Original article 79
Report of a family with craniofrontonasal syndrome Berk Özyılmaza, Alper Gezdiricic, Mustafa Özend and Önder Kalendererb Craniofrontonasal syndrome (CFNS, OMIM 304110) paradoxically presents a severe phenotype in heterozygous females and a mild or a normal phenotype in hemizygous males. Hypertelorism is seen in almost all of the female CFNS patients; craniosynostosis, facial asymmetry, and bifid nose are the other major clinical features. Most of the males are mildly affected, frequently only with hypertelorism. Here, we report a family with a G151S mutation in the EFNB1 gene. The mutation was identified in two severely affected sisters and paradoxically in their clinically unaffected father. The father on whom we report is the first male patient genetically proved to carry a CFNS-causing mutation and not presenting any signs nor
symptoms of CFNS. Clin Dysmorphol 24:79–83 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Clinical Dysmorphology 2015, 24:79–83 Keywords: cellular interference, craniofrontonasal syndrome, craniosynostosis, EFNB1, genotype phenotype, hemizygous, X-linked dominant Departments of aMedical Genetics, bOrthopedics and Traumatology, Tepecik Education and Research Hospital, Izmir, cDepartment of Medical Genetics, Kanuni Sultan Suleyman Training and Research Hospital, Istanbul and dDepartment of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey Correspondence to Berk Özyılmaz, MD, Department of Medical Genetics, Tepecik Education and Research Hospital, Yenisehir, Konak, Izmir, Turkey Tel: + 90 542 807 1057; fax: + 90 232 457 9651; e-mail: [email protected] Received 27 February 2014 Accepted 30 October 2014
Introduction Craniofrontonasal syndrome (CFNS, OMIM 304110) is an X-linked dominant disorder of craniofacial and skeletal development. CFNS paradoxically presents a severe phenotype in heterozygous females and a mild or a normal phenotype in hemizygous males. Although heterozygous females can have coronal craniosynostosis, craniofacial asymmetry, hypertelorism, cleft palate, bifid
Summary of the clinical features and differences between sexes
Table 1
System
Features
Cranial
Craniosinostosis Brachicephaly Frontal bossing Facial asymmetry Widow’s peak Thick, wiry hair Hypertelorism Telecanthus Down-slanting palpebral fissures Broad nasal root/bifid nasal tip Cleft lip/cleft palate Narrow-sloping shoulders Sprengel deformity Clavicle pseudoarthrosis Diaphragmatic hernia Short stature Asymmetric lower limb short. Fifth-finger clinodactyly Brittle-grooved nails Longitudinal splitting of nails Syndactyly Broad halluces/brachydactyly Normal intelligence Developmental delay Hypoplastic corpus callosum
Craniofacial
Thoracoabdominal
Skeletal
Neurologic
More frequent in female or male patients Females Females Females
Females Females and males Females
Females Males Males Females
0962-8827 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
nasal tip, Sprengel deformity, and coarse hair, males generally only show hypertelorism (Table 1). CFNS was clinically described by Cohen (1979) in a girl with coronal synostosis, shoulder movement difficulties, and digital anomalies with linear ridges in the nails. Pedigree analysis of reported families suggested that CFNS was inherited in an X-linked manner. In 2004, these data were confirmed by the demonstration of mutations in the EFNB1 gene, which was mapped to the Xq13.1 region (Twigg et al., 2004; Wieland et al., 2004). The EFNB1 gene encodes the transmembrane protein ephrin-B1, which acts as a ligand for Eph receptors and as a receptor for nearby ephrin-B1-expressing cells. This Eph/ephrin bidirectional signaling system guides cell migration in the frontonasal neural crest, morphogenesis of the palatal shelf, and formation of future coronal sutures (Twigg et al., 2004; Wieland et al., 2004; Bush and Soriano, 2010). The EFNB1 gene is located on the X chromosome, and is subject to random X-inactivation in females. In heterozygous females, random X-inactivation leads to mosaic distribution of the cells carrying mutant and wild-type EFNB1 genes (Wieland et al., 2005; Shotelersuk et al., 2006). Previously reported studies suggested that the severity of heterozygous females is associated with a patchy tissue distribution of EFNB1 functional and nonfunctional cells (Twigg et al., 2004; Wieland et al., 2005). A nonworking Eph/ephrin signaling system and inhibited communication in the boundaries between these tissue patches may provide a basis for the previously mentioned severe phenotype in females. This process was called ‘cellular interference’ (Wieland et al., 2005; Vasudevan et al., 2006; Twigg et al., 2013). DOI: 10.1097/MCD.0000000000000067
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80 Clinical Dysmorphology 2015, Vol 24 No 2
In hemizygous males, all cells carry the mutant EFNB1 gene, no patchy tissue pattern is generated, no disrupted tissue boundaries form, and possibly functional redundancy of other ephrins improves the milder phenotype (Wieland et al., 2005; Vasudevan et al., 2006; Twigg et al., 2013). These data are also supported by the demonstration of a more severe phenotype in constitutionally mosaic males. EFNB1 mutation mosaicism in hemizygous males leads to abnormal cellular interactions similar to heterozygous females (Twigg et al., 2013). The EFNB1 gene contains five exons. Exon 1 encodes a signal peptide, exons 2 and 3 encode extracellular ephrin domains, and exon 5 encodes transmembrane/cytoplasmic domains. The roles of exon 2 and 3 are important because of receptor–ligand interaction function and they harbor most of the missense mutations (Wieland et al., 2005; Vasudevan et al., 2006; Torii et al., 2007; Twigg et al., 2013). The most frequent type of mutations found in CFNS are nonsense, frameshift, and splice site mutations resulting in premature termination codons (PTCs). These PTCs lead to a nonfunctional ephrin-B1 protein (Ramirez-Garcia et al., 2013). Here, we report a family with two sisters manifesting severe craniofacial abnormalities suggestive of CFNS. Members of the family were screened for EFNB1 mutations. Sequencing analysis showed a G151S mutation in the two severely affected sisters but also in their clinically unaffected father. Mildly affected males have been reported previously in the literature, but completely unaffected males have not been described.
Clinical report The family was referred to us for a clinical genetics opinnion because of two sisters affected with craniofacial abnormalities. In the family, the parents were fifthdegree relatives. They had had five children; two males were physically normal; two females had craniofacial abnormalities; and no detailed clinical evaluation could be carried out on the third female as she had been given up for adoption. However, she was described as having hypertelorism, bifid nose, and exotropia. Patient 1 was an 11-year-old female who had frontal bossing, facial asymmetry, hypertelorism, telecanthus, exotropia, down-slanting palpebral fissures, nystagmus, a broad-bifid nasal tip, and coarse hair. She had undergone surgical correction for plagiocephaly at the age of 9 years. She also had narrow-sloping shoulders, Sprengel’s deformity, fifth-finger clinodactyly, and longitudinal splits of her nails. MRI of the brain showed partial agenesis of the corpus callosum and dysplasia of the right cerebellar hemisphere. She is of normal intellect and attends a regular school (Fig. 1). Patient 2 was the 7-year-old sister of patient 1. She had frontal bossing, craniofacial asymmetry, hypertelorism, telecanthus, exotropia, down-slanting palpebral fissures,
nystagmus, a broad-bifid nasal tip, and a complete cleft lip and palate. She had fifth-finger clinodactyly with longitudinal splits in her nails (Fig. 1) and also had partial agenesis of the corpus callosum. She was developmentally normal. The father (Fig. 1) was clinically normal. His head circumference was within normal limits (head circumference: 57.5 cm) and he did not have hypertelorism nor telecanthus (inner canthal distance: 3 cm; interpupillary distance: 6.6 cm) (Oztürk et al., 2006). The mother was entirely normal.
Methods After receiving informed consent from the family, genomic DNA were extracted from peripheral blood (3 ml) of the patients and their family members according to the manufacturer’s instructions (Roche, Mannheim, Germany). Exons 1, 2, 3, 4, and 5 (entire coding region of EFNB1) and exon–intron boundaries were amplified using primers and conditions as described previously (Seven et al., 2013). The PCR products were purified and sequenced using an ABI 3500 genetic analyzer. The sequencing data were compared with the EFNB1 GenBank reference sequence (accession numbers: NM_004429. 4 and NG_008887.71 and ensembl number: ENST00000204961).
Results Bidirectional sequencing analysis of the PCR products showed that the two clinically affected sisters (patient 1 and patient 2) were heterozygous for the transversion c.451G > A (G151S). As a result of the mutation, glycine at position 151 was replaced by serine (G151S). Sequencing analysis of the clinically unaffected parents showed that the mutation was inherited from the father whereas no mutation was found in the mother (Fig. 1). The father was hemizygous for the c.451G > A (G151S) mutation. The two clinically unaffected male siblings were also analyzed and, as expected, no mutation was found in the EFNB1 gene.
Discussion When the literature is reviewed with the reports comparing affected females with unaffected/mildly affected males, the paradoxic phenotypic pattern of the syndrome becomes evident. Before the gene in which mutations cause CFNS was discovered, Feldman et al. (1997) reported phenotypic analysis of 40 patients from 12 different families with a clinical diagnosis of CFNS. They evaluated 28 female and 12 male patients and found that most of the males were mildly affected, notably with hypertelorism as their only sign. In contrast to the males, female patients presented with severe clinical features including craniosynostosis and facial asymmetry. For two female patients who were mildly affected, they speculated the role of
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Craniofrontonasal syndrome Özyılmaz et al. 81
Fig. 1
(a)
(b)
(e) I
II
7
III
( ) 5
3
G (c)
(d)
G
G
C
A
G
T
G
II.7
G
G
G
C
R
G
T
G
G
G
G
C
R
G
T
G
III.3
III.5
Clinical features and the mutation analysis of the patients. (a) Patient 1 showed frontal bossing, facial asymmetry, hypertelorism, telecanthus, exotropia, and a broad-bifid nasal tip. (b) Patient 1 with Sprengel deformity (asymmetric upward displacement of the scapula). (c) Patient 2 showed frontal bossing, craniofacial asymmetry, hypertelorism, telecanthus, exotropia, and a broad-bifid nasal tip. (d) The father does not present signs of CFNS. (e) Sequence analysis of the EFNB1 exon 3 showing heterozygous c.451G > A mutation from patient 1 (III.3) and patient 2 (III.5) and hemizygous c.451G > A mutation from the father (II.7). CFNS, craniofrontonasal syndrome.
favorable lyonization or germinal mosaicism (Feldman et al., 1997). In 1999, Pulleyn and colleagues reported two clinically diagnosed CFNS families and their haplotype analysis. Although the phenotypes of affected females in the first family were consistent with typical CFNS, the male patient was suggested to be clinically normal (Pulleyn et al., 1999). Although the authors suggested that he was a clinically unaffected male CFNS patient as he carried the putative affected marker (Xp22), these data turned out to be incorrect when the EFNB1 gene was mapped to Xq13.1 (Twigg et al., 2004; Wieland et al., 2004). In 2005, Wieland and colleagues reported the mutation analysis of nine unrelated families and 29 sporadic patients with CFNS. In one of the families mentioned, a mother, a daughter, and a son were found to be carrying the c.80C > G mutation. Although the mother and a daughter presented hypertelorism and orbital asymmetry, the son only showed hypertelorism (Wieland et al., 2005). In 2006, Vasudevan and colleagues reported two unrelated families with severely affected mothers reported to
have typical CFNS symptoms. Although the sons had no major craniofacial features, they both had congenital diaphragmatic hernia (Vasudevan et al., 2006). Twigg et al. (2006) analyzed 50 female and nine male patients. Six hemizygous males had mild phenotypes with only hypertelorism. The clinical features in female CFNS patients are described in Table 2. G151S is a missense mutation in exon 3 that has been identified previously in four patients by Twigg et al. (2006). All four patients reported previously were affected by craniosynostosis and one additionally had cleft lippalate (Twigg et al., 2004). As patient 1 in this article also presented with cleft lip-palate and this phenotype is rarely found in female patients (Table 2), it can be speculated that the G151S mutation might be associated with cleft lip and palate. We reviewed 10 male CFNS patients from the literature, none of whom had craniosynostosis nor facial asymmetry (Table 2). Occasionally, more serious symptoms such as cleft lip-palate, chest abnormalities, Sprengel deformity,
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0/8 (0) 0/8 (0)
In 1999, Pulleyn and colleagues reported a clinically unaffected male patient by analyzing linked markers in Xp22; however, that linkage data were proved to be incorrect when the EFNB1 gene was mapped to Xq13.2 (Feldman et al., 1997; Twigg et al., 2004; Wieland et al., 2004). Thus, among the 10 male CFNS cases reviewed here, the father on whom we report is the first male patient genetically proved to carry a CFNS-causing mutation and not to present any features of CFNS.
CFNS, craniofrontonasal syndrome; NDA, no data available. As no detailed clinical data were available, craniosynostosis, facial asymmetry, hypertelorism, and bifid nose were counted in ‘Typical CFNS’ description. Three female and two male patients reported previously by Vasudevan and colleagues were not included. Patients ‘unknown’ were not included. b
a
1/3 (33) 1/4 (25) 0/10 (0) 9/10 (90) 0/10 (0) 0/10 (0) 10 Male
Total [n/N (%)]
and diaphragmatic hernia can present in male CFNS patients.
0/4 (0)
2/3 (66)
0/6 0/2 0/1 8/65 (12) 0/6 2/2 0/1 18/59 (30) NDA 2/2 0/1 3/6 (50) (9) 6 2 1 71 This study
Male Female Male Female
0/6 2/2 0/1 59/68 (86)
0/6 2/2 0/1 24/24 (100)
6/6 2/2 0/1 24/24 (100)
0/6 2/2 0/1 24/24 (100) 0/10 (0)
0/6 1/2 0/1 6/65 (9)
NDA 0/2 0/1 0/2 (0)
NDA 2/2 0/1 8/24 (33)
NDA 0/2 0/1 0/6 (0)
NDA 6/45 NDA 11/39 1/2 NDA Twigg et al. (2006)b
Male 2 Female (50) 47
0/2 35/44
0/2 NDA
2/2 NDA
0/2 NDA
0/2 4/45
1/2 NDA
0/2 NDA
2/2 NDA
0/1 NDA 0/1 NDA NDA 1/4 1 4 Vasudevan et al. (2006)a
Male Female
0/1 4/4
0/1 4/4
1/1 4/4
0/1 4/4
0/1 NDA
0/1 NDA
0/1 4/4
NDA 0/4
2/18 5/18 NDA NDA 18 Wieland et al. (2005)a
Female
18/18
18/18
18/18
18/18
1/18
NDA
2/18
Brain abnormality Diaphragmatic hernia Chest abnormality Bifid nose Hypertelorism Craniosynostosis Patients Authors
Table 2
Review of the literature on CFNS features
Facial asymmetry
Craniofacial abnormalities
Cleft lippalate
Hair abnormality
Nail abnormality
Other abnormalities
Skeletal abnormality
82 Clinical Dysmorphology 2015, Vol 24 No 2
In the literature, this mild or normal phenotypic outcome has been explained by several hypotheses. So far, the ‘cellular interference’ explanation is supported by most authors. The mild or normal phenotypic outcome in hemizygous males is based on the idea of preserved boundaries between tissue patches in the craniofrontonasal region and the theory of functional redundancy of ephrin activity (Cohen, 1979; Wieland et al., 2005; Vasudevan et al., 2006; Twigg et al., 2013). We report on this family to emphasize the fact that males who have no manifestations of CFNS may, nonetheless, still be mutation carriers with a consequent risk to their daughters.
Acknowledgements Conflicts of interest
There are no conflicts of interest.
References Bush JO, Soriano P (2010). Ephrin-B1 forward signaling regulates craniofacial morphogenesis by controlling cell proliferation across Eph-ephrin boundaries. Genes Dev 24:2068–2080. Cohen MM Jr (1979). Craniofrontonasal dysplasia. Birth Defects Orig Artic Ser 15:85–89. Feldman GJ, Ward DE, Lajeunie-Renier E, Saavedra D, Robin NH, Proud V, et al. (1997). A novel phenotypic pattern in X-linked inheritance: craniofrontonasal syndrome maps to Xp22. Hum Mol Genet 6:1937–1941. Oztürk F, Yavas G, Inan UU (2006). Normal periocular anthropometric measurements in the Turkish population. Ophthalmic Epidemiol 13:145–149. Pulleyn LJ, Winter RM, Reardon W, McKeown C, Jones B, Hayward R, et al. (1999). Further evidence from two families that craniofrontonasal dysplasia maps to Xp22. Clin Genet 55:473–477. Ramirez-Garcia MA, Chacon-Camacho OF, Leyva-Hernandez C, CardenasConejo A, Zenteno JC (2013). A novel de novo EFNB1 gene mutation in a mexican patient with craniofrontonasal syndrome. Case Rep Genet 2013: doi: 10.1155/2013/349725. Seven M, Gezdirici A, Ulucan H, Karatas OF, Yosunkaya E, Yuksel A, Ozen M (2013). A novel EFNB1 mutation in a patient with craniofrontonasal syndrome and right hallux duplication. Gene 527:675–678. Shotelersuk V, Siriwan P, Ausavarat S (2006). A novel mutation in EFNB1, probably with a dominant negative effect, underlying craniofrontonasal syndrome. Cleft Palate Craniofac J 43:152–154. Torii C, Izumi K, Nakajima H, Takahashi T, Kosaki K (2007). EFNB1 mutation at the ephrin ligand-receptor dimerization interface in a patient with craniofrontonasal syndrome. Congenit Anom (Kyoto) 47:49–52. Twigg SR, Kan R, Babbs C, Bochukova EG, Robertson SP, Wall SA, et al. (2004). Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome. Proc Natl Acad Sci USA 101:8652–8657. Twigg SR, Matsumoto K, Kidd AM, Goriely A, Taylor IB, Fisher RB, et al. (2006). The origin of EFNB1 mutations in craniofrontonasal syndrome: frequent somatic mosaicism and explanation of the paucity of carrier males. Am J Hum Genet 78:999–1010.
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Craniofrontonasal syndrome Özyılmaz et al. 83
Twigg SR, Babbs C, van den Elzen ME, Goriely A, Taylor S, McGowan SJ, et al. (2013). Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes. Hum Mol Genet 22:1654–1662. Vasudevan PC, Twigg SR, Mulliken JB, Cook JA, Quarrell OW, Wilkie AO (2006). Expanding the phenotype of craniofrontonasal syndrome: two unrelated boys with EFNB1 mutations and congenital diaphragmatic hernia. Eur J Hum Genet 14:884–887.
Wieland I, Jakubiczka S, Muschke P, Cohen M, Thiele H, Gerlach KL, et al. (2004). Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome. Am J Hum Genet 74:1209–1215. Wieland I, Reardon W, Jakubiczka S, Franco B, Kress W, Vincent-Delorme C, et al. (2005). Twenty-six novel EFNB1 mutations in familial and sporadic craniofrontonasal syndrome (CFNS). Hum Mutat 26:113–118.
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Report of a family with craniofrontonasal syndrome Berk Özyılmaza, Alper Gezdiricic, Mustafa Özend and Önder Kalendererb Craniofrontonasal syndrome (CFNS, OMIM 304110) paradoxically presents a severe phenotype in heterozygous females and a mild or a normal phenotype in hemizygous males. Hypertelorism is seen in almost all of the female CFNS patients; craniosynostosis, facial asymmetry, and bifid nose are the other major clinical features. Most of the males are mildly affected, frequently only with hypertelorism. Here, we report a family with a G151S mutation in the EFNB1 gene. The mutation was identified in two severely affected sisters and paradoxically in their clinically unaffected father. The father on whom we report is the first male patient genetically proved to carry a CFNS-causing mutation and not presenting any signs nor
symptoms of CFNS. Clin Dysmorphol 24:79–83 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Clinical Dysmorphology 2015, 24:79–83 Keywords: cellular interference, craniofrontonasal syndrome, craniosynostosis, EFNB1, genotype phenotype, hemizygous, X-linked dominant Departments of aMedical Genetics, bOrthopedics and Traumatology, Tepecik Education and Research Hospital, Izmir, cDepartment of Medical Genetics, Kanuni Sultan Suleyman Training and Research Hospital, Istanbul and dDepartment of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey Correspondence to Berk Özyılmaz, MD, Department of Medical Genetics, Tepecik Education and Research Hospital, Yenisehir, Konak, Izmir, Turkey Tel: + 90 542 807 1057; fax: + 90 232 457 9651; e-mail: [email protected] Received 27 February 2014 Accepted 30 October 2014
Introduction Craniofrontonasal syndrome (CFNS, OMIM 304110) is an X-linked dominant disorder of craniofacial and skeletal development. CFNS paradoxically presents a severe phenotype in heterozygous females and a mild or a normal phenotype in hemizygous males. Although heterozygous females can have coronal craniosynostosis, craniofacial asymmetry, hypertelorism, cleft palate, bifid
Summary of the clinical features and differences between sexes
Table 1
System
Features
Cranial
Craniosinostosis Brachicephaly Frontal bossing Facial asymmetry Widow’s peak Thick, wiry hair Hypertelorism Telecanthus Down-slanting palpebral fissures Broad nasal root/bifid nasal tip Cleft lip/cleft palate Narrow-sloping shoulders Sprengel deformity Clavicle pseudoarthrosis Diaphragmatic hernia Short stature Asymmetric lower limb short. Fifth-finger clinodactyly Brittle-grooved nails Longitudinal splitting of nails Syndactyly Broad halluces/brachydactyly Normal intelligence Developmental delay Hypoplastic corpus callosum
Craniofacial
Thoracoabdominal
Skeletal
Neurologic
More frequent in female or male patients Females Females Females
Females Females and males Females
Females Males Males Females
0962-8827 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
nasal tip, Sprengel deformity, and coarse hair, males generally only show hypertelorism (Table 1). CFNS was clinically described by Cohen (1979) in a girl with coronal synostosis, shoulder movement difficulties, and digital anomalies with linear ridges in the nails. Pedigree analysis of reported families suggested that CFNS was inherited in an X-linked manner. In 2004, these data were confirmed by the demonstration of mutations in the EFNB1 gene, which was mapped to the Xq13.1 region (Twigg et al., 2004; Wieland et al., 2004). The EFNB1 gene encodes the transmembrane protein ephrin-B1, which acts as a ligand for Eph receptors and as a receptor for nearby ephrin-B1-expressing cells. This Eph/ephrin bidirectional signaling system guides cell migration in the frontonasal neural crest, morphogenesis of the palatal shelf, and formation of future coronal sutures (Twigg et al., 2004; Wieland et al., 2004; Bush and Soriano, 2010). The EFNB1 gene is located on the X chromosome, and is subject to random X-inactivation in females. In heterozygous females, random X-inactivation leads to mosaic distribution of the cells carrying mutant and wild-type EFNB1 genes (Wieland et al., 2005; Shotelersuk et al., 2006). Previously reported studies suggested that the severity of heterozygous females is associated with a patchy tissue distribution of EFNB1 functional and nonfunctional cells (Twigg et al., 2004; Wieland et al., 2005). A nonworking Eph/ephrin signaling system and inhibited communication in the boundaries between these tissue patches may provide a basis for the previously mentioned severe phenotype in females. This process was called ‘cellular interference’ (Wieland et al., 2005; Vasudevan et al., 2006; Twigg et al., 2013). DOI: 10.1097/MCD.0000000000000067
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80 Clinical Dysmorphology 2015, Vol 24 No 2
In hemizygous males, all cells carry the mutant EFNB1 gene, no patchy tissue pattern is generated, no disrupted tissue boundaries form, and possibly functional redundancy of other ephrins improves the milder phenotype (Wieland et al., 2005; Vasudevan et al., 2006; Twigg et al., 2013). These data are also supported by the demonstration of a more severe phenotype in constitutionally mosaic males. EFNB1 mutation mosaicism in hemizygous males leads to abnormal cellular interactions similar to heterozygous females (Twigg et al., 2013). The EFNB1 gene contains five exons. Exon 1 encodes a signal peptide, exons 2 and 3 encode extracellular ephrin domains, and exon 5 encodes transmembrane/cytoplasmic domains. The roles of exon 2 and 3 are important because of receptor–ligand interaction function and they harbor most of the missense mutations (Wieland et al., 2005; Vasudevan et al., 2006; Torii et al., 2007; Twigg et al., 2013). The most frequent type of mutations found in CFNS are nonsense, frameshift, and splice site mutations resulting in premature termination codons (PTCs). These PTCs lead to a nonfunctional ephrin-B1 protein (Ramirez-Garcia et al., 2013). Here, we report a family with two sisters manifesting severe craniofacial abnormalities suggestive of CFNS. Members of the family were screened for EFNB1 mutations. Sequencing analysis showed a G151S mutation in the two severely affected sisters but also in their clinically unaffected father. Mildly affected males have been reported previously in the literature, but completely unaffected males have not been described.
Clinical report The family was referred to us for a clinical genetics opinnion because of two sisters affected with craniofacial abnormalities. In the family, the parents were fifthdegree relatives. They had had five children; two males were physically normal; two females had craniofacial abnormalities; and no detailed clinical evaluation could be carried out on the third female as she had been given up for adoption. However, she was described as having hypertelorism, bifid nose, and exotropia. Patient 1 was an 11-year-old female who had frontal bossing, facial asymmetry, hypertelorism, telecanthus, exotropia, down-slanting palpebral fissures, nystagmus, a broad-bifid nasal tip, and coarse hair. She had undergone surgical correction for plagiocephaly at the age of 9 years. She also had narrow-sloping shoulders, Sprengel’s deformity, fifth-finger clinodactyly, and longitudinal splits of her nails. MRI of the brain showed partial agenesis of the corpus callosum and dysplasia of the right cerebellar hemisphere. She is of normal intellect and attends a regular school (Fig. 1). Patient 2 was the 7-year-old sister of patient 1. She had frontal bossing, craniofacial asymmetry, hypertelorism, telecanthus, exotropia, down-slanting palpebral fissures,
nystagmus, a broad-bifid nasal tip, and a complete cleft lip and palate. She had fifth-finger clinodactyly with longitudinal splits in her nails (Fig. 1) and also had partial agenesis of the corpus callosum. She was developmentally normal. The father (Fig. 1) was clinically normal. His head circumference was within normal limits (head circumference: 57.5 cm) and he did not have hypertelorism nor telecanthus (inner canthal distance: 3 cm; interpupillary distance: 6.6 cm) (Oztürk et al., 2006). The mother was entirely normal.
Methods After receiving informed consent from the family, genomic DNA were extracted from peripheral blood (3 ml) of the patients and their family members according to the manufacturer’s instructions (Roche, Mannheim, Germany). Exons 1, 2, 3, 4, and 5 (entire coding region of EFNB1) and exon–intron boundaries were amplified using primers and conditions as described previously (Seven et al., 2013). The PCR products were purified and sequenced using an ABI 3500 genetic analyzer. The sequencing data were compared with the EFNB1 GenBank reference sequence (accession numbers: NM_004429. 4 and NG_008887.71 and ensembl number: ENST00000204961).
Results Bidirectional sequencing analysis of the PCR products showed that the two clinically affected sisters (patient 1 and patient 2) were heterozygous for the transversion c.451G > A (G151S). As a result of the mutation, glycine at position 151 was replaced by serine (G151S). Sequencing analysis of the clinically unaffected parents showed that the mutation was inherited from the father whereas no mutation was found in the mother (Fig. 1). The father was hemizygous for the c.451G > A (G151S) mutation. The two clinically unaffected male siblings were also analyzed and, as expected, no mutation was found in the EFNB1 gene.
Discussion When the literature is reviewed with the reports comparing affected females with unaffected/mildly affected males, the paradoxic phenotypic pattern of the syndrome becomes evident. Before the gene in which mutations cause CFNS was discovered, Feldman et al. (1997) reported phenotypic analysis of 40 patients from 12 different families with a clinical diagnosis of CFNS. They evaluated 28 female and 12 male patients and found that most of the males were mildly affected, notably with hypertelorism as their only sign. In contrast to the males, female patients presented with severe clinical features including craniosynostosis and facial asymmetry. For two female patients who were mildly affected, they speculated the role of
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
Craniofrontonasal syndrome Özyılmaz et al. 81
Fig. 1
(a)
(b)
(e) I
II
7
III
( ) 5
3
G (c)
(d)
G
G
C
A
G
T
G
II.7
G
G
G
C
R
G
T
G
G
G
G
C
R
G
T
G
III.3
III.5
Clinical features and the mutation analysis of the patients. (a) Patient 1 showed frontal bossing, facial asymmetry, hypertelorism, telecanthus, exotropia, and a broad-bifid nasal tip. (b) Patient 1 with Sprengel deformity (asymmetric upward displacement of the scapula). (c) Patient 2 showed frontal bossing, craniofacial asymmetry, hypertelorism, telecanthus, exotropia, and a broad-bifid nasal tip. (d) The father does not present signs of CFNS. (e) Sequence analysis of the EFNB1 exon 3 showing heterozygous c.451G > A mutation from patient 1 (III.3) and patient 2 (III.5) and hemizygous c.451G > A mutation from the father (II.7). CFNS, craniofrontonasal syndrome.
favorable lyonization or germinal mosaicism (Feldman et al., 1997). In 1999, Pulleyn and colleagues reported two clinically diagnosed CFNS families and their haplotype analysis. Although the phenotypes of affected females in the first family were consistent with typical CFNS, the male patient was suggested to be clinically normal (Pulleyn et al., 1999). Although the authors suggested that he was a clinically unaffected male CFNS patient as he carried the putative affected marker (Xp22), these data turned out to be incorrect when the EFNB1 gene was mapped to Xq13.1 (Twigg et al., 2004; Wieland et al., 2004). In 2005, Wieland and colleagues reported the mutation analysis of nine unrelated families and 29 sporadic patients with CFNS. In one of the families mentioned, a mother, a daughter, and a son were found to be carrying the c.80C > G mutation. Although the mother and a daughter presented hypertelorism and orbital asymmetry, the son only showed hypertelorism (Wieland et al., 2005). In 2006, Vasudevan and colleagues reported two unrelated families with severely affected mothers reported to
have typical CFNS symptoms. Although the sons had no major craniofacial features, they both had congenital diaphragmatic hernia (Vasudevan et al., 2006). Twigg et al. (2006) analyzed 50 female and nine male patients. Six hemizygous males had mild phenotypes with only hypertelorism. The clinical features in female CFNS patients are described in Table 2. G151S is a missense mutation in exon 3 that has been identified previously in four patients by Twigg et al. (2006). All four patients reported previously were affected by craniosynostosis and one additionally had cleft lippalate (Twigg et al., 2004). As patient 1 in this article also presented with cleft lip-palate and this phenotype is rarely found in female patients (Table 2), it can be speculated that the G151S mutation might be associated with cleft lip and palate. We reviewed 10 male CFNS patients from the literature, none of whom had craniosynostosis nor facial asymmetry (Table 2). Occasionally, more serious symptoms such as cleft lip-palate, chest abnormalities, Sprengel deformity,
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0/8 (0) 0/8 (0)
In 1999, Pulleyn and colleagues reported a clinically unaffected male patient by analyzing linked markers in Xp22; however, that linkage data were proved to be incorrect when the EFNB1 gene was mapped to Xq13.2 (Feldman et al., 1997; Twigg et al., 2004; Wieland et al., 2004). Thus, among the 10 male CFNS cases reviewed here, the father on whom we report is the first male patient genetically proved to carry a CFNS-causing mutation and not to present any features of CFNS.
CFNS, craniofrontonasal syndrome; NDA, no data available. As no detailed clinical data were available, craniosynostosis, facial asymmetry, hypertelorism, and bifid nose were counted in ‘Typical CFNS’ description. Three female and two male patients reported previously by Vasudevan and colleagues were not included. Patients ‘unknown’ were not included. b
a
1/3 (33) 1/4 (25) 0/10 (0) 9/10 (90) 0/10 (0) 0/10 (0) 10 Male
Total [n/N (%)]
and diaphragmatic hernia can present in male CFNS patients.
0/4 (0)
2/3 (66)
0/6 0/2 0/1 8/65 (12) 0/6 2/2 0/1 18/59 (30) NDA 2/2 0/1 3/6 (50) (9) 6 2 1 71 This study
Male Female Male Female
0/6 2/2 0/1 59/68 (86)
0/6 2/2 0/1 24/24 (100)
6/6 2/2 0/1 24/24 (100)
0/6 2/2 0/1 24/24 (100) 0/10 (0)
0/6 1/2 0/1 6/65 (9)
NDA 0/2 0/1 0/2 (0)
NDA 2/2 0/1 8/24 (33)
NDA 0/2 0/1 0/6 (0)
NDA 6/45 NDA 11/39 1/2 NDA Twigg et al. (2006)b
Male 2 Female (50) 47
0/2 35/44
0/2 NDA
2/2 NDA
0/2 NDA
0/2 4/45
1/2 NDA
0/2 NDA
2/2 NDA
0/1 NDA 0/1 NDA NDA 1/4 1 4 Vasudevan et al. (2006)a
Male Female
0/1 4/4
0/1 4/4
1/1 4/4
0/1 4/4
0/1 NDA
0/1 NDA
0/1 4/4
NDA 0/4
2/18 5/18 NDA NDA 18 Wieland et al. (2005)a
Female
18/18
18/18
18/18
18/18
1/18
NDA
2/18
Brain abnormality Diaphragmatic hernia Chest abnormality Bifid nose Hypertelorism Craniosynostosis Patients Authors
Table 2
Review of the literature on CFNS features
Facial asymmetry
Craniofacial abnormalities
Cleft lippalate
Hair abnormality
Nail abnormality
Other abnormalities
Skeletal abnormality
82 Clinical Dysmorphology 2015, Vol 24 No 2
In the literature, this mild or normal phenotypic outcome has been explained by several hypotheses. So far, the ‘cellular interference’ explanation is supported by most authors. The mild or normal phenotypic outcome in hemizygous males is based on the idea of preserved boundaries between tissue patches in the craniofrontonasal region and the theory of functional redundancy of ephrin activity (Cohen, 1979; Wieland et al., 2005; Vasudevan et al., 2006; Twigg et al., 2013). We report on this family to emphasize the fact that males who have no manifestations of CFNS may, nonetheless, still be mutation carriers with a consequent risk to their daughters.
Acknowledgements Conflicts of interest
There are no conflicts of interest.
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