BMPR-IB in all GDF-5 initiated signals, e.g. induction of the osteogenic marker alkaline phosphatase (ALP) by GDF-5 is observed in the murine pro-chondrogenic cell line ATDC5, which does not express BMPR-IB and thus in this case BMPR-IA can functionally replace BMPR-IB. Furthermore, in this cell line the concentration for half-maximal ALP induction is about 10fold lower than for BMP-2, which correlates very nicely with the difference in BMPR-IA affinity of both BMP factors (Nickel et al., 2005). In contrast, the mouse osteoblastic cell line MC3T3 or the mouse myoblastic cell line C2C12, which express BMPR-IA but not BMPR-IB, do not respond to GDF-5 in the alkaline phosphatase expression assay (but at the same time respond to BMP-2) (Nishitoh et al., 1996). Besides the fact that in the context of the developing joint BMPR-IA might not be the correct signaling receptor for GDF-5, the spatially highly defined expression pattern of GDF-5 and the two BMP type I receptors in the junction between the growth plate and the developing joint suggests that at sites of high GDF-5 concentration only BMPR-IB is highly expressed whereas BMPR-IA expression is rather low (see Fig. 3) ((Wolfman et al., 1997, Zou et al., 1997, Sakou et al., 1999, Storm & Kingsley, 1999, Yi et al., 2001, Settle et al., 2003, Minina et al., 2005) for review see (Pogue & Lyons, 2006)).

All BDA2 causing BMPR-IB mutations are located in the cytoplasmic kinase domain. One exchange - isoleucine 200 to lysine (I200K) - is placed within the so-called GS (glycine/serine-rich) box, which is phosphorylated upon ligand binding and hetero-oligomerization of the type I and type II receptors (see Fig. 6A-C). Structural analysis of the kinase domains of the

Figure 6. The kinase domain of the BMP receptor IB. A) Ribbon representation of a model of the BMPR-IB kinase domain (adapted from PDB entry 3MDY, (Chaikuad et al., 2010a)). The elements important in kinase activity and or BMP signaling are indicated. Glycine/serine-rich (GS-)box: yellow; L45-loop for SMAD subgroup specificity: purple; phosphate binding loop: cyan; activation loop: green; active site with Asp332 in stick representation: magenta; NANDOR-region regulation downstream signal activation: red. B) Magnification of the GS-box with the relevant serine and threonine residues that become phosphorylated during BMP type I receptor activation shown as sticks. The location of Ile200 mutated in BDA2 is indicated. C) Isoleucine 200, mutated to lysine in BDA2, is surrounded by hydrophobic residues. Threonine 199, which is required to become first phosphorylated to allow for further phosphorylation events in the GS-box, is located in close proximity, suggesting that mutation

Figure 6. The kinase domain of the BMP receptor IB. A) Ribbon representation of a model of the BMPR-IB kinase domain (adapted from PDB entry 3MDY, (Chaikuad et al., 2010a)). The elements important in kinase activity and or BMP signaling are indicated. Glycine/serine-rich (GS-)box: yellow; L45-loop for SMAD subgroup specificity: purple; phosphate binding loop: cyan; activation loop: green; active site with Asp332 in stick representation: magenta; NANDOR-region regulation downstream signal activation: red. B) Magnification of the GS-box with the relevant serine and threonine residues that become phosphorylated during BMP type I receptor activation shown as sticks. The location of Ile200 mutated in BDA2 is indicated. C) Isoleucine 200, mutated to lysine in BDA2, is surrounded by hydrophobic residues. Threonine 199, which is required to become first phosphorylated to allow for further phosphorylation events in the GS-box, is located in close proximity, suggesting that mutation

I200K might also act via abrogating the initial activating phosphorylation at Thr199. D) Magnification into the NANDOR domain of BMPR-IB. The mutated residue Arg486 is located at the solvent-accessible surface, thus mutations R486W and R486Q (shown in grey) very likely do not cause conformational alterations. This suggests that the NANDOR domain constitutes a binding interface for so far unknown proteins involved in the receptor activation.

BMP receptor BMPR-IB (PDB entry 3MDY, (Chaikuad et al., 2010a)), of the TGF-P receptor TGFPR-I (Huse et al., 1999) or the Activin type I receptor ActR-I (PDB entry 3H9R, (Chaikuad et al., 2010b)) show that the GS-box domain in the inactivated state consists of two antiparallel a-helices. Functional analysis of the TGFPR-I receptor kinase revealed that phosphorylation of all conserved serine and threonine residues in the consensus motif (T/S)SGSGSG placed in the loop between the two helices is absolutely required for downstream signaling (Wieser et al., 1995) and SMAD protein binding (Huse et al., 2001). More importantly, threonine residue Thr200 in TGFPR-I (equivalent to Thr199 in BMPR-IB) adjacent to this consensus motif is absolutely conserved between TGF-P type I receptors and is crucial for ligand-dependent receptor activation. Mutagenesis showed that phosphorylation of this particular threonine residue is a pre-requisite for further phosphorylation of the GS-box motif located N-terminally of this residue (Wieser et al., 1995). In the BDA2 associated mutation I200K in BMPR-IB the direct neighbor of Thr199 is exchanged from a hydrophobic isoleucine to a polar lysine residue. As the isoleucine is rather buried in this motif, the exchange might lead to local unfolding or the Ile to Lys substitution is such drastic that the recognition by the kinase responsible for phosphorylation of Thr199 and thus subsequent receptor activation is impeded (see Fig. 6A-C). In vitro kinase assays indeed revealed a complete loss of kinase activity of BMPR-IB carrying the I200K mutation (Lehmann et al., 2003).

The other mutations in BMPR-IB associated with BDA2, R486Q or R486W, are located in the so-called NANDOR region (for non-activating non-down-regulating) (see Fig. 6A/D). This region at the C-terminus of the kinase domain is highly conserved between TGF-P type I receptors but placed quite distantly from the regulatory important regions such as the GS-box or the L45-loop, which mediate binding to R-SMAD proteins upon receptor activation or the active site of the kinase domain. Studies on the TGF-P receptors TGFPR-I (Garamszegi et al., 2001) and TSR-I (Alk1) (Ricard et al., 2010) show that mutations within this domain abrogate type I receptor endocytosis and signal transduction as R-SMAD proteins are not phosphorylated by these receptor mutants. In BMPR-IB the exchange of the surface-accessible arginine 486 by either glutamine or tryptophan diminished not only SMAD1/5/8 phosphorylation, but also led to strongly decreased expression of alkaline phosphatase in C2C12 cells transfected with BMPR-IB. This signaling-impaired phenotype could also be confirmed in a more physiological assay measuring chondrocyte differentiation in virally transduced chicken limb-bud micromass cultures (Lehmann et al., 2003, Lehmann et al., 2006). The effects of these mutations on downstream SMAD-dependent and SMAD independent signaling pathways as well as receptor endocytosis suggests that this region likely constitutes a binding site for not yet identified signaling components required for general receptor activation.

Skeletal malformation diseases have also been linked to mutations in the BMP signaling modulator Noggin, which directly binds to various BMP as well as GDF ligands and, when harboring mutations interfering with ligand binding, can cause skeletal malformations of the brachydactyly type. Noggin initially identified as a dorsalizing factor expressed in the Spemann organizer (Smith & Harlan d, 1992) was found to be an efficient BMP antagonist, which - by binding to the BMP ligands in the extracellular space with extremely high affinity in the picomolar range - can completely abrogate receptor binding and thus BMP signaling (Holley et al., 1996, Zimmerman et al., 1996). Despite its role in establishing a long-range BMP-4 morphogen gradient for dorsal-ventral patterning during gastrulation, Noggin also has functions later in development of the embryo (for a recent review see (Krause et al., 2011)). Noggin knockout mice are embryonically lethal and show a complex phenotype (McMahon et al., 1998), however it is important to note that mice being heterozygous for the Noggin null mutation develop normally (Brunet et al., 1998). This suggests that the defects seen upon Noggin deletion do not result from gene dosage effects. Due to its expression in the ectoderm, loss of Noggin resulted in a severe neural tube phenotype with a failure of neural tube closure and a dramatic reduction in the amount of posterior neural tissue. As Noggin seems essential for ventral cell fates in the CNS development, motor neurons and ventral interneurons were lacking (McMahon et al., 1998). Besides the neural abnormalities Noggin knockout mice showed also a drastically altered skeletal development (Brunet et al., 1998, Tylzanowski et al., 2006). All skeletal elements are affected with the severity of the axial defects increasing towards the posterior direction. However, analysis for ossification shows that the time point for ossification in these elements seems unchanged. These observations suggest that the loss of Noggin in the knockout mice affects cartilage development. The ablation of Noggin also affects limb development, with null mice having shorter limbs and fusions of various joints. By the use of a heterozygous transgene, where the Noggin gene has been replaced by lacZ, expression of Noggin in the developing limb could be analyzed in detail (Brunet et al., 1998), showing that Noggin is strongly expressed in cartilage zones later forming bone, but is expressed at low levels or is absent in hypertrophic cartilage or joint cavities where GDF-5 expression is usually high. Analysis of the NOG-/- mice shows a massive overgrowth of cartilage in the limb, indicating that in wildtype mice Noggin represses the growth of these tissues in a negative feedback loop manner. It is known that in addition to GDF-5 a number of other BMPs, e.g. BMP-2, BMP-4, BMP-6 and BMP-7 are expressed in the limb and even the developing joints (Lyons et al., 1989, Brunet et al., 1998). Differential signaling of these different BMPs is required to induce apoptosis in interdigital tissues (Macias et al., 1997) and in Drosophila sharp zones of activity of the fly BMP-homolog DPP, which do not necessarily correlate with the local DPP concentration, trigger local cell death to define joints (Manjon et al., 2007). The locally highly variable expression of Noggin in the developing limb could provide for such a BMP activity modulating mechanism as in vivo Noggin inhibition of BMP signaling has distinct BMP specificity profiles (Zimmerman et al., 1996, Seemann et al., 2009, Song et al., 2010). The important regulatory role of Noggin as an BMP antagonist is also highlighted by the fact that the Noggin gene is a mutational hotspot in several skeletal malformation diseases of the brachydactyly type BDB as well as the more severe multiple synostosis syndrome (SYNS1), proximal symphalangism (SYM1), tarsal-carpal coalition (TCC) or SABTT (stapes ankylosis with broad thumbs and toes) syndromes (for a recent review see (Potti et al., 2011)).

3.3. Noggin a BMP interacting hub during limb and joint formation

Structure analysis of the complex of BMP-7 bound to Noggin provided insights into the molecular mechanism how Noggin antagonizes BMP signaling (Groppe et al., 2002). The homodimeric Noggin embraces the BMP ligand and simultaneously blocks type I and type II receptor binding via its C-terminal four-stranded P-sheet structure resembling a fingerlike structure as found in BMPs itself and a N-terminal peptide segment called clip (see Fig. 7). Whereas the type II receptor-binding epitope of BMP-7 is blocked by the large and structured C-terminal part, type I receptor binding is only inhibited by the small clip segment (Gln28 to Asp39 of human Noggin). Very few polar interactions, mainly between the polar main chain atoms of the Noggin clip and residues from BMP-7, stabilize this interaction. In addition to the polar interactions, Pro35 of Noggin, which is found mutated in several skeletal malformation diseases (Gong et al., 1999, Dixon et al., 2001, Mangino et al., 2002, Lehmann et al., 2007, Hirshoren et al., 2008), points into a hole in the type I receptor-binding epitope of BMP-7 formed by hydrophobic residues thereby mimicking a key interaction in the BMP ligand-type I receptor interaction (Hatta et al., 2000, Kirsch et al., 2000, Kotzsch et al., 2009).

The disease-causing mutations in Noggin known today can be clustered into three regions: the mutations located in the clip (P35A/S/R, A36P, P37R, P42A/R; (Gong et al., 1999, Dixon et al., 2001, Mangino et al., 2002, Debeer et al., 2004, Lehmann et al., 2007, Hirshoren et al., 2008, Oxley et al, 2008)), the P-sheet domain (E48K, P42A;P50R, R167G, L203P, R204L, W205C, W217G, I220N, Y222D/C, and P223L; (Gong et al, 1999, Dixon et al., 2001, Takahashi et al, 2001, Kosaki et al., 2004, van den Ende et al., 2005, Weekamp et al., 2005, Dawson et al., 2006, Lehmann et al., 2007, Oxley et al., 2008, Emery et al., 2009)) or the dimerization domain (C184Y, P187S, G189C, M190V, and C232Y; (Gong et al., 1999, Takahashi et al., 2001, Lehmann et al., 2007, Oxley et al., 2008, Rudnik-Schoneborn et al., 2010)). The molecular mechanisms by which these mutations disrupt proper function of Noggin can be classified in part. Mutations of prolines or from other residues to proline, e.g. P42R, P50R, P187S, L203P, or P223L, will potentially lead to misfolding of the Noggin mutant, such that local structures cannot be maintained leading to a secondary loss of other Noggin-BMP interactions or to lower dimer stability (and hence to decreased secretion) if these exchanges occur in the dimerization domain (see Fig. 7) (e.g. P187S, (Lehmann et al., 2007)). Some mutations in Noggin involving proline residues and occurring in the clip region disrupt BMP-Noggin hydrogen bonds, e.g. A36P, P37R or introduce steric hindrance by replacing the proline residue for geometrically non-fitting amino acids, e.g. P35A, P35S, or P35R. Various amino acid exchanges observed in the P-sheet domain substituting a hydrophobic residue for a polar, e.g. I220N, or replacing a large hydrophobic amino acid in the hydrophobic core with a smaller one, e.g. W205C, W217G, Y222C, probably cause local unfolding and thus weaken the Noggin:BMP binding. The amino acid residues Glu48, Arg167 and Arg204 together form a hydrogen bond network, thus mutation of any of these

Figure 7. BMP inhibition by the modulator Noggin. A) Ribbon representation of the BMP-7:Noggin complex (PDB entry 1M4U, (Groppe et al., 2002)). The dimeric Noggin (grey and light green) consists of three domains: the clip region located at the N-terminus, the C-terminal finger or P-sheet domain and a dimerization domain. By embracing the BMP ligand through the clip region and the C-terminal finger domain Noggin effectively blocks binding of type I and type II receptors thereby antagonizing BMP signaling. Mutations in Noggin identified in skeletal malformation diseases are shown as spheres color-coded according to their location in the aforementioned domains (green: clip region; cyan: finger/@-sheet domain; magenta: dimerization domain). B) Magnification into mutationally affected interactions between residues of the Noggin clip region and BMP-7 (shown as grey van der Waals surface representation). Mutation of the indicated residues (Pro35, Ala36, Pro37, and Pro42 are shown as stick representations with C-atoms in green) likely alters the conformation of the Noggin clip or disrupts polar interactions (indicated by stippled magenta lines) between Noggin and BMPs. C) Magnification into the interface between the Noggin finger domain and BMP-7. Residues in Noggin involved in skeletal malformation diseases upon mutation are shown as sticks (C-atoms are colored in cyan). Most mutations likely affect local folding of the finger domain thereby attenuating or disrupting Noggin binding to BMPs. D) Magnification into the dimerization domain of Noggin. Residues involved in disease-causing mutations are shown as sticks with the C-atoms colored in magenta. Mutation of most of the residues displayed will likely interfere with dimerization of Noggin, e.g. mutation of either Cys184 or Cys232 will directly disrupt the intermolecular disulfide bond or possibly shuffle the disulfide bond pattern in the dimerization domain.

three residues will disrupt this network likely causing local structure changes in the P-sheet domain of Noggin. Furthermore, all three charged residues are buried upon binding to BMP ligands, thus mutations resulting in unbalanced charges will probably lead to electrostatic repulsion upon ligand binding. The mutations in Noggin's dimerization domain, e.g. C184Y, P187S, G189C, M190V, or C232W, all will very likely disturb efficient dimerization either by disrupting the intermolecular disulfide bond through the formation of non-native intramolecular disulfide pairs or through interfering with the homodimer interface (see Fig. 7D) (Marcelino et al., 2001, Lehmann et al., 2007).

Interestingly, mutations in Noggin represent a rather heterogeneous picture of skeletal malformations with different digits being affected and from a mild phenotype, e.g. BDB2 to more severe traits, e.g. SYM1 or SYNS1 (Lehmann et al., 2007, Potti et al., 2011). A direct correlation between the location of the mutation in Noggin and the severity of the malformation seems not apparent although mutations in the clip domain are diagnosed more frequently with BDB2 and mutations in the dimerization domain usually result in SYM1 or SYNS1 disease (Potti et al., 2011). From a structural point of view these possible differences might be explained due to the fact that destabilizing changes in the clip region of Noggin might affect only certain BMPs. Analysis of in vitro binding of BMP-7 to the Noggin mutant P35R showed a rather small 7-fold decrease in BMP binding affinity (Groppe et al., 2002). For BMPs that exhibit high affinities for their type I receptors, e.g. BMP-2, BMP-4 or GDF-5 the weakened binding of the clip of Noggin to these ligands might allow for a competition mechanism in which the receptor binding to a Noggin:BMP complex subsequently strips off the antagonist. For those BMPs that have low binding affinities to their type I receptors, e.g. BMP-5, BMP-6 and BMP-7 even the decreased binding of the Noggin clip to the ligand is still sufficient to block receptor binding and hence signaling of these BMPs. The mutations in the P-sheet region of Noggin, however, should affect all BMP ligands similarly and the severity of the phenotype should principally correlate with the loss of BMP binding affinity. The amino acid substitutions in the Noggin dimerization domain are expected to exhibit the strongest phenotype as these mutations strongly affect dimerization and secretion efficiency of the Noggin protein. Even if a monomeric Noggin variant protein might be secreted, its binding to BMPs as a monomer will be severely impaired due to the loss of avidity. Thus the mutations in the clip of Noggin might only affect a subset of the different BMPs present in the developing joint thereby causing a distinct phenotype, whereas the other Noggin mutations more likely resemble the phenotype of a Noggin null mutation. With respect to the direct effect of Noggin on GDF-5 it is important to note that in mice even though the strongest expression of GDF5 mRNA is found in the joint, Noggin mRNA here is absent at these late stages of joint development. Thus it is unclear at which timepoints the BMP antagonist Noggin directly modulates GDF-5 during joint formation in vivo (Brunet et al., 1998). Furthermore, it has been shown that the loss of Noggin in homozygous null mice leads to a strong downregulation of the GDF5 mRNA message (Brunet et al., 1998), which would be compatible with the observed effect in loss-of-function Noggin mutants.

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 Table 1 and Fig. 8).

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

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