Role of the extracellular matrix and growth factors in skull morphogenesis and in the pathogenesis of craniosynostosis

The complex and largely obscure regulatory processes that underlie ossification and fusion of the sutures during skull morphogenesis are dependent on the conditions of the extracellular microenvironment. The concept that growth factors are involved in the pathophysiology of craniosynostosis due to premature fusion of skull bone sutures, is supported by recent genetic data. Crouzon and Apert syndromes, for example, are characterized by point mutations in the extracellular or transmembrane domains of fibroblast growth factor-2 receptor. In primary cultures of periosteal fibroblasts and osteoblasts obtained from Apert and Crouzon patients, we observed that Crouzon and Apert cells behaved differently with respect to normal cells as regards the expression of cytokines and extracellular matrix (ECM) macromolecule accumulation. Further modulation of ECM components observed after the addition of cytokines provides support for an autocrine involvement of these cytokines in Crouzon and Apert phenotype. Changes in ECM composition could explain the altered osteogenic process and account for pathological variations in cranial development. We suggest that a correlation exists between in vitro phenotype, clinical features and genotype in the two craniosynostotic syndromes. New research into signal transduction pathways should establish further connections between the mutated genotype and the molecular biology of the cellular phenotype.     

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.     

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     

Craniosynostosis genetics: The mystery unfolds

Craniosynsostosis syndromes exhibit considerable phenotypic and genetic heterogeneity. Sagittal synostosis is common form of isolated craniosynostosis. The sutures involved, the shape of the skull and associated malformations give a clue to the specific diagnosis. Crouzon syndrome is one of the most common of the craniosynostosis syndromes. Apert syndrome accounts for 4.5% of all craniosynostoses and is one of the most serious of these syndromes. Most syndromic craniosynostosis require multidisciplinary management. The following review provides a brief appraisal of the various genes involved in craniosynostosis syndromes, and an approach to diagnosis and genetic counseling.     

Genomic Screening of Fibroblast Growth-Factor Receptor 2 Reveals a Wide Spectrum of Mutations in Patients with Syndromic Craniosynostosis

It has been known for several years that heterozygous mutations of three members of the fibroblast growth-factor-receptor family of signal-transduction molecules-namely, FGFR1, FGFR2, and FGFR3-contribute significantly to disorders of bone patterning and growth. FGFR3 mutations, which predominantly cause short-limbed bone dysplasia, occur in all three major regions (i.e., extracellular, transmembrane, and intracellular) of the protein. By contrast, most mutations described in FGFR2 localize to just two exons (IIIa and IIIc), encoding the IgIII domain in the extracellular region, resulting in syndromic craniosynostosis including Apert, Crouzon, or Pfeiffer syndromes. Interpretation of this apparent clustering of mutations in FGFR2 has been hampered by the absence of any complete FGFR2-mutation screen. We have now undertaken such a screen in 259 patients with craniosynostosis in whom mutations in other genes (e.g., FGFR1, FGFR3, and TWIST) had been excluded; part of this screen was a cohort-based study, enabling unbiased estimates of the mutation distribution to be obtained. Although the majority (61/62 in the cohort sample) of FGFR2 mutations localized to the IIIa and IIIc exons, we identified mutations in seven additional exons-including six distinct mutations of the tyrosine kinase region and a single mutation of the IgII domain. The majority of patients with atypical mutations had diagnoses of Pfeiffer syndrome or Crouzon syndrome. Overall, FGFR2 mutations were present in 9.8% of patients with craniosynostosis who were included in a prospectively ascertained sample, but no mutations were found in association with isolated fusion of the metopic or sagittal sutures. We conclude that the spectrum of FGFR2 mutations causing craniosynostosis is wider than previously recognized but that, nevertheless, the IgIIIa/IIIc region represents a genuine mutation hotspot.     

Comparative study of normal, Crouzon, and Apert craniofacial morphology using finite element scaling analysis

Finite element scaling analysis is used to study differences in morphology between the craniofacial complex of normal individuals and those affected with the syndromes of Apert and Crouzon. Finite element scaling quantifies the differences in shape and size between forms without reference to any fixed, arbitrary registration point or orientation line and measures the amount of form change required to deform one object into another. Two-dimensional coordinates of landmarks digitized from annual sets of cephalometric radiographs were used in the analysis. A simple tabulation shows no difference in variances between the normal and pathological samples. A test of mean differences depicts the Apert and Crouzon morphologies as significantly different from normal. The Apert palate differs from normal in shape in the older age groups analyzed, and palatal size differences are most common at the posterior nasal spine. The Apert pituitary fossa and basi-occiput are significantly larger than normal. The Crouzon pituitary fossa is also larger than normal, but the difference is not always significant. The typical morphology of the Crouzon nose is due more to differences in shape than size. The Crouzon basi-occiput is significantly smaller than normal. An age association of the differences between the normal and pathological craniofacies was found in Apert syndrome but not in Crouzon syndrome. Apert syndrome is characterized by a more homogeneous pattern of craniofacial dysmorphology from 6 months to 18 years of age than Crouzon syndrome.     

No evidence of genetic heterogeneity in Crouzon craniofacial dysostosis

Crouzon craniofacial dysostosis (CFD) is an autosomal dominant form of craniosynostosis characterized by an abnormal skull shape, with hypertelorism, prominent eyes and midfacial retrusion. Recently, a gene for CFD has been mapped to chromosome 10q25-q26 and mutations in exon B of the fibroblast growth factor receptor 2 (FGFR2) gene have been identified. Here, we report the mapping of a CFD gene to chromosome 10q by close linkage to probe AFMa197wbl at locus D10 S1483 in six unrelated families of French ancestry (Z _max = 4.69 at = 0) and provide additional evidence of genetic homogeneity of this condition. In addition, we report a novel mutation in exon B of the FGFR2 gene (Cys 342 Trp) in familial CFD and describe recurrent mutations at codon 342 as a particularly frequent event in CFD. Since mutations in the extracellular domain of the FGFR2 gene are observed in a few clinically distinct craniosynostosis syndromes (CFD, Jackson-Weiss, Apert and Pfeiffer), the present study gives support to the variable clinical expression of FGFR2 mutations in humans.     

Mutational identification of fibroblast growth factor receptor 1 and fibroblast growth factor receptor 2 genes in craniosynostosis in Indian population

OBJECTIVE:

The Objective of this study was to identify the association of mutation of fibroblast growth factor receptor 1 (FGFR1), FGFR2 genes with syndromic as well as non-syndromic craniosynostosis in Indian population.

MATERIALS AND METHODS:

Retrospective analysis of our records from January 2008 to December 2012 was done. A total of 41 cases satisfying the inclusion criteria and 51 controls were taken for the study. A total volume of 3 ml blood from the patient as well as parents was taken. Deoxyribonucleic acid extracted using phenol chloroform extraction method followed by polymerase chain reaction-restriction fragment length polymorphism method.

RESULTS:

There were 33 (80.4%) non-syndromic cases of craniosynostosis while 8 (19.5%) were syndromic. Out of these 8 syndromic cases, 4 were Apert syndrome, 3 were Crouzon syndrome and 1 Pfeiffer syndrome. Phenotypically the most common non-syndromic craniosynostosis was scaphocephaly (19, 57.7%) followed by plagiocephaly in (14, 42.3%). FGFR1 mutation (Pro252Arg) was seen in 1 (2.4%) case of non-syndromic craniosynostosis while no association was noted either with FGFR1 or with FGFR2 mutation in syndromic cases. None of the control group showed any mutation.

CONCLUSION:

Our study proposed that FGFR1, FGFR2 mutation, which confers predisposition to craniosynostosis does not exist in Indian population when compared to the western world.     

FGFR2 exon IIIa and IIIc mutations in Crouzon, Jackson-Weiss, and Pfeiffer syndromes: evidence for missense changes, insertions, and a deletion due to alternative RNA splicing.

Fibroblast growth factor receptor 2 (FGFR2) mutations have been associated with the craniosynostotic conditions Crouzon, Jackson-Weiss, and Pfeiffer syndromes. Previously, mutations were described in the exons IIIa and IIIc, which form the extracellular, third immunoglobulin-like domain (IgIII) and adjacent linker regions, both of which are normally involved in ligand binding. For all three conditions, mutations were found in exon IIIc. Only in Crouzon syndrome were mutations identified in exon IIIa. In this study, 39 cases with one of these three conditions were screened for exon IIIa or IIIc mutations. Eleven mutations are reported in 17 unrelated cases. Mutations in exon IIIa are identified for not only Crouzon but also Jackson-Weiss and Pfeiffer syndromes. Four mutations in either exon IIIa or exon IIIc reported only in Crouzon syndrome are present also in one of the other two syndromes. Two insertions, one in exon IIIa in a Crouzon syndrome patient and the other in exon IIIc in a Pfeiffer syndrome patient, were observed. The latter mutation has the same alternative RNA splicing effect as a reported synonymous mutation for Crouzon syndrome. A missense mutation was detected in one Pfeiffer syndrome family in which two members had craniosynostosis without limb anomalies. The inter- and intrafamilial variability in expression of FGFR2 mutations suggests that these three syndromes, presumed to be clinically distinct, are instead representative of a spectrum of related craniosynostotic and digital disorders.     

Jackson-Weiss syndrome: identification of two novel FGFR2 missense mutations shared with Crouzon and Pfeiffer craniosynostotic disorders

Jackson-Weiss syndrome is a rare skeletal disorder characterized by craniosynostosis associated with foot malformations. This condition is inherited as an autosomal dominant trait with complete penetrance and wide phenotypic heterogeneity. Mutations in the fibroblast growth factor receptor 2 (FGFR2) gene have been recently identified as causes of this syndrome and of at least four other craniosynostotic disorders, namely the Apert, Beare-Stevenson cutis gyrata, Crouzon and Pfeiffer syndromes. We report two novel FGFR2 missense mutations associated with phenotypes consistent with Jackson-Weiss syndrome. Both nucleotide changes predict a serine for cysteine-342 substitution in the second half of the third immunoglobulin-like domain. The replacement of Cys-342 with arginine has previously been reported in one of the three Jackson-Weiss cases investigated. Interestingly, both Cys342Ser and Cys342Arg substitutions have been found to be associated with the Crouzon and Pfeiffer phenotypes; a phenotypic heterogeneity, Crouzon vs Jackson-Weiss clinical features, has been also observed for Gln289Pro and Ala344Gly amino-acid changes. This finding indicates the genetic homogeneity of the “heterogeneous” Jackson-Weiss phenotype and a common molecular basis for these apparently “clinically distinct” craniosynostotic disorders. Received: 13 February 1997 / Accepted: 10 June 1997     

Mutation associated with Crouzon syndrome causes ligand-independent dimerization and activation of FGF receptor-2

FGF signaling is clearly important for proper bone development, and several autosomally dominant forms of genetic bone disorders have been mapped to FGF receptors 1, 2, and 3. We have studied the biological effects of the most commonly mutated cysteine residue in FGFR-2 which is detected in individuals with Crouzon syndrome, an autosomally dominant trait which causes premature fusion of the skull bones (craniosynostosis). This Crouzon mutation replaces the cysteine at position 342 with tyrosine, thus disrupting the formation of the third immunoglobulin (Ig)-like loop in the extracellular portion of the receptor. By transfecting mutated and wild-type receptors into a variety of cell lines, we have shown that the C342Y mutation in FGFR-2 produces a receptor which is constitutively activated and capable of transforming NIH3T3 cells and preventing the differentiation of C2 myoblasts in the absence of ligand. Constitutive activation appears to result from the ability of this receptor to form stable interreceptor dimers which involve disulfide bonds between the remaining free cysteine in the mutant receptor. The altered conformation of the third Ig-like domain in the mutated receptor also results in a drastically reduced ability to bind FGF-1 or FGF-2 and in a reduced level of receptor glycosylation. Thus it appears that Crouzon syndrome results from constitutive activation of FGFR-2 and that uncontrolled FGF signaling produces alterations of intramembranous bone development and premature closing of cranial sutures. J. Cell. Physiol. 172:117–125, 1997. © 1997 Wiley-Liss, Inc.     

Clustering of FGFR2 gene mutations inpatients with Pfeiffer and Crouzon syndromes (FGFR2-associated craniosynostoses)

A cohort of 36 unrelated German patients with craniosynostosis syndromes of the Crouzon and Pfeiffer type were analyzed for FGFR mutations. Mutations in FGFR2 were identified in 25 Crouzon and 5 Pfeiffer syndrome patients, whereas no sequence alterations were found in the remaining patients, even after screening of the relevant parts of FGFR1, FGFR3, and TWIST. Mutations in FGFR2 clustered at two critical cysteine residues, 278 and 342, which were involved in 18 of 30 cases (60%). These two mutational hot spots, therefore, are prime targets for an efficient mutation-screening strategy. The spectrum of mutations overlapped the two syndromes and thus reflected the phenotypic similarities observed in both patient groups. In 21 families, the origin of the mutation could be traced by analyzing parents and relatives. Eleven mutations arose de novo, indicating a high mutation rate for FGFR2. In the 10 familial cases, the clinical presentation varied considerably within the pedigree, but both syndromes "bred true," i.e., a Pfeiffer syndrome phenotype was never observed in a Crouzon syndrome family and vice versa.     

A novel insertion in the FGFR2 gene in a patient with Crouzon phenotype and sacrococcygeal tail

BACKGROUND: Mutations in the FGFR2 gene are present in several syndromes with craniosynostosis, such as Pfeiffer's, Apert's, and Crouzon's. CASE: We report a case of craniosynostosis (Crouzon phenotype) with tracheal anomalies and a sacrococcygeal tail. In addition, the patient shows dolichoplagiocephaly, prominent occiput, proptosis, mild facial asymmetry, strabismus, small umbilical hernia, and syndactyly of the second and third toes. CONCLUSIONS: Molecular analysis of the FGFR2 gene in this patient revealed a 12-bp insertion (GAGGAGACCTAG) at nucleotide 824. This is an in-frame mutation that adds four amino acid residues to the immunoglobulin IIIa (IgIIIa) domain of the putative protein. This is the first report of an in-frame insertion in exon 8 of FGFR2 in a child with Crouzon's syndrome, tracheal anomalies, and a tail.     

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.     

Mesodermal expression of Fgfr2S252W is necessary and sufficient to induce craniosynostosis in a mouse model of Apert syndrome

Coordinated growth of the skull and brain are vital to normal human development. Craniosynostosis, the premature fusion of the calvarial bones of the skull, is a relatively common pediatric disease, occurring in 1 in 2500 births, and requires significant surgical management, especially in syndromic cases. Syndromic craniosynostosis is caused by a variety of genetic lesions, most commonly by activating mutations of FGFRs 1-3, and inactivating mutations of TWIST1. In a mouse model of TWIST1 haploinsufficiency, cell mixing between the neural crest-derived frontal bone and mesoderm-derived parietal bone accompanies coronal suture fusion during embryonic development. However, the relevance of lineage mixing in craniosynostosis induced by activating FGFR mutations is unknown. Here, we demonstrate a novel mechanism of suture fusion in the Apert Fgfr2(S252W) mouse model. Using Cre/lox recombination we simultaneously induce expression of Fgfr2(S252W) and β-galactosidase in either the neural crest or mesoderm of the skull. We show that mutation of the mesoderm alone is necessary and sufficient to cause craniosynostosis, while mutation of the neural crest is neither. The lineage border is not disrupted by aberrant cell migration during fusion. Instead, the suture mesenchyme itself remains intact and is induced to undergo osteogenesis. We eliminate postulated roles for dura mater or skull base changes in craniosynostosis. The viability of conditionally mutant mice also allows post-natal assessment of other aspects of Apert syndrome.     

Interleukin secretion, proteoglycan and procollagen alpha(1)(I) gene expression in Crouzon fibroblasts treated with basic fibroblast growth factor

The present study provides the first evidence that fibroblasts obtained from patients affected by Crouzon syndrome, a rare craniosynostosis, despite mutations in the high-affinity bFGF receptor retain their capacity to respond to bFGF. The growth factor reduces IL-1 secretion, downregulates biglycan and procollagen alpha(1)(I), and increases betaglycan expression. Since betaglycan is a co-receptor for bFGF signalling, an alternative signal transduction pathway is suggested in Crouzon fibroblasts, to explain the documented changes in ECM macromolecule production.