Introduction

Craniofrontonasal syndrome (CFNS) (OMIM #304110) has first been described in 1979 by Cohen [1]. It is an X-linked disorder caused by mutations in the Ephrin B1 gene (EFNB1), which is located on the short arm of the X chromosome (Xp13.1) [2]. The paucity of male CFNS patients has been described in the literature [3]. It is known that heterozygous females are more severely affected than hemizygous males [4,5,6]. In females, characteristic abnormalities include severe hypertelorism, depressed nasal bridge, and coronal synostosis. Occasionally, cleft lip and palate, diaphragmatic hernias, and corpus callosum agenesis or dysgenesis are present. Hemizygous males show no or mild signs, such as hypertelorism. Longitudinal ridging of the nails, syn- or poly-dactyly, and wiry hair commonly occur in CFNS [2, 5,6,7].

EFNB1 codes for Ephrin B1, which is a transmembrane protein of 346 amino acids (ENST00000204961.4). Ephrin B1 binds to Eph receptor kinases [8]. Eph/ephrin complexes play crucial roles in neural development and plasticity, as well as morphogenesis through formation of boundaries [9, 10]. Twigg et al. [11] proved that the Efnb1 expression is particularly high in murine neural crest cells. While the complete loss of Efnb1 in mice is perinatally lethal, female Efnb1+/− mutants exhibit dysmorphic features characteristic of CNFS, which are absent in hemizygous males [12]. This phenomenon might be explained by difficulties establishing boundaries in the mosaic state arising from X inactivation intermingling EFNB1-negative and EFNB1-positive cells [13]. In hemizygous males, the function of EFNB1 might be taken over by a related ephrin.

To date, a total of 116 mutations in EFNB1 have been described, the majority of which are missense mutations, followed by the occurrence of small deletions and splice-site mutations. Here, we report on four additional patients from four different families who carry four novel EFNB1 mutations and demonstrate clinical variability in CFNS.

Materials and methods

Sanger sequencing

Written informed consent of the patients was acquired prior to genetic testing. DNA was obtained from the whole-blood samples. The five exons and exon–intron boundaries of EFNB1 (NM_004429) were amplified using oligonucleotide primers. Primer pairs were 5′-AGAAGAGCGACACCGAAGC-3′ and 5′- AGACCTCCCCACATGCACT-3′ yielding a 379-bp product for exon 1, 5′- CCTGAGGCTGACCATCTTCT-3′ and 5′- GTTAAGCCCAGGGAGAGAGC-3′ resulting in a 357-bp product for exon 2, 5′-TGGGAGTTTCTGGGTAATGC-3′ and 5′-CTGTTCCAAAGGTCAAACAGG-3′ yielding a 223-bp product for exon 3, 5′-ATGACTGAGGGCACCTATGC-3′ and 5′-GGGCCTAACAAGGTGACAGA-3′ yielding a 250-bp product for exon 4, and 5′-GCCTGAAATCTGCTGTGTGT-3′ and 5′-AAATACAAAGGTGGGCACAG-3′ yielding a 585-bp product for exon 5. The PCR products were sequenced on an ABI3730xl DNA Analyzer (Applied Biosystems).

Mutation analysis

The sequencing data were compared with the EFNB1 cDNA reference sequence GenBank accession number NM_004429 and analyzed using the software Geneious [14]. Further in silico analysis of the mutations was performed by the prediction tools MutationTaster [15] and Human Splicing Finder [16].

Ephrin ectodomain 3D model

The ectodomain structure of the closely related Ephrin B2 (1iko) was uploaded to the CCP4 software [17, 18]. The residues Ile63, Cys64, and Cys101 were projected as ball-and-stick structures onto the ribbon-type protein model.

Results

Clinical presentation

The clinical findings of the four unrelated female patients are summarized in Table 1 and the corresponding images are shown in Figs. 1 and 2. All patients described in this paper are the first children of Indian origin born to nonconsanguineous parents, who did not show any signs of CFNS.

Table 1 Clinical features and genotype of four individuals with craniofrontonasal syndrome
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Fig. 1
figure 1

Variable craniofacial features of four female CFNS patients. All patients present with hypertelorism, depressed nasal bridge, and low-set ears. Patient 1 (a) and patient 2 (b) present with anteverted nares, patient 2 additionally with a high palate and a cleft upper lip. Patient 3 (c) and patient 4 (d) show downslanting palpebral fissures. Patient 3 additionally presents with midface retrusion, anterior open bite, and short neck, patient 4 with cleft upper lip and orbital dystopia

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Fig. 2
figure 2

Additional skeletal findings. Upper and lower extremities of patient 1 (a–c) and patient 4 (d–h). Patient 1 presents with a brachydactyly and a broad thumb, b bilateral undertoeing, and c a medially deviated third toe of the left foot. Patient 2 presents with d complete III–IV syndactyly of the right hand (operated) with fused nails at the fourth finger and e partial cutaneous III/IV syndactyly of the left hand. g Longitudinal ridging of the left big toe. f Radiographs revealed an osseous duplication of the middle and distal phalanges of the fourth finger of the left hand, h high ridging scapula with elongated clavicles, and i lateral view of skull shows severe craniostenosis with copper beaten appearance

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Patient 1 was referred for dysmorphic evaluation and developmental delay. Hypothyroidism was diagnosed in the newborn period and treated with 25 μg thyroxine. On examination at the age of 6 months, she presented with a weight of 5.5 kg (<3rd centile), length of 64 cm (3rd centile), and head circumference of 39.5 cm (<3rd centile). Head control was attained. She had a coarse face, a hoarse voice, a small anterior fontanel, significant hypertelorism, bilateral epicanthic folds, bilateral low-set ears, macrostomia, a tongue tie, a thick upper midline frenulum, a microform cleft upper lip, and a short neck. She had an extremely flat nasal bridge, anteverted nares, and a midline crease of the tip of the nose (Fig. 1a). Brachydactyly and hyperelasticity of finger joints were evident (Fig. 2a). The inspection of the feet revealed a bilateral splintering of the big toes (Fig. 2b, c) and a medially deviated third toe with bilateral undertoeing (Fig. 2c). Magnetic resonance imaging (MRI) showed agenesis of corpus callosum.

Patient 2 was born at term by normal delivery (birth weight 3.85 kg (75−90th centile)) and started to crawl at 5 months of age. Clinical examination demonstrated a head circumference of 39 cm (3rd centile), a small anterior fontanel, a coarse face, hypertelorism, downslanting palpebral fissures, brachycephaly, a small cleft of the upper lip, low-set overfolded pinna, a high-arched palate, and a short neck (Fig. 1b). No interdigital webbing or abnormalities of fingers and toes were present. A brain MRI at the age of 5 months revealed a corpus callosum (callosal) dysgenesis. She had a unicoronal synostosis (anterior plagiocephaly) and underwent fronto-orbital advancement and hypertelorism correction. At 2 ½ years of age, she had a weight of 10 kg (<3rd centile), height 85 cm (3rd centile), and head circumference of 44 cm (<3rd centile). She could walk without support but had unclear speech.

Patient 3 had an unremarkable family history. She showed normal development and intelligence. On examination at the age of 4 ½ years, her height was measured at 98 cm (3rd centile) and the head circumference at 48 cm (<3rd centile). Examination of the face revealed plagiocephaly and severe hypertelorism, a broad nasal root and tip, antimongoloid slant of the eyes, prognathism, and an anterior open bite (Fig. 1c). Cranial imaging showed unilateral left-sided coronal craniostenosis. She underwent fronto-orbital advancement for correction of unilateral coronal synostosis and facial bipartition for correction of hypertelorism. The neck was short and showed webbing, the chest was flattened, and the nipples were low placed. The patient was unable to elevate the shoulders completely, with prominent wasting of infraclavicular muscles. Dysmorphic features of the hands included a complete III/IV syndactyly of the left hand and a partial cutaneous syndactyly of the fingers 2–5 on the right hand. Longitudinal ridging of big toes and other toes was detected and significantly more distinct on the left side.

Patient 4 presented with a broad nasal root and tip, downslanting palpebral fissures, severe hypertelorism, and anterior open bite with normal intelligence (Fig. 1d). She had a webbed neck, dropping of shoulders, pectus excavatum, and low-lying and asymmetrical nipples. The inspection of the extremities revealed complete cutaneous III–IV syndactyly of the right hand, which was surgically treated (Fig. 2d), partial cutaneous III/IV syndactyly of the left hand (Fig. 2e), and longitudinal ridging of third fingers of both hands. Longitudinal ridging of the big toes and other toes was also evident and more distinct on the left side (Fig. 2g). Radiographs revealed an osseous duplication of middle and distal phalanges of the fourth finger of the right hand (Fig. 2f). She had left postal axial polydactyly, which was excised, and left-sided cleft lip and palate, which was surgically repaired during infancy. She had a large fossa ovalis, an atrial septal defect which was corrected by device closure at 9 ½ years of age. The patient also had a high-ridging scapula with elongated clavicles (Fig. 2h) and severe craniostenosis with copper-beaten appearance (Fig. 2i). Bicoronal synostoses with brachycephaly were evident and she underwent hypertelorism correction by facial bipartition technique and maxillary distraction.

In all four cases, CFNS was the suspected diagnosis, but for patient 2, the differential diagnosis of Teebi-type hypertelorism (OMIM #145420) was also considered.

Mutation analysis

Since the clinical findings were suggestive of CFNS, we amplified EFNB1 by PCR and analyzed the gene by Sanger sequencing. This revealed four heterozygous mutations in exons 1 and 2 of EFNB1 in the index patients, which were not listed in ExAC, gnomAD, or the 1000 genomes project. Investigation of the available parent DNAs revealed wild-type sequences. An overview of the Sanger sequencing and segregation testing is shown in Fig. 4, and bioinformatic pathogenicity predictions are summarized in Supplementary Table 1.

The heterozygous mutation c.186_188delCAT (p.Ile63del) in exon 2 detected in patient 1 affects a highly conserved amino acid in the extracellular ephrin domain next to a cysteine residue forming a disulfide bridge (Fig. 3a, b). The mutation was confirmed by Sanger sequencing, but could not be detected in the index’ mother (Fig. 4a). DNA of the father was not available. The variant is ranked pathogenic due to high amino acid conservation, but no splice alterations are predicted (ACMG class: likely pathogenic).

Fig. 3
figure 3

Consequences of the p.Ile63del mutation. a Alignment of the p.Ile63del mutation (red box) identified in patient 1 and the previously found single amino acid deletion p.Val51del (blue box). The adjacent Cys64 cysteine residue is marked in orange. Note the high evolutionary conservation of the sequence harboring both mutations. b A 3D model of the EFNB1 ectodomain showing the secondary structure as ribbon and side chains of the amino acids Ile63 and Cys64 as ball and stick (green = carbon, red = oxygen, and blue = nitrogen). The deleted Ile63 lies at the end of a β-sheet (gray arrow) and the adjacent cysteine forms an essential disulfide bridge (yellow), which—by prediciton—will get lost as a consequence of the deletion

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Fig. 4
figure 4

Pedigrees and segregation of mutations found in CFNS patients. Sanger validation and segregation of the EFNB1 mutations c.186_188delCAT in patient 1 (a), c.404_405insTACATTAC (p.S136Tfs*26) patient 2 (b), c.196_197insC (p.Arg66Profs*9) in patient 3 (c), and c.42_43delG (p.Ala15Argfs*31) in patient 4 (d)

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Patient 2 carries the mutation EFNB1 c.404_405insTACATTAC (p.Ser136Thrfs*26) (Fig. 4b). In silico analysis by the Human Splicing Finder indicates a possible alteration of splicing (ACMG class: pathogenic). In patient 3, the mutation EFNB1 c.196_197insC (p.Arg66Profs*9) was detected (Fig. 4c). No DNA from other family members was available. According to in silico analysis with the Human Splicing Finder, the original exonic splicing enhancer site is altered or broken in the setting of the mutation, most likely causing an alteration of splicing (ACMG class: likely pathogenic). The mutation c.42_43delG (p.Ala15Argfs*31) was found in patient 4 (Fig. 4d). Sequencing of the parents revealed a wild-type sequence. The activation of an exonic cryptic donor splice site is predicted by the Human Splicing Finder (ACMG class: pathogenic).

Discussion

In this study, we describe four female individuals from four different Indian families with characteristic stigmata of CFNS. By a targeted screening approach through Sanger sequencing, we found four novel disease-causing mutations in the EFNB1 gene, three of them are frameshift mutations and one is a small deletion.

All mutations described were neither annotated in ExAC nor in the 1000 genomes project. The heterozygous mutation EFNB1 c.186_188delCAT (p.Ile63del) in patient 1 is located in the highly conserved extracellular ephrin domain, which is crucial for receptor ligand recognition and complex formation [19]. This mutation is the only one of our series, for which no significant effect on splicing is predicted. The only single amino acid deletion reported so far is c.151_153delGTG (p.Val51del) described by Twigg et al. [3], which is in the vicinity of p.Ile63del. The two female carriers of this mutation exclusively revealed typical facial features and coronal craniosynostosis. The phenotype of patient 1 is considerably more severe, especially due to corpus callosum agenesis and developmental delay. In how far the latter might also be attributable to hypothyroidism is currently unknown. The strong effect of the p.Ile63del mutation is surprising, since it deletes one of the two consecutive isoleucine residues. We hypothesize that due to this deletion, the cysteine at position 64 loses its ability to form a disulfide bridge with cysteine 101, which is a strong stabilizer of the tertiary protein structure. The variant most similar to c.404_405insTACATTAC (p.Ser136Thrfs*26) found in patient 2 is c.296_297delTCinsGGTGCTCG (p.Thr100Valfs*62) reported by Inoue et al. [20]. The affected patient had hypertelorism, depressed nasal bridge, bifid nasal tip, bicoronal synostosis, and bilateral cleft lip and palate. No mutations similar to c.196_197insC (p.Arg66Profs*9) and c.42_43delG (p.Ala15Argfs*31) detected in patients 3 and 4, respectively, have been reported to date.

Mutations with premature termination codons (PTCs) are known to cause nonsense-mediated mRNA decay (NMD) or lead to severely truncated, instable proteins. In some cases, an escape of NMD is possible. We predict that the frameshift mutations described in patients 2, 3, and 4 lead to alternative splicing and result in NMD, with an overall loss-of-function effect. The mutations p.Val51del and p.Ile63del demonstrate how sensitive the Ephrin B1 protein is toward alterations of such kind and support the prediction that even if alternative splicing leads to in-frame products, the resulting larger deletions will also have a loss-of-function effect. Chacon-Camacho et al. [21] analyzed the truncating mutation EFNB1 c.445_449delGAGGG in exon 3 at the expression level and detected a severe decrease in the expression level of EFNB1 mRNA, which confirmed the degradation by NMD. Since no patient RNA was available for our studies, we could not perform investigations on the RNA level.

The facial changes in patients 3 and 4 are more pronounced than in patients 1 and 2. On the other hand, patients 1 and 2 had corpus callosum anomalies. An explanation for the phenotype variability could be the random X inactivation in females, which has been described in CFNS patients [22]. In heterozygous females, the X inactivation causes a somatic mosaicism, in which cells with defective EFNB1 on their active X chromosome are functionally EFNB1 null mutants. In this mosaic pattern, a higher share of mutated cells brings about a more severe phenotype and can thus explain the range in CFNS severity. Carrel and Willard [23] showed that the escape of X inactivation could contribute to the interindividual phenotypic differences of heterozygous females.

Some syndromes caused by mutations in different genes have overlapping features with CFNS. Three types of frontonasal dysplasia (FND) are caused by mutations in different genes. Type 1 (FND1, OMIM #136760) is caused by mutations in ALX3, Type 2 by mutations in ALX4 (FND2, OMIM #613451), and Type 3 by mutations in ALX1 (FND3, OMIM #613456) [24]. Acrofacial dysostosis (AFD1, OMIM #154400), also known as Nager syndrome, is caused by mutations in SF3B4. In addition to craniofacial features similar to CFNS, they have characteristic upper-extremity deformities, but no lower-extremity involvement [25]. Acromelic frontonasal dysostosis (OMIM #603671) is another rare syndrome similar to CFNS caused by heterozygous mutations in ZSWIM6. It comprises craniofacial malformations similar to CFNS (e.g., FND and corpus callosum agenesis), as well as nonfacial traits, e.g., brain and limb malformations (tibial hemimelia and preaxial polydactyly) [26].

In conclusion, CFNS can be accounted as part of a spectrum disorder of craniofacial and limb anomalies. Our findings broaden the spectrum of EFNB1 mutations and demonstrate that there is no obvious genotype–phenotype correlation, since the deletion of a single N-terminal amino acid has the same effect as mutations leading to PTCs.