@article{KniesSchusterAmezianeetal.2012, author = {Knies, Kerstin and Schuster, Beatrice and Ameziane, Najim and Rooimans, Martin and Bettecken, Thomas and de Winter, Johan and Schindler, Detlev}, title = {Genotyping of Fanconi Anemia Patients by Whole Exome Sequencing: Advantages and Challenges}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77985}, year = {2012}, abstract = {Fanconi anemia (FA) is a rare genomic instability syndrome. Disease-causing are biallelic mutations in any one of at least 15 genes encoding members of the FA/BRCA pathway of DNA-interstrand crosslink repair. Patients are diagnosed based upon phenotypical manifestationsand the diagnosis of FA is confirmed by the hypersensitivity of cells to DNA interstrand crosslinking agents. Customary molecular diagnostics has become increasingly cumbersome, time-consuming and expensive the more FA genes have been identified. We performed Whole Exome Sequencing (WES) in four FA patients in order to investigate the potential of this method for FA genotyping. In search of an optimal WES methodology we explored different enrichment and sequencing techniques. In each case we were able to identify the pathogenic mutations so that WES provided both, complementation group assignment and mutation detection in a single approach. The mutations included homozygous and heterozygous single base pair substitutions and a two-base-pair duplication in FANCJ, -D1, or - D2. Different WES strategies had no critical influence on the individual outcome. However, database errors and in particular pseudogenes impose obstacles that may prevent correct data perception and interpretation, and thus cause pitfalls. With these difficulties in mind, our results show that WES is a valuable tool for the molecular diagnosis of FA and a sufficiently safe technique, capable of engaging increasingly in competition with classical genetic approaches.}, subject = {Medizin}, language = {en} } @article{WeisSchoenVictoretal.2011, author = {Weis, Eva and Schoen, Holger and Victor, Anja and Spix, Claudia and Ludwig, Marco and Schneider-Raetzke, Brigitte and Kohlschmidt, Nicolai and Bartsch, Oliver and Gerhold-Ay, Aslihan and Boehm, Nils and Grus, Franz and Haaf, Thomas and Galetzka, Danuta}, title = {Reduced mRNA and Protein Expression of the Genomic Caretaker RAD9A in Primary Fibroblasts of Individuals with Childhood and Independent Second Cancer}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74777}, year = {2011}, abstract = {Background: The etiology of secondary cancer in childhood cancer survivors is largely unclear. Exposure of normal somatic cells to radiation and/or chemotherapy can damage DNA and if not all DNA lesions are properly fixed, the mis-repair may lead to pathological consequences. It is plausible to assume that genetic differences, i.e. in the pathways responsible for cell cycle control and DNA repair, play a critical role in the development of secondary cancer. Methodology/Findings: To identify factors that may influence the susceptibility for second cancer formation, we recruited 20 individuals who survived a childhood malignancy and then developed a second cancer as well as 20 carefully matched control individuals with childhood malignancy but without a second cancer. By antibody microarrays, we screened primary fibroblasts of matched patients for differences in the amount of representative DNA repair-associated proteins. We found constitutively decreased levels of RAD9A and several other DNA repair proteins in two-cancer patients, compared to onecancer patients. The RAD9A protein level increased in response to DNA damage, however to a lesser extent in the twocancer patients. Quantification of mRNA expression by real-time RT PCR revealed lower RAD9A mRNA levels in both untreated and 1 Gy c-irradiated cells of two-cancer patients. Conclusions/Significance: Collectively, our results support the idea that modulation of RAD9A and other cell cycle arrest and DNA repair proteins contribute to the risk of developing a second malignancy in childhood cancer patients.}, subject = {Medizin}, language = {en} } @article{SchneiderPliushchElHajjetal.2010, author = {Schneider, Eberhard and Pliushch, Galyna and El Hajj, Nady and Galetzka, Danuta and Puhl, Alexander and Schorsch, Martin and Frauenknecht, Katrin and Riepert, Thomas and Tresch, Achim and Mueller, Annette M. and Coerdt, Wiltrud and Zechner, Ulrich and Haaf, Thomas}, title = {Spatial, temporal and interindividual epigenetic variation of functionally important DNA methylation patterns}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68371}, year = {2010}, abstract = {DNA methylation is an epigenetic modification that plays an important role in gene regulation. It can be influenced by stochastic events, environmental factors and developmental programs. However, little is known about the natural variation of genespecific methylation patterns. In this study, we performed quantitative methylation analyses of six differentially methylated imprinted genes (H19, MEG3, LIT1, NESP55, PEG3 and SNRPN), one hypermethylated pluripotency gene (OCT4) and one hypomethylated tumor suppressor gene (APC) in chorionic villus, fetal and adult cortex, and adult blood samples. Both average methylation level and range of methylation variation depended on the gene locus, tissue type and/or developmental stage. We found considerable variability of functionally important methylation patterns among unrelated healthy individuals and a trend toward more similar methylation levels in monozygotic twins than in dizygotic twins. Imprinted genes showed relatively little methylation changes associated with aging in individuals who are >25 years. The relative differences in methylation among neighboring CpGs in the generally hypomethylated APC promoter may not only reflect stochastic fluctuations but also depend on the tissue type. Our results are consistent with the view that most methylation variation may arise after fertilization, leading to epigenetic mosaicism.}, subject = {Medizin}, language = {en} }