Epileptiform activity alters gene expression in the central nervous system, a phenomenon that has been studied extensively in animal models. Here, we asked whether also in vitro models of seizures are in principle suitable to investigate changes in gene expression due to epileptiform activity and tested this hypothesis mainly in rodent and additionally in some human brain slices. We focused on three genes relevant for seizures and epilepsy: FOS proto-oncogene (c-Fos), inducible cAMP early repressor (Icer) and mammalian target of rapamycin (mTor). Seizure-like events (SLEs) were induced by 4-aminopyridine (4-AP) in rat entorhinal-hippocampal slices and by 4-AP/8 mM potassium in human temporal lobe slices obtained from surgical treatment of epilepsy. SLEs were monitored simultaneously by extracellular field potentials and intrinsic optical signals (IOS) for 1–4 h, mRNA expression was quantified by real time PCR. In rat slices, both duration of SLE exposure and SLE onset region were associated with increased expression of c-Fos and Icer while no such association was shown for mTor expression. Similar to rat slices, c-FOS induction in human tissue was increased in slices with epileptiform activity. Our results indicate that irrespective of limitations imposed by ex vivo conditions, in vitro models represent a suitable tool to investigate gene expression. Our finding is of relevance for the investigation of human tissue that can only be performed ex vivo. Specifically, it presents an important prerequisite for future studies on transcriptome-wide and cell-specific changes in human tissue with the goal to reveal novel candidates involved in the pathophysiology of epilepsy and possibly other CNS pathologies.
Abnormal epigenetic regulation has been implicated in oncogenesis. Mutations in the DNA methyltransferase 3A (DNMT3A) gene were recently demonstrated in acute myeloid leukemia (AML) as a candidate for the initiating of lesions in AML with adverse clinical outcome. Using direct sequencing, identification of the DNMT3A mutations was done in 320 AML patients. Additionally, we analyzed NPM1, FLT3-ITD, FLT3-D835, IDH1 and IDH2 mutations by PCR and DNA sequencing. The retrospective analysis of diagnostic bone marrow samples was performed in AML patients treated in our institution. In all AML patients double induction therapy containing of “7+3” therapy followed by 4 cycles of high dose AraC consolidation was implicated in accordance with therapeutic standards of our institution. We identified DNMT3A mutations in 22% of AML patients. The most common of mutations affect amino acid R822 in exon 23. The DNMT3A mutation was highly enriched in the group of patients with intermediate-risk profile (35%, P<0.01). Unlikely FLT3, DNMT3A mutations were absent in all AML patients with favorable-risk group (P<0.001). AML patients with DNMT3A mutations were older (P=0.05), had higher WBC and platelet counts (P=0.02 for both comparison) and higher relapse rate (P=0.01). The median overall survival among AML patients with DNMT3A mutations was significantly shorter than among patients without such mutation (13.3 months versus 31.3 months, P<0.01). Occurrence of DNMT3A mutations was associated with presence of other common mutations, such as NPM1, FLT3-ITD, FLT3-D835, IDH1 and IDH2. Correlations of the DNMT3A mutations with NPM1 and FLT3-ITD mutations were significant (P<0.001). Our results indicate that DNMT3A mutations are highly recurrent in AML patients with intermediate-risk profile. DNMT3A mutations are highly correlated with NPM1/FLT3-ITD mutations and are associated with an unfavorable prognosis. The discovery of recurrent mutations in DNMT3A gene may provide a new prognostic marker for the risk stratification for AML patients. Disclosures: No relevant conflicts of interest to declare.
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