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Methyl-CpG-binding domain protein enriched genome-wide sequencing (MBD-Seq) is a robust and powerful method for analyzing methylated CpG-rich regions with complete genome-wide coverage. In chronic lymphocytic leukemia (CLL), the role of CpG methylated regions associated with transcribed long noncoding RNAs (lncRNA) and repetitive genomic elements are poorly studied. Based on MBD-Seq, we characterized the global methylation profiling of high CpG rich regions in different CLL prognostic subgroups based on IGHV mutational status. Our study identified 5800 hypermethylated and 12570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal controls. Out of total cllDMGs 40% of hypermethylated and 60% of hypomethylated genes were mapped to noncoding RNAs. In addition, we found that the major repetitive elements such as short interspersed elements (SINE) and long interspersed elements (LINE) have high percentage of cllDMRs (differentially methylated regions) in IGHV subgroups compared to normal controls. Finally, two novel lincRNAs (hypermethylated CRNDE and hypomethylated AC012065.7) were validated in an independent CLL sample cohort (48 samples) compared with 6 normal sorted B cell samples using quantitative pyrosequencing analysis. The methylation levels showed inverse correlation to the gene expression levels analyzed by real-time quantitative PCR. Notably, survival analysis revealed that hypermethylation of CRNDE and hypomethylation of AC012065.7 correlated with inferior outcome. Thus, our comprehensive methylation analysis by MBD-Seq provided novel hyper- and hypo-methylated long noncoding RNAs, repetitive elements along with protein coding genes as potential epigenetic-based CLL-signature genes involved in disease pathogenesis and prognosis. |