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Mutations that Cause Osteoglophonic Dysplasia Define Novel Roles for FGFR1 in Bone Elongation

2005; Elsevier BV; Volume: 76; Issue: 2 Linguagem: Inglês

10.1086/427956

1537-6605

Kenneth E. White, J. M. Segura Cabral, Siobhan I. Davis, Tonya Fishburn, W. Evans, Shoji Ichikawa, Joanna Fields, Xijie Yu, Nick J. Shaw, Neil McLellan, C McKeown, David Fitzpatrick, Kai Yu, David M. Ornitz, Michael J. Econs,

Connective tissue disorders research

Activating mutations in the genes for fibroblast growth factor receptors 1–3 (FGFR1–3) are responsible for a diverse group of skeletal disorders. In general, mutations in FGFR1 and FGFR2 cause the majority of syndromes involving craniosynostosis, whereas the dwarfing syndromes are largely associated with FGFR3 mutations. Osteoglophonic dysplasia (OD) is a “crossover” disorder that has skeletal phenotypes associated with FGFR1, FGFR2, and FGFR3 mutations. Indeed, patients with OD present with craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as the rhizomelic dwarfism and nonossifying bone lesions that are characteristic of the disorder. We demonstrate here that OD is caused by missense mutations in highly conserved residues comprising the ligand-binding and transmembrane domains of FGFR1, thus defining novel roles for this receptor as a negative regulator of long-bone growth. Activating mutations in the genes for fibroblast growth factor receptors 1–3 (FGFR1–3) are responsible for a diverse group of skeletal disorders. In general, mutations in FGFR1 and FGFR2 cause the majority of syndromes involving craniosynostosis, whereas the dwarfing syndromes are largely associated with FGFR3 mutations. Osteoglophonic dysplasia (OD) is a “crossover” disorder that has skeletal phenotypes associated with FGFR1, FGFR2, and FGFR3 mutations. Indeed, patients with OD present with craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as the rhizomelic dwarfism and nonossifying bone lesions that are characteristic of the disorder. We demonstrate here that OD is caused by missense mutations in highly conserved residues comprising the ligand-binding and transmembrane domains of FGFR1, thus defining novel roles for this receptor as a negative regulator of long-bone growth. The fibroblast growth factors (FGFs) and their receptors (FGFR1–4) play key roles in skeletal development. The FGFRs are part of the tyrosine kinase receptor family and comprise an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular tyrosine kinase region (Ruta et al. Ruta et al., 1989Ruta M Burgess W Givol D Epstein J Neiger N Kaplow J Crumley G Dionne C Jaye M Schlessinger J Receptor for acidic fibroblast growth factor is related to the tyrosine kinase encoded by the fms-like gene (FLG).Proc Natl Acad Sci USA. 1989; 86: 8722-8726Crossref PubMed Scopus (154) Google Scholar; Ornitz and Itoh Ornitz and Itoh, 2001Ornitz DM Itoh N Fibroblast growth factors [review].Genome Biol. 2001; 2: 3005Crossref Google Scholar). Activating mutations in FGFR1–3 result in skeletal disorders. FGFR1 and FGFR2 mutations cause syndromes involving craniosynostosis, such as Pfieffer, Crouzon, and Apert syndromes, whereas the dwarfing syndromes, such as achondroplastic and hypochondroplastic dwarfism, are associated with FGFR3 mutations (Muenke et al. Muenke et al., 1994Muenke M Schell U Hehr A Robin N Losken HW Schinzel A Pulleyn LJ Rutland P Reardon W Malcolm S Winter RM A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome.Nat Genet. 1994; 8: 269-274Crossref PubMed Scopus (526) Google Scholar; Reardon et al. Reardon et al., 1994Reardon W Winter RM Rutland P Pulleyn LJ Jones BM Malcolm S Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome.Nat Genet. 1994; 8: 98-103Crossref PubMed Scopus (589) Google Scholar; Shiang et al. Shiang et al., 1994Shiang R Thompson LM Zhu YZ Church DM Fielder TJ Bocian M Winokur ST Wasmuth JJ Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia.Cell. 1994; 78: 335-342Abstract Full Text PDF PubMed Scopus (1021) Google Scholar). Osteoglophonic dysplasia (OD [MIM 166250]) is an autosomal dominant disorder that shares characteristics with both the craniosynostosis syndromes and the dwarfing syndromes. OD is characterized by craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as by rhizomelic dwarfism and nonossifying bone lesions. However, the genetic basis for the unique skeletal phenotype associated with OD is unknown. A kindred with OD that consisted of a father and two sons was recognized because of skeletal complications and progressive weakness in the family members. The proband (patient 1) and the affected father (patient 2) were evaluated (fig. 1a). Patient 1 possessed a distinct OD facial phenotype characterized by craniosynostosis, severe nasal maxillary hypoplasia, telechanthus, and prominent supraorbital ridge (fig. 1a). Patient 1 also had growth retardation, with a peak stature of 40 in (101.6 cm) and a weight of 100 lb (45.36 kg). The proband’s father and brother (fig. 1a) had the same skeletal syndrome (the father’s peak stature was 48 in [121.92 cm]; see fig. 1b). They also had shortened necks, broad and shortened thumbs, brachydactyly, and generalized osteopenia. In addition, patients 1 and 2 never had tooth eruption, as documented by radiographs. The direct sequencing of all exons in FGFR1–4, as well as the novel decoy receptor gene FGFR5, by use of DNA samples from this family, revealed a heterozygous 1115G→A missense mutation within exon 10 of FGFR1 (fig. 1c). This change replaces a tyrosine with a cysteine residue at amino acid position 372 (Y372C), which maps to the extracellular juxtamembrane region of FGFR1. Unaffected family members were negative for the mutation, and no other nucleotide substitutions in FGFR1 were found. In addition, this change disrupts an RsaI restriction site and was not found in RFLPs from 880 control alleles (not shown). Tyrosine at position 372 is conserved among all FGFRs. The analogous mutations that result in unpaired cysteine residues in FGFR2 (Y375C) and FGFR3 (Y373C) cause Beare-Stevenson cutis gyrata syndrome (characterized by furrowed skin, acanthosis nigricans, craniosynostosis, digital anomalies, and early death [Hall et al. Hall et al., 1992Hall BD Cadle RG Golabi M Morris CA Cohen Jr, MM Beare-Stevenson cutis gyrata syndrome.Am J Med Genet. 1992; 44: 82-89Crossref PubMed Scopus (49) Google Scholar; Przylepa et al. Przylepa et al., 1996Przylepa KA Paznekas W Zhang M Golabi M Bias W Bamshad MJ Carey JC Hall BD Stevenson R Orlow S Cohen Jr, MM Jabs EW Fibroblast growth factor receptor 2 mutations in Beare-Stevenson cutis gyrata syndrome.Nat Genet. 1996; 13: 492-494Crossref PubMed Scopus (159) Google Scholar]) and thanatophoric dwarfism type I (characterized by craniosynostosis, short ribs and bones of the extremities, reduced vertebral bodies, and neonatal lethality [Rousseau et al. Rousseau et al., 1996Rousseau F el Ghouzzi V Delezoide AL Legeai-Mallet L Le Merrer M Munnich A Bonaventure J Missense FGFR3 mutations create cysteine residues in thanatophoric dwarfism type I (TD1).Hum Mol Genet. 1996; 5: 509-512Crossref PubMed Scopus (146) Google Scholar]), respectively. It is interesting that, although these mutations are analogous to FGFR2 mutations that cause the skin disorders mentioned above, these patients do not have dermatologic findings. Follow-up analysis of this kindred was not possible because the patients are deceased. Patient 1 died at age 28 years, presumably from a pulmonary embolism due to extended immobilization; patient 2 died at age 59 years, from respiratory distress under similar immobilizing conditions; and the brother of patient 1 died at age 24 years, from pneumonia. An unrelated patient with OD (patient 3), was found to have severe craniosynostosis, midface hypoplasia (fig. 2a), right choanal atresia, and mild rhizomelic shortening of the limbs, which became more pronounced with further growth. In radiological analysis, severe femur metaphyseal lesions were present (fig. 2b), and, similar to the findings for patient 1, marked demineralization of the long bones (fig. 2b), the mandible, and the maxilla was observed. Patient 3 developed swelling that arose from the lower gums, with a histologic appearance resembling giant-cell granulomata. The swelling did not respond to vinblastine or methotrexate, and, ultimately, the growth of the lesions was suppressed by intravenous bisphosphonate treatment. The karyotype of patient 3 was normal, and DNA analysis was negative for Pfeiffer syndrome mutations and Muenke and Saethre-Chotzen syndromes mutations—FGFR1 P252R and FGFR3 P250R, respectively (Muenke et al. Muenke et al., 1997Muenke M Gripp KW McDonald-McGinn DM Gaudenz K Whitaker LA Bartlett SP Markowitz RI et al.A unique point mutation in the fibroblast growth factor receptor 3 gene (FGFR3) defines a new craniosynostosis syndrome.Am J Hum Genet. 1997; 60: 555-564PubMed Google Scholar; Paznekas et al. Paznekas et al., 1998Paznekas WA Cunningham ML Howard TD Korf BR Lipson MH Grix AW Feingold M Goldberg R Borochowitz Z Aleck K Mulliken J Yin M Jabs EW Genetic heterogeneity of Saethre-Chotzen syndrome due to Twist and FGFR mutations.Am J Hum Genet. 1998; 62: 1370-1380Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). We then identified a heterozygous T→A mutation (929T→A) in exon 9 of FGFR1 (fig. 2c), which resulted in an asparagine-to-isoleucine (N330I) mutation. This change creates a Tsp509I restriction site, which was not found in 200 control individuals by RFLP analysis (not shown). Furthermore, the parents of patient 3 were negative for the mutation, which indicates that it was a de novo DNA mutation. N330 is a predicted glycosylation site, and homologous mutations in FGFR2 (N331I) and FGFR3 (N328I) cause Crouzon syndrome (Steinberger et al. Steinberger et al., 1996Steinberger D Mulliken JB Muller U Crouzon syndrome: previously unrecognized deletion, duplication, and point mutation within FGFR2 gene.Hum Mutat. 1996; 8: 386-390Crossref PubMed Scopus (25) Google Scholar) and hypochondroplasia (Winterpacht et al. Winterpacht et al., 2000Winterpacht A Hilbert K Stelzer C Schweikardt T Decker H Segerer H Spranger J Zabel B A novel mutation in FGFR-3 disrupts a putative N-glycosylation site and results in hypochondroplasia.Physiol Genomics. 2000; 2: 9-12Crossref PubMed Scopus (54) Google Scholar), respectively. Of note, exon 9 includes the sequence encoding the C-terminal portion of D3, a domain that plays a role in FGF binding and activation (Ornitz et al. Ornitz et al., 1996Ornitz DM Xu J Colvin JS McEwen DG MacArthur CA Coulier F Gao G Goldfarb M Receptor specificity of the fibroblast growth factor family.J Biol Chem. 1996; 271: 15292-15297Crossref PubMed Scopus (1362) Google Scholar). Exon 9 is present only in the “c” isoform of FGFR1, indicating that specific changes in FGFR1c will lead to OD. Similar to patients 1–3, patient 4 had clinical features at age 2.5 years that were consistent with OD and included prominent eyes, a short nose, palpable cranial sutures, pansutural craniosynostosis (confirmed by radiograph and CT scans; not shown), pectus excavatum, and delayed tooth eruption (fig. 3a). Her head circumference was in the 3rd percentile, and she had growth retardation (percentile 90% of achondroplastic dwarfism cases (Rousseau et al. Rousseau et al., 1994Rousseau F Bonaventure J Legeal-Mallet L Pelet A Michel-Rozet J Maroteaux P Merrer ML Munnich A Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia.Nature. 1994; 371: 252-254Crossref PubMed Scopus (704) Google Scholar; Shiang et al. Shiang et al., 1994Shiang R Thompson LM Zhu YZ Church DM Fielder TJ Bocian M Winokur ST Wasmuth JJ Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia.Cell. 1994; 78: 335-342Abstract Full Text PDF PubMed Scopus (1021) Google Scholar; Ikegawa et al. Ikegawa et al., 1995Ikegawa S Fudushima Y Isomura M Takada F Nakamura Y Mutations of the fibroblast growth factor receptor-3 gene in one familial and six sporadic cases of achondroplasia in Japanese patients.Hum Genet. 1995; 96: 309-311Crossref PubMed Scopus (53) Google Scholar). In examination of the biochemistries of the patients with OD, parallel observations were made in several cases. Patient 1 and his father, patient 2, were hypophosphatemic (1.0 mg/dl; normal, 2.7–4.5 mg/dl) secondary to renal phosphate wasting (tubular maximum phosphate transport/glomerular filtration rates, 0.9 mg/dl for patient 1 and 0.3 mg/dl for patient 2; normal, 2.5–4.5 mg/dl). This family also had 1,25-dihydroxyvitamin D concentrations that were inappropriately low, given the degree of hypophosphatemia (10 pg/ml; normal, 25–45 pg/ml). In a similar manner, patient 3 became hypophosphatemic (3.2 mg/dl; normal for children, 4.5–5.5 mg/dl) at age 3 years as a result of isolated renal phosphate wasting, with normal serum calcium, 25-hydroxyvitamin D, and alkaline phosphatase concentrations. In this patient, parathyroid hormone (PTH) levels fluctuated above the normal range, with unknown etiology, early in life. Although PTH was suppressed during the period of bisphosphonate treatment for lesions (aged 3–3.5 years), patient 3 remained hypophosphatemic. It is interesting that isolated renal phosphate wasting has not been a reported finding in OD. We previously determined that a known phosphaturic factor, FGF23, is produced by the nonossifying lesions in some patients with fibrous dysplasia of bone and that elevated circulating concentrations of FGF23 are positively correlated with phosphate wasting in these patients (Riminucci et al. Riminucci et al., 2003Riminucci M Collins MT Fedarko NS Cherman N Corsi A White KE Waguespack S Gupta A Hannon T Econs MJ Bianco P Gehron Robey P FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting.J Clin Invest. 2003; 112: 683-692Crossref PubMed Scopus (500) Google Scholar). Similarly, the hypophosphatemia and disturbed vitamin D metabolism observed in OD may be caused by increased FGF23 production within the characteristic radiolucent OD lesions (Beighton et al. Beighton et al., 1980Beighton P Cremin BJ Kozlowski K Osteoglophonic dwarfism.Pediatr Radiol. 1980; 10: 46-50Crossref PubMed Scopus (20) Google Scholar). We therefore tested serum levels of FGF23 in patient 3, which were indeed elevated (101 pg/ml; control mean ± SD, 30 ± 20 pg/ml). Of note, patient 4 had normal plasma phosphate concentrations and a normal serum FGF23 concentration (32.6 pg/ml). Indeed, the lesional burden is far greater in patient 3 than in patient 4 (figs. 2b and 3b), which is inversely correlated with the serum phosphate concentrations in these patients (not shown). Patients 1 and 2 are deceased; therefore, we were unable to determine their serum FGF23 values. However, because this family, parallel to patient 3, had the characteristic OD skeletal phenotype and renal phosphate wasting, we would hypothesize that serum FGF23 was elevated. To determine whether the OD mutations were activating mutations, wild-type FGFR1c and Y372C FGFR1c were assayed for ligand-independent and ligand-dependent activity. The FGFR1 Y372C missense mutation was introduced into the FGFR1 cDNA by use of a nested-PCR site-directed mutagenesis approach (Yang et al. Yang et al., 1994Yang X-F Fournier H Dion N Crine P Boileau G Site-directed mutagenesis and transfection methods in the study of prohormone processing.Neuroprotocols. 1994; 5: 157-168Google Scholar). Wild-type and mutant receptors were cotransfected with an osteocalcin FGF response element promoter-luciferase reporter into the osteogenic MC3T3 cell line (Newberry et al. Newberry et al., 1996Newberry EP Boudreaux JM Towler DA The rat osteocalcin fibroblast growth factor (FGF)-responsive element: an okadaic acid-sensitive, FGF-selective transcriptional response motif.Mol Endocrinol. 1996; 10: 1029-1040Crossref PubMed Scopus (36) Google Scholar). In the absence of exogenous FGF, the basal activity of Y372C FGFR1 was elevated 9-fold, as compared with that of wild-type FGFR1 (P<.01), which indicates that the mutation causes ligand-independent activation of FGFR1 (fig. 4a). Both wild-type and Y372C FGFR1 responded to increasing concentrations of FGF2, a known ligand for FGFR1c (Ornitz et al. Ornitz et al., 1996Ornitz DM Xu J Colvin JS McEwen DG MacArthur CA Coulier F Gao G Goldfarb M Receptor specificity of the fibroblast growth factor family.J Biol Chem. 1996; 271: 15292-15297Crossref PubMed Scopus (1362) Google Scholar). At all concentrations tested, FGF2-mediated activity was significantly higher for the Y372C FGFR1 mutant compared with the wild type (fig. 4a). In summary, OD is caused by activating mutations in highly conserved residues of FGFR1, within a limited region comprising the D3 domain, a linker region, and the initial transmembrane domain. Previously characterized mutations in FGFR1–3-associated skeletal dysplasias lead to inappropriate receptor signaling through covalent and noncovalent receptor dimerization and activation (Neilson and Friesel Neilson and Friesel, 1996Neilson KM Friesel R Ligand-independent activation of fibroblast growth factor receptors by point mutations in the extracellular, transmembrane, and kinase domains.J Biol Chem. 1996; 271: 25049-25057Crossref PubMed Scopus (134) Google Scholar; Li et al. Li et al., 1997Li Y Mangasarian K Mansukhani A Basilico C Activation of FGF receptors by mutations in the transmembrane domain.Oncogene. 1997; 14: 1397-1406Crossref PubMed Scopus (65) Google Scholar), increased ligand-binding affinity (Anderson et al. Anderson et al., 1998Anderson J Burns HD Enriquez-Harris P Wilkie AO Heath JK Apert syndrome mutations in fibroblast growth factor receptor 2 exhibit increased affinity for FGF ligand.Hum Mol Genet. 1998; 7: 1475-1483Crossref PubMed Scopus (210) Google Scholar; Ibrahimi et al. Ibrahimi et al., 2001Ibrahimi OA Eliseenkova AV Plotnikov AN Yu K Ornitz DM Mohammadi M Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome.Proc Natl Acad Sci USA. 2001; 98: 7182-7187Crossref PubMed Scopus (157) Google Scholar), altered specificity for FGF binding (Yu et al. Yu et al., 2000Yu K Herr AB Waksman G Ornitz DM Loss of fibroblast growth factor receptor 2 ligand-binding specificity in Apert syndrome.Proc Natl Acad Sci USA. 2000; 97: 14536-14541Crossref PubMed Scopus (207) Google Scholar), and direct activation of the FGFR tyrosine kinase domains (Neilson and Friesel Neilson and Friesel, 1996Neilson KM Friesel R Ligand-independent activation of fibroblast growth factor receptors by point mutations in the extracellular, transmembrane, and kinase domains.J Biol Chem. 1996; 271: 25049-25057Crossref PubMed Scopus (134) Google Scholar). In contrast, inactivating mutations in FGFR1 are responsible for autosomal dominant Kallmann syndrome, characterized by hypogonadism and anosmia (Dod et al. Dode et al., 2003Dode AC Levilliers J Dupont JM De Paepe A Le Dû N Soussi-Yanicostas N Coimbra RS et al.Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome.Nat Genet. 2003; 33: 463-465Crossref PubMed Scopus (604) Google Scholar); thus, it is highly likely that the OD N330I and C379R mutations, in addition to the Y372C substitution, are gain-of-function mutations. It is thought that activation of FGFR3 is responsible for negative regulation of bone elongation, because Fgfr3 null mice have increased long-bone length (Deng et al. Deng et al., 1996Deng C Wynshaw-Boris A Zhou F Kuo A Leder P Fibroblast growth factor receptor 3 is a negative regulator of bone growth.Cell. 1996; 84: 911-921Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar) and because of the relatively common occurrence of activating mutations in FGFR3-related achondroplasia (Naski et al. Naski et al., 1996Naski MC Wang Q Xu J Ornitz DM Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia.Nat Genet. 1996; 13: 233-237Crossref PubMed Scopus (410) Google Scholar). The most common FGFR1-activating mutation (P252R) causes the craniosynostosis disorder Pfeiffer syndrome (Muenke et al. Muenke et al., 1994Muenke M Schell U Hehr A Robin N Losken HW Schinzel A Pulleyn LJ Rutland P Reardon W Malcolm S Winter RM A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome.Nat Genet. 1994; 8: 269-274Crossref PubMed Scopus (526) Google Scholar); thus, FGFR1 has been associated primarily with flat bone growth and skull formation. Because the Fgfr1 null mouse is neonatal lethal (Deng et al. Deng et al., 1994Deng CX Wynshaw-Boris A Shen MM Daugherty C Ornitz DM Leder P Murine FGFR-1 is required for early postimplantation growth and axial organization.Genes Dev. 1994; 8: 3045-3057Crossref PubMed Scopus (618) Google Scholar) and because of the rarity of OD, the role of FGFR1 in long-bone growth has not been fully recognized. In long bones, FGFR1 expression is spatially separated from that of FGFR3, as the epiphyseal growth plate is formed. Prehypertrophic and hypertrophic chondrocytes, osteoblasts, and perichondrium express FGFR1, whereas FGFR3 is found in proliferating chondrocytes and adult osteoblasts (Peters et al. Peters et al., 1992Peters KG Werner S Chen G Williams LT Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse.Development. 1992; 114: 233-243Crossref PubMed Google Scholar; Deng et al. 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Together with the expression profile of FGFR1 in bone, the exon 9 mutation (N330I) demonstrates that the activation of the FGFR1c isoform has profound effects on long bones, possibly by regulation of skeletal formation through suppression of chondrocyte or osteoblast function. It is interesting that patients 1 and 2 had respiratory difficulties, which is not a typical characteristic of other FGFR syndromes. The patients did not have rib deformities; thus, there are two things that most likely affected respiratory function. The hypophosphatemia and marked reduction in calcitriol concentration may have led to weakness, which resulted in poor inspiratory effort with atelectasis, thereby predisposing the patients to pneumonia. In addition, it is likely that immobilization led to a pulmonary embolus in patient 1 and, possibly, in his father (patient 2) and/or brother. In summary, OD is a disorder that shares skeletal characteristics with both the craniosynostoses and the dwarfing syndromes. OD is caused by activating mutations in FGFR1, which thus reveals novel critical functions of the FGFR1 receptor in the modulation of bone elongation. We are very grateful to the kindreds for their participation and for permission to present family photographs. We also thank David Weaver, M.D., for assistance in characterizing the craniofacial features of OD; Mack Harrell, M.D., and Dawn Vickers, R.N., for taking many of the original patient photographs and obtaining blood samples; and Jean Kirk, M.D., for facilitating the sample processing. The authors also greatly appreciate the scientific advice from Moosa Mohammadi, Ph.D., and from Omar Ibrahimi, given during the course of these studies. This work was supported by National Institutes of Health grant DK063934 (to K.E.W.), training grant T32 AR007033 (to K.Y.), grant HD39952 (to D.M.O.), and grants AR42228, AG18397, AR02095, and AR47866 (to M.J.E.). The authors would also like to acknowledge the support of the Indiana Genomics Initiative, supported in part by Lilly Endowment.