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. 2004 Jun 8;101(23):8652-7.
doi: 10.1073/pnas.0402819101. Epub 2004 May 27.

Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome

Stephen R F Twigg  1 , Rui Kan, Christian Babbs, Elena G Bochukova, Stephen P Robertson, Steven A Wall, Gillian M Morriss-Kay, Andrew O M Wilkie
Affiliations

Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome

Stephen R F Twigg et al. Proc Natl Acad Sci U S A. .

Abstract

Craniofrontonasal syndrome (CFNS) is an X-linked developmental disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Females have frontonasal dysplasia and coronal craniosynostosis (fusion of the coronal sutures); in males, hypertelorism is the only typical manifestation. Here, we show that the classical female CFNS phenotype is caused by heterozygous loss-of-function mutations in EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases. In mice, the orthologous Efnb1 gene is expressed in the frontonasal neural crest and demarcates the position of the future coronal suture. Although EFNB1 is X-inactivated, we did not observe markedly skewed X-inactivation in either blood or cranial periosteum from females with CFNS, indicating that lack of ephrin-B1 does not compromise cell viability in these tissues. We propose that in heterozygous females, patchwork loss of ephrin-B1 disturbs tissue boundary formation at the developing coronal suture, whereas in males deficient in ephrin-B1, an alternative mechanism maintains the normal boundary. This is the only known mutation in the ephrin/Eph receptor signaling system in humans and provides clues to the biogenesis of craniosynostosis.

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Figures

Fig. 1.
Fig. 1.
Clinical features of CFNS. (a) Facial view showing marked hypertelorism, divergent squint, and central nasal groove (subject ID, 1593; age, 1 year). (b) Three-dimensional computed tomographic skull reconstruction (subject ID, 3167; age, 8 months) showing right unicoronal synostosis, lateral displacement of orbits, and central defect between frontal bones. Note bony ridge at site of obliterated right coronal suture (arrowhead); the left coronal suture is patent (arrow). f, frontal bone; p, parietal bone. (c) Longitudinal splitting of the nails is frequent.
Fig. 2.
Fig. 2.
Selected heterozygous mutations of EFNB1 in females with CFNS. Each figure part shows, from top to bottom, a normal DNA sequence chromatogram, mutant DNA sequence chromatogram(s), and corresponding restriction digest confirmation. Pedigree symbols (shown in black for affected individuals) are aligned vertically with corresponding lanes of the gel. N, normal control. (a) Frame-shifting deletion 246delG in subject 369 (sequenced with reverse primer) and confirmation by HhaI digest, showing creation of a new restriction site. (b) De novo nonsense mutation 196C→ T in subject 344, which abolishes an AvaI site. (c) The recurrent mutation 451G→ A, present in four unrelated families, is transmitted by an affected mother to her daughter and has arisen de novo in one parent–child trio. Also shown is the adjacent mutation 452G→ T in subject 372 that abolishes the same AciI site.
Fig. 3.
Fig. 3.
Comparative amino acid sequence alignment of human (h), mouse (m), chick (c), zebrafish (z), and Xenopus (x) ephrin-B1; human ephrin-B2 and ephrin-B3; and C. elegans VAB-2. Above the human ephrin-B1 sequence, the identities of missense substitutions are shown in red single-letter codes, together with the positions of the single-nucleotide deletion (▵), nonsense (•), and donor and acceptor splice site mutations (> and <, respectively). Regions of secondary structure in murine ephrin-B2 are indicated at the top of each sequence block. Interacting regions in the complex with murine EphB2 (21) are color-coded at the bottom of each sequence block. Note that the targets of missense mutations at L98, Q115, and P119 are amino acids that are predicted to interact with the Eph receptor at the major (dimerization) interface: L98 and Q115 form hydrogen bonds while P119 inserts into a hydrophobic pocket, forming both van der Waals contacts and a hydrogen bond (21).
Fig. 4.
Fig. 4.
RNA in situ hybridization of Efnb1 in mouse embryos at E9.5 (a) and E10.5 (b). Arrow in b marks the boundary between a high level of expression in the region of the telencephalon compared with the adjacent diencephalon, and it indicates the level of the section shown in c. e, eye. (c) Section of an E10.5 embryo showing high Efnb1 transcript levels in the neuroepithelium (nep) of the telencephalon and adjacent neural crest-derived mesenchyme (nc) but not in the diencephalon (d) or cranial mesoderm (m). e, eye. Arrow shows the boundary between neural crest-derived and mesoderm-derived cranial mesenchyme, which marks the position of the future coronal suture (23). Scale bars indicate 1 mm (a and b) and 200 μm(c).
Fig. 5.
Fig. 5.
X-inactivation patterns in the AR gene of CFNS subjects. Mean and range of values obtained from three independent PCRs is shown. Pairs of affected mothers and daughters are aligned at the same position on the horizontal axis and indicated by ▴ and ▵ for mothers and daughters, respectively. Triangles point up or down, respectively, according to whether the mutant or the wild-type allele is preferentially inactivated. •, Phase-unknown cases.
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