Duplication of the EFNB1 gene in familial hypertelorism: imbalance in ephrin-B1 expression and abnormal phenotypes in humans and mice

Abstract

Familial hypertelorism, characterized by widely spaced eyes, classically shows autosomal dominant inheritance (Teebi type), but some pedigrees are compatible with X-linkage. No mechanism has been described previously, but clinical similarity has been noted to craniofrontonasal syndrome (CFNS), which is caused by mutations in the X-linked EFNB1 gene. Here we report a family in which females in three generations presented with hypertelorism, but lacked either craniosynostosis or a grooved nasal tip, excluding CFNS. DNA sequencing of EFNB1 was normal, but further analysis revealed a duplication of 937 kb including EFNB1 and two flanking genes: PJA1 and STARD8. We found that the X chromosome bearing the duplication produces ∼1.6-fold more EFNB1 transcript than the normal X chromosome and propose that, in the context of X-inactivation, this difference in expression level of EFNB1 results in abnormal cell sorting leading to hypertelorism. To support this hypothesis, we provide evidence from a mouse model carrying a targeted human EFNB1 cDNA, that abnormal cell sorting occurs in the cranial region. Hence, we propose that X-linked cases resembling Teebi hypertelorism may have a similar mechanism to CFNS, and that cellular mosaicism for different levels of ephrin-B1 (as well as simple presence/absence) leads to craniofacial abnormalities.

Figure 4

Quantification of relative allelic transcription at EFNB1 (A) and XIST (B) by Pyrosequencing. In each figure, the part of the cDNA sequence analysed is shown at the top, with the amplification primer sequences in bold, sequencing primers in lower case, and the polymorphic base(s) enclosed by square brackets. Below, the dispensation order and allele-specific bases for comparison are indicated, with a table listing the allele-specific incorporation of bases at each dispensation (denoted by subscripted numbers). The lower part shows pyrograms obtained using genomic DNA from hemizygous males carrying each of the alleles under investigation, genomic DNA from subject I-2, genomic DNA from a normal female and cDNA generated from peripheral blood from subject I-2. The allele-specific peaks used for quantification are marked with arrows and raw measurements of comparative ratios indicated as percentages.

Figure 1

Pedigree and phenotype of individuals heterozygous for EFNB1 duplication. A: Pedigree showing the immediate family of the proband (arrow). Filled symbols represent individuals shown to carry the duplication. Under each symbol, haplotypes of loci assayed in EFNB1 and XIST are shown together with duplication status of each X chromosome (see key). Note that the X chromosome bearing the duplication (marked in red in the duplicated individuals and in the nonduplicated sisters of I-2) is identified by a C allele at the EFNB1 and XIST SNPs. na indicates that no DNA was available for analysis. B,C: Facial appearance of the proband, aged 10 years. D: Proband's mother (II-2) aged 41 years and E, grandmother (I-2) aged 63 years.

Figure 2

Molecular and cytogenetic characterization of X-chromosome duplication including EFNB1. A: MLPA analysis of the proband III-3 (lower panel) compared with a normal female control (upper panel). The estimated dose of EFNB1 exons 1–5 in the proband is ∼1.5 compared with 31 autosomal amplicons. B: Plot showing extent of duplication determined by array CGH. Probes apparently duplicated in the proband are shown in green, indicating a duplication size of ∼900 kb. C: FISH with BAC clone RP11-30P7. This clone spans the EFNB1 gene and hybridizes to the X chromosomes only, with one chromosome showing a brighter signal, consistent with a tandem duplication of the EFNB1 region. D: Duplex PCR assay for duplication junction (lower fragment) and GLI2 amplification control (upper fragment). Pedigree symbols are aligned above corresponding lanes; H₂O, water control. na indicates that no DNA was available for analysis.

Figure 3

DNA sequence characterization of X chromosome duplication. The upper panel shows positions and directions of transcription of genes in the region and the size and extent of the duplicated segment (rectangular box). Below, the DNA sequence chromatogram spanning the breakpoint and alignment of this sequence compared to the normal sequences at the telomeric and centromeric limits of the duplication. The arrowheads indicate the range of possible positions of the breakpoint within the six-nucleotide identity (shaded) shared by both normal sequences. [Color figures can be viewed in the online issue, which is available at http://www.wiley.com/humanmutation.]

Figure 5

Analysis of Efnb1^Lox/+ mice. A: 3D Micro-CT scan of an adult Efnb1^Lox/+ C57BL6/J skull. B–E: E12.5 limb buds: B: GFP in wt; C: EphB2 expression (β-galactosidase staining) in wt. D: GFP in Efnb1^Lox/+; E, EphB2 expression in Efnb1^Lox/+. F,G: Transverse section through E12.5 heads showing GFP in wt (F) and Efnb1^Lox/+ (G). H,I: higher magnification of areas in rectangles in F and G, respectively (white arrowhead shows abnormal boundary). Abbreviations: LAM, lambdoid suture; COR, coronal suture; FNS, frontonasal suture; PFS, posterior frontal suture; AFS, anterior frontal suture.