Inherited FGFR2 mutation in a Chinese patient with Crouzon syndrome and luxation of bulbus oculi provoked by trauma: a case report.

BACKGROUND: Crouzon syndrome (CS), which results from fibroblast growth factor receptor 2 mutations, is associated with craniosynostosis, exophthalmos, and other symptoms. Herein, we report the genetic abnormalities detected in a Chinese family with autosomal dominant CS, combined with luxation of the eyeball. This luxation was a consequence of the trauma to the shallow orbits. CASE PRESENTATION: The proband was a 4-year-old boy. He accidentally fell, following which luxation of the bulbus oculi occurred immediately. Computed tomography and magnetic resonance imaging clearly revealed ocular proptosis. Upon physical examination, the proband, his father, and grandfather had ocular proptosis, shallow orbits, and mid-face hypoplasia. However, their hands and feet were clinically normal. Genomic DNA was extracted from the peripheral blood through a polymerase chain reaction performed for the target sequence. Genetic assessments revealed a heterozygous missense mutation (c.1012G > C, p.G338R) in exon 10 of the human FGFR2, cosegregated with the disease phenotype in this family. These findings confirmed the diagnosis of CS. DISCUSSION: CS is usually caused by FGFR2 mutations. While there are a few reports of luxation of the bulbus oculi in Chinese families with CS, the ocular proptosis, shallow orbits, combined with luxation of eyeball after trauma observed in this patient were particularly interesting. Our findings enhance the current knowledge of traumatic luxation concomitant with CS.

Crouzon syndrome mouse model exhibits cartilage hyperproliferation and defective segmentation in the developing trachea.

Crouzon syndrome is the result of a gain-of-function point mutation in FGFR2. Mimicking the human mutation, a mouse model of Crouzon syndrome (Fgfr2342Y) recapitulates patient deformities, including failed tracheal cartilage segmentation, resulting in a cartilaginous sleeve in the homozygous mutants. We found that the Fgfr2C342Y/C342Y mutants exhibited an increase in chondrocytes prior to segmentation. This increase is due at least in part to over proliferation. Genetic ablation of chondrocytes in the mutant led to restoration of segmentation in the lateral but not central portion of the trachea. These results suggest that in the Fgfr2C342Y/C342Y mutants, increased cartilage cell proliferation precedes and contributes to the disruption of cartilage segmentation in the developing trachea.

Developmental processes regulate craniofacial variation in disease and evolution.

Variation in development mediates phenotypic differences observed in evolution and disease. Although the mechanisms underlying phenotypic variation are still largely unknown, recent research suggests that variation in developmental processes may play a key role. Developmental processes mediate genotype-phenotype relationships and consequently play an important role regulating phenotypes. In this review, we provide an example of how shared and interacting developmental processes may explain convergence of phenotypes in spliceosomopathies and ribosomopathies. These data also suggest a shared pathway to disease treatment. We then discuss three major mechanisms that contribute to variation in developmental processes: genetic background (gene-gene interactions), gene-environment interactions, and developmental stochasticity. Finally, we comment on evolutionary alterations to developmental processes, and the evolution of disease buffering mechanisms.

Molecular analysis of FGFR 2 and associated clinical observations in two Chinese families with Crouzon syndrome.

Crouzon syndrome, a dominantly inherited disorder and the most common type of craniosynostosis syndrome, is caused by mutations in the fibroblast growth factor receptor 2 (FGFR 2) gene, and characterized by craniosynostosis, shallow orbits, ocular proptosis, midface hypoplasia and a curved, beak­like nose. The purpose of the present study was to investigate the fibroblast growth factor receptor 2 (FGFR 2) gene in two Chinese families with Crouzon syndrome and to characterize the associated clinical features. Two families underwent complete ophthalmic examination, and three patients in two families were diagnosed with Crouzon syndrome. Genomic DNA was extracted from leukocytes of peripheral blood samples, which were collected from the family members and 200 unrelated control subjects from the same population. Exons 8 and 10 of the FGFR 2 gene were amplified using polymerase chain reaction analysis and were directly sequenced. Ophthalmic examinations, including best­corrected visual acuity, slit­lamp examination, fundus examination and Computerized Tomography scans, and physical examinations were performed to exclude systemic diseases. These patients were affected with shallow orbits and ocular proptosis, accompanied by midface hypoplasia, craniosynostosis, strabismus or papilloedema, with clinically normal hands and feet. A heterozygous FGFR 2 missense mutation, c.811­812insGAG (p.273insGlu) in exon 8 was identified in the affected individual, but not in the unaffected family members or the normal control individuals in family 1. In family 2, another heterozygous FGFR 2 missense mutation, c.842A>G (P.Tyr281Cys or Y281C), in exon 8 was identified in the affected boy and his mother, but not in the unaffected family members or the normal control individuals. Although FGFR 2 gene mutations and polymorphisms have been reported in various ethnic groups, particularly in the area of osteology, the present study reported for the first time, to the best of our knowledge, the identification of two novel FGFR 2 gene mutations in Chinese patients with Crouzon syndrome.

The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects.

The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/ß cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5-9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2-35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function.

Deficiency of zebrafish fgf20a results in aberrant skull remodeling that mimics both human cranial disease and evolutionarily important fish skull morphologies.

The processes that direct skull remodeling are of interest to both human-oriented studies of cranial dysplasia and evolutionary studies of skull divergence. There is increasing awareness that these two fields can be mutually informative when natural variation mimics pathology. Here we describe a zebrafish mutant line, devoid of blastema (dob), which does not have a functional fgf20a protein, and which also presents cranial defects similar to both adaptive and clinical variation. We used geometric morphometric methods to provide quantitative descriptions of the effects of the dob mutation on skull morphogenesis. In combination with "whole-mount in situ hybridization" labeling of normal fgf20a expression and assays for osteoblast and osteoclast activity, the results of these analyses indicate that cranial dysmorphologies in dob zebrafish are generated by aberrations in post-embryonic skull remodeling via decreased osteoblasotgenesis and increased osteoclastogenesis. Mutational effects include altered skull vault geometries and midfacial hypoplasia that are consistent with key diagnostic signs for multiple human craniofacial syndromes. These phenotypic shifts also mimic changes in the functional morphology of fish skulls that have arisen repeatedly in several highly successful radiations (e.g., damselfishes and East-African rift-lake cichlids). Our results offer the dob/fgf20a mutant as an experimentally tractable model with which to examine post-embryonic skull development as it relates to human disease and vertebrate evolution.

ALX4 dysfunction disrupts craniofacial and epidermal development.

Genetic control of craniofacial morphogenesis requires a complex interaction of numerous genes encoding factors essential for patterning and differentiation. We present two Turkish families with a new autosomal recessive frontofacial dysostosis syndrome characterized by total alopecia, a large skull defect, coronal craniosynostosis, hypertelorism, severely depressed nasal bridge and ridge, bifid nasal tip, hypogonadism, callosal body agenesis and mental retardation. Using homozygosity mapping, we mapped the entity to chromosome 11p11.2-q12.3 and subsequently identified a homozygous c.793C-->T nonsense mutation in the human ortholog of the mouse aristaless-like homeobox 4 (ALX4) gene. This mutation is predicted to result in a premature stop codon (p.R265X) of ALX4 truncating 146 amino acids of the protein including a part of the highly conserved homeodomain and the C-terminal paired tail domain. Although the RNA is stable and not degraded by nonsense-mediated RNA decay, the mutant protein is likely to be non-functional. In a skin biopsy of an affected individual, we observed a hypomorphic interfollicular epidermis with reduced suprabasal layers associated with impaired interfollicular epidermal differentiation. Hair follicle-like structures were present but showed altered differentiation. Our data indicate that ALX4 plays a critical role both in craniofacial development as in skin and hair follicle development in human.

Crouzon's syndrome: tooth morphological and microanalytical evaluation.

AIM: To assess the morphological and microanalytical structure of teeth in Crouzon's Syndrome. METHODS: A 21 year old patient with this condition was evaluated to demonstrate tooth morphology in this syndrome. Assessment of 4 teeth from this patient consisted of morphological analysis in SEM as well as microanalysis of elements. Sections were created in order to assess any defects of the enamel, dentine, root, and any demineralisation. RESULTS: Micro-analysis using x-ray techniques revealed lower levels of calcium and magnesium from this patient with Crouzon's as compared with samples from a normal healthy patient of comparable age. Furthermore, in the subject's teeth notable amounts of lead were found. CONCLUSION: Histological images revealed morphological abnormalities especially in dentine. Furthermore, in the dentine levels of calcium and magnesium were lowered according to the microanalytical tests.

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.

Transmembrane helix heterodimerization in lipid bilayers: probing the energetics behind autosomal dominant growth disorders.

Here, we show that the energetics of transmembrane helix heterodimer formation can be characterized in liposomes using Förster resonance energy transfer (FRET). We present the theory and the protocol for measuring the free energy of heterodimerization, and the total (hetero and homo-dimeric) dimer fraction. We use the presented methodology to determine the propensity for heterodimer formation between wild-type fibroblast growth factor receptor 3 (FGFR3) transmembrane domain and the Ala391Glu mutant, linked to Crouzon syndrome with acanthosis nigricans.

Crouzon's syndrome: differential in vitro secretion of bFGF, TGFbeta I isoforms and extracellular matrix macromolecules in patients with FGFR2 gene mutation.

In the Crouzon's syndrome the cranial morphogenic processes are altered due to the early fusion of cranial sutures. We analysed the phenotype of cultured fibroblasts from normal subjects and from Crouzon patients with a specific fibroblast growth factor receptor 2 mutation resulting in a Cys 342 Tyr substitution within the third immunoglobulin domain. Crouzon fibroblasts differed from normal fibroblasts in their extracellular matrix macromolecule accumulation. In Crouzon fibroblasts glycosaminoglycans and fibronectin were decreased and type I collagen increased. As transforming growth factors beta (TGF beta) and basic fibroblasts growth factor (bFGF) together regulate extracellular matrix deposition, we evaluated TGF beta(1), TGF beta(3) and bFGF production by Crouzon and normal fibroblasts. TGF beta(1), TGFb(3) and bFGF levels were lower while TGF beta(1) mRNA transcripts were higher in Crouzon cells. As the increased TGF beta(1) gene expression did not translate into a parallel increase of secreted TGF beta(1), control of TGF beta(1) secretion may be mainly post-transcriptional. Furthermore, adding bFGF increased TGF beta(1) and TGF beta(3) secretion, suggesting the drop may be due to the altered signal transduction of bFGF. These innovative data suggest the in vitro differences between normal and Crouzon fibroblasts may be due to an imbalance in TGF beta and bFGF levels which alters the microenvironment where morphogenesis takes place.

Basic fibroblast growth factor: effects on matrix remodeling, receptor expression, and transduction pathway in human periosteal fibroblasts with FGFR2 gene mutation.

The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.

[Complex diagnosis of congenital cranial dysostosis in children]. / Kompleksnaia diagnostika vrozhdennykh kraniodizostozov u detei.

Ten patients (aged 3-15 years) with congenital cranial dysostosis were examined by a pediatrician, geneticist, gastroenterologist, neuropathologist, ophthalmologist, endocrinologist, and orthopaedist. In addition to the clinical signs characteristic of hereditary multiple developmental defects, the study revealed changes in the jaws and temporomandibular joint and local factors promoting the progress of deformations of the jaws. Manifest and inapparent pathological changes and dysfunctions in gastrointestinal organs were paralleled by dysfunctions of the central and autonomic nervous systems, risk of maxillofacial and general deformations, and signs of congenital disorders in calcium, lactic acid, and pyridoxine metabolism. The results necessitate analyses of the blood and urine and development of new methods for the diagnosis of congenital cranial dysostosis and improvement of methods for the correction of this condition.

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.

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.

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.

A regulatory role of fibroblast growth factor in the expression of decorin, biglycan, betaglycan and syndecan in osteoblasts from patients with Crouzon's syndrome.

Bone development is controlled by the autocrine and/or paracrine effects of regulatory molecules. We previously showed that the phenotype of fibroblasts obtained from patients affected by Crouzon's syndrome, an autosomal dominant disease characterized by pathological skull bone development, differed from that of normal cells and was regulated by interleukin treatments. The changes in the relative concentrations of extracellular macromolecules (glycosaminoglycans-GAG, collagen and fibronectin) were associated with abnormal interleukin secretion that affected the microenvironment where the osteogenic processes take place. Mutations in human fibroblast growth factor receptors are now thought to be involved in Crouzon's syndrome. Since coactivation of interleukins and basic fibroblast growth factor (bFGF) is probably implicated in morphogenetic and osteogenic processes and heparan sulphate proteoglycans have a critical role in regulating bFGF activity, the phenotypes of normal and Crouzon osteoblasts were studied and the effects of bFGF on the expression of bFGF, procollagen alpha1 (I), and proteoglycan (PG) genes for biglycan, decorin, betaglycan and syndecan analyzed. Specific human cDNA probes were used to screen the relative levels of mRNA by Northern analysis. Spontaneous or bFGF-modulated release of interleukins was also assayed. The bFGF gene transcript was detected only in Crouzon osteoblasts. We showed for the first time that Crouzon osteoblasts, despite a mutation in the FGF receptor, still responded to exogenous bFGE In fact, the growth factor induced changes in the GAG profile and in the levels of mRNA coding for PG and procollagen alpha1 (I) and down-regulated heparan sulfate GAG chains. ELISA showed that bFGF-induced interleukin secretion differed in normal and Crouzon osteoblasts. The observed differences in PG core protein, procollagen alpha1 (I) and bFGF could be associated with the Crouzon bone phenotype and also should provide further understanding on the molecular basis of the diseased state of bone.

The mutations in FGFR2-associated craniosynostoses are clustered in five structural elements of immunoglobulin-like domain III of the receptor.

Exons 5 and 7 of the fibroblast growth factor receptor 2 (FGFR2) gene code for immunoglobulin-like domain III (IgIII) and for the region connecting the second and the third Ig domain of the receptor. Numerous mutations in these two exons have been shown to cause various craniosynostotic syndromes. Here, we describe three previously unrecognized mutations at amino acid positions 276, 301, and 314, in one nonspecific craniosynostosis and in two Crouzon patients. We also present a polypeptide model of IgIII of FGFR2. The known mutations involve five distinct structural elements of the receptor. The changes within these elements affect receptor function by various mechanisms, including altered dimerization, truncation, increased mobility between Ig domains, disintegration of IgIII, and alteration of the ligand-binding site.

Effects of interleukins on Crouzon fibroblast phenotype in vitro. Release of cytokines and IL-6 mRNA expression.

In Crouzon's syndrome, the cranial morphogenic processes are altered due to the early fusion of the facial and basal cranial bones. Evidence that Crouzon fibroblasts have an altered phenotype which is modulated by interleukin 1 (IL-1) and interleukin 6 (IL-6) is provided. [3H]Thymidine incorporation and cell count studies showed that Crouzon fibroblasts have an accelerated proliferation rate. [3H]Glucosamine incorporation studies, followed by chromatography analysis of culture medium samples, revealed that the various classes of glycosaminoglycans (GAG) secreted into the medium were differently distributed in Crouzon and normal fibroblasts. Crouzon fibroblasts secreted less total GAG, particularly hyaluronic acid (HA) and heparan sulfate (HS), but a relatively greater quantity of chondroitin sulfate. Type I and III collagen were raised in Crouzon fibroblast medium whereas the concentration of fibronectin was lower than in normal cells. Interleukin treatment induced changes in cell growth and neosynthesis of extracellular matrix macromolecules. Both IL-1 and IL-6 stimulated proliferation of Crouzon fibroblasts, whereas only IL-6 increased [3H]thymidine incorporation in normal cells. IL-1 provided a drop in HA and a rise in GAG sulfates in normal fibroblasts, but caused an opposite effect in Crouzon fibroblasts. Type I collagen and fibronectin secretions are differently modulated by the cytokines in the two populations. Moreover, level of IL-1 able to stimulate [3H] thymidine incorporation into mouse thymocytes, and level of IL-1 receptor antagonist (IL-1ra), as determined by ELISA, were higher in pathological than in normal fibroblasts. Also evidence that levels of IL-1 alpha and IL-6 proteins measured by ELISA and also IL-6 mRNA expression are enhanced in Crouzon fibroblasts is provided. These novel data suggested that, in Crouzon's syndrome, the modifications in the relative concentrations of the extracellular matrix (ECM) components associated with the abnormal cytokine networks may alter the balance of the microenvironment where the morphogenic events take place.