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The molecular mechanisms leading to brain tumors still remain unclear. Nevertheless, there is increasing evidence that epigenetic effects play crucial roles in tumor development and progression. Thereby, 5-hydroxymethylcytosine (5hmC) represents a further base modification of cytosine besides 5-methylcytosine (5mC). In addition to the role of 5hmC as an intermediate in demethylation, 5hmC is of reasonable importance for cellular control. Previous studies showed that loss of 5hmC is a hallmark of human malignancies, e.g. in glioma, melanoma, and myeloid tumors. In myeloid malignancies studies showed that loss of 5hmC was due to mutations within ten-eleven-translocation (TET) genes, enzymes being responsible for conversion of 5mC to 5hmC. Nevertheless, till date there are no genetic characterization data of TET enzymes available for glioma. In this study, we genetically characterized TET2 and TET3 alterations in 50 human gliomas (WHO-Grade II-IV) and in 19 healthy brain samples. We identified 7 genetic alterations within TET2 (p.V218M, p.G355N, p.P363L, p.L1721W, p.P1723S, p.I1762V, p.H1778R). Additionally, we performed quantification of 5hmC amount and added functional prediction analysis of identified TET alterations to evaluate the biological impact of these alterations on the hydroxymethylome. An analysis of TET3 showed no non-synonymous alterations. In summary, we did not find correlations of TET alterations with 5hmC amount. Thus, our data emphasize that, in contrast to leukemia, loss of 5hmC in glioma is not caused by TET gene alterations. Moreover, other disturbances, such as disrupted gene expressions or functional inhibitions of TET proteins may be responsible for the aberrant epigenome of human glioma.

Background: Recent research indicates that long non-coding RNAs (lncRNA) represent a new family of RNAs that is of fundamental importance for controlling transcription and translation. Thereby, there is increasing evidence that lncRNAs are also important in tumourigenesis. Thereby valid expression profiling using quantitative PCR requires suitable, stably expressed normalisers to achieve reliable and reproducible data. However, no systematic analysis of suitable references in lncRNA studies in human glioma has been performed yet., Methods: In this study, we investigated 90 lncRNAs in 30 tissue specimen for the expression stability in human diffuse astrocytoma (WHO-Grade II), anaplastic astrocytoma (WHO-Grade III) and glioblastoma (WHO-Grade IV) both alone as well as in comparison with normal white matter. Our identification procedure included a rigorous bioinformatical selection process that resulted in the inclusion of only highly abundant, equally expressed lncRNAs for further analysis. Additionally, lncRNAs were classified according to their stability value using the NormFinder algorithm., Results: We identified 24 appropriate normalisers suitable for studies in diffuse astrocytoma, 22 for studies in anaplastic astrocytoma and 12 for studies in glioblastoma. Comparing all three glioma entities 7 lncRNAs showed stable expression levels. Addition of normal brain tissue resulted in only 4 suitable lncRNAs., Conclusions: Our findings indicate that 4 lncRNAs (HOXA6as, H19 upstream conserved 1 and 2, Zfhx2as and BC200) are suitable as normalisers in glioma and normal brain. These lncRNAs may thus be regarded as universal references being applicable for the accurate normalisation of lncRNA expression profiling in various glioma (WHO-Grades II-IV) alone and in combination with brain tissue. This enables to perform valid longitudinal studies, e.g. of glioma before and after malignisation to identify changes of lncRNA expressions probably driving malignant transformation.