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| Status |
Public on Feb 21, 2015 |
| Title |
Genome-wide DNA methylation maps in chronic lymphocytic leukemia cells determined by next-generation sequencing (RRBS) |
| Organism |
Homo sapiens |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
Chronic lymphocytic leukemia (CLL) is a biologically and clinically heterogeneous disease. The somatic hypermutation status of the immunoglobulin heavy chain variable (IGHV) genes has been identified as one of the most robust prognostic markers in CLL. Patients with unmutated IGHV status (U-CLL) typically experience an inferior outcome compared to those whose clones express mutated IGHV genes (M-CLL). We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from a group of 43 CLL patients using reduced representation bisulfite sequencing (RRBS). Using base-pair resolution methylation sequencing, 2323 differentially methylated regions between CLL and normal B-cells (CLL-specific DMRs) and 569 between M-CLL and U-CLL samples (IGHV-specific DMRs) were identified in the CLL genomes. The IGHV-specific DMRs are mostly unique when compared to the CLL-specific DMRs. Less than 10% of the IGHV-specific DMRs are located in promoter regions; however, more than half of these overlap with known DNase I hypersensitive sites, enhancer regions marked by histone modification (H3K4Me1 and H3K27Ac), and transcription factor binding sites in the ENCODE datasets, which indicates that these DMRs contain regulatory sequences. Distinctive DNA methylation patterns were observed in M-CLL and U-CLL samples. Overall, U-CLL was found to contain 50% more hypermethylated regions than M-CLL samples. The hypermethylated loci observed in the U-CLL samples also appear to be hypermethylated in normal naïve B-cells as compared memory B-cells, suggesting that M-CLL and U-CLL differ in differentiation status corresponding to normal B-cell differentiation stages. RNA-seq analysis performed using matched samples (n=34), in which both DNA methylation and gene expression data were available, demonstrated excellent correlation between DNA methylation and gene expression. Several genes whose expression status was previously shown to be associated with CLL prognosis such as ZAP70, CRY1, LDOC1, SEPT10, LAG3, and LPL were differentially methylated in the promoter regions between M-CLL and U-CLL samples indicating that DNA methylation plays an important role in defining the gene expression patterns of these prognostic genes. We further validated 9 genes with IGHV-specific DMRs in the promoter regions using bisulfite pyrosequencing, and the results demonstrated excellent correlation between differential methylation and IGHV mutation status. These novel differentially methylated genes could be developed into biomarkers for CLL prognosis. In addition, DNA hypomethylation was observed in a significant number of genes involved in lymphocyte activation such as PDCD1, NFAT1, and CD5. DNA hypomethylation was observed in the proximal promoter and far up-stream enhancer regions of CD5, an important cell surface marker that uniquely identifies CLL. Overall, the DNA methylation landscape in CLL patients indicates that CLL B cells possess an active B-cell phenotype; at the same time, U-CLL and M-CLL are faithfully committed to their lineage resembling either naïve or memory B-cells. In summary, this comprehensive DNA methylation analysis has identified a large number of novel epigenetic changes in CLL patients. The results from this study will further advance our understanding of the epigenetic contribution to molecular subtypes in CLL.
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| Overall design |
To perform a genome-wide analysis of DNA methylation in CLL, we applied the Reduced Representation Bisulfite Sequencing (RRBS) to CD19+ B-cells isolated from normal control and CLL peripheral blood samples. The genomic DNA from each sample was digested with the methylation-insensitive restriction enzyme MspI (restriction site, CCGG) and ligated to Illumina sequencing adaptors containing methylated cytosine residues. The ligated MspI fragments were size-selected, treated with sodium bisulfite, and amplified by PCR. The PCR products were purified and sequenced using Illumina Hiseq2000 sequencer with a read length of 100bp. 32 CLL B-cell samples, 10 normal control samples including one each of normal CD19+, CD19+/ IgD+ naïve, and CD19+/CD27+ memory B-cell sample. We generated 20-30 million Illumina sequencing reads for each sample.
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| Contributor(s) |
Shi H |
| Citation(s) |
27461342 |
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| Submission date |
Feb 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Huidong Shi |
| E-mail(s) |
[email protected]
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| Phone |
706-721-6000
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| Organization name |
Augusta University
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| Department |
Georgia Cancer Center
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| Lab |
2125 K
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| Street address |
1120 15th Street, CN2138
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| City |
Augusta |
| State/province |
GA |
| ZIP/Postal code |
30912 |
| Country |
USA |
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| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
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| Samples (42)
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| This SubSeries is part of SuperSeries: |
| GSE66167 |
Transcriptome analysis and Genome-wide DNA methylation maps in chronic lymphocytic leukemia cells determined by next-generation sequencing |
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| Relations |
| BioProject |
PRJNA276004 |
| SRA |
SRP055387 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE66121_RAW.tar |
943.7 Mb |
(http)(custom) |
TAR (of BED) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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