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Blood, Vol. 113, Issue 12, 2795-2804, March 19, 2009
Genome-wide epigenetic analysis delineates a biologically distinct immature acute leukemia with myeloid/T-lymphoid features
Blood Figueroa et al.
113: 2795
Supplemental materials for: Figueroa et al
Gene expression arrays and analysis: Gene expression data were obtained using Affymetrix Human Genome 133 Plus2.0 GeneChips. mRNA isolation, labeling, hybridization, and quality control were carried out as described previously.1 Raw data were processed using the Robust Multi-Averaging (RMA) algorithm.2 Data are available in the NCBI Gene Expression Omnibus database (accession number pending) Quantitative DNA methylation analysis by MassARRAY EpiTyping: Validation of HELP findings was performed by MALDI-TOF mass spectrometry using EpiTyper by MassARRAY (Sequenom, CA) on bisulfite-converted DNA as previously described.3 MassArray primers were designed to cover the flanking HpaII sites for a given HAF, as well as any other HpaII sites found up to 2,000 bp upstream of the downstream site and up to 2,000 bp downstream of the upstream site, in order to cover all possible alternative sites of digestion (Primer sequences available as supplementary data). Statistical analysis: Enrichment for HpaII fragments overlapping with CpG islands or CG clusters in the gene signatures vs. that of the whole HELP array was calculated using a proportion test in the Statistix software (Tallahassee, FL). Gene network and gene ontology analysis: Ingenuity Pathway Analysis (IPA) software was used to carry out network composition analysis. HpaII amplifiable fragments on the HELP microarray were annotated to the nearest gene up to a maximum distance of 5 kb from the transcription start site. Gene ontology analysis was performed using DAVID,4 with the entire HELP microarray as the background reference against which enrichment of level 5 GO
categories was determined. Sequence analysis: Sequence retrieval: Sequences were downloaded from the UCSC Genome Browser, selecting the 2k upstream of the reported 5’ end. Only genes that were found in the RefSeq annotations were downloaded. 327 RefSeq sequences were retrieved for the genes that were differentially methylated, and 2422 RefSeq sequences were retrieved for the set of control sequences. For the CEBPAsil vs. normal CD34+ cells, 750 RefSeq sequences were retrieved for the signature genes and 3086 for the controls. Repeat element analysis: RepeatMasker 3.1.6 was used to find all human repeats in the genes from the CEBPAsil vs. CEBPAmut signature and the CEBPAsil vs. normal CD34+ cells signature, and in two randomly selected sets of control sequences. The number of times each element was found was calculated, and the different proportions between each set were estimated, using a Fisher Exact test. Motif analysis: FIRE (Finding Informative Regulatory Elements)5 was used to detect motifs that were able to distinguish between a) the CEBPAsil vs. CEBPAmut signature genes, and a group of control sequences, and b) the CEBPAsil vs. normal CD34+ signature and a set of control sequences. In vitro cultures and thymidine incorporation studies: Ficoll separated leukemia cells vitally cryopreserved in 7.5% dimethylsulfoxide were thawed and analyzed by flow cytometry on a LSR II (Becton Dickinson, Franklin Lakes, NJ USA) using fluorochrome labeled CD34, CD7, and CD11b (Becton Dickinson). Tritiated thymidine incorporation was carried out serum-free using hematopoietic growth factors as described in 6,7. REFERENCES 1. Valk PJ, Verhaak RG, Beijen MA, et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med. 2004;350:1617–1628.
2. Irizarry RA, Hobbs B, Collin F, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003;4:249–264.
3. Ehrich M, Nelson MR, Stanssens P, et al. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc Natl Acad Sci U S A. 2005;102:15785–15790.
4. Dennis G, Jr., Sherman BT, Hosack DA, et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003;4:P3.
5. Elemento O, Slonim N, Tavazoie S. A universal framework for regulatory element discovery across all genomes and data types. Mol Cell. 2007;28:337–350.
6. Delwel R, Salem M, Pellens C, et al. Growth regulation of human acute myeloid leukemia: effects of five recombinant hematopoietic factors in a serum-free culture system. Blood. 1988;72:1944–1949.
7. Rombouts WJ, Lowenberg B, van Putten WL, Ploemacher RE. Improved prognostic significance of cytokine-induced proliferation in vitro in patients with de novo acute myeloid leukemia of intermediate risk: impact of internal tandem duplications in the Flt3 gene. Leukemia. 2001;15:1046–1053.
Files in this Data Supplement:
- Table S1. ADULT T-ALL samples - Erasmus MC Immunology cohort (XLS, 20.5 KB)
- Table S2. Genes differentially methylated between CEBPAsil and CEBPAmut leukemias (XLS, 55 KB)
- Table S3. Genes differentially methylated between CEBPAsil cases and normal CD34+ bone marrow cells (XLS, 91 KB)
- Table S4. Genes abeerantly methylated in both CEBPAsil and CEBPAmut leukemias (XLS, 16.5 KB)
- Table S5. Genes differentially methylated between CEBPAsil leukemias and a selection of T ALL cases (XLS, 26.5 KB)
- Table S6. Stimulation index (XLS, 13.5 KB)
- Table S7. qRT-PCR and MassARRAY primers (XLS, 24.5 KB)
- Figure S1. Validation of HELP by MassARRAY EpiTyper (JPG, 29.9 KB)
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Dot plot showing correlation between log2 (HpaII/MspI) ratios (x axis) and percent methylation as determined by MassARRAY EpiTyper (y axis) for 8 different HpaII amplifiable fragments in seven randomly selected samples (3 CEBPAsil, 2 CEBPAmut, and 2 T ALL cases).
- Figure S2. Top scoring networks generated by the DNA hypermethylation signature of CEBPAsil leukemias using Ingenuity Pathway Analysis (JPG, 144 KB)
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Genes differentially methylated between the two groups of AML were involved in the CEBPA, NFkB, and BML signaling networks. Genes colored in grey were present in the differentially methylated signature while genes in white represent other genes interacting with genes from the signature.
- Figure S3. CEBPA methylation status by HELP in CEBPAsil cases and normal CD 34+ hematopoietic cells (JPG, 51.6 KB)
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Representation of the positioning of the four probe sets relative to the genomic localization of the CEBPA locus and its CpG island on chromosome 19. HELP methylation values for each sample are represented in one row; the y axis represents centered log2 (HpaII/MspI) ratios and positive values correspond to hypomethylated fragments, while a negative deflection reflects a methylated fragment. The first 8 rows correspond to the CEBPAsil cases (in red) and the remaining rows to the normal CD 34+ hematopoietic cells (in green).
- Figure S4. Principal component analysis of gene expression data (JPG, 34 KB)
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Unsupervised analysis of gene expression data of both AML subgroups using principal component analysis. CEBPAsil are represented as red dots while CEBPAmut cases are in blue.
- Figure S5. Validation of genes differentially methylated in the two leukemia subgroups (JPG, 162 KB)
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(A) Heatmap representing percent cytosine methylation determined by MassARRAY EpiTyper on 7 randomly selected samples: 3 CEBPAsil (left) and 4 CEBPAmut cases (right). P values were calculated using a T test to determine the statistical difference between the average methylation levels across each of the genes. (B) qRT-PCR to validate low expression of differentially methylated genes in 2 CEBPAsil (left) as well as 4 CEBPAmut cases (right). Expression levels were calculated as percentages of expression of the PBGD housekeeping gene. Measurements were performed in duplicates and averaged. Amplification of a specific product in a positive control sample was confirmed by agarose gel electrophoresis and dissociation curve. Control samples used were: NB4 cell line (NFIB, TFAP2C, FGF5, HGF), primary brain tissue (BMP7, NELL1), or SKOV3 cell line (SFRP1, BMP6, CD40), depending on the presence of expression of the particular gene studied. The black dashed line represents the threshold percentage of PBGD expression expected based on the microarray data at the threshold used to define “low expression,” i.e. log2(100). Calculated as follows: 100/(average PBGD signal intensities in linear scale probe set 203040_s_at for these six samples) * 100 = 15%. Every value below this line therefore corresponds to the set definition of low expression.
- Figure S6. CEBPA methylation status by HELP in T ALL cases (JPG, 34.4 KB)
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Representation of the positioning of the four HpaII Amplifiable fragments relative to the genomic localization of the CEBPA locus and its CpG island on chromosome 19. HELP methylation values for each T ALL sample are represented in each row; the y axis represents centered log2 (HpaII/MspI) ratios and positive values correspond to hypomethylated fragments, while a negative deflection reflects a methylated fragment.
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