Contributions
Abstract: PB1853
Type: Publication Only
Background
CLL is the most common leukemia in Western countries, but rare in Asia including Korea (5/100,000 vs. 0.5/100,000 per year), even though the incidence is increasing in Korea (0.5/100,000 in 1999-2001 to more than 1.2/100,000 in 2005-2010). Epigenetic research in Asian CLL is very rare and restricted to a few genes.
Aims
We hypothesized that CLL in Korea shows unique features compared to CLL in Western countries in terms of epigenetics. We performed Methyl-CpG Binding Domain Sequencing on CLL patients for the second time in the world and for the first time in Asia.
Methods
Nine Korean CLL patients and 5 age-matched Korean healthy individuals were included in the study. All the 9 patients showed TP53 mutation, and 1 patient showed IGHV hypermutation. One patient with IGHV hypermutation was excluded from the further analysis. B lymphoid cells were extracted using magnetic bead sorting technology. MBD-seq was performed using Invitrogen MethylMiner Methylated DNA Enrichment Kit and Illumina HiSeq 2000 platforms. The windows with FDR<0.01 were deemed as differentially methylated. The list of differentially methylated genes (DMGs) was then finally obtained. Functional enrichment analysis was performed by the clusterProfiler program with respect to Gene Ontology (GO) Biological Process and KEGG pathways, with significance criteria of p-value < 0.05. For GO analysis, the options minGSSize of 20 and maxGSSize of 1,000 were applied.
Results
Our approach with MBD-seq enables a comprehensive genome-wide interrogation of differentially methylated regions (DMRs) in CLL. There were more hypomethylated regions (2,062 windows) than hypermethylated regions (777 windows). Distal intergenic and intron regions contain the largest number of DMRs followed by promoter, 3'UTR and exon. As to the proportion of the DMRs, promoters contained the highest proportion of DMRs (around 0.08%), followed by 3'UTR, 5'UTR, downstream, distal intergenic region, exon and intron. A supervised hierarchical clustering clearly distinguished CLL patients from healthy individuals. The top forty differentially methylated genes (DMGs) include ERBIN, LINC00273, AGBL5, MIR4537, HCN1, LHPP, KIF16B, MYH3, LMNTD1 and CWH43. Immune process and cancer-related GO terms were dysregulated in CLL by DNA methylation either by hypo- or hyper-methylation. The dysregulated GO terms include Ras protein signal transduction, lymphocyte differentiation, immune system development, hematopoietic or lymphoid organ develoment, lymphocyte activation and positive regulation of apoptotic process. Dysregulated KEGG pathways included those that are directly related to immune process and cancer, mainly by hypomethylation. Most KEGG pathways were dysregulated by differential methylation in introns. The dysregulated KEGG pathway includes gastric cancer pathway, hepatocellular carcinoma pathway, CML pathway, breast cancer pathway Wnt signaling pathway, Ras signaling pathway, AML patway and transcriptional misregulation in cancer pathway. The reason why CLL pathway was not listed on top dysregulated KEGG pathways is simply because it is missing from KEGG pathway database content.
Conclusion
MBD-sequencing revealed that CLL patients have a distinct methylation profile from that of healthy individuals. Genes involved in cancer, immunity, lymphoid differentiation and major signaling pathways were dysregulated suggesting the role of dysregulated methylation in the pathogenesis of CLL. A further analysis will provide an important insight into the difference in methylation between Asian CLL and Western CLL.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Methylation, Signaling
Abstract: PB1853
Type: Publication Only
Background
CLL is the most common leukemia in Western countries, but rare in Asia including Korea (5/100,000 vs. 0.5/100,000 per year), even though the incidence is increasing in Korea (0.5/100,000 in 1999-2001 to more than 1.2/100,000 in 2005-2010). Epigenetic research in Asian CLL is very rare and restricted to a few genes.
Aims
We hypothesized that CLL in Korea shows unique features compared to CLL in Western countries in terms of epigenetics. We performed Methyl-CpG Binding Domain Sequencing on CLL patients for the second time in the world and for the first time in Asia.
Methods
Nine Korean CLL patients and 5 age-matched Korean healthy individuals were included in the study. All the 9 patients showed TP53 mutation, and 1 patient showed IGHV hypermutation. One patient with IGHV hypermutation was excluded from the further analysis. B lymphoid cells were extracted using magnetic bead sorting technology. MBD-seq was performed using Invitrogen MethylMiner Methylated DNA Enrichment Kit and Illumina HiSeq 2000 platforms. The windows with FDR<0.01 were deemed as differentially methylated. The list of differentially methylated genes (DMGs) was then finally obtained. Functional enrichment analysis was performed by the clusterProfiler program with respect to Gene Ontology (GO) Biological Process and KEGG pathways, with significance criteria of p-value < 0.05. For GO analysis, the options minGSSize of 20 and maxGSSize of 1,000 were applied.
Results
Our approach with MBD-seq enables a comprehensive genome-wide interrogation of differentially methylated regions (DMRs) in CLL. There were more hypomethylated regions (2,062 windows) than hypermethylated regions (777 windows). Distal intergenic and intron regions contain the largest number of DMRs followed by promoter, 3'UTR and exon. As to the proportion of the DMRs, promoters contained the highest proportion of DMRs (around 0.08%), followed by 3'UTR, 5'UTR, downstream, distal intergenic region, exon and intron. A supervised hierarchical clustering clearly distinguished CLL patients from healthy individuals. The top forty differentially methylated genes (DMGs) include ERBIN, LINC00273, AGBL5, MIR4537, HCN1, LHPP, KIF16B, MYH3, LMNTD1 and CWH43. Immune process and cancer-related GO terms were dysregulated in CLL by DNA methylation either by hypo- or hyper-methylation. The dysregulated GO terms include Ras protein signal transduction, lymphocyte differentiation, immune system development, hematopoietic or lymphoid organ develoment, lymphocyte activation and positive regulation of apoptotic process. Dysregulated KEGG pathways included those that are directly related to immune process and cancer, mainly by hypomethylation. Most KEGG pathways were dysregulated by differential methylation in introns. The dysregulated KEGG pathway includes gastric cancer pathway, hepatocellular carcinoma pathway, CML pathway, breast cancer pathway Wnt signaling pathway, Ras signaling pathway, AML patway and transcriptional misregulation in cancer pathway. The reason why CLL pathway was not listed on top dysregulated KEGG pathways is simply because it is missing from KEGG pathway database content.
Conclusion
MBD-sequencing revealed that CLL patients have a distinct methylation profile from that of healthy individuals. Genes involved in cancer, immunity, lymphoid differentiation and major signaling pathways were dysregulated suggesting the role of dysregulated methylation in the pathogenesis of CLL. A further analysis will provide an important insight into the difference in methylation between Asian CLL and Western CLL.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Methylation, Signaling