|dc.description.abstracteng||CHARGE syndrome is a complex congenital malformation disorder, caused in more about two-thirds of the cases by heterozygous loss of function mutations in the chromodomain helicase DNA-binding protein 7 (CHD7). The acronym CHARGE reflects the main clinical features seen in the most CHARGE syndrome patients namely coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia and ear anomalies/deafness. Because of the phenotypic spectrum, it was supposed that the CHARGE syndrome belongs to the neurocristopathies. In addition, the essential role of CHD7 for the formation and migration of neural crest cells (NCCs), which give rise to many tissues of the embryo including craniofacial and heart structures, was recently demonstrated. Two murine NCC lines, the trunk JoMa cells and the cranial O9-1 cells, were characterized for the expression of NCC and other molecular marker genes as well as Chd7 by RT-PCR analysis. The results revealed that the two cell lines do not differ
much in the expression of the tested marker genes for NCCs, neurons, glial cells,
smooth muscle cells, melanocytes and chondrocytes as well as Chd7. The nuclear expression of Chd7 was validated by western blot analysis and for O9-1 cells also by immunocytochemical staining. The results of the western blot analysis demonstrated a strong Chd7 expression in O9-1 cells in comparison with JoMa cells. This strengthens the assumption that the symptoms seen in CHARGE syndrome are caused by a dysfunction of the cranial NCCs and not of the trunk NCCs.
Proper NCC guidance through the embryo depends on guidance cues and the large family of the semaphorins thereby plays an important role. Subsequent experiments were performed with the NCC line O9-1. Using RT-PCR analysis, the expression of semaphorin receptors and for the first time of class 3 semaphorins was identified in NCCs. Sema3a expression was proven by immunocytochemical staining and western blot analysis. Further experiments were carried out to check the localization of Sema3a in the different cell compartments and a possible Sema3a secretion into the cell culture medium. Western blot analysis revealed both the expression in the nucleus and the cytoplasm and the secretion into the medium. Moreover, the proteolytic cleavage and dimerization of Sema3a could be identified as described for class 3 semaphorins. Recently, the described regulatory effect of Chd7 on Sema3a in Xenopus laevis embryos and mouse embryos could be confirmed in O9-1 cells through the Sema3a downregulation by siRNA-mediated depletion of Chd7. Further experiments revealed a co-immunoprecipitation of Chd7 with Sema3a, indicating the association of these two proteins in the same protein complex.
A further part of the present thesis focused on the identification of possible mutations in the SEMA3A gene of CHD7-positive CHARGE syndrome patients. SEMA3A mutations are hypothesized to negatively modulate the phenotype of CHARGE patients and are known to be involved in the pathogenesis of the allelic disorder Kallmann syndrome, which is suggested to represent the mild end of the phenotypic abnormalities seen in CHARGE syndrome. The non-synonymous SEMA3A mutation c.2002A>G (p.I668V) was identified in a severe affected CHARGE syndrome patient. This finding might strengthens the hypothesis that SEMA3A act as modifier in CHARGE patients with a pathogenic mutation in the CHD7 gene contributing to a more severe phenotype in these patients due to digenetic inheritance. It would also explain the high clinical inter- and intra-familial variability of CHARGE syndrome. The new identified mutation and the already published variant p.R66W were characterized with overexpressing plasmids in the human
HEK293 cell line on protein level. The obtained results showed no effect of the
mutations on expression, proteolytic processing, dimerization or secretion of SEMA3A.
By co-immunoprecipitation experiments it was demonstrated that CHD7 interacts with SEMA3A WT and the two SEMA3A variants, p.R66W and p.I668V. Yeast-two hybrid assay indeed revealed no direct interaction between CHD7 and SEMA3A with or without mutation.
In summary, the results of the present thesis are another step towards understanding the pathomechanism behind the neurocristopathy CHARGE syndrome.||de