**Meet the editors**

David N. Cooper is Professor of Human Molecular Genetics at Cardiff University in the UK. His research interests are largely focused upon elucidating the mechanisms of mutagenesis underlying human genetic disease, but include genotype–phenotype relationships in various inherited conditions, as well as human evolutionary and population genetics. He has published

over 350 papers in the field of human molecular genetics and curates the Human Gene Mutation Database (http://www.hgmd.org), a comprehensive database of mutations causing human inherited disease. Professor Cooper is European Editor of Human Genetics and Editor of the Genetics & Disease section of Wiley's Encyclopedia of Life Sciences.

Jian-Min Chen is Director of Research at the Etablissement Française du Sang-Bretagne, Brest, France. He obtained his M.D. in 1993 from the Academy of Military Medical Sciences, Beijing, China and his Ph.D in 2002 from the Université de Bretagne Occidentale, Brest, France. He joined the INSERM team headed by Prof. Claude Férec in 1998 and took up his present position in

2008. He is interested in the genetic basis of chronic pancreatitis and cystic fibrosis as well as the mechanisms of mutagenesis underlying human genetic disease.

Contents

**Preface IX** 

Chapter 1 **Missense Mutation in AR-CGD 1** 

M. Yavuz Köker and Hüseyin Avcilar

**Molecular Mechanisms Behind Skeletal Malformation 11**  Tina V. Hellmann, Joachim Nickel and Thomas D. Mueller

**in Correlation to Its Phenotype – VHL as a Model 75** 

**of Some Biomarkers in Colorectal Cancer 91**  Mihaela Tica, Valeria Tica, Alexandru Naumescu, Mihaela Uta, Ovidiu Vlaicu and Elena Ionica

**Non-Syndromic Congenital Heart Disease 119** 

**-HCO3 -**

George Seki, Shoko Horita, Masashi Suzuki, Osamu Yamazaki and Hideomi Yamada

 **Cotransporter NBCe1 167** 

Chapter 2 **Missense Mutations in GDF-5 Signaling:** 

Chapter 3 **Missense Mutation in the** *LDLR* **Gene: A Wide Spectrum in the Severity of Familial Hypercholesterolemia 55**  Mathilde Varret and Jean-Pierre Rabès

Chapter 4 **Missense Mutation in Cancer** 

Chapter 5 **Genotype-Phenotype Disturbances** 

Chapter 6 **Genetic Causes of Syndromic and** 

Akl C. Fahed and Georges M. Nemer

Chapter 7 **The Prototype of Hereditary Periodic Fevers: Familial Mediterranean Fever 149**  Afig Berdeli and Sinem Nalbantoglu

Chapter 8 **Pathophysiological Roles of Mutations in** 

**the Electrogenic Na<sup>+</sup>**

Suad AlFadhli

## Contents

#### **Preface XI**



## Preface

Just over 30 years ago, the first heritable human gene mutations were characterized at the DNA level: gross deletions of the human α-globin and β-globin gene clusters giving rise to α- and β-thalassaemia (1978) and a nonsense mutation in the human βglobin (*HBB*) gene causing β-thalassaemia (1979). With the number of known germline mutations in human nuclear genes either underlying or associated with inherited disease now exceeding 125,000 in ~5,000 different genes (Human Gene Mutation Database; http://www.hgmd.org; October 2012), the characterization of the spectrum of human germ-line mutations is proceeding apace. Such information is beginning to shed new light on longstanding questions such as the nature of disease predisposition, the determinants of the genotype-phenotype relationship, the molecular basis of reduced penetrance and the measurement of the human gene mutation rate, as well as posing profound questions pertaining to how we conceptualise genetic disease. Thus, from data generated by the 1000 Genomes Project, it has become clear that an average human genome typically contains ~100 loss-of-function variants, with ~20 genes being completely inactivated. In addition, even apparently healthy individuals harbour many tens or even hundreds of potentially deleterious variants in their genomes whose impact on the phenotype is usually still unknown.

Mutations are also likely to play a role in many complex genetic diseases (such as heart disease, neuropsychiatric disease or diabetes). These are conditions that do not display simple Mendelian patterns of inheritance even although genes may exert an important influence; hence close relatives of individual patients will often have an increased risk of developing the condition. These disorders are thought to be due to the combined effects of genetic variants at multiple gene loci, interacting with the environment. Complex disease has a very significant impact on human health because of the high population incidence of these conditions (unlike most Mendelian disorders which tend to be individually rare).

The advent of next-generation sequencing has also made possible the detailed characterization of whole cancer genomes, allowing for the first time a comprehensive assessment of the lexicon of somatic mutations driving tumorigenesis in a given cell or tissue. It has become clear that cancer genomes often constitute an intricate patchwork of clustered, or even overlapping, somatic lesions. Next-generation sequencing has the major advantage of being capable of simultaneously detecting genome/exome-wide,

#### XII Preface

deletions, insertions, copy number alterations and translocations as well as nucleotide substitutions (including hot-spot mutations in known cancer-related genes). Such studies are transforming our knowledge of oncogenic pathways and providing novel molecular targets of use in diagnosis, prognostic and therapeutic contexts. The ultimate goal is to provide a personalized treatment regime for both solid tumours and hematologic malignancies by tailoring health care to the individual patient using their own genetic information. Significant challenges remain to be addressed, such as the dissection of intra-tumoral DNA sequence heterogeneity and the development of powerful new bioinformatic tools with which to differentiate reliably between driver and passenger mutations.

It should be abundantly clear from the above that the study of mutation is relevant to all of us, not just the minority of individuals who may be afflicted in a very immediate way by inherited disease or cancer. In this volume, the interested reader will find 14 chapters on different aspects of mutation as it impacts human genetic disease. It is hoped that this book will not only be of practical assistance to those scientists and clinicians already working in the field but will also serve to encourage others to make their own unique contribution to understanding the nature and pathological consequences of mutation in the human genome.

**David N. Cooper**

Department of Medical Genetics, Haematology and Pathology, Cardiff University School of Medicine, Institute of Medical Genetics Building Heath Park, Cardiff, Wales

#### **Jian-Min Chen**

Directeur de Recherche, Institut National de la Santé et de la Recherche Médicale (INSERM), U613, Brest, France; Etablissement Français du Sang (EFS)- Bretagne, Brest, France

**Chapter 1** 

## **Missense Mutation in AR-CGD**

M. Yavuz Köker and Hüseyin Avcilar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/35758

#### **1. Introduction**

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by impairment of intracellular microbicidal activity of phagocytes. Mutations in one of four known nicotinamide adenine dinucleotide phosphate (NADPH) -oxidase components preclude generation of superoxide and related antimicrobial oxidants, leading to the phenotype of CGD. Defects in gp91-phox, encoded by *CYBB* gene, lead to X-linked CGD and have been reported to be responsible for approximately 65% of all CGD cases. The autosomal gene in CGD are *CYBA*, encoding p22-phox, *NCF2*, encoding p67-phox, *NCF1*, encoding p47-phox, and *NCF4*, encoding p40-phox (figure 1) (1,2). The mutation in these genes, respectively, abolishes the activity of the oxidase and leads to autosomal recessive chronic granulomatous disease (AR-CGD) which is approximately 35% of all CGD cases (table 1).

## **2. Phenotype- genotype correlation in CGD**

Identification of specific mutations in CGD patients may help to clarify some of the variability in clinical severity seen in this disorder and shows genotype-phenotype correlation. In general, X-CGD patients follow a more severe clinical course than patients with an AR-CGD and exhibit in the first years of life. AR-CGD patients follow a milder clinical course, especially p47-phox defect, and mostly seen in first and second decade of life. AR-CGD patients with missense mutations usually exhibit a mild clinical course, associated with a residual activity of p47-phox and also p22 and p67-phoxs. However, the level of superoxide generation does not always correlate with the clinical course. Some patients suffer from severe and recurrent infections despite having neutrophils with 10–20% of normal oxidase activity (1). Within our study with 40 AR-CGD families, we could not define a direct correlation between the molecular defect and the clinical course of the disease. Either truncations (nonsense and frameshift mutations) or missense mutations could have resulted in severe influence on phenotype.

© 2012 Köker and Avcilar, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Missense Mutation in AR-CGD 3

membrane and proline rich domains [3, 4]. *CYBA* gene encoding p22-phox has 19 different missense mutations in 65 mutated alleles and has more missense mutation than other NADPH oxidase subunit genes (table 2a) (figure 2). Mutations in the *CYBA* gene have been updated by [Roos et al., (2)] and are reviewed in Human Gene Mutation Database, (HGMD;

P22-phox has a key role in the interaction of NADPH-oxidase subunits and any difference in amino acid pattern of this phox protein may change the globular conformation of this protein due to the difference in the electrophoretic characteristic of new amino acid, which

**Figure 2.** Missense mutations in *CYBA* gene, encoding p22-phox; *CYBA* gene contains 6 exons. P22 phox contains trans-membrane and proline rich domains. Missense mutation points in *CYBA* gene and

Missense mutation points may have an important role in the interaction with other subunits, so the amino acid change in that regions may change the property of interactions and prevents or decreases the complex formation, so the activity of NADPH oxidase was

Total numbers of alleles which have missense mutations are 65 of 173 mutated alleles in *CYBA* gene and the percentage of missense mutations in that mutated alleles are %37,5 (table 2a) (2). Percentage of missense and all mutations of *CYBA* gene in the overall

change in encoded 195 aa. of p22-phox represented in the figure (2).

abolished.

http://www.hgmd.cf.ac.uk/ac/all.php).

prevents the complex formation with other subunits.

**Figure 1.** NADPH oxidase enzyme subunits and complex in activation phase.


**Table 1.** The molecular characteristic of NADPH Oxidase components.

#### **3.** *CYBA* **(cytochrome** *b* **alfa chain) gene**

Cytochrome *b* is comprised of a light a-chain and a heavy b-chain. This gene encodes the light, alpha subunit which has been proposed as a primary component of the microbicidal oxidase system of phagocytes. Mutations in this gene are associated with AR-CGD that is characterized by the failure of activated phagocytes to generate superoxide, which is important for the microbicidal activity of these cells. http://www.genecards.org/cgibin/carddisp.plgene=*CYBA*.

In about 5% of the CGD patients, the disease is caused by mutations in the cytochrome *b* alfa chain *(CYBA)* gene. The *CYBA* gene encoding p22-phox which contains 195 amino acid, is localized on chromosome 16q24, has a size of about 8,5 and contains six exons and transmembrane and proline rich domains [3, 4]. *CYBA* gene encoding p22-phox has 19 different missense mutations in 65 mutated alleles and has more missense mutation than other NADPH oxidase subunit genes (table 2a) (figure 2). Mutations in the *CYBA* gene have been updated by [Roos et al., (2)] and are reviewed in Human Gene Mutation Database, (HGMD; http://www.hgmd.cf.ac.uk/ac/all.php).

2 Mutations in Human Genetic Disease

**Figure 1.** NADPH oxidase enzyme subunits and complex in activation phase.

**Table 1.** The molecular characteristic of NADPH Oxidase components.

**3.** *CYBA* **(cytochrome** *b* **alfa chain) gene** 

bin/carddisp.plgene=*CYBA*.

**Components gp91 phox p22 phox p47 phox p67 phox** Gene CYBB CYBA NCF1 NCF2 Chromosome Xp21.1 16q24 7q11.23 1q25 Number of Exon 13 6 11 16 The length of bp 30kb 8.5kb 15.3kb 40kb Genotype X91, X-linked R A22, OR A47, OR A67, OR Incidence % %60 %5 %30 %5 The length of aa. chain 570 195 390 526

Localization membrane membrane cytosol cytosol

Cytochrome *b* is comprised of a light a-chain and a heavy b-chain. This gene encodes the light, alpha subunit which has been proposed as a primary component of the microbicidal oxidase system of phagocytes. Mutations in this gene are associated with AR-CGD that is characterized by the failure of activated phagocytes to generate superoxide, which is important for the microbicidal activity of these cells. http://www.genecards.org/cgi-

In about 5% of the CGD patients, the disease is caused by mutations in the cytochrome *b* alfa chain *(CYBA)* gene. The *CYBA* gene encoding p22-phox which contains 195 amino acid, is localized on chromosome 16q24, has a size of about 8,5 and contains six exons and transP22-phox has a key role in the interaction of NADPH-oxidase subunits and any difference in amino acid pattern of this phox protein may change the globular conformation of this protein due to the difference in the electrophoretic characteristic of new amino acid, which prevents the complex formation with other subunits.

**Figure 2.** Missense mutations in *CYBA* gene, encoding p22-phox; *CYBA* gene contains 6 exons. P22 phox contains trans-membrane and proline rich domains. Missense mutation points in *CYBA* gene and change in encoded 195 aa. of p22-phox represented in the figure (2).

Missense mutation points may have an important role in the interaction with other subunits, so the amino acid change in that regions may change the property of interactions and prevents or decreases the complex formation, so the activity of NADPH oxidase was abolished.

Total numbers of alleles which have missense mutations are 65 of 173 mutated alleles in *CYBA* gene and the percentage of missense mutations in that mutated alleles are %37,5 (table 2a) (2). Percentage of missense and all mutations of *CYBA* gene in the overall

mutations of AR-CGD is %6.3 and %16.8, respectively (table 3 and 4). Most prevalent missense mutations points in *CYBA* gene are c.70G>A, c.268C>T and c.354C>A which cause p.Gly24Arg, p.Arg90Trp and p.Ser118Arg in p22-phox, respectively (table 5).

Missense Mutation in AR-CGD 5

missense and all mutations (including delta-GT mutation) of *NCF1* gene in the overall mutations of AR-CGD is %0.6 and %66.4, respectively (table 3 and 4). So, this high percentage due to the high number of delta-GT mutation in exon 2 of *NCF1* gene and is more than all the mutations in AR-CGD (table 5). This deletion points is hot-spot mutation

The neutrophil cytosolic factor 2 (*NCF2)* gene encoding p67-phox is localized on chromosome 1q25, has a size of about 40 kb and contains 16 exons and TRP1-4, AD, SH3a, PB1 and SH3b domains (figure 4) (7, 8). *NCF2* gene, encoding p67-phox, has 41 different missense mutations in 171 mutated alleles (table 2c) (2, 4, 5, 6). Mutations in the *NCF2* gene have been published by [Roos et al., (2)] and are reviewed in Human Gene Mutation Database, (HGMD; http://www.hgmd.cf.ac.uk/ac/all.php). P67-phox has a major role in the interaction of NADPH-oxidase subunits in cytoplasm and any difference in amino acid pattern of this phox protein may prevent the complex formation with other subunits

**Figure 4.** Missense mutations in NCF2 gene, encoding p67-phox; *NCF2* gene contains 16 exons. P67 phox contains TRP1-4, AD, SH3a, PB1 and SH3b domains. Mutation points in *NCF2* gene and change in

Total numbers of mutated alleles leading AR-CGD in *NCF2* gene are 171 and 41 of them are missense and percentage of missense mutations in that mutated alleles are %24 (table 2c) (2). Percentage of missense and all mutations of *NCF2* gene in the overall mutations of AR-CGD is %4 and %16.6, respectively (table 3 and 4). Most prevalent missense mutations points in

*NCF2* gene is c.279C>G which causes p.Asp93Glu in p67-phox (table 5).

encoded 526 aa. of p67-phox represented in the figure (2).

region for *NCF1* gene.

(figure 1).

**5.** *NCF2* **(Neutrophil Cytosolic Factor 2) gene** 

## **4.** *NCF1* **(Neutrophil Cytosolic Factor 1) gene**

In about 25% of the CGD patients, the disease is caused by mutations in the neutrophil cytosolic factor 1 (*NCF1*) gene on chromosome 7q11.23, which encodes p47phox, one of the structural components of the NADPH oxidase and has a size of about 40 kb and contains 11 exons (5, 6). The protein encoded by this gene is a 47 kDa cytosolic subunit of neutrophil NADPH oxidase and is required for activation of the latent NADPH oxidase and contains 390 amino acids and PX, SH3a, SH3b and polybasic domains (figure 3).

**Figure 3.** Missense mutations in *NCF1* gene, encoding p47-phox; *NCF1* gene contains 11 exons. p47 phox contains PX, SH3a, SH3b and polybasic domains. Mutation points in *NCF1* gene and change in encoded 390 aa. of p47-phox represented in the figure (2).

 A very common mutation found in these patients is a GT deletion in a GTGTrepeat sequence at the beginning of exon 2 of NCF1 (c.75\_76delGT) gene (5, 7). *NCF1* gene encoding p47-phox has only 4 different missense mutation in 6 alleles (table 2b) (figure 3) (2). P47-phox has an important role in the interaction of cytoplasmic NADPH-oxidase subunits and any difference in amino acid pattern of this phox protein may abolish the complex formation with other subunits.

Total numbers of alleles which have missense mutations are 6 of 63 (other than delta-GT mutation in exon 2, in more than 620 alleles) mutated alleles in *NCF1* gene and the percentage of missense mutations in that mutated alleles are %9,5. The percentage of missense and all mutations (including delta-GT mutation) of *NCF1* gene in the overall mutations of AR-CGD is %0.6 and %66.4, respectively (table 3 and 4). So, this high percentage due to the high number of delta-GT mutation in exon 2 of *NCF1* gene and is more than all the mutations in AR-CGD (table 5). This deletion points is hot-spot mutation region for *NCF1* gene.

#### **5.** *NCF2* **(Neutrophil Cytosolic Factor 2) gene**

4 Mutations in Human Genetic Disease

mutations of AR-CGD is %6.3 and %16.8, respectively (table 3 and 4). Most prevalent missense mutations points in *CYBA* gene are c.70G>A, c.268C>T and c.354C>A which cause

In about 25% of the CGD patients, the disease is caused by mutations in the neutrophil cytosolic factor 1 (*NCF1*) gene on chromosome 7q11.23, which encodes p47phox, one of the structural components of the NADPH oxidase and has a size of about 40 kb and contains 11 exons (5, 6). The protein encoded by this gene is a 47 kDa cytosolic subunit of neutrophil NADPH oxidase and is required for activation of the latent NADPH oxidase and contains

**Figure 3.** Missense mutations in *NCF1* gene, encoding p47-phox; *NCF1* gene contains 11 exons. p47 phox contains PX, SH3a, SH3b and polybasic domains. Mutation points in *NCF1* gene and change in

 A very common mutation found in these patients is a GT deletion in a GTGTrepeat sequence at the beginning of exon 2 of NCF1 (c.75\_76delGT) gene (5, 7). *NCF1* gene encoding p47-phox has only 4 different missense mutation in 6 alleles (table 2b) (figure 3) (2). P47-phox has an important role in the interaction of cytoplasmic NADPH-oxidase subunits and any difference in amino acid pattern of this phox protein may abolish the

Total numbers of alleles which have missense mutations are 6 of 63 (other than delta-GT mutation in exon 2, in more than 620 alleles) mutated alleles in *NCF1* gene and the percentage of missense mutations in that mutated alleles are %9,5. The percentage of

p.Gly24Arg, p.Arg90Trp and p.Ser118Arg in p22-phox, respectively (table 5).

390 amino acids and PX, SH3a, SH3b and polybasic domains (figure 3).

**4.** *NCF1* **(Neutrophil Cytosolic Factor 1) gene** 

encoded 390 aa. of p47-phox represented in the figure (2).

complex formation with other subunits.

The neutrophil cytosolic factor 2 (*NCF2)* gene encoding p67-phox is localized on chromosome 1q25, has a size of about 40 kb and contains 16 exons and TRP1-4, AD, SH3a, PB1 and SH3b domains (figure 4) (7, 8). *NCF2* gene, encoding p67-phox, has 41 different missense mutations in 171 mutated alleles (table 2c) (2, 4, 5, 6). Mutations in the *NCF2* gene have been published by [Roos et al., (2)] and are reviewed in Human Gene Mutation Database, (HGMD; http://www.hgmd.cf.ac.uk/ac/all.php). P67-phox has a major role in the interaction of NADPH-oxidase subunits in cytoplasm and any difference in amino acid pattern of this phox protein may prevent the complex formation with other subunits (figure 1).

Total numbers of mutated alleles leading AR-CGD in *NCF2* gene are 171 and 41 of them are missense and percentage of missense mutations in that mutated alleles are %24 (table 2c) (2). Percentage of missense and all mutations of *NCF2* gene in the overall mutations of AR-CGD is %4 and %16.6, respectively (table 3 and 4). Most prevalent missense mutations points in *NCF2* gene is c.279C>G which causes p.Asp93Glu in p67-phox (table 5).


Missense Mutation in AR-CGD 7

**In the all mutations of AR-CGD** 

**Different missense / different all mutations in that gene** 

**Total mutations %**

**%**

c.305G>C p.Arg102Pro R102P 1(1) c.323A>T p.Asp108Val D108V 1(2) c.383C>T p.Ala128Val **A128V** 1(2) c.409T>A p.Trp137Arg W137R 1(2) c.419C>G p.Ala140Asp A140D 1(1) c.[479A>T; 481A>G] p.AspLys160\_161ValGlu **DK160\_161VE** 1(1) c.505C>G p.Gln169Glu **Q169E** 1(2) c.551G>C p.Arg184Pro **R184P** 1(2) c.605C>T p.Ala202Val **A202V** 2(4) c.1256A>T p.Asn419Ile **N419I** 1(2) 17 different alleles 41 alleles

(c)

**Alleles with mutations&**

**# % # % Missense** 

*CYBA* **65 37,5 173 100** 6.3 **16.8** *NCF1* **6 9,5\* 63 +620\* 100** 0.6 **66.4** *NCF2* **41 24 171 100** 4 **16.6** *NCF4* **1 50 2 100** 0.1 **0.2** In AR-CGD **113 27.6 409+620\* 100** 11 **100**

**Table 3.** Distribution of number and percentage of missense and all mutations in genes (*CYBA, NCF1,* 

*CYBA* **19 55 %34.6** *NCF1* **4 23 %17.4** *NCF2* **17 54 %31.5** *NCF4* **1 2 %50** In AR-CGD **41 134 %30**

**Table 4.** Number and percentage of different missense mutations in genes (*CYBA, NCF1, NCF2* and

**Total # of different mutations&**

**Table 2.** (a) Missense Mutation in *CYBA* gene. (b) Missense Mutation in *NCF1* gene. (c) Missense

**Alleles with missense mutations** 

Mutation in *NCF2* gene.

\*: (delta-GT mutations in exon 2, not included)

*NCF2* and *NCF4*) of AR-CGD.

**Autosomal Gene** 

*NCF4*) of AR-CGD.

**&:** Including nonsense, missense, splice site, deletion and others.

**# of different missense mutations**

**Autosomal Gene** 

#### (a)


(b)



(c)

**Table 2.** (a) Missense Mutation in *CYBA* gene. (b) Missense Mutation in *NCF1* gene. (c) Missense Mutation in *NCF2* gene.


\*: (delta-GT mutations in exon 2, not included)

6 Mutations in Human Genetic Disease

**Nucleotide change Amino acid change Amino acid # of families (alleles)** c.2T>A p.Met1Lys **M1K** 1(2) c.70G>A p.Gly24Arg **G24R 9(14)** c.71G>A p.Gly24Glu **G24E** 1(2) c.74G>T p.Gly25Val **G25V** 1(1) c.152T>A p.Leu51Gln **L51Q** 1(1) c.155T>C p.Leu52Pro L52P 1(2) c.158A>T p.Glu53Val E53V 1(1) c.164C>G p.Pro55Arg Q55R 1(2) c.268C>T p.Arg90Trp R90W **8(14)**  c.268C>G p.Arg90Gly **R90G** 1(2) c.269G>A p.Arg90Gln R90Q 2(3) c.269G>C p.Arg90Pro R90P 1(2) c.281A>G p.His94Arg **H94R** 1(2) c.354C>A p.Ser118Arg **S118R 4(8)**  c.370G>T p.Ala124Ser **A124S** 1(2) c.371C>T p.Ala124Val **A124V** 1(1) c.373G>A p.Ala125Thr **A125T** 1(2) c.385G>A p.Glu129Lys **E129K** 1(2) c.467C>A p.Pro156Gln **P156Q** 1(2) 19 different alleles 65 alleles

(a)

**Nucleotide change Amino acid change Amino acid # of families (alleles)** c.125G>A p.Arg42Gln **R42Q 3(3)**  c.730G>A p.Glu244Lys **E244K** 1(1) c.784G>A p.Gly262Ser **G262S** 1(1) c.789G>C p.Trp263Cys **W263C** 1(1) 4 different alleles 6 alleles

(b)

**Nucleotide change Amino acid change Amino acid # of families (alleles)** c.1A>G p.Met1Val **M1V** 1(1) c.125A>G p.Asn42Ser **N42S** 1(2) c.130G>C p.Gly44Arg **G44R** 2(4) c.130G>T p.Gly44Cys **G44C** 1(2) c.230G>A p.Arg77Gln **R77Q** 3(3) c.233G>A p.Gly78Glu G78E 1(2) c.279C>G p.Asp93Glu D93E **4(8)** 

**&:** Including nonsense, missense, splice site, deletion and others.

**Table 3.** Distribution of number and percentage of missense and all mutations in genes (*CYBA, NCF1, NCF2* and *NCF4*) of AR-CGD.


**Table 4.** Number and percentage of different missense mutations in genes (*CYBA, NCF1, NCF2* and *NCF4*) of AR-CGD.


Missense Mutation in AR-CGD 9

**Author details** 

M. Yavuz Köker

Hüseyin Avcilar

**8. References** 

**Acknowledgement** 

1990;86:1729–37.

(2006).

1681. (1996)

*Kökbiotek Company, Kayseri, Turkey* 

*Oxford University Press*, 2007; 37: 525–49.

*Cells, Molecules, and Diseases*, 44: 291-299, (2010).

Disease. *Eur J of Clin Invest,* 39(4): 311-319*,* (2009).

(NCF1). *Am J Hum Genet* 1990;47:483–92.

*Erciyes BM Transplant Centre, Division of Immunology, University of Erciyes, Kayseri, Turkey* 

Roos D, Kuijpers TW, Curnutte *JT.* Chronic granulomatous disease. In: Primary Immunodeficiencies, 2nd edition (Editors: Ochs HD., Smith CIE., Puck JM.), New York:

Dirk Roos, Doug Kuhns, Anne Maddalena, Jacinta Bustamante, Caroline Kannengiesser, Martin de Boer, Karin van Leeuwen, M. Yavuz Köker, Baruch Wolach, Joachim Roesler, John I. Gallin and Marie-José Stasia. Hematologically Important Mutations: The Autosomal Recessive Forms of Chronic Granulomatous Disease (Second Update). *Blood* 

Dinauer MC, Pierce EA, Bruns GAP, Curnutte JT, Orkin SH. Human neutrophil cytochrome b light chain (p22phox): gene structure, chromosomal localization and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. *J Clin Invest* 

M.Y. Köker, K. van Leeuwen, M. de Boer, F. Çelmeli, A. Metin, T.T. Özgür, İ. Tezcan, Ö. Sanal, D. Roos. Six Different *CYBA* Mutations Including Three Novel Mutations in Ten Families from Turkey, Resulting in Autosomal Recessive Chronic Granulomatous

Roos D, de Boer M, Koker MY, Dekker J, Singh-Gupta V, Ahlin A, Palmblad J, Sanal O, Kurenko-Deptuch M, Jolles S, Wolach B. Chronic granulomatous disease caused by mutations other than the common GT deletion in *NCF1*, the gene encoding the p47 (phox) component of the phagocyte NADPH oxidase. *Hum Mutat,* 27(12):1218-1229,

Roos D, de Boer M, Kuribayashi F, Meischl C, Weening RS, Segal AW, Ahlin A, Nemet K, Hossle JP, Bernatowska-Matuszkiewicz E, Middleton- Price H. Mutations in the Xlinked and autosomal recessive forms of chronic granulomatous disease. *Blood* 87:1663–

Franke U, Hsieh CL, Foellmer BE, Lomax KJ, Malech HL, Leto TL. Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23

This study is kindly supported by TÜBİTAK with project number 110S252.

**Table 5.** Most prevalent missense mutation in the genes of AR-CGD.

## **6.** *NCF4* **(Neutrophil Cytosolic Factor 2) gene**

*NCF4* gene encoding p40-phox with 339 amino acids is localized on chromosome 22q13.1 has a size of about 4,4 kb and contains 10 exons. P40-phox interacts primarily with p67 phox. Up to know, the first mutation in *NCF4* gene was founded in a family with compound heterozygote mutations and one of the mutations was a missense with c.314G>A in one allele, which causes change in p.Arg105Gln amino acid in the structure of p40-phox (2, 9).

## **7. Conclusion**

19 different alleles in *CYBA* gene, 4 different alleles in *NCF1* gene, 17 different alleles in NCF2 gene and one allele in *NCF4* gene have missense mutations which cause change in amino acid patterns of NADPH oxidase subunits and results in AR-CGD. The percentage of missense mutations in the overall mutations of AR-CGD is %11 (table 3). One of the most prevalent missense mutations in AR-CGD is in *CYBA* gene with c.70G>A, in 14 alleles of 9 families, which causes p.Gly24Arg in p22-phox (table 5).

In p22-phox the first interaction with p67-phox occur in B part (domain) which is located between 81-91 amino acids in p22-phox. There are 4 different missense mutations (in 21 alleles of *CYBA* gene) change amino acid (arginine) at position 90. So, this position is highly susceptible to any conformational changes which may prevent the interaction with p67 phox. So, the change in the molecular structure of this part may abolish the stability and function of p22-phox and latent NADPH oxidase could not be activated leading to AR-CGD. P22-phox has more different missense mutation than other NADPH oxidase components. The ratio of the number of different missense mutation and the number of amino acid in the chain is approximately 19/195 (%9.74). The different missense mutation to overall amino acid chain length in p67-phox is 17/526 (%3.23). But, the ratio in p47-phox is 4/390 (%1). This result shows that p67-phox has 3 times and p22-phox has approximately 10 times high incidence of different missense mutations than p47-phox in their primary amino acid structure. The underlying reason for this may be the highly specific interaction and function of p22-phox which is vulnerable to any change in the globular structure of protein.

#### **Author details**

8 Mutations in Human Genetic Disease

**7. Conclusion** 

**Autosomal Gene Nucleotide change Aa change Number of family (alleles)** *CYBA* c.70G>A p.Gly24Arg 9(14) *"* c.268C>T p.Arg90Trp 8(14) *"* c.354C>A p.Ser118Arg 4(8) *NCF1* c.125G>A p.Arg42Gln **3(3)** *NCF2* c.279C>G p.Asp93Glu 4(8)

*NCF4* gene encoding p40-phox with 339 amino acids is localized on chromosome 22q13.1 has a size of about 4,4 kb and contains 10 exons. P40-phox interacts primarily with p67 phox. Up to know, the first mutation in *NCF4* gene was founded in a family with compound heterozygote mutations and one of the mutations was a missense with c.314G>A in one allele, which causes change in p.Arg105Gln amino acid in the structure of p40-phox (2, 9).

19 different alleles in *CYBA* gene, 4 different alleles in *NCF1* gene, 17 different alleles in NCF2 gene and one allele in *NCF4* gene have missense mutations which cause change in amino acid patterns of NADPH oxidase subunits and results in AR-CGD. The percentage of missense mutations in the overall mutations of AR-CGD is %11 (table 3). One of the most prevalent missense mutations in AR-CGD is in *CYBA* gene with c.70G>A, in 14 alleles of 9

In p22-phox the first interaction with p67-phox occur in B part (domain) which is located between 81-91 amino acids in p22-phox. There are 4 different missense mutations (in 21 alleles of *CYBA* gene) change amino acid (arginine) at position 90. So, this position is highly susceptible to any conformational changes which may prevent the interaction with p67 phox. So, the change in the molecular structure of this part may abolish the stability and function of p22-phox and latent NADPH oxidase could not be activated leading to AR-CGD. P22-phox has more different missense mutation than other NADPH oxidase components. The ratio of the number of different missense mutation and the number of amino acid in the chain is approximately 19/195 (%9.74). The different missense mutation to overall amino acid chain length in p67-phox is 17/526 (%3.23). But, the ratio in p47-phox is 4/390 (%1). This result shows that p67-phox has 3 times and p22-phox has approximately 10 times high incidence of different missense mutations than p47-phox in their primary amino acid structure. The underlying reason for this may be the highly specific interaction and function

of p22-phox which is vulnerable to any change in the globular structure of protein.

**Table 5.** Most prevalent missense mutation in the genes of AR-CGD.

**6.** *NCF4* **(Neutrophil Cytosolic Factor 2) gene** 

families, which causes p.Gly24Arg in p22-phox (table 5).

M. Yavuz Köker *Erciyes BM Transplant Centre, Division of Immunology, University of Erciyes, Kayseri, Turkey* 

Hüseyin Avcilar *Kökbiotek Company, Kayseri, Turkey* 

### **Acknowledgement**

This study is kindly supported by TÜBİTAK with project number 110S252.

#### **8. References**


M.Y. Köker, Ö. Sanal, K. van Leeuwen, M. de Boer, A. Metin, Türkan Patroğlu, T.T. Özgür, İ. Tezcan, , D. Roos. Four Different *NCF2* Mutations in Six Families from Turkey and an Overview of *NCF2* Gene Mutations. *Eur J of Clin Invest,* 39(10): 942-951, (2009).

**Chapter 2** 

© 2012 Mueller et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Missense Mutations in GDF-5 Signaling:** 

**Molecular Mechanisms Behind** 

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

Members of the large transforming growth factor β (TGF-β) 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 ligandreceptor 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-β 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-β member, but we usually see that even a particular TGF-β 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-β members (Hoodless *et al.*, 1996, Nakao *et al.*, 1997). After liganddependent 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

**Skeletal Malformation** 

http://dx.doi.org/10.5772/35195

**1. Introduction** 

Additional information is available at the end of the chapter

J.D. Matute, A.A. Arias, N.A.M. Wright, I. Wrobel, C.C.M. Waterhouse, X.J. Li, C.C. Marchal, N.D. Stull, D.B. Lewis, M. Steele, J.D. Kellner, W. Yu, S.O. Meroueh, W.M. Nauseef,, M.C. Dinauer, A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity. *Blood* 114 (2009) 3309–3315.

**Chapter 2** 

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

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**

10 Mutations in Human Genetic Disease

M.Y. Köker, Ö. Sanal, K. van Leeuwen, M. de Boer, A. Metin, Türkan Patroğlu, T.T. Özgür, İ. Tezcan, , D. Roos. Four Different *NCF2* Mutations in Six Families from Turkey and an

Overview of *NCF2* Gene Mutations. *Eur J of Clin Invest,* 39(10): 942-951, (2009). J.D. Matute, A.A. Arias, N.A.M. Wright, I. Wrobel, C.C.M. Waterhouse, X.J. Li, C.C. Marchal, N.D. Stull, D.B. Lewis, M. Steele, J.D. Kellner, W. Yu, S.O. Meroueh, W.M. Nauseef,, M.C. Dinauer, A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil

NADPH oxidase activity. *Blood* 114 (2009) 3309–3315.

Members of the large transforming growth factor β (TGF-β) 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 ligandreceptor 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-β 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-β member, but we usually see that even a particular TGF-β 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-β members (Hoodless *et al.*, 1996, Nakao *et al.*, 1997). After liganddependent 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

© 2012 Mueller et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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-β 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 cotranscription factor (see Fig. 1) (Heldin *et al.*, 1997, Miyazono, 2000, Massague *et al.*, 2005).

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 13

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 extracelIular 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

**1.1. The multitude of biological functions of TGF-**β **members is established by a** 

Analysis of the patterning function of TGF-β 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-β 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

I-SMADs compete with activated R-SMADs for interaction with SMAD4.

**highly complex regulatory "cross-reactive" signaling network** 

signaling network with established equilibrium of cross-interacting partners.

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

**1.2. What can be learned from individual gene deletions?** 

**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 extracelIular 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.

12 Mutations in Human Genetic Disease

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-β 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 cotranscription 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

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

Analysis of the patterning function of TGF-β 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-β 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-β ligands, receptors, modulator proteins, etc.) and the loss of individual or combinations of genes of the TGF-β signaling network were analyzed in detail in hetero- as well as in homozygous situations (Zhao, 2003). Surprisingly, given the importance of TGF-β 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.

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 15

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

**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

B) Representation of the bony elements of the human autopod subdivided into the bones of the wrist

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, Francis-West *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

zeugopod. Most distally, the autopod forms the bones of the hand.

(carpals), palm (metacarpals) and digits (phalanges).

defined limb elements.

#### **2. The role of GDFs in limb development**

Astonishingly, within the complex machinery of TGF-β 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-β 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.

14 Mutations in Human Genetic Disease

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-β ligands, receptors, modulator proteins, etc.) and the loss of individual or combinations of genes of the TGF-β signaling network were analyzed in detail in hetero- as well as in homozygous situations (Zhao, 2003). Surprisingly, given the importance of TGF-β 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.

Astonishingly, within the complex machinery of TGF-β 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-β 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*

**2. The role of GDFs in limb development** 

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, Francis-West *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).

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 17

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 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.

Kingsley, 1996).

limb.

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 &

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

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* 

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

expression can be found in regions of the future interphalangeal joints.

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

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 19

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*-/- 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

For GDF-7 function the effects in *GDF7*-/- 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,

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

of specific joints (Settle *et al.*, 2003).

contribute to joint formation *in vivo*.

**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/β-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).

18 Mutations in Human Genetic Disease

**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

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/β-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

and the dorsal and ventral ectoderm establishing a dorso-ventral signal, thereby generating a

halted (Settle *et al.*, 2003). In contrast to *GDF5*-/- mice, which had fusions restricted to synovial joint, *GDF6*-/- 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*-/- 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.

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 21

**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

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-β 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).

disease family″, Copyright 2011, with permission from John Wiley and Sons.

### **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-β 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-β/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

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 21

required but not sufficient for joint formation.

**3. Disorders in limb development** 

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

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-β 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-β/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-β 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-β/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-β/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.

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 23

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-β 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

**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 10 fold 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

behind these disease-causing mutations.

#### **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-β 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**

22 Mutations in Human Genetic Disease

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-β/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-β/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 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 10 fold 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)).

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 25

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

BMP receptor BMPR-IB (PDB entry 3MDY, (Chaikuad *et al.*, 2010a)), of the TGF-β receptor TGFβR-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 α-helices. Functional analysis of the TGFβR-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 TGFβR-I (equivalent to Thr199 in BMPR-IB) adjacent to this consensus motif is absolutely conserved between TGF-β 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

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-β type I receptors but placed quite distantly from the regulatory important regions such as the GSbox 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-β receptors TGFβR-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 surfaceaccessible 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

proteins involved in the receptor activation.

carrying the I200K mutation (Lehmann *et al.*, 2003).

general receptor activation.

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/serinerich) 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

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.

24 Mutations in Human Genetic Disease

Lyons, 2006)).

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 &

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/serinerich) 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

BMP receptor BMPR-IB (PDB entry 3MDY, (Chaikuad *et al.*, 2010a)), of the TGF-β receptor TGFβR-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 α-helices. Functional analysis of the TGFβR-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 TGFβR-I (equivalent to Thr199 in BMPR-IB) adjacent to this consensus motif is absolutely conserved between TGF-β 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-β type I receptors but placed quite distantly from the regulatory important regions such as the GSbox 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-β receptors TGFβR-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 surfaceaccessible 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 & Harland, 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), Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 27

proximal symphalangism (SYM1), tarsal-carpal coalition (TCC) or SABTT (stapes ankylosis

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 β-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 receptorbinding 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,

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 β-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 β-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

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** 

Kotzsch *et al.*, 2009).

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**

26 Mutations in Human Genetic Disease

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 & Harland, 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),

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 β-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 receptorbinding 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 β-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 β-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

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 29

three residues will disrupt this network likely causing local structure changes in the β-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.

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 β-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

7D) (Marcelino *et al.*, 2001, Lehmann *et al.*, 2007).

loss-of-function Noggin mutants.

**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 βsheet 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 β-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).

28 Mutations in Human Genetic Disease

**Figure 7.** BMP inhibition by the modulator Noggin. A) Ribbon representation of the

disulfide bond pattern in the dimerization domain.

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 βsheet 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

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 β-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).

Missense Mutations in GDF-5 Signaling: Molecular Mechanisms Behind Skeletal Malformation 31

susceptibility

susceptibility

susceptibility

Grebe type

Grebe type

Grebe type

Acromesomelic dysplasia, DuPan syndrome

heterozygous Brachydactyly type A2 # 112600 (Ploger *et al.*,

heterozygous Brachydactyly type C # 113100 (Thomas *et* 

**disease OMIM # reference** 
