Phenotypic diversity in patients with craniosynostoses unrelated to Apert syndrome: the role of fibroblast growth factor receptor gene mutations

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1 J Neurosurg (Pediatrics 1) 102:23 30, 2005 Phenotypic diversity in patients with craniosynostoses unrelated to Apert syndrome: the role of fibroblast growth factor receptor gene mutations SUSUMU ITO, M.D., KEN ICHI SEKIDO, M.D., HIROSHI KANNO, M.D., HIRONOBU SATO, M.D., MASAAKI TANAKA, M.D., KAZUO YAMAGUCHI, M.D., AND ISAO YAMAMOTO, M.D. Department of Neurosurgery, Yokohama City University Medical Center and Yokohama City University School of Medicine; Department of Neurosurgery, Kanagawa Children s Medical Center, Yokohama; and Department of Neurosurgery, Shonan Hospital, Yokosuka, Japan Object. The goal of this study was to elucidate the genotype phenotype relationship in syndromic craniosynostoses by analyzing the mutations of the fibroblast growth factor receptor (FGFR) gene and its clinical manifestations in patients, particularly those in atypical cases. Methods. Twenty patients with craniosynostoses unrelated to Apert syndrome were enrolled in this study. The phenotypes indicated the following syndromes: 12 patients with unrelated Crouzon syndrome, including nine sporadic and three familial cases; two with sporadic Pfeiffer syndrome; and one with Antley Bixler syndrome. The Crouzon phenotype was subdivided into three clinical forms: regular, top, and bottom ones. Two patients who demonstrated craniofacial anomalies and bilateral elbow joint contractures were categorized as having an unspecified craniosynostosis. Three cases of unclassifiable cloverleaf skull malformation were also analyzed. Fourteen mutations of the FGFR2 gene were identified in these patients; seven of the 10 cysteine-related mutations were substitutions of codon 342 in the third immunoglobulin-like domain of this gene. The phenotypes of these seven cases were three of regular Crouzon, two of unspecified craniosynostosis, and one each of top Crouzon and unclassifiable cloverleaf skull malformation. In addition, four of the seven patients were found to have the same genotype (Cys342Arg). The phenotypes of these patients, however, were quite variable, ranging from regular Crouzon to unclassifiable cloverleaf skull malformation. Conclusions. The phenotypes of patients with craniosynostoses unrelated to Apert syndrome proved quite variable, even in cases in which patients demonstrated the same genotype. In view of the phenotypic diversity evident in cases in which the same mutation in the FGFR2 gene is present, it is possible that other disease-modifying genetic factors exist to control the abnormal gain-of-function that accompanies FGFR signaling. KEY WORDS craniosynostosis fibroblast growth factor receptor mutation phenotype genotype pediatric neurosurgery SYNDROMIC Abbreviations used in this paper: FGFR = fibroblast growth factor receptor; PCR = polymerase chain reaction; VP = ventriculoperitoneal. J. Neurosurg. (Pediatrics 1) / Volume 102 / January, 2005 craniosynostoses are generally classified based on patients clinical manifestations. Some patients, however, demonstrate features different from the typical phenotypes; therefore, it is sometimes difficult to make a definite diagnosis on the basis of physical examination alone. Jackson, et al., 12 have expressed uncertainty regarding whether to describe an unclassifiable phenotype as a new syndrome or as a variation of a previously described genetic disorder. Since 1994 mutations of the FGFR gene have been identified in craniosynostotic syndromes. 6 8,10,11,13 16,19 23,25 28,30, 31,33,35 In Apert syndrome, the mutations are relatively localized to some specific areas of the FGFR2 gene; 13,21,31,35 therefore, molecular genetic investigation makes it possible to differentiate this syndrome from the others. The same mutation of the FGFR2 gene, however, causes the different phenotypes evident in Crouzon, Pfeiffer, and Jackson Weiss syndromes. 8,14,19,20,25,28,30 Consequently, analysis of this gene has not necessarily assisted the classification of these syndromes. The mechanism by which the same mutation can cause different clinical manifestations has not fully been elucidated. In this study, we analyzed FGFR1 and FGFR2 genes in 20 patients with craniosynostoses unrelated to Apert syndrome, that is, Crouzon, Pfeiffer, and Antley Bixler syndromes, and several unclassifiable cases. We investigated the clinical manifestations and abnormalities of the FGFR genes to evaluate the genotype phenotype relationships in these patients, focusing particularly on the atypical clinical features. 23

2 S. Ito, et al. Patient Population Clinical Material and Methods Twenty patients with syndromic craniosynostoses were enrolled in this study. Twelve suffered from unrelated Crouzon syndrome (nine sporadic and three familial cases), two from sporadic Pfeiffer syndrome, and one from Antley Bixler syndrome. Two patients demonstrated craniofacial anomalies and bilateral elbow joint contractures; these were categorized as cases of unspecified craniosynostosis. Three patients with unclassifiable cloverleaf skull malformation were also examined. Patient age at the last follow-up examination ranged from 13 months to 24 years. All patients were Japanese and had been referred to Kanagawa Children s Medical Center. This study was approved by the ethical committee of Yokohama City University, and informed consent was obtained from all patients and/or their families before study participation. Patients were classified according to the results of clinical examinations and radiological studies by using the following criteria. Crouzon syndrome is a disorder consisting of craniosynostosis, maxillary hypoplasia, and ocular proptosis without limb anomaly. In this study, cases of Crouzon syndrome were subdivided into three different clinical forms regular, top, and bottom forms using the categories described by Tessier. 34 In the regular type of Crouzon syndrome, exophthalmos and maxillary hypoplasia are similar in degree. In top Crouzon syndrome, exophthalmos is present, but maxillary hypoplasia is absent or mild. In the bottom Crouzon phenotype, maxillary hypoplasia is severe in comparison with the degree of the ocular proptosis. Pfeiffer syndrome is characterized mainly by craniosynostosis, broad thumbs, and broad great toes. Antley Bixler syndrome is characterized by craniosynostosis, dysplastic ears, arachnodactyly, radiohumeral synostosis, femoral bowing, and joint contractures. 2 The unclassifiable cloverleaf skull malformation is diagnosed by trilobular configuration of the cranium, facial and skeletal anomalies, and hydrocephalus without distinct manifestations of Pfeiffer or other craniofacial syndromes. 4,9 Procedure for DNA Analysis Genomic DNA was extracted from peripheral blood samples and PCRs were performed for the regions including exon 5 of the FGFR1 gene and exons 3a and 3c of the FGFR2 gene. The primers and PCR conditions for exon 5 of the FGFR1 gene and exon 3c of the FGFR2 gene were the same as those reported by Muenke, et al., 16 and Reardon, et al., 25 respectively. The forward primer for exon 3a of the FGFR2 gene, 5 -GGTCTCTCATTCTCCCATC CC-3, was the same as that described by Slaney, et al., 31 and the reverse primer, 5 -GGTACCTTTAGATTCA GAAAG-3, was newly designed to obtain a 270-bp PCR product including the entire region of exon 3a. The PCR conditions for exon 3a were as follows: 94 C 1 minute, 60.5 C 1 minute, and 72 C 1 minute for 40 cycles. The PCR products underwent single-strand conformation polymorphism/heteroduplex analysis to detect mutations. Radiolabeled PCR products were obtained by adding 0.1 l [ 35 S]deoxyadenosine triphosphate to each reaction. The double-strand PCR products were denatured at 98 C for 5 minutes and immediately cooled on ice. The PCR products were electrophoresed on mutation-detection-enhancement gels (Hydrolink; AT Biochemical, Malvern, PA) with 10% glycerol at 6 W for 15 hours. The gels were then dried and exposed to x-ray films and imaging plates (Fuji Photo Film, Tokyo, Japan). When abnormal single-strand conformation polymorphism and/or heteroduplex patterns were detected, the PCR products were sequenced in both forward and reverse directions by using a 33 P terminator cycle sequence method (Amersham Biosciences Corp., Piscataway, NJ). The gels were also dried and exposed to x-ray films and imaging plates. The imaging plates were analyzed using a BAS-2500 system (Fuji Photo Film). The numbering of nucleotides and amino acids for the FGFR2 were the same as that reported by Oldridge, et al. 19 To confirm the 1036:T C mutation in exon 3c of the FGFR2 gene, we used an amplification refractory mutation system. 18 The forward primer for the normal allele was 5 - ACGCTGGGGAATATACGT-3 and that for the mutant allele was 5 -ACGCTGGGGAATATACGC-3. A common reverse primer was used, 5 -AAAAAACCCAGAGA GAAAGAACAGTATA-3. The PCR conditions were 94 C 1 minute, 63.5 C 30 seconds, and 72 C 1 minute for 30 cycles. The PCR products were electrophoresed on agarose gels to confirm the 109-bp fragments. Results We identified 14 mutations of the FGFR2 gene in these 20 patients (Table 1). Ten of these 14 mutations were related to cysteine residues and seven were substitutions of the codon 342 cysteine residue in the third immunoglobulin-like domain of the gene. In one patient with Pfeiffer syndrome, the one patient with Antley Bixler syndrome, two patients with Crouzon syndrome, and two with cloverleaf skull malformation no mutation was detected in exon 5 in the FGFR1 gene or in exon 3a or 3c of the FGFR2 gene. Genotype Phenotype Relationships Crouzon Syndrome. In 10 of the 12 patients with Crouzon syndrome, three mutations were in exon 3a, and seven were in exon 3c of the FGFR2 gene. All these mutations caused amino acid changes. In the 10 patients with Crouzon syndrome in whom mutations were detected, nine demonstrated regular phenotypes and one demonstrated the top Crouzon type. These patients had no limb anomalies. In the top Crouzon type (Case 10), the facial abnormality was minimal (Table 2) the clinical appearance resembled nonsyndromic oxycephaly (Fig. 1). The mutation in this patient was Cys342- Phe (1037:G T) (Table 1). Other abnormalities and/or symptoms in these patients were present: one patient suffered from occult spinal dysraphism and spinal lipoma (Case 8); one, precocious puberty (Case 5); two, mental retardation (Cases 8 and 10); and one, epilepsy (Case 10). Four of 10 patients with Crouzon syndrome demonstrated progressive hydrocephalus, which was treated with the placement of a VP shunt; one of these patients in whom syringomyelia also developed underwent placement of a syringosubarachnoid shunt (Case 9) (Table 24 J. Neurosurg. (Pediatrics 1) / Volume 102 / January, 2005

3 Phenotypic diversity in craniosynostoses with FGFR gene mutations TABLE 1 Summary of phenotypes and mutations of FGFR2 gene in 14 patients studied* Case No. Age (yrs), Sex Sporadic/Familial Phenotype Mutation of FGFR2 Gene , F s regular Crouzon Tyr281Cys (854:A G) , M f regular Crouzon Gln289Pro (878:A C) , M s regular Crouzon Gln289Pro ( 878:A C) , M s regular Crouzon Gly338Arg (1024:G C) , M s regular Crouzon Cys342Arg (1036:T C) , F s regular Crouzon Cys342Arg (1036:T C) , F s regular Crouzon Cys342Tyr (1037:G A) , F s regular Crouzon Ser347Cys (1052:C G) , F s regular Crouzon Ser354Cys (1073:C G) , M f top Crouzon Cys342Phe (1037:G T) , M s Pfeiffer splice site (952-1:G C) , F s unspecified Cys342Ser (1037:G C) , F s unspecified Cys342Arg (1036:T C) , M s cloverleaf skull Cys342Arg, Thr341Thr malformation (1036:T C, 1035:G T) * f = familial; s = sporadic. 2). Three patients underwent linear craniectomy in early infancy (Cases 1, 7, and 8), and nine patients underwent frontoorbital advancements one or two times between the age of 9 months and 12.9 years. In eight of nine patients with regular Crouzon syndrome, plastic surgeons performed a Le Fort III osteotomy in patients who at the time of surgery ranged in age from 5 to 18.4 years. Pfeiffer Syndrome. In one patient with Pfeiffer phenotype (Case 11), the mutation was an acceptor splice-site one (952-1:G C) in the FGFR2 gene (Table 1). This patient FIG. 1. Case 10. Radiograph obtained in a 4-year-old patient with the top Crouzon phenotype, demonstrating a very mild maxillary hypoplasia. J. Neurosurg. (Pediatrics 1) / Volume 102 / January, 2005 demonstrated brachycephaly, ocular proptosis, and a depressed nasal bridge. Bilateral broad thumbs with syndactyly involving the third and fourth fingers were also observed. In addition, bilateral broad great toes with syndactyly involving the second, third, and fourth toes were compatible with the typical phenotype (Table 2). This patient underwent multiple cranial operations until he reached the age of 5.8 years; he later underwent Le Fort III osteotomy at the age of 11.4 years. Unspecified Craniosynostosis. In this study two patients suffered from unspecified craniosynostosis; in one (Case 12), the mutation of the FGFR2 gene was Cys342Ser (1037:G C) (Table 1). This patient suffered from bilateral radiohumeral synostosis, hydrocephalus, and hearing disturbance; however, she had no hand or foot anomalies (Fig. 2) (Table 2). This patient underwent frontoorbital advancement at the ages of 2 months and 6 years, VP shunt placement at the age of 10 months, and a Le Fort III osteotomy twice, at the age of 12.2 years and again at 16.8 years. In the other patient with unspecified craniosynostotis and bilateral elbow joint contractures (Case 13), the mutation was Cys342Arg (1036:T C), the same as in two of the patients with the regular Crouzon phenotype (Cases 5 and 6) (Fig. 3, Table 1). Radiological studies of this patient revealed a narrow anterior skull base, remarkably elevated sphenoidal wings, and bilateral radial head dislocation (Fig. 4, Table 2). At the age of 2 months she underwent bilateral frontoorbital advancement, performed using distraction devices, and at the age of 5 months VP shunt placement was undertaken to treat progressive hydrocephalus. Unclassifiable Cloverleaf Skull Malformation. In one patient with unclassifiable cloverleaf skull malformation (Case 14), two point mutations (1036:T C and 1035:G T) were detected in exon 3c of the FGFR2 gene (Fig. 5). The 1036:T C mutation caused an amino acid substitution (Cys342Arg); however, the 1035-point mutation was a silent one (Thr341Thr) (Table 1). Consequently, the amino acid substitution (Cys342Arg) was the same as that identified in two patients with regular Crouzon syn- 25

4 S. Ito, et al. TABLE 2 Summary of facial and other abnormalities in 14 patients studied* Case No. Facial Anomaly Other Abnormalities Progressive Hydrocephalus 1 MH, proptosis none yes 2 MH, proptosis none no 3 MH, proptosis none no 4 MH, proptosis none no 5 MH, proptosis precocious puberty no 6 MH, proptosis none yes 7 MH, proptosis none no 8 MH, proptosis occult spinal dysraphism (S3 5), spinal yes lipoma, mental retardation 9 MH, proptosis syringomyelia, Chiari I malformation, yes sinus pericranii 10 mild proptosis mental retardation, epilepsy no 11 MH, proptosis broad thumbs, broad great toes, no syndactyly in hands & feet 12 MH, proptosis bilat radiohumeral synostosis, yes hearing disturbance 13 mild MH, proptosis elbow joint contractures yes 14 MH, proptosis elbow joint ankylosis, coccygeal tail, yes congenital pyroric stenosis, Chiari I malformation, coanal atresia, hearing disturbance * MH = maxillary hypoplasia. drome (Cases 5 and 6) and in one patient with unspecified craniosynostosis (Case 13). The patient in Case 14 demonstrated exophthalmos and a narrow palate. Magnetic resonance imaging revealed a trilobular shape of the head and marked hydrocephalus as well as a prominent coccygeal tail. The patient also suffered from bilateral elbow joint contractures and radioulnar synostosis (Fig. 6, Table 2); nevertheless, no abnormalities were evident in the hands or feet and, therefore, this case was classified as one of cloverleaf skull malformation. The boy underwent multiple operations for craniosynostosis, hydrocephalus, congenital pyloric stenosis, upper-airway narrowing, and Chiari I malformation as well as medical treatment for an idiopathic thrombocytopenia. Discussion Since 1994, mutations of the FGFR gene have been reported in cases of Crouzon, 8,14,19,20,25,28 Apert, 13,21,31,35 Pfeiffer, 14,16,28,30 and Jackson Weiss syndromes. 8,10,14,20 Other craniofacial disorders related to the FGFR gene have also been described in Saethre Chotzen, 6,7,22 thanatophoric dwarfism, 27,33 Beare Stevenson cutis gyrata syndrome, 23 and one type of coronal synostosis. 15,26 Mutations of the TWIST and MSX2 genes were found to be associated with Saethre Chotzen syndrome 6,7,22 and Boston-type craniosynostosis, 11 respectively. Neilson and Friesel 17 postulated one possible molecular mechanism of syndromic craniosynostosis: a mutation of the FGFR gene that is related to a cysteine residue leads to ligand-independent dimerization of the receptor, which in turn results in constitutive activation of intracellular tyrosine kinase. Abnormal gain-of-function FGFR may cause early closure of cranial sutures. Recently, Warren, et al., 36 reported that both Crouzon (Cys342Tyr) and Apert (Ser252Trp) FGFR2 mutations suppressed the production FIG. 2. Case 12. Radiographs obtained in a 4.8-year-old patient with unspecified craniosynostosis. Left: Note the marked midface hypoplasia resembling regular Crouzon syndrome. Right: Radiohumeral synostosis is clearly demonstrated. 26 J. Neurosurg. (Pediatrics 1) / Volume 102 / January, 2005

5 Phenotypic diversity in craniosynostoses with FGFR gene mutations FIG. 3. Results of amplification refractory mutation system analysis for the 1036: T C mutation in the FGFR2 gene. M = PCR products for mutant allele; n = PCR products for normal allele; 1 = healthy control patient; 2 = Case 5; 3 = Case 6; 4 = Case 13. of noggin, a protein that prevents cranial suture fusion. They argued that syndromic FGFR-related craniosynostoses may be the result of abnormal downregulation of noggin production. In Crouzon, Pfeiffer, and Jackson Weiss syndromes, amino acid changes related to cysteine residues in the FGFR2 were frequently reported. 8,10,14,16,19,20, 25,28,30 In our study, 70% of the mutations were related to cysteine residues in patients with Crouzon syndrome as well. In most patients with Apert syndrome, the mutations of the FGFR2 gene were relatively localized in the anterior part of the third immunoglobulin-like domain, and correlated with the typical phenotype. 13,21,31,35 In craniofacial syndromes unrelated to the Apert syndrome, however, the same missense mutation caused different phenotypes. For example, one of the most common mutations, Cys342Arg in the FGFR2 gene, was able to cause any phenotype of Crouzon, 25 Pfeiffer, 14,28,30 or Jackson Weiss syndrome. 20 Other mutations of codon 342 in the FGFR2 gene, for example, Cys342Ser and Cys342Try, could also cause either the Crouzon or Pfeiffer phenotype. 8,14,19,25,28 In our investigation, the same missense mutation (Cys342Arg) resulted in wider varieties of phenotypes than previously reported, including those for regular Crouzon syndrome, unspecified craniosynostosis with elbow joint abnormalities, and unclassifiable cloverleaf skull malformation. In Crouzon syndrome, we grouped patients according to Tessier s classification. 34 The phenotypes of the 10 patients with confirmed mutation were nine regular and one top Crouzon type. In the case of the patient with the top Crouzon phenotype (Case 10), whose facial anomaly was milder than that in the patient with the regular Crouzon phenotype, the diagnosis may have been confused with nonsyndromic oxycephaly. There have been few reports citing differential diagnoses between the oxycephaly and the top Crouzon type in relation to FGFR2 gene mutations. In the two patients with craniosynostosis and abnormalities of elbow joints (Cases 12 and 13), mutations of the FGFR2 gene (Cys342Ser, Cys342Arg) were also identified. Elbow joint contractures have been reported in Apert, Antley Bixler, and Pfeiffer syndromes. 1,2,5,28 In our study, however, the two patients had normal hands and feet and showed no other clinical features compatible with the aforementioned syndromes. We could not categorize these patients, therefore. Pulleyn, et al., 24 also reported an unclas- J. Neurosurg. (Pediatrics 1) / Volume 102 / January, 2005 FIG. 4. Case 13. Radiographs obtained in a patient with unspecified craniosynostosis. Upper: Note the elevated sphenoidal wings. Lower: Radial head dislocation is demonstrated. 27

6 S. Ito, et al. FIG. 5. Sequence data for exon 3c of the FGFR2 gene. Left: Healthy control. Right: 1036: T C and 1035: G T mutation in Case 14. sifiable case with elbow joint abnormalities. In one patient with unclassifiable cloverleaf skull malformation (Case 14), a mutation of the FGFR2 gene was detected; this was the same mutation found in two patients with regular Crouzon syndrome and one with unspecified craniosynostosis (Cys342Arg). Cloverleaf skull malformation, which is characterized by a trilobular configuration of the cranium, facial and skeletal anomalies, and hydroceph- FIG. 6. Case 14. Imaging results for a patient with a cloverleaf skull malformation. Upper: Cranial magnetic resonance images demonstrating marked exophthalmos, trilobular shape of the head, and hydrocephalus. Lower Left: An x-ray study demonstrating radioulnar synostosis. Lower Right: Spinal magnetic resonance image revealing prominent coccygeal tail (arrow). 28 J. Neurosurg. (Pediatrics 1) / Volume 102 / January, 2005

7 Phenotypic diversity in craniosynostoses with FGFR gene mutations alus, 9 derives from heterogeneous causes and has been reported to be associated with various other syndromes such as Pfeiffer, thanatophoric dysplasia, Beare Stevenson cutis gyrata syndrome, and others. 4,5,9,33 Particularly in Pfeiffer Type 2 syndrome, a cloverleaf skull is a principal clinical manifestation in addition to broad thumbs, broad great toes, and elbow ankylosis. 5 In the patient in our study (Case 14), no abnormalities in the hands and feet were detected; therefore, we could not classify this case as one of Pfeiffer syndrome. The mechanism by which the same genotype causes different phenotypes has not been elucidated fully. Before advances occurred in molecular genetic analysis, intrafamilial phenotypic variability was reported in Pfeiffer 29 and Jackson Weiss syndromes, 12 and, more recently, in unclassifiable craniosynostosis. 32 Rutland, et al., 28 reported that identical mutations in the FGFR2 gene caused both Pfeiffer and Crouzon syndromes. They gave one possible explanation for the result that sequence polymorphism in another part of the same gene affected the phenotypic expression of the mutant allele. Britto, et al., 3 emphasized that subtle differences in the patterns of FGF/FGFR expression played an important role in the clinical manifestation of craniofacial syndromes. In addition, other mechanisms related to the control of suture fusion were also studied, including transforming growth factor, homeobox-containing genes, hedgehog gene, TWIST gene, and neural epidermal growth factor like gene. 37 In view of the phenotypic diversity within the same mutation in the FGFR2 gene, it appears possible that other disease-modifying genetic factors may exist to control the abnormal gain-of-function FGFR signaling. Conclusions The phenotypes of patients with craniosynostoses unrelated to Apert syndrome varied greatly, even in cases in which patients demonstrated the same genotype. Particularly in patients with atypical clinical manifestations, it is sometimes difficult to make a definitive diagnosis of a known, specific syndrome. Descriptions of clinical features as well as evaluations of genetic abnormalities should be used, therefore, to facilitate further investigations of syndromic craniosynostoses. Acknowledgments We thank Dr. Keiichi Kondo, Yoko Ibuka, Teruyo Watanabe, and Kyoko Arai for their technical assistance. We also gratefully acknowledge the support of Drs. Katsuyuki Torikai, Shinji Kobayashi, Mitsuo Masuno, and Kenji Kurosawa. References 1. Anderson PJ, Hall CM, Evans RD, Hayward RD, Jones BM: The elbow in syndromic craniosynostosis. J Craniofacial Surg 9: , Antley R, Bixler D: Trapezoidocephaly, midfacial hypoplasia and cartilage abnormalities with multiple synostoses and skeletal fractures. Birth Defects Orig Artic Ser 11: , Britto JA, Evans RD, Hayward RD, Jones BM: From genotype to phenotype: the differential expression of FGF, FGFR and TGF genes characterizes human cranioskeletal development J. Neurosurg. (Pediatrics 1) / Volume 102 / January, 2005 and reflects clinical presentation in FGFR syndromes. Plast Reconstr Surg 108: , Cohen MM Jr: Pfeiffer syndrome update, clinical subtypes, and guidelines for differential diagnosis. 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8 S. Ito, et al. syndrome, due to TWIST and FGFR mutations. Am J Hum Genet 62: , Przylepa KA, Paznekas W, Zhang M, Giolabi M, Bias W, Bamshad MJ, et al: Fibroblast growth factor receptor 2 mutations in Beare-Stevenson cutis gyrata syndrome. Nat Genet 13: , Pulleyn LJ, Reardon W, Wilkes D, Rutland P, Jones BM, Hayward R, et al: Spectrum of craniosynostosis phenotypes associated with novel mutations at the fibroblast growth factor receptor 2 locus. Eur J Hum Genet 4: , Reardon W, Winter RM, Rutland P, Pulleyn LJ, Jones BM, Malcolm S: Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet 8:98 103, Renier D, El-Ghouzzi V, Bonaventure J, le Merrer ML, Lajeunie E: Fibroblast growth factor receptor 3 mutation in nonsyndromic coronal synostosis: clinical spectrum, prevalence and surgical outcome. J Neurosurg 92: , Rousseau F, el Ghouzzi V, Delezoide AL, Legeai-Mallet L, le Merrer ML, Munnich A: Missense FGFR3 mutations create cysteine residues in thanatophoric dwarfism type I (TD1). Hum Mol Genet 5: , Rutland P, Pulleyn LJ, Reardon W, Baraitser M, Hayward R, Jones B, et al: Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes. Nat Genet 9: , Sanchex HM, De Negrotti TC: Variable expression in Pfeiffer syndrome. J Med Genet 18:73 75, Schell U, Hehr A, Feldman GJ, Robin NH, Zackai EH, de Die- Smulders C, et al: Mutations in FGFR1 and FGFR2 cause familial and sporadic Pfeiffer syndrome. Hum Mol Genet 4: , Slaney SF, Oldridge M, Hurst JA, Moriss-Kay GM, Hall CM, Poole MD, et al: Differential effects of FGFR2 mutations on syndactyly and cleft palate in Apert syndrome. Am J Hum Genet 58: , Steinberger D, Reinhartz T, Unsöld R, Muller U: FGFR2 mutation in clinically nonclassifiable autosomal dominant craniosynostosis with pronounced phenotypic variation. Am J Med Genet 66:81 86, Tavormina PL, Shiang R, Thompson LM, Zhu YZ, Wilkin DJ, Lachman RS, et al: Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nat Genet 9: , Tessier P: Craniofacial surgery in syndromic craniosynostosis, in Cohen MM Jr (ed): Craniosynostosis. Diagnosis, Evaluation, and Management. New York: Raven Press, 1986, pp Wilkie AO, Slaney SF, Oldridge M, Poole MD, Ashworth GJ, Hockley AD, et al: Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nat Genet 9: , Warren SM, Brunet LJ, Harland RM, Economides AN, Longaker MT: The BMP antagonist noggin regulates cranial suture fusion. Nature 422: , Warren SM, Greenwald JA, Spector JA, Bouletreau P, Mehrara BJ, Longaker MT: New development in cranial suture research. Plast Reconstr Surg 107: , 2001 Manuscript received March 6, Accepted in final form July 6, This study was supported in part by a grant to Susumu Ito, M.D., from the Japan Spina Bifida and Hydrocephalus Research Foundation. Address reprint requests to: Susumu Ito, M.D., Department of Neurosurgery, Yokohama City University Medical Center, 4 57 Urahune-cho, Minami-ku, Yokohama, , Japan. sinito@mars.dti.ne.jp. 30 J. Neurosurg. (Pediatrics 1) / Volume 102 / January, 2005