Meclozine Facilitates Proliferation and Differentiation of Chondrocytes by Attenuating Abnormally Activated FGFR3 Signaling in Achondroplasia

Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.     

The cytoplasmic tyrosine kinase Pyk2 as a novel effector of fibroblast growth factor receptor 3 activation

Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes thanatophoric dysplasia types I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. The molecular pathways exploited by FGFR3 to stimulate abnormal proliferation during neoplasia are unclear. The nonreceptor protein-tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) has been shown previously to regulate apoptosis in multiple myeloma cells. Here we describe a novel interaction between FGFR3 and Pyk2, mediated by the juxtamembrane domain of FGFR3 and the kinase domain of Pyk2. Within the FGFR family, Pyk2 also interacted significantly with FGFR2. Overexpression of Pyk2 alone led to its spontaneous activation and tyrosine phosphorylation, resulting in activation of Stat5B, indicated by the reporter GFP-Stat5B. These effects were completely dependent upon Tyr(402), the autophosphorylation site of Pyk2, which allows recruitment of Src family members for further activating phosphorylations at other sites on Pyk2. In the presence of activated FGFR3, the activation of Pyk2 itself became independent of Tyr(402), indicating that FGFR3 activation circumvents the requirement for c-Src recruitment at Tyr(402) of Pyk2. We also examined the role of the tyrosine phosphatase Shp2 in antagonizing Pyk2 activation. Taken together, these results suggest that signaling pathways regulated by FGFR3 may converge with Pyk2-dependent pathways to provide maximal activation of Stat5B.     

Overexpression of Spry1 in chondrocytes causes attenuated FGFR ubiquitination and sustained ERK activation resulting in chondrodysplasia

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.     

A Src homology 3-binding sequence on the C terminus of Sprouty2 is necessary for inhibition of the Ras/ERK pathway downstream of fibroblast growth factor receptor stimulation

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.     

Direct binding of PP2A to Sprouty2 and phosphorylation changes are a prerequisite for ERK inhibition downstream of fibroblast growth factor receptor stimulation

In the context of fibroblast growth factor (FGF) signaling, Sprouty2 (Spry2) is the most profound inhibitor of the Ras/ERK pathway as compared with other Spry isoforms. An exclusive, necessary, but cryptic PXXPXR motif in the C terminus of Spry2 is revealed upon stimulation. The activation of Spry2 appears to be linked to sequences in the N-terminal half of the protein and correlated with a bandshifting seen on SDS-PAGE. The band-shifting is likely caused by changes in the phosphorylation status of key Ser and Thr residues following receptor stimulation. Dephosphorylation of at least two conserved Ser residues (Ser-112 and Ser-115) within a conserved Ser/Thr sequence is accomplished upon stimulation by a phosphatase that binds to Spry2 around residues 50-60. We show that human Spry2 co-immunoprecipitates with both the catalytic and the regulatory subunits of protein phosphatase 2A (PP2A-C and PP2A-A, respectively) in cells upon FGF receptor (FGFR) activation. PP2A-A binds directly to Spry2, but not to Spry2Delta50-60 (Delta50-60), and the activity of PP2A increases with both FGF treatment and FGFR1 overexpression. c-Cbl and PP2A-A compete for binding centered around Tyr-55 on Spry2. We show that there are at least two distinct pools of Spry2, one that binds PP2A and another that binds c-Cbl. c-Cbl binding likely targets Spry2 for ubiquitin-linked destruction, whereas the phosphatase binding and activity are necessary to dephosphorylate specific Ser/Thr residues. The resulting change in tertiary structure enables the Pro-rich motif to be revealed with subsequent binding of Grb2, a necessary step for Spry2 to act as a Ras/ERK pathway inhibitor in FGF signaling.     

The pleiotropic effects of fibroblast growth factor receptors in mammalian development

In recent years the study of fibroblast growth factor receptors (FGFRs) in normal development and human genetic disorders has increased our understanding of some complex cellular processes. At least fifteen genetic disorders result from mutations within FGFR genes including skeletal dysplasias such as Apert syndrome and achondroplasia. In vitro experiments and the generation of animal models indicate that these mutations result in activation of the receptors and that FGFRs act as negative regulators of bone growth. FGFRs also play a role in wound healing and cancer. In this article, we review the expression of FGFRs in human development, the phenotypes resulting from FGFR mutations, and recent data identifying pathways downstream of the activated receptors.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

K644E/M FGFR3 mutants activate Erk1/2 from the endoplasmic reticulum through FRS2 alpha and PLC gamma-independent pathways

Fibroblast growth factor receptors 3 (FGFR3) with K644M/E substitutions are associated to the severe skeletal dysplasias: severe achondroplasia with developmental delay and achanthosis nigricans(SADDAN) and thanatophoric dysplasia(TDII). The high levels of kinase activity of the FGFR3-mutants cause uncompleted biosynthesis that results in the accumulation of the immature/mannose-rich, phosphorylated receptors in the endoplasmic reticulum (ER) and STATs activation. Here we report that FGFR3 mutants activate Erk1/2 from the ER through an FRS2-independent pathway: instead, a multimeric complex by directly recruiting PLCgamma, Pyk2 and JAK1 is formed. The Erk1/2 activation from the ER however, is PLCgamma-independent, since preventing the PLCgamma/FGFR3 interaction by the Y754F substitution does not inhibit Erks. Furthermore, Erk1/2 activation is abrogated upon treatment with the Src inhibitor PP2, suggesting a role played by a Src family member in the pathway from the ER. Finally we show that the intrinsic kinase activity by mutant receptors is required to allow signaling from the ER. Overall these results highlight how activated FGFR3 exhibits signaling activity in the early phase of its biosynthesis and how segregation in a sub-cellular compartment can affect the FGFR3 multi-faceted capacity to recruit specific substrates.     

Bent bone dysplasia-FGFR2 type, a distinct skeletal disorder, has deficient canonical FGF signaling

Fibroblast growth factor receptor 2 (FGFR2) is a crucial regulator of bone formation during embryonic development. Both gain and loss-of-function studies in mice have shown that FGFR2 maintains a critical balance between the proliferation and differentiation of osteoprogenitor cells. We have identified de novo FGFR2 mutations in a sporadically occurring perinatal lethal skeletal dysplasia characterized by poor mineralization of the calvarium, craniosynostosis, dysmorphic facial features, prenatal teeth, hypoplastic pubis and clavicles, osteopenia, and bent long bones. Histological analysis of the long bones revealed that the growth plate contained smaller hypertrophic chondrocytes and a thickened hypercellular periosteum. Four unrelated affected individuals were found to be heterozygous for missense mutations that introduce a polar amino acid into the hydrophobic transmembrane domain of FGFR2. Using diseased chondrocytes and a cell-based assay, we determined that these mutations selectively reduced plasma-membrane levels of FGFR2 and markedly diminished the receptor's responsiveness to extracellular FGF. All together, these clinical and molecular findings are separate from previously characterized FGFR2 disorders and represent a distinct skeletal dysplasia.     

Fibroblast growth factor signaling controlling bone formation: An update

Fibroblast Growth Factors (FGFs) regulate prenatal and postnatal bone formation through activation of FGF receptors (FGFR) and downstream signaling events. During the last decade, major advances have been made in our understanding of the mechanisms by which FGF/FGFR signaling controls osteoprogenitor cell replication and osteoblast differentiation and function. The analysis of the phenotype induced by FGF invalidation and mutations in FGFR allowed to delineate key FGF signaling pathways that regulate osteoblastogenesis. Molecular genomic studies led to identify target genes that are controlled by FGF/FGFR signaling and govern osteoblasts. The analysis of intracellular signaling pathways showed the importance of functional crosstalks between FGF signaling and other pathways in the regulation of bone formation. These recent progresses in the mechanisms underlying FGF/FGFR signaling may provide a molecular basis for developing therapeutic strategies in human skeletal dysplasias.     

A novel mutation in FGFR3 causes camptodactyly, tall stature, and hearing loss (CATSHL) syndrome

Activating mutations of FGFR3, a negative regulator of bone growth, are well known to cause a variety of short-limbed bone dysplasias and craniosynostosis syndromes. We mapped the locus causing a novel disorder characterized by camptodactyly, tall stature, scoliosis, and hearing loss (CATSHL syndrome) to chromosome 4p. Because this syndrome recapitulated the phenotype of the Fgfr3 knockout mouse, we screened FGFR3 and subsequently identified a heterozygous missense mutation that is predicted to cause a p.R621H substitution in the tyrosine kinase domain and partial loss of FGFR3 function. These findings indicate that abnormal FGFR3 signaling can cause human anomalies by promoting as well as inhibiting endochondral bone growth.     

Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf.

Several genetic studies in Drosophila have shown that the dSprouty (dSpry) protein inhibits the Ras/mitogen-activated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context.     

Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway

Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2-Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.     

Tyrosine phosphorylation of Sprouty2 enhances its interaction with c-Cbl and is crucial for its function

Mammalian Sprouty (Spry) proteins are now established as receptor tyrosine kinase-induced modulators of the Ras/mitogen-activated protein kinase pathway. Specifically, hSpry2 inhibits the fibroblast growth factor receptor (FGFR)-induced mitogen-activated protein kinase pathway but conversely prolongs activity of the same pathway following epidermal growth factor (EGF) stimulation, where activated EGF receptors are retained on the cell surface. In this study it is demonstrated that hSpry2 is tyrosine-phosphorylated upon stimulation by either FGFR or EGF and subsequently binds endogenous c-Cbl with high affinity. A conserved motif on hSpry2, together with phosphorylation on tyrosine 55, is required for its enhanced interaction with the SH2-like domain of c-Cbl. A hSpry2 mutant (Y55F) that did not exhibit an enhanced binding with c-Cbl failed to retain EGF receptors on the cell surface. Furthermore, individually mutating hSpry2 residues 52-59 to alanine indicated a tight correlation between their affinity for c-Cbl binding and their inhibition of ERK2 activity in the FGFR pathway. We postulate that tyrosine phosphorylation "activates" hSpry2 by enhancing its interaction with c-Cbl and that this interaction is critical for its physiological function in a signal-specific context.     

Enhanced signaling and morphological transformation by a membrane-localized derivative of the fibroblast growth factor receptor 3 kinase domain.

Fibroblast growth factor (FGF) receptors (FGFRs) are membrane-spanning tyrosine kinase receptors that mediate regulatory signals for cell proliferation and differentiation in response to FGFs. We have previously determined that the Lys650-->Glu mutation in the activation loop of the kinase domain of FGFR3, which is responsible for the lethal skeletal dysplasia thanatophoric dyplasia type II (TDII), greatly enhances the ligand-independent kinase activity of the receptor. Here, we demonstrate that expression of this construct induces a c-fos promoter construct approximately 10-fold but does not lead to proliferation or morphological transformation of NIH 3T3 cells. In contrast, the isolated kinase domain of activated FGFR3, targeted to the plasma membrane by a myristylation signal, is able to stimulate c-fos expression by 40-fold, induce proliferation of quiescent cells, and morphologically transform fibroblasts. This result suggests that the extracellular and transmembrane domains of FGFRs exert a negative regulatory influence on the activity of the kinase domain. Targeting of the activated kinase domain to either the cytoplasm or the nucleus does not significantly affect biological signaling, suggesting that signals from FGFR3 resulting in mitogenesis originate exclusively from the plasma membrane. Furthermore, our novel observation that expression of a highly activated FGFR3 kinase domain is able to morphologically transform fibroblasts suggests that dysregulation of FGFR3 has the potential to play a role in human neoplasia.     

The Pathogenic A391E Mutation in FGFR3 Induces a Structural Change in the Transmembrane Domain Dimer

Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.     

Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2

Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGF-dependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when co-expressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels.     

FGFR3 induces degradation of BMP type I receptor to regulate skeletal development

Fibroblast growth factors (FGFs) and their receptors (FGFRs) play significant roles in vertebrate organogenesis and morphogenesis. FGFR3 is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia, one of the most common dwarfisms in humans, but the molecular mechanism remains elusive. In this study, we found that chondrocyte-specific deletion of BMP type I receptor a (Bmpr1a) rescued the bone overgrowth phenotype observed in Fgfr3 deficient mice by reducing chondrocyte differentiation. Consistently, using in vitro chondrogenic differentiation assay system, we demonstrated that FGFR3 inhibited BMPR1a-mediated chondrogenic differentiation. Furthermore, we showed that FGFR3 hyper-activation resulted in impaired BMP signaling in chondrocytes of mouse growth plates. We also found that FGFR3 inhibited BMP-2- or constitutively activated BMPR1-induced phosphorylation of Smads through a mechanism independent of its tyrosine kinase activity. We found that FGFR3 facilitates BMPR1a to degradation through Smurf1-mediated ubiquitination pathway. We demonstrated that down-regulation of BMP signaling by BMPR1 inhibitor dorsomorphin led to the retardation of chondrogenic differentiation, which mimics the effect of FGF-2 on chondrocytes and BMP-2 treatment partially rescued the retarded growth of cultured bone rudiments from thanatophoric dysplasia type II mice. Our findings reveal that FGFR3 promotes the degradation of BMPR1a, which plays an important role in the pathogenesis of FGFR3-related skeletal dysplasia.