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.     

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     

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.     

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.     

Craniosynostosis and congenital tracheal anomalies in an infant with Pfeiffer syndrome carrying the W290C FGFR2 mutation

Pfeiffer syndrome (OMIM 101600) is an autosomal dominant disorder characterized by craniosynostosis, midface hypoplasia, ocular proptosis and digital malformations. We report on a type II Pfeiffer female infant with craniosynostosis, hydrocephalus, and characteristic craniofacial and digital abnormalities. The patient had a history of airway difficulty. Bronchoscopy at age four months revealed low tracheal stenosis and fibrous cartilaginous rings. She underwent tracheostomy for the treatment of cyanotic episodes. Molecular analysis revealed a de novo missense mutation c.870 G>T (TGG>TGT) in the FGFR2 gene that predicts a substitution of cysteine for tryptophan at the codon 290, (W290C). There is phenotypic heterogeneity of tracheal anomalies due to FGFR2 mutations. A review of the literature shows that Pfeiffer patients with the similar tracheal abnormalities can be caused by different FGFR2 mutations and, likewise, the patients with the same FGFR2 mutation may manifest different kinds of tracheal anomalies. Tracheal anomalies may occur in Pfeiffer patients and cause morbidity and mortality because of airway obstruction. Recognition and detailed evaluation of tracheal anomalies should be included in the early diagnostic workup for severe Pfeiffer patients.     

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     

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.     

Paternal origin of FGFR2 mutations in sporadic cases of Crouzon syndrome and Pfeiffer syndrome

Crouzon syndrome and Pfeiffer syndrome are both autosomal dominant craniosynostotic disorders that can be caused by mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. To determine the parental origin of these FGFR2 mutations, the amplification refractory mutation system (ARMS) was used. ARMS PCR primers were developed to recognize polymorphisms that could distinguish maternal and paternal alleles. A total of 4,374 bases between introns IIIa and 11 of the FGFR2 gene were sequenced and were assayed by heteroduplex analysis, to identify polymorphisms. Two polymorphisms (1333TA/TATA and 2710 C/T) were found and were used with two previously described polymorphisms, to screen a total of 41 families. Twenty-two of these families were shown to be informative (11 for Crouzon syndrome and 11 for Pfeiffer syndrome). Eleven different mutations in the 22 families were detected by either restriction digest or allele-specific oligonucleotide hybridization of ARMS PCR products. We molecularly proved the origin of these different mutations to be paternal for all informative cases analyzed (P=2. 4x10-7; 95% confidence limits 87%-100%). Advanced paternal age was noted for the fathers of patients with Crouzon syndrome or Pfeiffer syndrome, compared with the fathers of control individuals (34. 50+/-7.65 years vs. 30.45+/-1.28 years, P<.01). Our data on advanced paternal age corroborates and extends previous clinical evidence based on statistical analyses as well as additional reports of advanced paternal age associated with paternal origin of three sporadic mutations causing Apert syndrome (FGFR2) and achondroplasia (FGFR3). Our results suggest that older men either have accumulated or are more susceptible to a variety of germline mutations.     

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.     

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.     

Further evidence of association between mutations in FGFR2 and syndromic craniosynostosis with sacrococcygeal eversion

BACKGROUND: Pfeiffer syndrome (PS; OMIM #101600) is an autosomal dominant disorder characterized by craniosynostosis, midface hypoplasia, broad thumbs, brachydactyly, broad great toes, and variable syndactyly. CASE: We report a case of PS (type 3) with tracheal and visceral involvement and sacrococcygeal eversion. The patient shows facial dysmorphism with macrocephaly, dolichocephaly, and trigonocephaly, and an asymmetric skull, bilateral and severe exophthalmia with shallow orbits and ocular hypertelorism, downslanting palpebral fissures, constant strabismus, short anterior cranial base, and midface hypoplasia. CONCLUSIONS: Molecular analysis of the FGFR2 gene in this patient revealed a point mutation (c.890G>C NM_000141). This mutation leads to the substitution of the residue tryptophan at position 290 to cysteine in the protein (p.Try290Cys). These data reinforce the hypothesis that the p.Trp290Cys mutation is more often associated with a severe and poor prognosis of PS. Furthermore they suggest that the presence of sacrococcygeal defects is not associated with any specific FGFR2 mutation.     

Analysis of the mutational spectrum of the FGFR2 gene in Pfeiffer syndrome

Pfeiffer syndrome (PS) is one of the classical craniosynostosis syndromes correlated with specific mutations in the human fibroblast growth factor receptor (FGFR) genes, FGFR1 and FGFR2. In this study, we set out to examine the exons in FGFR2 most commonly associated with mutations in PS, exons IIIa and IIIc, in a panel of 78 unrelated individuals with PS by the most sensitive method (direct DNA sequencing). We have identified a total of 18 different mutations among 40 patients; eight of these mutations have not been previously described. The mutational spectrum displays a non-random character with the frequent involvement of cysteine codons. Received: 6 January 1999 / Accepted: 10 March 1999     

Pfeiffer syndrome caused by haploinsufficient mutation of FGFR2

Mutations of the fibroblast growth factor receptors (FGFRs) cause several dominantly inherited congenital skeletal disorders and syndromes. Recently, these mutations have been suggested to cause either ligand-independent activation of the receptor or a dominant negative inactivation. The analysis of two Japanese patients with Pfeiffer syndrome and postaxial polydactyly of the hand now shows that both carried the same 1119-2A-to-G transition of the FGFR2 gene and this nonsense mutation caused skipping of exon 9(B) and haploinsufficiency of FGFR2.     

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.     

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.     

De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome.

Apert syndrome, one of five craniosynostosis syndromes caused by allelic mutations of fibroblast growth-factor receptor 2 (FGFR2), is characterized by symmetrical bony syndactyly of the hands and feet. We have analyzed 260 unrelated patients, all but 2 of whom have missense mutations in exon 7, which affect a dipeptide in the linker region between the second and third immunoglobulin-like domains. Hence, the molecular mechanism of Apert syndrome is exquisitely specific. FGFR2 mutations in the remaining two patients are distinct in position and nature. Surprisingly, each patient harbors an Alu-element insertion of approximately 360 bp, in one case just upstream of exon 9 and in the other case within exon 9 itself. The insertions are likely to be pathological, because they have arisen de novo; in both cases this occurred on the paternal chromosome. FGFR2 is present in alternatively spliced isoforms characterized by either the IIIb (exon 8) or IIIc (exon 9) domains (keratinocyte growth-factor receptor [KGFR] and bacterially expressed kinase, respectively), which are differentially expressed in mouse limbs on embryonic day 13. Splicing of exon 9 was examined in RNA extracted from fibroblasts and keratinocytes from one patient with an Alu insertion and two patients with Pfeiffer syndrome who had nucleotide substitutions of the exon 9 acceptor splice site. Ectopic expression of KGFR in the fibroblast lines correlated with the severity of limb abnormalities. This provides the first genetic evidence that signaling through KGFR causes syndactyly in Apert 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.