Expression and functional analysis of EFNB1 mutations in craniofrontonasal syndrome

Autor :Roman Makarov
Herkunft :OvGU Magdeburg, Fakultät für Naturwissenschaften
Datum :26.05.2010
Dokumente :
Dataobject from HALCoRe_document_00008135
Typ :Dissertation
Format :Text
Kurzfassung :Ephrin-B1 protein forms signalling complexes with EphB receptor expressed in complementary cells. This complex was shown to work as a bi-directional signalling system, and ephrin-B1 was called “receptor-like protein”. Ephrin-B1 is encoded by EFNB1 gene. Mutations of this gene cause the X-linked disease called craniofrontonasal syndrome (CFNS). This disease shows an unusual phenotypic pattern of inheritance, it affects heterozygous females more severely than hemizygous males although EFNB1 is located on the X chromosome and has no homologue on the Y chromosome. This 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 with EphB-receptors expressing cells. Several patient derived EFNB1 mutations have been analysed in this work, which consists of two parts. First one premature termination codon (PTC)-causing mutations were analysed: frameshift mutation c.377_384delTCAAGAAG, frameshift mutation c.614_615delCT (PTC in exon 4) and splice-site mutation c.406+2T>C (PTC in intron 2 or exon 3). All three mutations were predicted in silico to result in a truncated, soluble ephrin-B1 protein. Such protein would cause distant interaction with EphB-receptors. In the second part of this work the impact of missense mutations c.161C>T/p.P54L and c.332C>T/p.T111I on cell behaviour and reverse ephrin-B1 cell signalling was investigated. The role of these mutations was analysed in a cell culture model using NIH3T3 fibroblasts. This cell line was chosen because it does not express Efnb1, the mouse homologue of EFNB1 gene. Both missense mutations are located in the extracellular ephrin domain that is involved in Eph-ephrin-B1 recognition and higher order complex formation. Reverse signalling of ephrin-B1 involves phosphorylation of several conserved tyrosine residues of the cytoplasmic tail. Two of them, Tyr324 and Tyr329 in human ephrin-B1, were shown to be the most important for signal transduction. To analyse the impact of missense mutations in ephrin-B1 signalling the phosphorylation of these two amino acids in mutant and wild type ephrin-B1 was monitored after the EphB2-Fc stimulation. It was reported previously, that ephrin-B1 is involved in STAT3 signalling pathway. Also, it is known that STAT3 controls the expression of TWIST1 gene and MSX2 is also involved in STAT3 signalling. According to that, the change of expression level of MSX2 and TWIST1 due to ephrin-B1 stimulation was monitored. Mutation c. .377_384delTCAAGAAG showed a strong decrease of the mutant transcript level, mutations c.614_615delCT and c.406+2T>C showed the presence of the mutant RNA but not the mutant protein. Mutation p.P54L showed no phosphorylation of the Tyr324/329, wild type and p.T111I mutation showed the difference in the phosphorylation timing. Also wild type and p.T111I expressing cells showed patches formation after the EphB2-Fc stimulation whereas p.P54L expressing cells remained to be scattered. MSX2 and TWIST1 genes showed only a minor change of the transcription level in response to the ephrin-B1 stimulation with EphB2. Four from the five investigated EFMB1 mutations appeared to have rather loss-of-function.
Schlagwörter :EFNB1, PTC-causing mutations, real-time PCR, mRNA decay, craniofrontonasal syndrome, ephrin-B1
Rechte :Dieser Text ist urheberrechtlich geschützt
Größe :79 S.
Erstellt am :30.06.2010 - 11:27:05
Letzte Änderung :30.06.2010 - 11:27:39
MyCoRe ID :HALCoRe_document_00008135
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