A novel mutation in FGFR-3 disrupts a putative N-glycosylation site and results in hypochondroplasia

ANDREAS WINTERPACHT1,2, KATJA HILBERT1, CHRISTIANE STELZER1, THORSTEN SCHWEIKARDT3, HEINZ DECKER3, HUGO SEGERER4, JÜRGEN SPRANGER1 and BERNHARD ZABEL1

1 Children's Hospital, University of Mainz, D-55101 Mainz
2 Institute of Human Genetics, University of Hamburg, D-22529 Hamburg
3 Institute for Molecular Biophysics, University of Mainz, D-55128 Mainz
4 St. Hedwig Hospital, D-93006 Regensburg, Germany


    ABSTRACT
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Winterpacht, Andreas, Katja Hilbert, Christiane Stelzer, Thorsten Schweikardt, Heinz Decker, Hugo Segerer, Jürgen Spranger, and Bernhard Zabel. A novel mutation in FGFR-3 disrupts a putative N-glycosylation site and results in hypochondroplasia. Physiol. Genomics 2: 9–12, 2000.—Fibroblast growth factor receptor 3 (FGFR3) is a glycoprotein that belongs to the family of tyrosine kinase receptors. Specific mutations in the FGFR3 gene are associated with autosomal dominant human skeletal disorders such as hypochondroplasia, achondroplasia, and thanatophoric dysplasia. Hypochondroplasia (HCH), the mildest form of this group of short-limbed dwarfism disorders, results in ~60% of cases from a mutation in the intracellular FGFR3-tyrosine kinase domain. The remaining cases may either be caused by defects in other FGFR gene regions or other yet unidentified genes. We describe a novel HCH mutation, the first found outside the common mutation hot spot of this condition. This point mutation, an N328I exchange in the extracellular Ig domain III of the receptor, seems to be unique as it affects a putative N-glycosylation site that is conserved between different FGFRs and species. The amino acid exchange itself most probably has no impact on the three-dimensional structure of the receptor domain, suggesting that the phenotype is the result of altered receptor glycosylation and its pathophysiological consequences.

glycosylation; tyrosine kinase receptor; chondrodysplasia; glycoprotein


    INTRODUCTION
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HYPOCHONDROPLASIA (HCH) (MIM*146000) is an autosomal dominant skeletal dysplasia that resembles achondroplasia but is phenotypically less severe and mostly not evident before 2 yr of age. The clinical findings, which include some specific radiological features, consist of short stature with rhizomelic shortening of the limbs and relative macrocephaly. HCH is caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene (1) and is allelic with other dwarfism and craniosynostosis syndromes, like achondroplasia, thanatophoric dysplasia, and Muenke syndrome (for review, see Ref. 25). FGFR3 is a member of the tyrosine kinase receptor family. The four known members (FGFR1–4) of this family play important roles in the regulation of proliferation, differentiation, and angiogenesis, as well as other processes involved in growth and development. Structurally, they comprise three extracellular immunoglobulin-like domains (Ig-like domains I–III), one transmembrane domain, and a split intracellular tyrosine kinase domain. FGFR3 is expressed during skeletal growth and endochondral ossification (for review, see Refs. 7, 13, 26). FGFR3 seems to have a specific role as negative regulator of bone growth, and the known FGFR3 mutations result in a ligand independent activation of the receptor (15, 23, 24). Up to now, two different amino acid conversions in the FGFR3 gene have been identified in HCH patients: an N540K exchange, which is present in about 40–60% of the patients (1, 6, 18, 21), and an I538V exchange, which has been reported in one case (4). Both exchanges affect the first portion of the split intracellular tyrosine kinase domain of the receptor and, like the other FGFR3 mutations, cause a ligand independent activation of the receptor (19). So far, screening of the complete FGFR3 gene in the remaining HCH patients did not reveal any other mutation, indicating genetic heterogeneity of this disorder, which was confirmed by studies showing no linkage of some HCH families to the FGFR3 locus at chromosome 4p16.3 (21).

Here, we report the identification and characterization of the first mutation located in the extracellular, ligand-binding domain of FGFR3 in a patient with HCH. We present evidence that this represents a unique FGFR3 mutation, as it disrupts a putative N-glycosylation site and the phenotype most likely results from a change in receptor glycosylation. The mutation may therefore serve as a model to with which to investigate the effect of glycosylation on proper FGF receptor function.


    METHODS
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 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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Mutation screening.
Exons 1–18 of the FGFR3 gene were amplified on the genomic level from DNA extracted from blood lymphocytes using a recently established primer set (27). PCR products were sequenced directly using the Dye Deoxy cycle sequencing kit (ABI) and a model ABI377 sequencing apparatus.

Molecular modeling.
A provisional homology model for the normal and mutant IgIII domain was built on the basis of the known three-dimensional structure of the FV domain of the antibody (Protein Data Bank no. 1A6U) from Mus musculus, strain C57BL/6, using the program Modeller 4 (22).


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After approval by the appropriate institutional review board, we investigated a patient without the known FGFR3 mutations but with all the criteria of HCH. The 20-mo-old girl was presented because of her short stature. She was born with a length of 50 cm. Except for poor growth, her postnatal development was unremarkable. Her mother measures 150 cm, and her maternal grandparents measure 185 and 170 cm. Physical examination disclosed an alert and active girl with a height of 73 cm (5 cm below 3rd centile), a weight of 10.7 kg (90th centile for height), and a head circumference of 49.5 cm (97th centile). Body proportions were slightly abnormal with relatively short upper arms and thighs. Except for short stature, there were no physical abnormalities, and her psychomotor development was unremarkable. Routine laboratory values including serum alkaline phosphatase, insulin-like growth factor I (IGF-I), and IGF-binding proteins were normal. A skeletal survey showed alterations compatible with a diagnosis of HCH (Fig. 1). The mother declined to be physically examined.



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Fig. 1. Skeletal changes of the patient at 18 mo display typical HCH features: slightly shortened metacarpal I (A); iliac bones are slightly squared and the femoral necks short with wide physes (B); tibia is short and plump with wide metaphyses, and fibula is disproportionately long (C); interpediculate distances of the lower lumbar spine are smaller than those of the upper lumbar spine (D); lumbar vertebral bodies are slightly ovoid with minimal beaking and concave dorsal borders (E). R and L indicate right and left.

 
We screened the complete coding sequence of the FGFR3 gene for mutations on the genomic level using a recently established primer set (27). We identified an A-to-T transversion in exon 9 at position 1022 of the cDNA sequence (9) (Fig. 2). The mutation resulted in an asparagine-to-isoleucine conversion at amino acid position 328 in the IgIIIc domain. The same mutation was present in the patient's mother, who is most probably also affected, but not in DNA from the healthy father (Fig. 2) and in 50 normal probands (data not shown), which strongly confirms that the mutation is the cause of the clinical phenotype.



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Fig. 2. DNA sequence analysis of exon 9 of the fibroblast growth factor receptor 3 (FGFR3) gene. The heterozygous A-to-T transversion resulting in an Asn-to-Ile exchange is present in the proband and in the affected mother, but not in DNA from the healthy father and 50 unaffected probands (not shown). The mutation affects a putative N-glycosylation site (N-V-T).

 
To investigate whether the observed N328I exchange itself has a probable effect on the folding of the IgIII loop, we tried to build a provisional homology model for the normal and mutant IgIII domain (Fig. 3) on the basis of the known three-dimensional structures of immunoglobulin superfamily members (I-set immunoglobulin domains) using the program Modeller 4 (22).



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Fig. 3. Three-dimensional structures of the modeled IgIII domain of FGFR3 (blue) and the FV domain of the antibody (Protein Data Bank no. 1A6U) from Mus musculus, strain C57BL/6 (red): normal FGFR3 sequence (left) and mutant (N328I) FGFR3 sequence (right). Side chain of the normal and mutant amino acid is shown.

 
Similar models for Ig domains have already been used by others (3, 5, 26). Figure 3 demonstrates that the three-dimensional structures of the modeled IgIII domain of FGFR3 and the FV domain of the antibody (Protein Data Bank no. 1A6U) from M. musculus, strain C57BL/6, are in good accordance, especially in the three different loop regions of the IgIII domain where the mutant amino acid is located. This indicates a fairly high level of confidence for the predictions in this region. It is not possible to directly show the impact of a mutation on the tertiary protein structure by comparative modeling, because the structural alteration caused by the mutation can only be calculated for the immediate perimeter around the mutant amino acid. Nevertheless, on the basis of the model, a major structural rearrangement seems to be unlikely, if one accounts for the position of the mutant amino acid, which is located in a loop at the outer surface of the protein, with its side chain oriented to the surroundings. A mutation to proline or glycine would certainly disturb the given structure of the loop, but the observed isoleucine should have no such effect. The data therefore indicate that the substitution of an asparagine residue itself is probably not sufficient to cause the clinical phenotype. This is further supported by the crystal structure of FGF2 bound to the IgII and IgIII domain of FGFR1, which has been published recently by Plotnikov et al. (17). From these data it can be concluded that our model is correct and that the affected asparagine at position 328 (position 330 in FGFR1) is not involved in ligand-receptor or receptor-receptor interaction.


    DISCUSSION
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of mutations have been identified in different parts of the FGFR1–3 genes in patients with dwarfism (FGFR3) and craniosynostosis syndromes (FGFR1–3) (for review, see Ref. 25). Most of the mutations in the extracellular ligand binding domains (FGFR2 and FGFR3) destroy or create cysteine or histidine residues and thereby disrupt normal folding of the IgIII loops, which possibly mimics ligand binding by constitutively dimerizing and activating the receptor (25). Other mutations involve conserved residues that are predicted to play a key structural role in the immunoglobulin fold or introduce proline residues that cause gross rearrangements in the protein structure (for review, see Ref. 26). Interestingly, so far no conversion of an amino acid into a leucine or isoleucine residue has been observed in any of the FGFRs in dwarfism and craniosynostosis syndromes, although these exchanges should be expected at a high frequency (26). This suggests that they would result in a different or no obvious phenotype (26). In the present case the isoleucine substitutes an asparagine which is part of a putative N-glycosylation site (N-V-T). The asparagine as well as the glycosylation consensus site at this position is strongly conserved between all four FGFRs and different vertebrate species (data not shown), indicating that this site is of functional importance. Different forms of the receptor with varying degrees of N-glycosylation have been described (9, 10), and there is evidence that the affected site, which is only present in the common IIIc splice variant of the receptor, is indeed glycosylated (14). For these obvious reasons and on the basis of the present data as well as a comparison with the work of Plotnikov et al. (17), it is tempting to speculate that the phenotypic effect of the N328I mutation may be due to a disruption of the putative N-glycosylation site (N-V-T), resulting in altered glycosylation and changes in the overall structure of the receptor.

Several disorders are associated with an altered glycosylation pattern of specific proteins (for review, see Ref. 2), but only very few are caused by changes of possible N-glycosylation sites (11, 16, 20, 26). In a patient with Crouzon's syndrome, an S257P exchange in the FGFR2 gene was described (26) that disturbed the first putative N-glycosylation site in the IgIIIa exon. The mutant amino acid directly flanks the highly conserved homeo-interaction domain (13) and introduces a proline residue that strongly interferes with the correct folding of the domain. Therefore, it can be assumed that this mutation, in contrast to the described N328I exchange, may influence correct FGFR function by a direct effect on the receptor structure, rather than by the disruption of an N-glycosylation site. Interestingly, it has recently been shown that mutations in the FGFR2 gene which result in constitutive activation of the receptor and a loss of ligand-binding property are less glycosylated compared with their wild-type counterparts (12). Raffioni et al. (19) could show that mutant FGFR3 (K650E and to a lesser extend N540K) leads to a reduction of the mature glycosylated form of the receptor and a ligand-independent phosphorylation of the less glycosylated precursor form of the receptor in the cells. Although these two forms occur in normal cells, phosphorylation of the lower molecular weight form is normally not observed.

The present case is the first report of a mutation in the extracellular part of the FGFR3 resulting in HCH and is one of the very few examples of mutant putative N-glycosylation sites linked to a clinical phenotype. The data suggest that correct glycosylation plays a crucial role in the normal and abnormal functioning of FGF receptors and that changes in the glycosylation pattern may therefore significantly contribute to a clinical phenotype. Experiments to address this issue are planned and will include the in vitro mutagenesis of additional glycosylation sites, the expression of these constructs in cells, and the subsequent measurement of receptor activity using standard assays. In addition, it should be reinvestigated whether other HCH cases without the common N540K mutation are due to similar mutations in the extracellular part of the receptor and/or altered glycosylation of the receptor. In any case, the mutation described here represents an excellent model with which to study the effect of glycosylation on normal and disturbed FGF receptor function and the associated disorders, an aspect which has not been investigated in more detail, so far.


    ACKNOWLEDGMENTS
 
We would like to thank J. Busch for excellent technical assistance.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to A. Winterpacht and B. Zabel).

Address for reprint requests and other correspondence: A. Winterpacht, Institute of Human Genetics, Univ. of Hamburg, Butenfeld 42, D-22529 Hamburg, Germany (E-mail: winterpacht{at}uke.uni-hamburg.de).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


    REFERENCES
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 INTRODUCTION
 METHODS
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 DISCUSSION
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