6 The second mechanism now is the addition of methyl groups to the position 5 carbon of the cytosine pyrimidine ring when it is followed by a guanine in the DNA sequence (CpG site), known as DNA methylation. In a normal cell, the CpG sites scattered throughout the genome are heavily methylated and the dense regions of CpG sites (CpG islands) located in approximately 50% of all human genes are unmethylated if the gene is expressed. In cancer cells this situation is reversed, with the scattered CpG sites becoming hypomethylated and the promoter CpG islands of genes such as tumor suppressors becoming hypermethylated, leading to genomic instability and decreased expression of these genes.
7 Genome-wide studies looking at DNA methylation patterns are now also being engaged to characterize leukemia genomes, with the goal of improved diagnostic accuracy (classification) and ultimately the discovery of novel therapeutic strategies. In this study, the methylation profiles of the subgroups of AML associated with a more favourable outcome were investigated and associated with their respective gene expression profiles. This was done in two stages, initially the profiles of samples which had favourable cytogenetics (t(15; 17) and t(8; 21)) versus samples with NK-AML were analyzed. Then to assist with further stratifying the NK-AML, the methylation profiles of samples with a NPM1 mutation versus wild type NPM1 samples were analyzed. Distinct methylation profiles associated with prognostic groups were identified and this may aid in the classification of AML, particularly in the NK-AML subclass and also in the identification of therapeutic targets.
Materials and Methods Research study subject samples Bone marrow aspirate samples from 19 acute myeloid leukemia research study subjects (subjects) were obtained at diagnosis and before treatment. The study design was approved and ethical approval obtained before starting. Informed consent was given by all subjects. Mononuclear cells were isolated using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden). For DNA isolation aliquots of cells from each subject were pelleted, DNA extracted as described below, and the DNA stored at ?80 ��C until used. For RNA isolation, aliquots of cells from each subject were lysed in buffer RLT +1% ��-mercaptoethanol, RNA extracted as described below and stored at ?80 ��C until used.
DNA isolation Genomic DNA from subject samples was isolated using DNeasy blood and tissue kit. DNA was eluted in AE buffer (Qiagen, Crawley, UK). Human genomic DNA (Novagen, Merck Chemicals Ltd., Nottingham, UK) was used as control DNA for subject sample Dacomitinib comparison within the methylation analysis. RNA isolation RNA was extracted from subject samples using Ampi-Lute total RNA purification kit (Qiagen). RNA was eluted in RNase-free water.