Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide DNA methylation profiling predicts relapse in childhood B-cell acute lymphoblastic leukemia

ABSTRACT: Although the 5-year survival of childhood Acute Lymphoblastic Leukemia (ALL) exceeds 80%, a group of patients presents poor prognosis due to early relapse. To date, treatment strategies are defined by cytogenetically-based subtype categorization. However, ALL patients without chromosomal translocations associated with poor prognosis lack diagnostic markers to adjust specific therapies. DNA methylation alteration is a frequent event in cancer with high potential in diagnosis, prognosis and prediction of drug response. Hence, we aimed to characterize childhood B-ALLs without Philadelphia (BCR-ABL) and MLL translocations based on their DNA methylation profile of more than 450,000 CpG sites to improve accuracy in prognosis and treatment strategies. DNA was quantified by Quant-iT PicoGreen dsDNA Reagent (Invitrogen) and the integrity was analyzed in a 1.3% agarose gel. Bisulfite conversion of 600 ng of each sample was performed according to the manufacturer's recommendation for the Illumina Infinium Assay. Effective bisulfite conversion was checked for three controls that were converted simultaneously with the samples. 4 ul of bisulfite-converted DNA were used to hybridize on Infinium HumanMethylation 450 BeadChip, following the Illumina Infinium HD Methylation protocol. Chip analysis was performed using the Illumina HiScan SQ fluorescent scanner. The intensities of the images were extracted using GenomeStudio (2010.3) Methylation module (1.8.5) software. The methylation score of each CpG is represented as a beta value.

ORGANISM(S): Homo sapiens

SUBMITTER: Antonio Gómez

PROVIDER: E-GEOD-39141 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genome-wide DNA methylation profiling predicts relapse in childhood B-cell acute lymphoblastic leukaemia.

Sandoval Juan J Heyn Holger H Méndez-González Jesús J Gomez Antonio A Moran Sebastian S Baiget Montserrat M Melo Montserrat M Badell Isabel I Nomdedéu Josep F JF Esteller Manel M

British journal of haematology 20121030 3

PMID: 23110451

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Project description:Human aging implies many phenotypic modifications and an increased susceptibility to many common diseases, phenomena that cannot be fully explained by the constrained genetic setting. An alternative pathway that could explain the age-associated alterations is epigenetic drifts. To address this issue, we performed Whole Genome Bisulfite Sequencing (WGBS) of a newborn and of a centenarian. The centenarian DNA exhibited a significant loss in DNA methylation and a poor correlation in the methylation status of neighboring CpGs throughout the genome. From a gene-regulatory region standpoint, we observed that the DNA hypomethylation events occurred mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. Most importantly, we extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray. The 450K DNA methylation approach enabled validation of the WGBS data and revealed additional age-related DNA methylation events. Interestingly, we also observed the DNA methylation fingerprint characteristic of a healthy aged cell in samples from patients with premature-aging disorders such as Hutchinson-Gilford progeria and Werner syndrome. Overall, we suggest that defects in the physiological DNA methylation profile contribute to the process of human aging. As the disease associated cells were immortalized B cells, the effect of EBV immortalization was determined in advance to the study using immortalized and naive B cells. DNA was quantified by Quant-iT PicoGreen dsDNA Reagent (Invitrogen) and the integrity was analyzed in a 1.3% agarose gel. Bisulfite conversion of 600 ng of each sample was perform according to the manufacturer's recommendation for Illumina Infinium Assay. Effective bisulphite conversion was checked for three controls that were converted simultaneously with the samples. 4 ul of bisulfite converted DNA were used to hybridize on Infinium HumanMethylation 450 BeadChip, following Illumina Infinium HD Methylation protocol. Chip analysis was performed using Illumina HiScan SQ fluorescent scanner. The intensities of the images are extracted using GenomeStudio (2010.3) Methylation module (1.8.5) software. Methylation score of each CpG is represented as beta value.

Project description:In the current study, we have performed a high-throughput CpG methylation analysis of well characterized and defined populations of human adipose-derived stem cells (hASCs) before and after in vitro induction of osteogenic and myogenic differentiation that allows identifying DNA methylation- regulated differentiation genes. We have also address the extent of the epigenetic programming of hASCs- derived differentiated cells by comparing the methylation profiling of these cells with their somatic counterparts from primary tissues. Finally, we also compared the patterns of CpG methylation of hASCs (and their derivatives) with the methylation profiles of myosarcoma and osteosrcoma cell lines. All the CpG methylation studies have been performed with the Infinium 27K methylation arrays (from Illumina). DNA from adipose M-bM-^@M-^Sderived stem cells (n=4), in vitro induced myocytes (n=3), in vitro induced osteocytes (n=3), primary osteocytes obtained from ribs (n=1), primary myocytes (n=1), osteosarcoma cell line (MG63) and myosarcoma cell lines (Te 32.T and RD) was isolated applying the QIAampM-BM-. DNA Mini Kit (Qiagen Iberia, Spain). Microarray- based DNA methylation profiling was performed with the HumanMethylation27 BeadChip Infinium Methylation ArraysM-BM-. (Illumina, Inc.).The panel was designed to compare the DNA methylation status of each group of samples, which allow interrogating 27,578 CpG loci covering 14,495 genes at single-nucleotide resolution by typing bisulfite-converted DNA. The sequences included in the panel are derived from the well-annotated NCBI CCDS database (Genome Build 36) and is supplemented with more than 1,000 cancer-related genes described in published literature. Probe content has been enriched to deeply cover more than 150 well-established cancer genes known to show differential methylation patterns. Methylation array content also targets the promoter regions of 110 miRNA genes. Methylation arrays were performed as follows. Briefly, bisulfite conversion of 1 M-NM-<g of genomic DNA was done using the CpGenomicTM DNA Modification Kit (Intergen Company, Purchase, NY, USA). After sodium bisulfite treatment, the remaining assay steps used Infinium technology and using Illumina-supplied reagents and conditions. A thermocycling program with a short denaturation step included for bisulfite conversion (16 cycles of 95M-BM-:C for 30 seconds followed by 50M-BM-:C for 1 hour) was performed to improve bisulfite conversion efficiency. After bisulfite conversion, each sample was whole-genome amplified (WGA) and enzymatically fragmented. The bisulfite-converted WGA-DNA samples were purified and applied to the BeadChips. During hybridization, the WGA-DNA molecules anneal to locus-specific DNA oligomers linked to individual bead types. The two bead types correspond to each CpG locus -one to the methylated and the other to the unmethylated state. Allele-specific primer annealing is followed by single-base extension using DNP- and Biotin-labeled dNTPs. Both bead types for the same CpG locus will incorporate the same type of labelled nucleotide, determined by the base preceding the interrogated cytosine in the CpG locus, and therefore will be detected in the same colour channel. After extension, the array is fluorescently stained, scanned, and the intensities of the unmethylated and methylated bead types measured. DNA methylation values, described as beta values, are recorded for each locus in each sample via BeadStudio software. DNA methylation beta values are continuous variables between 0 (completely unmethylated) and 1 (completely methylated), representing the ratio of the intensity of the methylated bead type to the combined locus intensity.

Dataset Information

Genome-wide DNA methylation profiling predicts relapse in childhood B-cell acute lymphoblastic leukemia

Publications

Genome-wide DNA methylation profiling predicts relapse in childhood B-cell acute lymphoblastic leukaemia.

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