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. ¹⁹⁹²; Przylepa et al. ¹⁹⁹⁶]) and thanatophoric dwarfism type I (characterized by craniosynostosis, short ribs and bones of the extremities, reduced vertebral bodies, and neonatal lethality [Rousseau et al. ¹⁹⁹⁶]), 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. ¹⁹⁹⁷; Paznekas et al. ¹⁹⁹⁸). 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. ¹⁹⁹⁶) and hypochondroplasia (Winterpacht et al. ²⁰⁰⁰), 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. ¹⁹⁹⁶). 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 <0.4) with bilateral flexion contractures of her fingers. Furthermore, a skeletal survey of patient 4 showed multiple lesions in the metaphyseal regions of the long bones (fig. 3b), although they were not as severe as those of patient 2.

In sequence analysis of FGFR1 in patient 4, a heterozygous T→C mutation (1135T→C; C379R) was identified in exon 10 (fig. 3c), which resulted in a cysteineto-arginine change. This substitution interrupts an HpyCH4V restriction site, and the change was not found in 100 control individuals by RFLP analysis. Furthermore, both parents were negative for the mutation, which indicates that it was a de novo substitution (not shown). Cysteine at position 379 is predicted to reside within the FGFR1 transmembrane domain, and mutations in FGFR3 at the position homologous to C379 (G380R) are responsible for >90% of achondroplastic dwarfism cases (Rousseau et al. ¹⁹⁹⁴; Shiang et al. ¹⁹⁹⁴; Ikegawa et al. ¹⁹⁹⁵).

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. ²⁰⁰³). 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. ¹⁹⁸⁰). 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 and33b), 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. ¹⁹⁹⁴). 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. ¹⁹⁹⁶). 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. ¹⁹⁹⁶). 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 ¹⁹⁹⁶; Li et al. ¹⁹⁹⁷), increased ligand-binding affinity (Anderson et al. ¹⁹⁹⁸; Ibrahimi et al. ²⁰⁰¹), altered specificity for FGF binding (Yu et al. ²⁰⁰⁰), and direct activation of the FGFR tyrosine kinase domains (Neilson and Friesel ¹⁹⁹⁶). In contrast, inactivating mutations in FGFR1 are responsible for autosomal dominant Kallmann syndrome, characterized by hypogonadism and anosmia (Dod et al. ²⁰⁰³); 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. ¹⁹⁹⁶) and because of the relatively common occurrence of activating mutations in FGFR3-related achondroplasia (Naski et al. ¹⁹⁹⁶). The most common FGFR1-activating mutation (P252R) causes the craniosynostosis disorder Pfeiffer syndrome (Muenke et al. ¹⁹⁹⁴); thus, FGFR1 has been associated primarily with flat bone growth and skull formation. Because the Fgfr1 null mouse is neonatal lethal (Deng et al. ¹⁹⁹⁴) 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. ¹⁹⁹²; Deng et al. ¹⁹⁹⁶; Delezoide et al. ¹⁹⁹⁸; Xiao et al. ²⁰⁰⁴). 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.