Abstract
Introduction:
Craniosynostosis, the premature fusion of cranial sutures, is usually divided into 2 major categories: syndromic and nonsyndromic. Mutations in the FGFR1, FGFR2, FGFR3, TWIST1, and EFNB1 genes cause the common craniosynostosis syndromes Muenke, Crouzon and Crouzon with acanthosis nigricans, Apert, Pfeiffer, Saethre-Chotzen, and Craniofrontonasal. Overlapping features among craniosynostosis syndromes, phenotypic heterogeneity even within the same syndrome, especially in the case of Muenke syndrome, and inadequate clinical evaluation can lead to misdiagnosis, which molecular testing can help clarify.
Objective:
The aim of this study is to investigate the underlying genetic cause in 46 patients with syndromic or nonsyndromic craniosynostosis by direct sequencing and/or microdeletion/microduplication analysis of the FGFR1-3, TWIST1, and EFNB1 genes.
Results:
Genetic analysis identified 3 novel mutations, c.413T>C - p.(Leu138Pro) [p.(L138P)] in TWIST1, the previously reported c.373G>A - p.(Glu125Lys) [p.(E125K)], and c.717dupA - p.(Leu240IlefsTer79) [p.(L240fs)] mutation in EFNB1 gene as well as 6 previously known mutations and a heterozygous TWIST1 gene deletion. The 2 novel mutations within EFNB1 gene arose de novo, but the novel mutation p.(L138P) within TWIST1 gene was inherited from the patient’s father, who was found to be mosaic for the mutation. To our knowledge, this is the first case of mosaicism described for TWIST1 gene.
Conclusions:
The contribution of molecular genetic analysis to the diagnosis of patients with syndromic craniosynostosis was useful because some were originally misdiagnosed. Conversely, thorough clinical evaluation can guide molecular testing and result in a correct diagnosis.
Introduction
Craniosynostosis, the premature fusion of cranial sutures, has an overall prevalence of 1 in 2100 to 2500 births (Johnson and Wilkie, 2011). Based on the presence or absence of other clinical features, craniosynostosis is usually divided into 2 major categories: syndromic and nonsyndromic. In addition to the craniofacial deformities, syndromic cases present with primary defects also affecting the upper and lower limbs, the heart, and the central nervous system. In nonsyndromic cases, craniosynostosis is the sole primary defect, whereas secondary manifestations can be present as a consequence of early suture fusion. Nonsyndromic synostosis, accounting for 70% of the cases, can be classified according to the fused suture, namely sagittal, coronal, metopic, and lambdoid (Passos-Bueno et al., 2008). Genetic lesions in the FGFR1, FGFR2, FGFR3, TWIST1, and EFNB1 genes, accounting for approximately 25% of craniosynostosis (Wilkie et al., 2006), are the cause of common craniosynostosis syndromes Muenke, Crouzon and Crouzon with acanthosis nigricans, Apert, Pfeiffer, Saethre-Chotzen, and craniofrontonasal syndrome (CFNS). FGFR2 mutations cause the majority of Crouzon and Pfeiffer cases. Although a genotype-phenotype correlation is observed in many cases, several mutations have been associated with both syndromes. Crouzon syndrome with acanthosis nigricans is characterized by the FGFR3 p.A391E substitution, and less than 10% of Pfeiffer syndrome patients are found to carry the FGFR1 p.P252R mutation. Muenke syndrome is defined by the presence of FGFR3 p.P250R mutation, and more than 98% of Apert syndrome cases are caused by 2 specific FGFR2 missense mutations, p.S252W (66%) or p.P253R (32%). Mutations, and more rarely deletions of TWIST1 and EFNB1 genes, cause Saethre-Chotzen and CFNS, respectively (Johnson and Wilkie, 2011). All of the above-mentioned syndromes share a great number of clinical manifestations, some of which are also features of several cases of nonsyndromic craniosynostosis. Commonly overlapping features among craniosynostosis syndromes include the combination of synostotic sutures resulting in an abnormally shaped skull, hypoplasia of the upper and middle third of the face, exophthalmos, and other skeletal malformations, the most common of which are related to the digits. Overlapping clinical manifestations in addition to the heterogeneity found even within the same syndrome, especially in the case of Muenke, can make it very difficult to distinguish craniosynostosis cases in practice and give a correct clinical diagnosis. Therefore, molecular testing is a necessity in these cases in order to achieve molecular diagnosis.
The aim of this study was to explore the underlying genetic cause of syndromic and nonsyndromic craniosynostosis in 46 patients in Greece and Cyprus.
Patients and Methods
Patients
Forty-six children were included in our study who were born within the years 1987-2011 and who presented to the Hellenic Craniofacial Center (HCC) and the Department of Genetics and Molecular Biology, “MITERA” General, Maternity and Children Hospital, Athens. They were referred from Greece and Cyprus and were diagnosed with nonsyndromic or syndromic craniosynostosis of variable severity (Table 1). All patients with premature suture fusion and no previous molecular diagnosis referred to the HCC were selected for this study. Diagnosis for all patients was made by the head surgeon of the HCC. All patients underwent at least 1 major craniofacial surgical procedure in order to treat the craniofacial deformities and/or concomitant functional disorders. We also included family members in the study, whenever available. The research was approved by the Scientific and Ethical Committee of Faculty of Biology, National and Kapodistrian University of Athens, and is in accordance with the 1964 Declaration of Helsinki. Informed consent was obtained from all patients or the patients’ parents.
Numbering and Clinical Description of All Probands Based on Initial Evaluation Before Genetic Testing.
Abbreviations: ?, possible; bil, bilateral; CFNS, craniofrontonasal syndrome; L, left; NAI, nonavailable information; NS, nonsyndromic; PS, possibly syndromic; R, right.
aPatients 1 and 3 are sister and brother, patients 14 and 15 are female monozygotic twins.
Molecular Genetic Analysis
Blood samples from probands and their family members, when available, were drawn for diagnostic purposes after obtaining informed consent. Genomic DNA was extracted using QIamp DNA mini kit (Qiagen, Madison, WI).
Based on the flow diagram for molecular diagnosis of craniosynostosis suggested by Johnson and Wilkie (2011), patients were screened according to their initial clinical evaluation for mutations in exons 8 (IIIa), 10 (IIIc), 14, and 16 of FGFR2 gene, exon 1 (ORF) of TWIST1 gene, all 5 exons of EFNB1, the p.P250R and p.A391E mutations in the FGFR3 gene and the p.P252R in FGFR1 gene, as well as for microdeletions/microduplications in the above-mentioned genes and, additionally, in the MSX2, ALX1, ALX3, ALX4, and RUNX2 genes. Polymerase chain reaction (PCR) amplification of FGFR2 exons 8 (IIIa), 10 (IIIc), 14 and 16, FGFR1 and FGFR3 exon 7, TWIST1 exon 1, and EFNB1 exons 1, 2, 4-5, was performed using previously described primers (Kan et al., 2002; Simsek et al., 2003; Wieland et al., 2004; Seto et al., 2007). PCR amplification of FGFR3 exon 10 was performed using either previously described primers (Shiang et al., 1994) or using primers 5′-TAGACTCACTGGCGTTACTG-3′ (forward) and 5′-CGGGCAGGCAGCTCAGAA-3′ (reverse). PCR amplification of EFNB1 exons 3-4 was performed using primers 5′-GCTGAAGCAGAATGGGAGTTTC-3′ (forward) and 5′-GCCTAACAAGGTGACAGAGCAA-3′ (reverse). For mutation screening, PCR products were directly sequenced on an ABI 3130xl DNA Analyser (Applied Biosystems, Foster City, CA) and/or cut by restriction enzymes and subsequently analyzed by electrophoresis on a 3% agarose gel. For the identification and/or confirmation of the following mutations FGFR1: p.P252R, FGFR2: p.S252W; p.P253R; 1084+3A>G, FGFR3: p.P250R; p.A391E and EFNB1: p.(E125K) and p.(L240fs), the corresponding PCR products were digested with the following restriction enzymes: MnlI, MboI, BglI, AccI (New England BioLabs, Ipswich, MA), BcnI (NciI), AciI (SsiI) (Fermentas), BpmI and MfeI (New England BioLabs), respectively. For microdeletion/microduplication analysis MRC-Holland SALSA MLPA KITs P080-B1 and P064-B3 (MRC-Holland, Amsterdam, Netherlands) were used. GenBank accession numbers are as follows: FGFR1: NG_007729, FGFR2: AF360695, FGFR3: AY768549, TWIST1: NG_008114, EFNB1: NG_008887.
Parentage was proven where necessary by trio (proband, father, mother) short tandem repeats (STR) analysis of chromosomes 13, 18, 21, and X using quantitative fluorescent PCR, as previously described (Bili et al., 2002).
Results
Molecular Genetic Analysis Results
Genetic analysis revealed pathogenic mutations in 13 patients, 3 of which were novel, the previously described clinically insignificant c.1150T>C (p.F384L) polymorphism in FGFR3 gene (Golla et al., 1997) in 2 patients and a heterozygous deletion of TWIST1 gene in 1 patient (Table 2). Genetic analysis was inconclusive for the remaining 33 patients with syndromic or nonsyndromic craniosynostosis participating in this study because no mutations were found in the genes tested.
Patients With Molecular Findings.a
aIn bold are shown causative genetic lesions as well as final molecular diagnosis that does not agree with the initial clinical evaluation, for patients 18, 42, 47, and 53.
bPatient 48 died postoperatively.
Novel Mutations
Both direct sequencing and amplified fragment length polymorphism (AFLP) analysis revealed that the CFNS patient (no. 40) was heterozygous for the novel frameshift mutation c.717dupA - p.(L240fs) in the EFNB1 gene (HGMD, last accessed January 2018) (Figure 1A). Digestion with MfeI, which recognizes and cleaves the mutant sequence leaving the normal sequence intact, was performed in order to confirm the mutation (Figure 1B). The mutation was shown to be de novo because it was not present in either of her parents. Parentage was proven by trio STR analysis. The other novel mutation within EFNB1 gene was the previously reported missense mutation c.373G>A - p.(E125K) found in patient 22. Clinical phenotype as well as detailed molecular analysis of patient 22, revealing the novel p.(E125K) has been described elsewhere (Apostolopoulou et al., 2012).
Νovel frameshift mutation c.717dupA - p.(Leu240IlefsTer79) in EFNB1 gene. DNA sequence chromatogram from the patient (heterozygote). Normal sequence and the site of adenine insertion are noted (A). Agarose gel electrophoresis of PCR products digested with MfeI (lane 1: U = undigested PCR product; lanes 2 through 4: 40 = patient 40, 40F = patient’s father, 40M = patient’s mother). The c.717 adenine duplication creates a MfeI site (B).
DNA sequencing analysis revealed that patient 47, initially diagnosed as Pfeiffer syndrome, was carrying the novel missense mutation c.413T>C - p.(L138P) in TWIST1 gene (HGMD, last accessed January 2018). Mutations in the TWIST1 gene are responsible for the Saethre-Chotzen syndrome. Analysis of her family revealed that the patient has inherited the p.L138P mutation from her unaffected father, who was mosaic for the mutation (Figure 2).
Novel missense mutation c.413T>C - p.(Leu138Pro) in TWIST1 gene. DNA sequence chromatograms from patient 47 (heterozygote, N/M) (A), her father (unaffected mosaic) (B), her brother (normal, N/N) (C), and her healthy mother (D).
Previously Reported Known Mutations
Four out of 5 Crouzon patients (nos. 10, 33, 34 and 44) were found to carry the previously described mutations in FGFR2 gene c.1025G>A (p.C342Y), 1084+3A>G, c.1061C>G (p.S354C) and c.1024T>C (p.C342R), respectively. The FGFR2 c.758C>G (p.P253R) mutation was found in the Apert twins (nos. 14 and 15), whereas the other 2 Apert patients (nos. 43 and 48) were carriers of the c.755C>G (p.S252W) mutation.
One patient (no. 18) who was initially designated as a case of nonsyndromic unicoronal synostosis was found to be a carrier of TWIST1 whole gene deletion. The deletion arose apparently de novo because none of his parents carried it. Deletions including TWIST1 gene account for at least 10% of Saethre-Chotzen syndrome cases (Passos-Bueno et al., 2008).
The FGFR3 c.749C>G (p.P250R) mutation, responsible for Muenke syndrome, was found in 2 patients (nos. 42 and 53), initially diagnosed with nonsyndromic and syndromic coronal synostosis, respectively. Although DNA from the parents of patient 42 was not available for testing, it is possible that he inherited the mutation from his affected mother, whose clinical presentation included asymmetric flatness of the forehead and moderate deformation of the orbits, compatible with unilateral coronal synostosis.
AFLP analysis of patient 53’s family showed that he inherited the mutation from his affected father, to whom it was passed through his mildly affected mother, the patient’s grandmother. The mutation was also present in his affected infant sister.
The clinically insignificant FGFR3 p.F384L polymorphism was found in the patient with syndromic trigonocephaly (no. 2) and was inherited from her unaffected father, as well as in one patient with Crouzon syndrome (no. 44), who also was a carrier of the pathogenic FGFR2 p.C342R mutation. No other genetic defect was revealed in patient 2; thus, genetic testing was inconclusive for this patient.
Clinical Phenotypes
Clinical phenotype findings of all patients participating in this study are summarized in Table 1. Crouzon patients (nos. 10, 33, 34, and 44), Apert patients (nos. 14, 15, 43, and 48), and Muenke patients (nos. 42 and 53) were found to carry known mutations in the FGFR2 and FGFR3 genes, as well as patient 18 with Saethre-Chotzen syndrome, who carries the TWIST1 gene deletion, were found to exhibit no unexpected or previously undescribed clinical findings. Detailed clinical description of patients 40 and 47, who harbor novel mutations in EFNB1 and TWIST1, respectively, is listed below in order to provide further useful data for better phenotype-genotype correlation.
Patient 40
The patient presented at the Hellenic Craniofacial Center at 1 month of age. She was the product of normal conception, uneventful gestation, and vaginal delivery at full term. Her abnormal morphology consists of turribrachycephaly caused by bilateral coronal synostosis, hypertelorism, strabismus, and a broad nose. The patient’s neck was short, and findings from the trunk included scoliosis, kyphosis, pectus excavatum, and aplasia of the breasts. The upper limbs exhibited restriction of shoulder movements, axillary pterygia, bilateral syndactyly of the third and fourth ray sparing the bones, and clinodactyly of the fifth finger, bilaterally (Figure 3). Preoperatively, clinical findings were confirmed by a 3-dimensional computed tomography scan (3D CT scan) and magnetic resonance imaging (MRI) study. She underwent occipital decompression and fronto-orbital advancement at the age of 6 months and 7 years, respectively. Bilateral correction of syndactyly was performed at the age of 13 years. Even though the patient was initially misdiagnosed, careful clinical evaluation pointed toward CFNS. The patient was followed up for 17 years postoperatively with no complications. However, she shows progressive signs of relapse, so additional revision operations cannot be excluded in the future. The patient showed mild learning and behavioral problems. The overall annual school performance was poor, and she failed several examinations. She was referred to a psychologist by the school board because of behavioral problems within the classroom.
Frontal (A) and lateral (B) view of patient 40. Frontal view of her left foot (C). Frontal (D) and lateral (E) view of the trunk and breasts. Anterior cranium bifidum, hypertelorism, and broad nose, the appearance of which is further aggravated by its bifid tip (A). Frontal flattening, low-set ears, thick and curly hair, and low-set hairline are also observed (B). Hallucal groove and mild syndactyly of the third and fourth ray limited to soft tissue web (C). The short neck is also associated with bilateral pterygium colli. Apart from the pectus excavatum, there is bilateral breast hypoplasia and low-set nipples (D, E).
Patient 47
The patient was referred to the Hellenic Craniofacial Center at 1 month of age. She was the product of normal conception, uneventful gestation, and vaginal delivery at full term. She was initially misdiagnosed as having Pfeiffer syndrome. The clinical examination revealed turribrachycephaly, large, bilateral ossification defects of the frontal bones causing frontal bossing and mild maxillary hypoplasia with concomitant flatness of the mid face. Preoperatively, clinical findings were confirmed by a 3D CT scan and MRI study. Additional findings included shallow orbits, hypertelorism, and low-set ears with narrow auditory canals. Stenosis of the lacrimal ducts was also confirmed (Figure 4). The patient underwent a fronto-orbital advancement procedure with removal of surplus interorbital bone at the age of 6 months. Recurrence of hypertelorism and flatness of the mid- and upper face necessitated a facial bipartition 7 years later. The patient was followed up for 5 years postoperatively with no complications, although she shows progressive signs of relapse, so additional revision operations cannot be excluded in the future. Her mental and intellectual development was normal according to school performance and teachers’ reports. No abnormalities of her physical and sexual development were detected.
Frontal (A) and lateral (B) view of patient 47. Hypertelorism accompanied by mild blepharophimosis, epicanthal folds, ptosis, and hypoplastic skin of the upper eyelids (A). Frontal bossing along with flattened and posteriorly displaced supraorbital rims, and low-set ears (B).
Discussion
Genetic analysis of 46 syndromic and nonsyndromic craniosynostosis patients in the Greek or Cypriot population revealed 9 different mutations in FGFR1-3, TWIST1, and EFNB1 genes, 3 of which were novel, a heterozygous deletion of TWIST1 gene and the clinically insignificant FGFR3 p.F384L polymorphism. The FGFR2 mutations p.C342Y, p.S354C, and p.C342R found in the Crouzon patients, both p.S252W and p.P253R found in the Apert patients, and the FGFR3 p.P250R mutation as well as the heterozygous deletion of whole TWIST1 gene found in 3 of the patients with coronal synostosis are well-documented genetic causes of syndromic craniosynostosis (reviewed in Passos-Bueno et al., 2008). The 1084+3A>G mutation in intron 10 of FGFR2 gene found in patient 33 with mild Crouzon syndrome is very rare, and it has been described 3 times before. Until now, this mutation has been linked to mild Crouzon in only 1 family (Kan et al., 2004). The 2 other cases in which FGFR2 1084+3A>G mutation has been reported include a patient with Pfeiffer syndrome (Cornejo-Roldan et al., 1999) and a clinically unclassified patient with a combination of sagittal and lambdoid synostosis (Mulliken et al., 1999). 1084+3A>G mutation results in activation of an upstream cryptic donor splice site, which leads to FGFR2 mRNA synthesis lacking 51 nucleotides in exon IIIc (Kan et al., 2004).
Novel Mutations in the Twist1 and Efnb1 Genes
Twist1
TWIST1 is a highly conserved basic helix-loop-helix (bHLH) transcription factor and its expression seems to negatively regulate osteoblast differentiation, maintaining osteoblastic cells in an osteoprogenitor-like state (Miraoui and Marie, 2010). TWIST1 can form homo- or heterodimers with other bHLH proteins such as E2A (E12 and E47) and binds to short conserved sequences called E-boxes (5′-CANNTG-3′) in the promoter of target genes. The HLH region, which consists of a short alpha helix (helix I) connected by a loop to a second, longer alpha helix (helix II) is very important for protein dimerization and DNA binding (El Ghouzzi et al., 2000; Connerney et al., 2006; Maia et al., 2012). Residue L138, which lies within the loop of bHLH motif, is highly conserved among TWIST1 proteins of different organisms such as mouse, frog, and Drosophila, as well as in other human HLH proteins, such as E12 and E47. The novel mutation that we found in the second proband with Saethre-Chotzen syndrome, leading to the replacement of hydrophobic leucine at position 138 of TWIST1 with proline, a nonhydrophobic secondary amine, commonly found in turns, could result in loss of DNA-binding capacity of TWIST1 dimers, thus impairing TWIST1 activity, as expected by other missense mutations within the HLH loop (El Ghouzzi et al., 2000, Maia et al., 2012). The female patient carrying the novel p.L138P mutation shows a severe phenotype with some Pfeiffer-like clinical features, such as low-set ears and hypertelorism, while her mosaic father is unaffected. To our knowledge, the patient’s father is the first case of mosaicism described for TWIST1 gene. Until now, when counseling concerning sibling recurrence risk in the case of de novo TWIST1 mutations, a figure of 2% is considered appropriate, but in cases of parental mosaicism, caution should be taken in genetic counseling as the figure could rise to 10%, as for siblings of females with CNFS (Johnson and Wilkie, 2011).
Efnb1
Ephrin-B1, encoded by EFNB1 gene, belongs to the B-subclass of ligands of Eph receptors (receptor tyrosine kinases) and consists of an ephrin domain, a transmembrane domain, a cytoplasmic domain and a PDZ domain (protein-protein interaction domain) (Kullander and Klein, 2002). Eph/ephrin signaling is important for the developmental processes of the embryo, including skeletal and craniofacial development (Arvanitis and Davy, 2008). A unique feature of Eph/ephrin interaction is the bidirectional signaling that results in forward signaling through Eph receptors and reverse signaling through ephrin ligands, activating pathways in both receptor-expressing and ligand-expressing cells, respectively (Edwards and Mundy, 2008). The novel p.(L240fs) mutation, found in the patient with CFNS, caused by an adenine duplication in position c.717 of EFNB1 gene, disrupts the open reading frame and creates a premature stop codon, thus destroying part of the transmembrane as well as the entire cytoplasmic domain of ephrin-B1. This truncated protein product, definitely lacking the ability of reverse signaling, is probably unstable, or even soluble. It is interesting to note that only frameshift mutations have been identified in exon 5 (Wallis et al., 2008). The female patient carrying the novel p.(L240fs) mutation shows a severe phenotype of CFNS syndrome, including not only craniofacial but also skeletal malformations, such as pterygium colli, scoliosis, kyphosis, pectus excavatum, aplasia of the breasts, axillary pterygia, and bilateral syndactyly.
The Significant Role of Molecular Diagnosis in Clinical Evaluation and Vice Versa
Commonly overlapping clinical features among different craniosynostosis syndromes on the one hand and great phenotypic heterogeneity even within the same syndrome, especially Muenke, on the other hand, as well as inadequate and sometimes misleading clinical evaluation create great difficulties in establishing proper diagnosis without the contribution of genetic testing. Molecular diagnosis, which will confirm or reject initial clinical evaluation, is highly important for prognosis, family counseling, and treatment. Repeat craniofacial surgery was required for 67% of children with genetic lesions in FGFR2, FGFR3 (p.P250R mutation), TWIST1, and EFNB1 genes, whereas only 6.25% of patients with chromosomal abnormalities or without a genetic diagnosis underwent or are scheduled for reoperation (this study). Likewise, meticulous clinical documentation of craniofacial patients is acknowledged as being an integral part of molecular testing because it supplies the appropriate guidance to narrow down the target range. Otherwise, molecular testing can become expensive and time consuming.
Value of Molecular Testing and Careful Clinical Evaluation
Patients’ molecular testing was based on the algorithm suggested by Johnson and Willkie (2011). According to the initial clinical evaluation a first line test was performed and if negative, more genes were tested. Regarding patient 47 who carries the novel p.L138P TWIST1 gene mutation, which is responsible for Saethre-Chotzen syndrome, the first-line test was negative because she was not screened for mutations in TWIST1 gene as a result of the initial clinical misdiagnosis as possible Pfeiffer syndrome. TWIST1 gene was screened as a second-line test. This was also the case for patient 18 with an initial misdiagnosis of nonsyndromic unicoronal synostosis who was found to carry a TWIST1 gene deletion. Based on these findings, we would like to propose that TWIST1 testing both by sequencing as well as by microdeletion analysis could be included for patients showing syndromic craniosynostosis with Pfeiffer-like features, as well as for apparent nonsyndromic unicoronal synostosis patients as second line testing, especially when they are tested negative for variants in the common genes, because clinical features are sometimes overlooked and result in misdiagnosis. As for Saethre-Chotzen syndrome, proper and definitive diagnosis for Muenke syndrome patients (nos. 42 and 53) was achieved only after genetic testing revealed p.P250R mutation in the FGFR3 gene. Thus, the contribution of molecular genetic analysis in the diagnosis of patients with syndromic craniosynostosis was extremely useful because some were originally misdiagnosed.
On the other hand, patient 40, with CFNS, who was found to carry the novel p.(L240fs) mutation within the EFNB1 gene, had been originally designated as a mild Apert syndrome case. Reevaluation of her clinical features suggested a possible diagnosis of CFNS, thus leading to sequence analysis of EFNB1 gene. Thorough clinical evaluation guided molecular testing, leading to correct diagnosis.
Once again, our results support the invaluable role of molecular testing as well as thorough clinical evaluation in the achievement of correct diagnosis in craniosynostosis patients.
Syndromic Craniosynostosis Cases have a Higher Positive Molecular Testing Rate Compared to Nonsyndromic
Irrespective of their initial clinical evaluation, which in some cases does not agree with genetic testing results, all cases (n=14) in which molecular diagnosis was successful were syndromic. Genetic diagnosis accounted for approximately 30% of all craniosynostosis cases. In accordance to the literature (Wilkie et al., 2010), cases with nonsyndromic sagittal (n=12), metopic (n=4), or lambdoid (n=3) synostoses had negative test results.
Clinical Heterogeneity in Muenke Syndrome
A typical example of clinical heterogeneity in Muenke syndrome is patient 53’s family. He and his infant sister presented with severe right coronal synostosis and life-threatening elevated intracranial pressure, which was addressed surgically with success. His father was characterized by brachycephaly due to bicoronal synostosis, whereas his grandmother had a very mild phenotype, possibly caused by right coronal synostosis. Both were never operated on mainly because they were undiagnosed until patient 53 was referred to the HCC. We should note that patient 53’s infant sister was born during this study. Although proper genetic counseling was given to the family, they chose not to proceed with prenatal diagnosis.
Conclusion
The novel TWIST1 and EFNB1 gene mutations described here resulted in a severe phenotype of Saethre-Chotzen syndrome and CFNS, respectively. All other syndromic cases with positive molecular analysis showed expected and well-described clinical features. For better phenotype-genotype correlation more cases must be described, especially for the novel mutations reported. High heterogeneity due to differences in penetrance and expressivity as well as overlapping clinical features observed in craniosynostosis cases often make correct clinical diagnosis extremely difficult. Thus, clinical evaluation and description of craniosynostosis patients should be made by a specialized medical team, which includes a molecular geneticist. Thorough clinical evaluation can guide molecular testing. In this study, it was only after the designation of patient 40 as possible CFNS that she was screened for mutations in the EFNB1 gene, revealing the novel p.(L240fs) mutation. Conversely, molecular testing proved to be an invaluable tool in completing clinical diagnosis. Muenke syndrome is a typical example of molecularly designated syndrome, because it is characterized by the presence of p.P250R mutation in the FGFR3 gene. Here we describe a whole family and an isolated patient with Muenke syndrome, designated as such only after they were tested positive for FGFR3 p.P250R mutation. Both Saethre-Chotzen syndrome patients described here, one carrying the novel p.L138P mutation and the other with the heterozygous deletion of TWIST1 gene, were discovered after molecular testing in the TWIST1 gene. Because of the initial clinical misdiagnosis in both of these Saethre-Chotzen syndrome patients, the TWIST1 gene was not tested at first. This is why TWIST1 testing could be useful for patients showing syndromic craniosynostosis with Pfeiffer-like features, as well as for apparent nonsyndromic unicoronal synostosis patients as a second-line testing when no mutations are found in FGFR1-3 genes. Therefore, molecular genetic analysis completes clinical diagnosis and is necessary to connect as many phenotypes as possible with their underlying genetic cause, that is, building the base for proper phenotype-genotype correlation, allowing correct genetic counseling and prediction of their clinical course.
Web Resources
HGMD-Human Genome Mutation Database, http://www.hgmd.cf.ac.uk/ac/all.php
GenBank, www.ncbi.nlm.nih.gov/Genbank/
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.