The impact of CFNS-causing <it>EFNB1 </it>mutations on ephrin-B1 function

• Main Authors: Wieacker Peter (Author), Tasic Velibor (Author), Gucev Zoran (Author), Steiner Bernhard (Author), Makarov Roman (Author), Wieland Ilse (Author)
• Format: Book
• Published: BMC, 2010-06-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.

Summary

<p>Abstract</p> <p>Background</p> <p>Mutations of <it>EFNB1 </it>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 <it>EFNB1</it>-expressing cell populations. <it>EFNB1 </it>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 <it>EFNB1 </it>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.</p> <p>Methods</p> <p>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.</p> <p>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 <it>in vitro </it>site-directed mutagenesis. Investigation of missense mutations was performed using the Western blot analysis and time-lapse microscopy.</p> <p>Results and Discussion</p> <p>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.</p> <p>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.</p> <p>Conclusions</p> <p>Pathogenic mechanisms in CFNS manifestation include impaired ephrin-B1 signalling combined with cellular interference.</p>

Item Description

10.1186/1471-2350-11-98
1471-2350