Institut für Humangenetik, Universitätsklinikum, Otto-von-Guericke-Universität, Magdeburg, Germany.
BMC Med Genet. 2010 Jun 17;11:98. doi: 10.1186/1471-2350-11-98.
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.
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.
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.
Pathogenic mechanisms in CFNS manifestation include impaired ephrin-B1 signalling combined with cellular interference.
EFNB1 突变导致 X 连锁的颅面畸形综合征（CFNS）。CFNS 的特征是一种不寻常的表型遗传模式，因为它使杂合子女性比半合子男性受到更严重的影响。这种性别依赖性遗传可以通过杂合子女性中随机的 X 失活和野生型和突变型 EFNB1 表达细胞群体之间的细胞干扰来解释。EFNB1 编码跨膜蛋白 ephrin-B1，它与互补细胞上表达的 Eph 受体酪氨酸激酶形成双向信号复合物。在这里，我们研究了导致截短的 ephrin-B1 蛋白或干扰 Eph/ephrin-B1 反向 ephrin-B1 信号的患者衍生 EFNB1 突变的影响。本工作研究了五种突变：无意义突变 c.196C>T/p.R66X、移码突变 c.614_615delCT、剪接位点突变 c.406+2T>C 和两个错义突变 p.P54L 和 p.T111I。这两个错义突变都位于细胞外的 Ephrin 结构域，该结构域参与 Eph-ephrin-B1 识别和更高阶复合物形成。
通过 DNA 的直接测序在原代患者成纤维细胞中检测到无意义突变 c.196C>T/p.R66X、移码突变 c.614_615delCT 和剪接位点突变 c.406+2T>C，并进一步通过 RT-PCR 和 Western blot 分析进行分析。在 NIH 3T3 成纤维细胞的细胞培养模型中分析错义突变 p.P54L 和 p.T111I 对细胞行为和反向 ephrin-B1 细胞信号的影响。这些细胞通过体外定点诱变生成的构建体进行转染。使用 Western blot 分析和延时显微镜检查来研究错义突变。
无意义突变 c.196C>T/p.R66X 和移码突变 c.614_615delCT 逃避无意义介导的 RNA 衰减（NMD），剪接位点突变 c.406+2T>C 导致内含子 2 的保留或外显子 2 中的隐蔽剪接位点的激活。然而，发现 c.614_615delCT 和 c.406+2T>C 突变与可溶性 ephrin-B1 蛋白的产生不兼容。p.R66X 突变的蛋白表达预计不太可能，但尚未进行研究。异位表达 p.P54L ephrin-B1 抵抗 Eph 受体介导的组织培养中的细胞簇形成和细胞内 ephrin-B1 Tyr324 和 Tyr329 磷酸化。表达 p.T111I 蛋白的细胞表现出与野生型表达细胞相似的反应，然而，Tyr324 和 Tyr329 的磷酸化减少。
CFNS 表现的发病机制包括受损的 ephrin-B1 信号传导和细胞干扰。
