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Hum Genet (1998) 102 : 145–150

© Springer-Verlag 1998


Daniela Steinberger · Gert Vriend · John B. Mulliken · Ulrich Müller

The mutations in FGFR2-associated craniosynostoses are clustered in five structural elements of immunoglobulin-like domain III of the receptor Received: 30 June 1997 / Accepted: 31 October 1997

Abstract Exons 5 and 7 of the fibroblast growth factor receptor 2 (FGFR2) gene code for immunoglobulin-like domain III (IgIII) and for the region connecting the second and the third Ig domain of the receptor. Numerous mutations in these two exons have been shown to cause various craniosynostotic syndromes. Here, we describe three previously unrecognized mutations at amino acid positions 276, 301, and 314, in one nonspecific craniosynostosis and in two Crouzon patients. We also present a polypeptide model of IgIII of FGFR2. The known mutations involve five distinct structural elements of the receptor. The changes within these elements affect receptor function by various mechanisms, including altered dimerization, truncation, increased mobility between Ig domains, disintegration of IgIII, and alteration of the ligandbinding site.

Introduction The craniosynostotic syndromes of Crouzon, Pfeiffer, Jackson-Weiss, and Apert are characterized by premature fusion of various calvarial sutures, frequently in association with maldevelopment of the limbs. Mutations in exons 5 and 7 (nomenclature of Johnson et al. 1991; exons 7 and 9 according to Givol and Yayon 1992) of fibroblast growth factor receptor 2 (FGFR2) are a common cause of these clinically defined syndromes. The two exons code

D. Steinberger · U. Müller (Y) Institut für Humangenetik, Justus-Liebig-Universität, Schlangenzahl 14, D-35392 Giessen, Germany Tel.: +49-641-99-41600, Fax: +49-641-99-41609; e-mail: [email protected] G. Vriend EMBL, Biocomputing Unit, Meyerhofstrasse 1, D-69117 Heidelberg, Germany J. B. Mulliken Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA

for the third extracellular immunoglobulin-like domain (IgIII, composed of IgIIIa and IgIIIc) and for the region connecting the second and the third domains of the receptor. A great variety of mutations has been described, but convincing genotype-phenotype correlations are not possible in Crouzon, Pfeiffer, Jackson-Weiss, and some clinically non-classifiable craniosynostotic syndromes. Identical mutations have been described in these syndromes (Jabs et al. 1994; Reardon et al. 1994; Rutland et al. 1995; Steinberger et al. 1995, 1996b; Ma et al. 1995; Park et al. 1995a; Schell et al. 1995; Oldridge et al. 1995; Meyers et al. 1996; Schwartz et al. 1996). Distinct mutations have only been detected in Apert syndrome at the two adjacent codons 252 and 253 of exon 5 of FGFR2 (Oldridge et al. 1995, 1997; Meyers et al. 1996; Wilkie et al. 1995; Park et al. 1995b; Slaney et al. 1996). The molecular consequences of most FGFR2 mutations on the structure of the receptor are not known. Although a 3-dimensional (3-D) model of IgIIIc of FGFR2 has been constructed previously (Bateman and Chothia 1995), only a few mutations were known at that time and could be mapped to this model. The functional consequence of several mutations has been shown in a Xenopus model. These mutations result in the constitutive activation of the receptor (Neilson and Friesel 1995, 1996). Given the important role of the affected domain in ligand binding (Johnson and Williams 1993), some of the mutations are thought to affect the normal interaction of the receptor with its ligand. In order to understand the sequence-structure-function relationship of the amino acids involved in the phenotypic changes, we have constructed a 3-D model. All mutations that we describe here and all previously reported mutations (Jabs et al. 1994; Reardon et al. 1994; Rutland et al. 1995; Steinberger et al. 1995, 1996a, b, 1997; Ma et al. 1995; Park et al. 1995a, b; Schell et al. 1995;Oldridge et al. 1995, 1997; Wilkie et al. 1995; Lajeunie et al. 1995; Gorry et al. 1995; Meyers et al. 1996; Schwartz et al. 1996; Slaney et al. 1996; Bellus et al. 1996; Pulleyn et al. 1996) cluster in a few well-defined topological regions of IgIII (splice variant IgIIIc).


Materials and methods Analysis of exons 5 and 7 (exons 7 and 9 according to Givol and Yayon 1992) of FGFR2 was essentially performed as described before (Oldridge et al. 1995). For the amplification of exon 5 (exon 7), we used primers formerly described by Slaney et al. (1996): 30 cycles of 94° C for 30 s, 61° C for 60 s, and 72° C for 30 s, preceded by 3 min denaturation at 94° C and followed by a final extension at 72° C for 7 min; or primers 5′ TGA CAG CCC TCT GGA CAA CAC ACC 3′ and 5′ TTT CCA CGT GCT TGA TCC 3′: 35 cycles of 94° C for 60 s, 59° C for 60 s, 72° C for 60 s, preceded by 3 min denaturation at 94° C and followed by a final extension at 72° C for 7 min. Amplification of exon 7 (exon 9) was performed exactly as described before (Steinberger et al. 1995) or with primers 5′ CCC TCC ACA ATC ATT CCT 3′ and CCC AGA GAG AAA GAA CAG TA 3′: 30 cycles of 94° C for 30 s, 63° for 60 s, and 72° C for 30 s, preceded by 3 min denaturation and followed by a final extension at 72° C for 10 min. The tertiary structure of immunoglobulin like chain III (splice variant IgIIIc) of FGFR2 was deduced based on the crystal structure of the BenceJones Ig variable domain resolved at 1.6 Å (Furey et al. 1983) and an alignment according to Bork et al. (1994). We used this alignment rather than the slightly different alignment suggested by Bateman and Chothia (1995), because the alignment with the Bence-Jones Ig variable domain gave a more complete model. Modeling was carried out with WHAT IF (Vriend 1990) and the protocol described by Chinea et al. (1995). Since all mutations are located in subunit III (splice variant IgIIIc) and the linker region of the extracellular domain of FGFR2, we only modeled this subunit. The model coordinates and all details concerning its construction are available upon request from G. V. (for further details, see or

Results and discussion Novel FGFR2 mutations in craniosynostosis We detected three point mutations in 3 patients with craniosynostosis; these mutations have not been described before. The mutations are listed in Table 1 and result in amino acid changes at positions 276, 301, and 314. The nucleotide changes at the corresponding nucleotide positions counting from ATG (see the Addendum below) are also given in Table 1. The patients in whom we detected mutations at positions 276 and 301 had typical Crouzon syndrome. The phenotype of the patient with the mutation at position 314 could not be assigned to any known craniosynostotic syndrome. Mutation 314 may be identical to the mutation G(+1)T of exon 7 described by Schell et al. (1995) as a splicing mutation. There is, however, neither experimental (Schell et al. 1995) nor observational

Table 1 Novel mutations in FGFR2 in patients with craniosynostotic syndromes (nomenclature according to Johnson et al. 1991) Nucleotide change

Effect on coding sequence



T826G A902G G940T

F276V Y301C A314S

5 5 7

Crouzon Crouzon Unknown craniosynostosis

(Krawczak et al. 1992) evidence of abnormal splicing as the result of an exchange of a G at position +1 of an exon. Therefore, we assigned the mutation the proper amino acid position and gave the amino acid change (A→S) as being caused by this mutation. Compilation of known FGFR2 mutations in craniosynostosis For FGFR2, we compiled all point mutations that have been described in the various craniosynostotic syndromes. The resulting amino acid changes and their positions are summarized in Table 2. In addition to the novel mutations described above, we also found several mutations at known positions; these are also listed in Table 2. Deduction of the three dimensional structure of IgIII In order to determine whether the FGFR2 mutations found in various craniosynostotic syndromes occur in distinct domains of IgIII (splice variant IgIIIc), we performed molecular modeling of the structure. The structure of Ig-like domain III of FGFR2 was deduced from the known crystal structure of the Bence-Jones protein lambda variable domain (Furey et al. 1983). Bork et al. (1994) have carried out an extensive analysis of the immunoglobulin fold. According to their investigations, the FGFR2 sequence belongs to what they call the “v-type” family, for which the Bence-Jones protein lambda variable domain (PDB entry: 2RHE) is the most appropriate structure to serve as a template. The percentage sequence identity between FGFR2 and 2RHE is low for homology modeling. However, the typical sequence pattern (Bork et al. 1994) for the v-type family is clearly present in FGFR2, including all key residues. These residues are the central two cysteines at positions 278 and 342, and the highly conserved tryptophan at 290. Therefore, the IgIII (splice variant IgIIIc) domain of FGFR2 could be modeled with sufficient reliability to draw qualitative conclusions regarding the relative locations of individual mutated residues. Some of the prominent features of the FGFR2 model are: a large surface-accessible polar pocket near the site anchored in the membrane; a large protrusion; a protruding tip in the putative direction of subunit II; and a long and deep cleft orthogonal to the direction of the strands, entirely across two sides of the molecule. These structural characteristics are schematically depicted in Figs. 1, 2a, b). Mapping of mutations onto the 3-D model of IgIII We mapped the positions where mutations are known to cause craniosynostoses (Table 2) onto this model. The mutations in FGFR2 were found not to be distributed randomly within the 3-D structure of IgIII (exon 5 and 7 mutations), but to occur at positions that appear to affect distinct structural elements.

147 Table 2 Compilation of FGFR2 mutations found in craniosynostosis (AS Apert syndrome, CS Crouzon syndrome, PS Pfeiffer syndrome, JWS Jackson-Weiss syndrome, UCS unspecified craniosynostosis) Mutation


Localisation (structural element)





S252F S252L S252/P253 252F/253S P253R



Meyers et al. (1996), Wilkie et al. (1995), Park et al. (1995b), Slaney et al. (1996), this study Oldridge et al. (1997) Oldridge et al. (1997) Oldridge et al. (1997)



S267P F276V C278F Q289P W290G/R/C K292E Y301C A314S D321A Y328C N331I G338E/R Y340H T341P C342R/S/Y/W/F


B D D C D A C, A C A B, A B A, B A C D

A344G A344P A344A S347C S351C S345C


E, D E, D E, D E C, E C




Meyers et al. (1996), Wilkie et al. (1995), Park et al. (1995b), Slaney et al. (1996), this study Oldridge et al. (1995) This study Oldridge et al. (1995), Meyers et al (1996), this study Oldridge et al. (1995) Meyers et al. (1996), Gorry et al. (1995) Park et al. (1995a), Oldridge et al. (1995), this study Steinberger et al. (1997) This study This study Lajeunie et al. (1995) Jabs et al. (1994) Steinberger et al. (1996a) Pulleyn et al. (1996) Jabs et al. (1994), Reardon et al. (1994), this study Rutland et al. (1995) Reardon et al. (1994), Rutland et al. (1995), Steinberger et al. (1995), Ma et al. (1995), Park et al. (1995a), Schell et al. (1995), Oldridge et al. (1995), Meyers et al. (1996), Schwartz et al. (1996), this study Jabs et al. (1994), Gorry et al. (1995), Park et al. (1995a) Meyers et al. (1996) Jabs et al. (1994), Reardon et al. (1994), Steinberger et al. (1996b) Jabs et al. (1994), Oldridge et al. (1995), this study Pulleyn et al. (1996) Reardon et al (1994), Park et al. (1995a), Oldridge et al. (1995), Gorry et al. (1995), this study Meyers et al. (1996)

Group A mutations affect the large surface-accessible polar pocket of IgIII (Figs. 1, 2b). The observed point mutations are located at positions 292, 321, 336, 337, 338, and 340. The polar pocket faces the membrane. Heparin in solution or heparin-like oligosaccharides on the cell surface (heparan sulfate proteoglycans, HSPGs) are prerequisites for dimerization of the receptor molecules (Yayon et al. 1991; Spivak-Kroizman et al. 1994). The membrane-faced polar pocket is a possible heparan-sulfate-binding site, and mutations are therefore likely to affect the dimerization process and, as a consequence, to alter normal ligand-receptor interactions. Group B (Figs. 1, 2b) includes mutations of amino acid residues that are in close proximity to the cell surface. They are at positions 267, 328, 331, and 359 and might disturb normal interactions of IgIII with membrane components, such as HSPGs. Group C (Figs. 1, 2b) mutations involve the cleft, orthogonal to the direction of the strands. This putative binding cleft could be required for receptor-ligand inter-

actions. Point mutations at positions 289, 301, 314, 341, 343, 351, and 354 potentially interfere with the putative binding function of this domain. Group D (Figs. 1, 2b) mutations involve the core of IgIII and include the central tryptophan (position 290), the phenylalanine at position 276, or either one of the two cysteines at positions 278 and 342. Mutations at these central positions of the domain may cause its disintegration. The likely result is a truncated receptor with the extracellular elements missing, but the intracellular domains being intact. This is compatible with experimental findings in Xenopus oocytes carrying the 342 mutation where constitutive stimulation of the tyrosine kinase of the receptor has been found (Neilson and Friesel 1995). Alternatively, replacement of phenylalanine 276, which is located just behind and above the polar pocket, might alter the conformation of this polar pocket. Finally, group E (Figs. 1, 2b) includes mutations of amino acids located at the tip (344, 347) or at the linker (252, 253) region between the 2nd and 3rd domain. They


Fig. 1 Artist’s rendition of IgIII of FGFR2 to emphasize the relative positions of the domains discussed in the text. Of the five domains described, only A, C, and E are visible. B points toward the membrane and D involves core residues

alter the IgII-IgIII contact. Three different mutations have been described at position 344, viz., the replacement of arginine by either glycine or proline and a deletion of 17 amino acids resulting from abnormal splicing (Del Gatto and Breathnach 1995). The single amino acid changes affect the shape of the tip, and the deletion includes the entire tip. The mutations might result in increased mobility between IgIII and IgII and thus may alter ligand binding. A similar effect is expected of the mutation at 347. Mutations at 253 (Pro→Arg) and 252 (Ser→Trp and Ser→Phe) result in Apert syndrome (Oldridge et al. 1995, 1997; Wilkie et al. 1995; Park et al. 1995b; Slaney et al. 1996), the most severe form of the FGFR2-associated craniosynostoses. Two different mutations that involve codon 252 (Ser252Leu, Ser252 and Pro253→Phe252 and Ser253) result in a milder phenotype different from Apert syndrome (Oldridge et al. 1997). All these mutations at codons 252 and 253 are likely to disturb the IgII/IgIII interface. A proline to arginine mutation can render the linker more flexible or more prone to proteolyis, resulting in faster degradation of the external components of the receptor. In our model the tip is located in close proximity to the cleft, and some residues in the core are also near the polar pocket. Although our interpretation of the location of the mutations relative to these structural elements is somewhat subjective, repetition of the modeling with telokin as a template (Bateman and Chothia 1995) results in the same conclusions for all but two mutations. These are Y301C and Y328C, and their location could be interpreted alternatively as influencing the polar pocket. Finally, our model is more complete, as residues 314 and

Fig. 2 a Ribbon-plot of IgIII of FGFR2. Red Strands, green and blue loops and turns. b CPK model of IgIII of FGFR2. Mutant residues are coloured: purple residues in or near the polar pocket (group A mutations), red residues at the outer surface pointing at the membrane (group B mutations), yellow residues lining the cleft (group C mutations), green core residues (group D mutations), blue residues likely to influence interaction with the second external domain (group E mutations)




321 are not visualized by the model of Bateman and Chothia (1995). Conclusions In conclusion, we have shown that mutations in IgIII (splice variant IgIIIc) and the IgII-IgIII interface that result in craniosynostoses are either deleterious to the structural integrity of the third extracellular domain of FGFR2 or are clustered in three distinct areas. The latter appear to affect ligand binding by various mechanisms, including dimerization and HSPG interactions. The surprising finding that these mutations are not distributed randomly in the model and that they are clustered in distinct regions allows the design of new experimental approaches and thereby improves our understanding of the etiology of craniosynostoses. Addendum The designation of nucleotide positions in the FGFR2 gene is inconsistent in the literature dealing with FGFR2related craniosynostotic syndromes. Thus, the Apert mutations at amino acid positions 252 and 253 are referred to as affecting nucleotide positions 934 and 937 (Wilkie et al. 1995). The nucleotide position of the common cysteine mutation at amino acid position 342 is given as 1036 (Reardon et al. 1994) Additional nucleotide positions are listed in relation to these two mutations. The discrepancies appear to arise because of counting variable numbers of nucleotides upstream of the ATG start codon. In the case of the Apert mutations, the nucleotide positions given are based on the work of Dionne et al. (1990) who sequenced 179 nucleotides upstream of ATG. The nucleotide number at codon 342 appears to rely mainly on the publication of Houssaint et al. (1990) who sequenced only 12 nucleotides upstream of ATG. Until the exact transcription start site is known, we suggest that nucleotides of the FGFR2 gene (BEK variant) are counted from ATG onwards, thus giving a nucleotide position of 1 for the first base of codon 1, of 754 for the first base of codon 252, and of 1024 for the first base of codon 342 (based on the sequence of Dionne et al. 1990). Acknowledgements We thank Sabine Kunze for technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Ste770/1–1)

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