The impact of CFNS-causing EFNB1 mutations on ephrin-B1 function

BMC Med Genet. 2010 Jun 17:11:98. doi: 10.1186/1471-2350-11-98.


Background: Mutations of EFNB1 cause the X-linked malformation syndrome craniofrontonasal syndrome (CFNS). CFNS is characterized by an unusual phenotypic pattern of inheritance, because it affects heterozygous females more severely than hemizygous males. This sex-dependent inheritance has been explained by random X-inactivation in heterozygous females and the consequences of cellular interference of wild type and mutant EFNB1-expressing cell populations. EFNB1 encodes the transmembrane protein ephrin-B1, that forms bi-directional signalling complexes with Eph receptor tyrosine kinases expressed on complementary cells. Here, we studied the effects of patient-derived EFNB1 mutations predicted to give rise to truncated ephrin-B1 protein or to disturb Eph/ephrin-B1 reverse ephrin-B1 signalling. Five mutations are investigated in this work: nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT, splice-site mutation c.406 + 2T > C and two missense mutations p.P54L and p.T111I. Both missense mutations are located in the extracellular ephrin domain involved in Eph-ephrin-B1 recognition and higher order complex formation.

Methods: Nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT and splice-site mutation c.406+2T > C were detected in the primary patient fibroblasts by direct sequencing of the DNA and were further analysed by RT-PCR and Western blot analyses.The impact of missense mutations p.P54L and p.T111I on cell behaviour and reverse ephrin-B1 cell signalling was analysed in a cell culture model using NIH 3T3 fibroblasts. These cells were transfected with the constructs generated by in vitro site-directed mutagenesis. Investigation of missense mutations was performed using the Western blot analysis and time-lapse microscopy.

Results and discussion: Nonsense mutation c.196C > T/p.R66X and frameshift mutation c.614_615delCT escape nonsense-mediated RNA decay (NMD), splice-site mutation c.406+2T > C results in either retention of intron 2 or activation of a cryptic splice site in exon 2. However, c.614_615delCT and c.406+2T > C mutations were found to be not compatible with production of a soluble ephrin-B1 protein. Protein expression of the p.R66X mutation was predicted unlikely but has not been investigated.Ectopic expression of p.P54L ephrin-B1 resists Eph-receptor mediated cell cluster formation in tissue culture and intracellular ephrin-B1 Tyr324 and Tyr329 phosphorylation. Cells expressing p.T111I protein show similar responses as wild type expressing cells, however, phosphorylation of Tyr324 and Tyr329 is reduced.

Conclusions: Pathogenic mechanisms in CFNS manifestation include impaired ephrin-B1 signalling combined with cellular interference.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Codon, Nonsense
  • Craniosynostoses / genetics*
  • Ephrin-B1 / biosynthesis
  • Ephrin-B1 / genetics*
  • Ephrin-B1 / metabolism
  • Ephrins / genetics
  • Ephrins / metabolism
  • Exons
  • Female
  • Frameshift Mutation
  • Heterozygote
  • Humans
  • Male
  • Mutagenesis, Site-Directed
  • Mutation*
  • Mutation, Missense
  • RNA Splice Sites
  • Receptors, Eph Family
  • Reverse Transcriptase Polymerase Chain Reaction
  • Syndrome
  • X Chromosome Inactivation


  • Codon, Nonsense
  • Ephrin-B1
  • Ephrins
  • RNA Splice Sites
  • Receptors, Eph Family