Abstract
We have extensively characterized the DNA methylomes of 139 patients with chronic lymphocytic leukemia (CLL) with mutated or unmutated IGHV and of several mature B-cell subpopulations through the use of whole-genome bisulfite sequencing and high-density microarrays. The two molecular subtypes of CLL have differing DNA methylomes that seem to represent epigenetic imprints from distinct normal B-cell subpopulations. DNA hypomethylation in the gene body, targeting mostly enhancer sites, was the most frequent difference between naive and memory B cells and between the two molecular subtypes of CLL and normal B cells. Although DNA methylation and gene expression were poorly correlated, we identified gene-body CpG dinucleotides whose methylation was positively or negatively associated with expression. We have also recognized a DNA methylation signature that distinguishes new clinico-biological subtypes of CLL. We propose an epigenomic scenario in which differential methylation in the gene body may have functional and clinical implications in leukemogenesis.
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Acknowledgements
We thank M. Seifert for advice about isolating normal mature B-cell subpopulations and C. Rozman, J. Valcárcel, R. Küppers and R. Siebert for their comments on the manuscript. We are grateful to S. Guijarro, S. Martín, C. Capdevila, M. Sánchez and L. Plà for excellent technical assistance; to M. Lozano for helping us obtain normal peripheral blood samples; and to N. Villahoz and C. Muro for excellent work in the coordination of the CLL Spanish Consortium. We are indebted to the Hospital Clínic de Barcelona–IDIBAPS Biobank-Tumor Bank and Hematopathology Collection for the sample procurement. We are also very grateful to the patients with CLL who have participated in this study. This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) through the Instituto de Salud Carlos III (ISCIII) (to E.C. and C.L.-O.) and the Red Temática de Investigación del Cáncer (RTICC) of the ISCIII (to E.C.), project SAF2009-08663 (to J.I.M.-S.) and project MCyT-BIO2007-666855 (to A.V.), as well as the European Union's Seventh Framework Programme through the Blueprint Consortium (grant agreement 282510) (to E.C. and I.G.) and the Botín Foundation (to C.L.-O.). J.I.M.-S. is supported by a Ramon y Cajal contract of the MINECO, M.K. by the Agència de Gestió d'Ajuts Universitaris i de Recerca (Generalitat de Catalunya), A.C.Q. by the Portuguese Fundação para a Ciência e a Tecnologia, S.B.-S. by an EMBO Long-Term Fellowship and S.E. by La Caixa.
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M.K. and A.C.Q. purified normal B cells, analyzed DNA methylation and gene expression arrays and integrated the data. S.H., M. Rubio, R.R. and D.G.P. processed and analyzed WGBS data. M. Bibikova, V.H., B.K. and J.-B.F. performed Infinium 450k microarray experiments and primary data analysis. A.N. performed mutational analysis of IGHV genes. G. Clot, M.K., A.C.Q., G. Castellano, A.M.-T. and N.V. performed statistical analysis. A.M.-T., N.V., M.A., M. Rozman, E.C. and A.L.-G. reviewed the pathologic and clinical data and confirmed diagnoses. I.B.-H., M. Bayes and M.G. performed WGBS library preparation and sequencing. M.P. and P.J. performed gene expression microarray experiments. S.B.-S., P.P., A.C.Q., L.H. and M.L.-G. performed gene expression and/or alternative splicing studies. S.B. performed copy-number analyses. G. Castellano, D.R., S.E. and A.V. functionally characterized differentially methylated regions. L.C., D.C., M.P. and M.A. coordinated or performed sample preparation and quality control. V.Q., X.S.P. and M.K. integrated DNA methylation and gene mutation data. M. Rubio, R.R., J.L.G., M.O., D.G.P. and A.V. were in charge of data management. I.G. coordinated sequencing efforts and performed primary data analysis. C.L.-O., E.C. and J.I.M.-S. conceived of the study. J.I.M.-S. led the experiments and wrote the paper with assistance from M.K., S.H., A.C.Q., C.L.-O. and E.C.
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M.Bibikova., V.H., B.K. and J.-B.F. are employees of Illumina, Inc.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–21 and Supplementary Tables 1–4, 9,10,12–14,16–18 (PDF 2662 kb)
Supplementary Table 5
Annotation of differentially methylated CpGs between CLL subgroups (XLS 517 kb)
Supplementary Table 6
Annotation of differentially methylated CpGs between U-CLL and CD5+NBC/NBC (XLS 4769 kb)
Supplementary Table 7
Annotation of differentially methylated CpGs between M-CLL and MBC (XLS 762 kb)
Supplementary Table 8
Lists of genes differentially methylated in distinct parts of the gene length in CLLs vs. controls by microarrays (XLS 297 kb)
Supplementary Table 11
Genes and CpGs showing a significant correlation between expression and DNA methylation levels (XLS 607 kb)
Supplementary Table 15
Differentially methylated CpGs located in the proximity of alternative splicing regions (XLS 146 kb)
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Kulis, M., Heath, S., Bibikova, M. et al. Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia. Nat Genet 44, 1236–1242 (2012). https://doi.org/10.1038/ng.2443
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DOI: https://doi.org/10.1038/ng.2443
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