Resented unknown genes, non-coding RNAs, hypothetical proteins, chromosomal loci, predicted open reading frames, and probes associated with sex-chromosomes were excluded from the downstream analysis. Among the differentially methylated probes, 53.8 of hypermethylated probes were located inside known gene bodies, 38.2 in the promoters, 2.6 in divergent promoters and 5.2 were located downstream of the known genes (Fig. 2a). The frequency distribution of the hypomethylated probes (Fig. 2b) was comparable to the hypermethylated probes. Of the differentially methylated probes, CpG sites were found in 73 of the hypermethylated probes and in 81 of the hypomethylated probes. The details pertaining to these probes, including the gene name, chromosomal location and distribution are provided in Additional file 1: Tables S2A and S2B.On the basis of gene functions, the CpG islands in the promoter regions of PRIMA-1 the tumor suppressor genes (KLF4, PTCH1, PAX5, PCDH10, RASSF10, IRX1, TBX5, ID4, SOX7, SLIT2) and the transcription factors (TWIST1, KLF4, TAL1, PAX2, PAX9, NR2F2, IRX4, MEIS1) were found to be hypermethylated. Approximately, 10 of the hypermethylated CpG promoters were located within the homeobox genes. Promoter regions of genes such as FOXD3, FOXE1, FOXG1, ID4, SLIT2, BNC1, SALL1, RIPK4, HAND2, SOX9, SOX11, NR2F2, TAL1, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12711626 SIM2, PAX9, and TBX2 were also found to be hypermethylated in sync with earlier reported results in CLL [16, 19, 25]. In addition, hypomethylation was observed in the promoter region of NFATC1 and inside gene body in NOTCH1, SFRP1, and GPS as has been reported in earlier studies in CLL [19, 25]. Using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis tool, the differentially methylated genes were evaluated for the overrepresented Gene Ontology (GO) categories and the most significant overrepresented GO biological processes were found to be related to regulation of transcription (p < 0.0001) [26, 27]. To identify the association of differential methylation profile with the IGHV mutation status, the methylation array data from 9 IGHV unmutated and 5 IGHV mutated cases was compared. This analysis elicited a distinct signature of 56 hypermethylated (p < 0.05, log2FC 0.25) and 2402 hypomethylated probes (p < 0.05, log2FC (-)0.25) in unmutated CLL. The hypermethylated probes were distributed across 46 genes and spanned PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8627573 promoter regions of 10 genes (Additional file 1: Table S3A).Similarly, the hypomethylated probes spread across 1332 genes and spanned promoter regions of 399 genes (Additional file 1: Table S3B). Differential methylation of several genes previously reported in the IGHV mutation based subgroups [NCOR2, KCNJ2, SIX3, CHRM1, [16]], [NRF1, CRY1, KCNJ2, SOX5 [28]] was also noticed in the present study. In addition, differential CpG promoter hypomethylation of genes already known to influence clinical outcome in other malignancies was observed and includes EMILIN2 [29], TBX5 [30], CBX8 [31],OLIG2 [32], and PCDH10 [33]. The DAVID database was used to identify biological pathways for the differentially methylated genes. Four of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including circadian rhythm pathway (p = 0.002), calcium signalling pathway (p = 0.03), axon guidance (p = 0.02), and gap junction pathway (p = 0.04) were found to be significantly affected in the IGHV unmutated Vs. mutated subgroup.Correlation of methylation and gene expression analysisTo inv.