Differential expression of fibroblast growth factor receptors in human digital development suggests common pathogenesis in complex acrosyndactyly and craniosynostosis

The Apert hand is characterized by metaphyseal fusions of the metacarpals and distal phalanges, symphalangism, and soft-tissue syndactyly. More subtle skeletal anomalies of the limb characterize Pfeiffer and Crouzon syndromes. Different mutations in the fibroblast growth factor receptor 2 (FGFR2) gene cause these syndromes, and offer the opportunity to relate genotype to phenotype. The expression of FGFR1 and of the Bek and KGFR isoforms of FGFR2 has, therefore, been studied in human hand development at 12 weeks by in situ hybridization. FGFRs are differentially expressed in the mesenchyme and skeletal elements during endochondral ossification of the developing human hand. KGFR expression characterizes the metaphyseal periosteum and interphalangeal joints. FGFR1 is preferentially expressed in the diaphyses, whereas FGFR2-Bek expression characterizes metaphyseal and diaphyseal elements, and the interdigital mesenchyme. Apert metaphyseal synostosis and symphalangism reflect KGFR expression, which has independently been quantitatively related ex vivo to the severity of clinical digital presentations in these syndromes. Studies in avian development implicate FGF signaling in preventing interdigital apoptosis and maintaining the interdigital mesenchyme. Herein is proposed that in human FGFR syndromes the balance of signaling by means of KGFR and Bek in digital development determines the clinical severity of soft-tissue and bony syndactyly.     

Analysis of phenotypic features and FGFR2 mutations in Apert syndrome.

A phenotypic and genotypic survey was conducted on 36 Apert syndrome patients. In all but one patient, an FGFR2 mutation, either S252W or P253R, was found in exon IIIa (exon U or 7). The frequency was 71% and 26%, for the mutations S252W and P253R, respectively. These mutations occur in the linker region between immunoglobulin-like domains II and III, which are involved in activation of the receptor by ligand binding and dimerization. The fact that one patient did not have a mutation in the same exon suggests further genetic heterogeneity in Apert syndrome. The frequencies of occurrence or means for measurements of 29 different clinical features (including severity of craniofacial features, syndactyly of the hands and feet, and multisystem involvement) were determined for all patients and for the two subgroups defined by their mutations. Comparison between the subgroups for the different clinical features was performed and suggested no statistically significant differences. These results are not unexpected, because the two common mutations for Apert syndrome alter FGFR2 at adjacent amino acids that are likely to have similar biological, and therefore phenotypic, consequences.     

Differential effects of FGFR2 mutations on syndactyly and cleft palate in Apert syndrome.

Apert syndrome is a distinctive human malformation characterized by craniosynostosis and severe syndactyly of the hands and feet. It is caused by specific missense substitutions involving adjacent amino acids (Ser252Trp or Pro253Arg) in the linker between the second and third extracellular immunoglobulin domains of fibroblast growth factor receptor 2 (FGFR2). We have developed a simple PCR assay for these mutations in genomic DNA, based on the creation of novel (SfiI) and (BstUI) restriction sites. Analysis of DNA from 70 unrelated patients with Apert syndrome showed that 45 had the Ser252Trp mutation and 25 had the Pro253Arg mutation. Phenotypic differences between these two groups of patients were investigated. Significant differences were found for severity of syndactyly and presence of cleft palate. The syndactyly was more severe with the Pro253Arg mutation, for both the hands and the feet. In contrast, cleft palate was significantly more common in the Ser252Trp patients. No convincing differences were found in the prevalence of other malformations associated with Apert syndrome. We conclude that, although the phenotype attributable to the two mutations is very similar, there are subtle differences. The opposite trends for severity of syndactyly and cleft palate in relation to the two mutations may relate to the varying patterns of temporal and tissue-specific expression of different fibroblast growth factors, the ligands for FGFR2.     

Apert syndrome with preaxial polydactyly showing the typical mutation Ser252Trp in the FGFR2 gene

The Apert syndrome is characterized by craniosynostosis and syndactyly of hands and feet. Although most cases are sporadic, an autosomal dominant mode of inheritance is well documented. Two mutations in the FGFR2 gene (Ser252Trp and Pro253Arg) account for most of the cases. We report a patient with a rare form of Apert syndrome with polydactyly. The proposita has turribrachycephaly. complete syndactyly of 2nd to 5th digits ("mitten hands" and cutaneous fusion of all toes). The X-rays revealed craniosynostosis of the coronal suture and preaxial polydactyly of hands and feet with distal bony fusion. Molecular analysis found a C755G transversion (Ser252Trp) in the FGFR2 gene. Only eight patients with Apert syndrome and preaxial polydactyly have been reported and this is the first case in which molecular diagnosis is available. On the basis of the molecular findings in this patient, polydactyly should be considered part of the spectrum of abnormalities in the Apert syndrome. This assertion would establish the need for a new molecular classification of the acrocephalopolysyndactylies.     

Apert syndrome mutant FGFR2 and its soluble form reciprocally alter osteogenesis of primary calvarial osteoblasts

Apert syndrome is characterized by craniosynostosis and syndactyly, and is predominantly caused by mutation of either S252W or P253W in the fibroblast growth factor receptor (FGFR) 2 gene. In this study, we characterized the effects of one of the mutations (S252W) using primary calvarial osteoblasts derived from transgenic mice, Ap-Tg and sAp-Tg, that expressed an Apert-type mutant FGFR2 (FGFR2IIIc-S252W; FGFR2IIIc-Ap), and the soluble form (extracellular domain only) of the mutant FGFR2 (sFGFR2IIIc-Ap), respectively. Compared to WT-derived osteoblasts, osteoblasts from Ap-Tg mouse showed a higher proliferative activity and enhanced differentiation, while those from sAp-Tg mouse exhibited reduced potential for proliferation and osteogenic differentiation. When transplanted with β-tricalcium phosphate (β-TCP) granules into immunodeficient mice, Ap-Tg-derived osteoblasts showed a higher bone forming capacity, whereas sAp-Tg-derived osteoblasts were completely deficient for this phenotype. Phosphorylation of extracellular signal-regulated kinase (ERK), MEK, PLCγ, and p38 was increased in Ap-Tg-derived osteoblasts, whereas phosphorylation of these signaling molecules was reduced in sAp-Tg-derived osteoblasts. Interestingly, when these experiments were carried out using osteoblasts from the mice generated by crossing Ap-Tg and sAp-Tg (Ap/sAp-Tg), which co-expressed FGFR2IIIc-Ap and sFGFR2IIIc-Ap, the results were comparable to those obtained from WT-derived osteoblasts. Taken together, these results indicate that osteoblasts expressing FGFR2IIIc-Ap proliferate and differentiate via highly activated MEK, ERK, and p38 pathways, while these pathways are suppressed in osteoblasts expressing sFGFR2IIIc-Ap. Our findings also suggest that altered FGFR2IIIc signaling in osteoblasts is mostly responsible for the phenotypes seen in Apert syndrome, therefore these osteoblast cell lines are useful tools for investigating the pathogenesis of Apert syndrome.     

Understanding the molecular basis of Apert syndrome

Apert syndrome, first described in 1906, is one of the most severe of the craniosynostosis syndromes and is further characterized by midface hypoplasia, syndactyly, and other visceral abnormalities. Affected individuals generally require lifelong management by a multidisciplinary team of health care specialists. Apert syndrome results almost exclusively from one or the other of two point mutations in fibroblast growth factor receptor 2. Tremendous scientific advances have been made recently in understanding the molecular basis for Apert syndrome through clinical genetic, biochemical, and structural approaches. In this review, the authors provide the clinician with a basic overview of these findings and their therapeutic implications.     

A soluble form of fibroblast growth factor receptor 2 (FGFR2) with S252W mutation acts as an efficient inhibitor for the enhanced osteoblastic differentiation caused by FGFR2 activation in Apert syndrome

Apert syndrome is an autosomal dominant disease characterized by craniosynostosis and bony syndactyly associated with point mutations (S252W and P253R) in the fibroblast growth factor receptor (FGFR) 2 that cause FGFR2 activation. Here we investigated the role of the S252W mutation of FGFR2 on osteoblastic differentiation. Osteoblastic cells derived from digital bone in two Apert patients with the S252W mutation showed more prominent alkaline phosphatase activity, osteocalcin and osteopontin mRNA expression, and mineralized nodule formation compared with the control osteoblastic cells derived from two independent non-syndromic polydactyly patients. Stable clones of the human MG63 osteosarcoma cells (MG63-Ap and MG63-IIIc) overexpressing a splice variant form of FGFR2 with or without the S252W mutation (FGFR2IIIcS252W and FGFR2IIIc) showed a higher RUNX2 mRNA expression than parental MG63 cells. Furthermore MG63-Ap exhibited a higher osteopontin mRNA expression than did MG63-IIIc. The enhanced osteoblastic marker gene expression and mineralized nodule formation of the MG63-Ap was inhibited by the conditioned medium from the COS-1 cells overexpressing the soluble FGFR2IIIcS252W. Furthermore the FGF2-induced osteogenic response in the mouse calvarial organ culture system was blocked by the soluble FGFR2IIIcS252W. These results show that the S252W mutation in the FGFR2 gene enhances the osteoblast phenotype in human osteoblasts and that a soluble FGFR2 with the S252W mutation controls osteoblast differentiation induced by the S252W mutation through a dominant negative effect on FGFR2 signaling in Apert syndrome.     

Molecular diagnosis of bilateral coronal synostosis.

The authors performed a prospective study evaluating molecular diagnosis in patients with bilateral coronal synostosis. The patients were divided into two groups: (1) those clinically classified as having Apert, Crouzon, or Pfeiffer syndrome and (2) those clinically unclassified and labeled as having brachycephaly. Blood samples were drawn for genomic DNA analysis from 57 patients from 1995 to 1997. Polymerase chain reactions were performed using primers flanking exons in FGFR 1, 2, and 3. Each exon was screened for mutations using single-strand confirmation polymorphism, and mutations were identified by DNA sequencing. Mutations in FGFR2 or FGFR3 were found in all patients (n = 38) assigned a phenotypic (eponymous) diagnosis. All Apert syndrome patients (n = 13) carried one of the two known point mutations in exon 7 of FGFR2 (Ser252Trp and Pro253Arg). Twenty-five patients were diagnosed as having either Crouzon or Pfeiffer syndrome. Five patients with Crouzon syndrome of variable severity had mutations in exon 7 of FGFR2. Fifteen patients (12 with Crouzon, 3 with Pfeiffer) had a mutation in exon 9 of FGFR2, many of which involved loss or gain of a cysteine residue. A wide phenotypic range was observed in patients with identical mutations, including those involving cysteine. Two patients labeled as having Crouzon syndrome had the Pro250Arg mutation in exon 7 of FGFR3. All three patients with the crouzonoid phenotype and acanthosis nigricans had the same mutation in exon 10 of FGFR3 (Ala391Glu). This is a distinct disorder, characterized by jugular foraminal stenosis, Chiari I anomaly, and intracranial venous hypertension. Mutations were found in 14 of 19 clinically unclassifiable patients. Three mutations were in exon 9, and one was in the donor splice site of intron 9 on FGFR2. The most common mutation discovered in this group was Pro250Arg in exon 7 of FGFR3. These patients (n = 10) had either bilateral or unilateral coronal synostosis, minimal midfacial hypoplasia with class I or class II occlusion, and minor brachysyndactyly. No mutations in FGFR 1, 2, or 3 were detected in five patients with nonspecific brachycephaly. In conclusion, a molecular diagnosis was possible in all patients (n = 38) given a phenotypic (eponymous) diagnosis. Different phenotypes observed with identical mutations probably resulted from modulation by their genetic background. A molecular diagnosis was made in 74 percent of the 19 unclassified patients in this series; all mutations were in FGFR2 or FGFR3. Our data and those of other investigators suggest that we should begin integrating molecular diagnosis with phenotypic diagnosis of craniosynostoses in studies of natural history and dysmorphology and in analyses of surgical results.     

Trp290Cys mutation in exon IIIa of the fibroblast growth factor receptor 2 (FGFR2) gene is associated with Pfeiffer syndrome

Pfeiffer syndrome is a skeletal disorder characterized by craniosynostosis associated with foot and hand anomalies. Mutations in the genes encoding fibroblast growth factor receptors 1 and 2 (FGFR1 and FGFR2) have recently been implicated in the aetiology of such a syndrome, as well as of other craniosynostotic conditions. We now report a novel missense mutation, a G to C transversion at position 1049 (exon IIIa) of FGFR2, detected in a patient with severe Pfeiffer clinical features. The mutation results in the substitution of a cysteine for tryptophan-290 in the third immunoglobulin-like domain and affects both spliceoforms of FGFR2. Mutations causing replacement of tryptophan-290 have also been reported previously in Crouzon syndrome, a similar but clinically distinct craniosynostotic disorder. This finding confirms the involvement of mutations of FGFR2 exon IIIa in Pfeiffer syndrome, and emphasizes both the extensive heterogeneity of the FGFR2 mutations that result in the Pfeiffer phenotype and the perturbations caused by unpaired cysteine residues in receptor dimerization and transduction of the FGFs signal. Received: 15 August 1996 / Revised: 19 October 1996     

FGF/FGFR signaling coordinates skull development by modulating magnitude of morphological integration: evidence from Apert syndrome mouse models

The fibroblast growth factor and receptor system (FGF/FGFR) mediates cell communication and pattern formation in many tissue types (e.g., osseous, nervous, vascular). In those craniosynostosis syndromes caused by FGFR1-3 mutations, alteration of signaling in the FGF/FGFR system leads to dysmorphology of the skull, brain and limbs, among other organs. Since this molecular pathway is widely expressed throughout head development, we explore whether and how two specific mutations on Fgfr2 causing Apert syndrome in humans affect the pattern and level of integration between the facial skeleton and the neurocranium using inbred Apert syndrome mouse models Fgfr2(+/S252W) and Fgfr2(+/P253R) and their non-mutant littermates at P0. Skull morphological integration (MI), which can reflect developmental interactions among traits by measuring the intensity of statistical associations among them, was assessed using data from microCT images of the skull of Apert syndrome mouse models and 3D geometric morphometric methods. Our results show that mutant Apert syndrome mice share the general pattern of MI with their non-mutant littermates, but the magnitude of integration between and within the facial skeleton and the neurocranium is increased, especially in Fgfr2(+/S252W) mice. This indicates that although Fgfr2 mutations do not disrupt skull MI, FGF/FGFR signaling is a covariance-generating process in skull development that acts as a global factor modulating the intensity of MI. As this pathway evolved early in vertebrate evolution, it may have played a significant role in establishing the patterns of skull MI and coordinating proper skull development.     

Apert syndrome with omphalocele: A case report

Apert syndrome is a genetic disorder known as acrocephalopolysyndactyly type 1 caused by mutations in the fibroblast growth factor receptor 2 and characterized by coronal craniosynostosis, symmetric bone and skin syndactyly of hands and feet, and craniofacial dysmorphic features. The estimated prevalence of this syndrome is 10 to 15.5 cases per 1,000,000 live births. Apert syndrome has considerable clinical variability. We present a case of Apert syndrome and associated features reported to the National Registry of Congenital Anomalies of Argentina (RENAC). The reported case had omphalocele, esophageal atresia, and mega cisterna magna. The last two signs were reported several times as part of the clinical presentation of Apert syndrome. To our knowledge, this is the second reported case diagnosed with Apert syndrome associated with omphalocele. Birth Defects Research (Part A), 100:726–729, 2014. © 2014 Wiley Periodicals, Inc.     

Mutation detection in FGFR2 craniosynostosis syndromes

Five autosomal dominant craniosynostosis syndromes (Apert, Crouzon, Pfeiffer, Jackson-Weiss and Crouzon syndrome with acanthosis nigricans) result from mutations in FGFR genes. Fourteen unrelated patients with FGFR2-related craniosynostosis syndromes were screened for mutations in exons IIIa and IIIc of FGFR2. Eight of the nine mutations found have been reported, but one patient with Pfeiffer syndrome was found to have a novel G-to-C splice site mutation at –1 relative to the start of exon IIIc. Of those mutations previously reported, the mutation C1205G was unusual in that it was found in two related patients, one with clinical features of Pfeiffer syndrome and the other having mild Crouzon syndrome. This degree of phenotypic variability shows that the clinical features associated with a specific mutation do not necessarily breed true. Received: 4 June 1996 / Revised: 3 September 1996     

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family

The human fibroblast growth factor receptor (FGFR) genes play important roles in normal vertebrate development. Mutations in the human FGFR2 gene have been associated with many craniosynostotic syndromes and malformations, including Crouzon, Pfeiffer, Apert, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes, and Kleeblaatschadel (cloverleaf skull) deformity. The mutations identified to date are concentrated in the previously characterized region of FGFR2 that codes for the extracellular IgIII domain of the receptor protein. The search for mutations in other regions of the gene, however, has been hindered by lack of knowledge of the genomic structure. Using a combination of genomic library screening, long-range PCR, and genomic walking, we have characterized the genomic structure of nearly the entire human FGFR2 gene, including a delineation of the organization and size of all introns and exons and determination of the DNA sequences at the intron/exon boundaries. Comparative analysis of the human FGFR gene family reveals that the genomic organization of the FGFRs is relatively conserved. Moreover, alignment of the amino acid sequences shows that the four corresponding proteins share 46% identity overall, with up to 70% identity between individual pairs of FGFR proteins. However, the FGFR2 gene contains an additional exon not found in other members of the family, and it also has much larger intronic sequences throughout the gene. Remarkable similarities in genomic organization, intron/exon boundaries, and intron sizes are found between the human and mouse FGFR2 genes. Knowledge gained from this study of the human FGFR2 gene structure may prove useful in future screening studies designed to find additional mutations associated with craniosynostotic syndromes, and in understanding the molecular and cell biology of this receptor family.     

FGF2 effects in periosteal fibroblasts bearing the FGFR2 receptor Pro253 Arg mutation

AIM: A growing number of mutations mapped in the receptor gene for fibroblast growth factor have been implicated in several cranial development disorders including the Apert and Crouzon syndromes. The present paper investigated cellular mechanisms underlying Apert phenotype, by analyzing the effects of FGF2 in primary cultures of Apert periosteal fibroblasts carrying the FGFR2 Pro253Arg mutation. RESULTS: FGF2 administration significantly decreased extracellular matrix production in mutant cells by stimulating degradative enzymatic activities. Gene expression analysis revealed that decorin and biglycan, two proteoglycans involved in collagen fibrillogenesis, were more expressed in mutant cells and down-regulated by FGF2. FGF2 receptor binding showed little differences in high affinity receptor counts between mutant and wild-type cells, while we showed for the first time that low affinity receptors are significantly fewer in mutant cells. Differences were found in Crouzon syndrome, where both high and low affinity receptor counts were up-regulated. CONCLUSIONS: The different mutation and low affinity receptor regulation in mutant receptors support the hypothesis that the impact on the activity of the ligand-receptor complex could allow distinct modes of FGF2 activation in Apert and Crouzon syndromes, which interfere with the FGFR2 signalling cascade.     

Predisposition for cysteine substitutions in the immunoglobulin-like chain of FGFR2 in Crouzon syndrome

Four cases of Crouzon syndrome, one familial and three sporadic, were investigated for mutations in exon B of the fibroblast growth factor receptor 2 (FGFR2) gene. In the familial case, a mutation was found at codon 340 that exchanged tyrosine for histidine. Mutations at codon 342, detected in the three sporadic cases, replaced a cysteine by another amino acid. While three of the mutations have been described before, the fourth mutation, a CG transversion at codon 342 in one of the sporadic cases, has not been recognized previously. Compilation of all exon B mutations in Crouzon syndrome described to date revealed that 6 of the 8 sporadic and 2 of the 9 familial cases have mutations in codon 342. These mutations caused the substitution of cysteine for another amino acid. Given that a mutation in codon 342 was found in 8 out of 17 cases and that in 9 cases the mutation occurred at five additional positions, codon 342 of exon B of the FGFR2 gene may be predisposed to mutations in Crouzon syndrome.     

FGFR3 mutation in thanatophoric dysplasia type 1 with bilateral cystic renal dysplasia: coincidence or a new association?

Thanatophoric dysplasia (TD) is a lethal dwarfism condition due to missense mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Examination of TD patients reveals mainly the involvement of the skeletal system and the brain, but also renal and cardiovascular anomalies have been described. We report the prenatal detection of TD type 1 (TD1) associated with bilateral cystic renal dysplasia (CRD) Potter's type II, in which the molecular analysis reveals the typical Arg248Cys substitution in the FGFR3 gene. CRD has not been previously described in TD or other conditions due to FGFR3 mutations, but occurs in Apert syndrome (due to FGFR2 mutations). The possible involvement of renal developmental defect in FGFR3 mutations is discussed.     

Therapeutic Effect of Nanogel-Based Delivery of Soluble FGFR2 with S252W Mutation on Craniosynostosis

Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIc^S252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIc^S252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIc^S252W, and FGFR2IIIb^S252W. Administration of sFGFR2IIIc^S252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2^S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIc^S252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2^S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIc^S252W could be effective for treating this disorder.