Estigate the possible influence of CpG methylation status on the expression level of the corresponding genes, the gene expression profiles were integrated withRani et al. Clinical Epigenetics (2017) 9:Page 6 ofFig. 2 Distribution of a hypermethylated and b hypomethylated probes in CLL Vs. CD19+ normal controlsthe DNA methylation profiles and co-analyzed. On comparing the data of CLL patients with healthy individuals, a negative correlation in CpG methylation and gene expression was observed for 211genes (Additional file 1: Table S4). Of these, 149 genes were hypermethylated and downregulated and 62 genes were hypomethylated and upregulated including AXIN2, ID4, EBF1, SOX4, SOX7, TAL1, PMEPA1, SPRY1, CDK6, and MEIS1. Pathway analysis using the genes having negative correlation for DNA methylation and gene expression in CLL Vs. normal CD19+ cells identified significant enrichment of three KEGG pathways which included PRIMA-1 p53 signalling pathway (p = 0.002), pathways in cancer (p = 0.005), and the cell cycle pathway (p = 0.007). A comparison of the CpG methylation and gene expression profiles among the IGHV unmutated Vs. mutated patients identified 64 differentially expressed genes (Fig. 3) including BMPR2, CRY1, FGFR2, FOSB,INPP4B, PLD5, PAX9, RGS2, RIC8B, and VIPR1 (Additional file 1: Table S5). Of the various genes found to be differentially methylated and/or differentially expressed, a total of 17 genes (Table 2) were validated using RQ-PCR on a cohort of 93 (22 female: 71 male) early stage CLL patients and pooled CD19+ B cells from 10 healthy volunteers. The criteria for selection of these genes was negative correlation between CpG promoter methylation and gene expression in CLL Vs. normal (MEIS1, PMEPA1, SOX7, SPRY1, CDK6, ID4, AXIN2, TNRC18) and in the IGHV unmutated Vs. mutated subgroup (CRY1, VIPR1, PAX9, RIC8B). Other genes selected for validation included NFATC1 (hypomethylated in CLL), TBX2, TSHZ3 (hypermethylated in CLL), SPRY2 (upregulated in CLL) and BIK (downregulated in CLL). We focused on these five genes as they had previously been shown in the literature to be epigenetically influenced in CLL PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/18111632 [NFATCFig. 3 Supervised hierarchical clustering of hypermethylated-dowregulated and hypomethylated -upregulated genes selected on the basis of significant log2FC values in IGHV mutated (n = 05) Vs. IGHV unmutated (n = 09) CLL. The Euclidean hierarchical clustering was performed using Gene Spring Gx software version 13.5 and is based on normalized intensity values. Each row represents an individual patient and each column represents a gene. A gradient color scale ranging between blue (hypermethylated and downregulated) and red (hypomethylated and upregulated) is includedRani et al. Clinical Epigenetics (2017) 9:Page 7 ofTable 2 Comparison of mRNA levels of selected genes (median Cq and median fold change) as assessed by real time RQ-PCR in CLL (n = 93; unmutated = 39; mutated = 54) and CD19+ sorted B-cells from healthy individuals (n = 10)Comparison of levels of mRNA expression of selected genes in CLL S.No. 1. Gene CRY1 CLL Vs. normal Group CLL 19+ Normal 2. MEIS1 CLL 19+ Normal 3. ID4 CLL 19+ Normal 4. TNRC18 CLL 19+ Normal 5. NFATC1 CLL 19+ Normal 6. CDK6 CLL 19+ PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9547713 Normal 7. VIPR1 CLL 19+ Normal 8. SPRY1 CLL 19+ Normal 9. PAX9 CLL 19+ Normal 10. PMEPA CLL 19+ Normal 11. TBX2 CLL 19+ Normal 12. TSHZ3 CLL 19+ Normal 13. BIK CLL 19+ Normal 14. SPRY2 CLL 19+ Normal 15. AXIN2 CLL 19+ Normal 16. SOX7 CLL 19+ Normal 17. RIC8B CLL 19+ Normal Me.
