Methylation and Hydroxymethylation Status of Tumor Suppressor Genes and Common Fragile Sites Genes in Acute Myeloid Leukemia

Background: 5-methylation (5-mC) is the predominant epigenetic mark in mammalian genomic DNA. When promoter region of certain gene is hypermethylated, the gene becomes transcription silent. Promoter of tumor suppressor genes (TSG) usually exists in CpG islands, and silencing of TSGs in cancer cells is often associated with hypermethylation. p15, CDH1 are frequently methylated in myeloid malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Common Fragile Site (CFS) is a fragile site on the chromosomes easy to produce gap and break, and it contains putative TSGs. FHIT, WWOX and PARK2 are the CFS genes known to be frequently methylated in solid tumors, but their status of hematologic malignancies has not been fully elucidated yet. 5-hydroxymethylaiton (5-hmC) is a newly discovered epigenetic modification that is presumably generated by oxidation of 5-mC by the TET family of cytosine oxygenases. Techniques identifying 5-mC cannot distinguish between 5-mC and 5-hmC, therefore 5-hmC status of the genes have not fully elucidated yet too. Recently it has been demonstrated that mutation of epigenetic modifiers (DNMT3A, TET2, IDH1/2) play important role on AML pathogenesis. We tried to clarify 5-mC and 5-hmC status of TSG p15, CDH1 and CFS genes FHIT, WWOX and PARK2 by using new techniques and the relationships with expression levels of epigenetic modifiers in AML.

Methods: BM samples obtained from 74 of AML patients are subjected to the study after informed consent. This study was approved by IRB of Gunma University Hospital. DNA, RNA were extracted from BM mononuclear cells. Methylation specific PCR (MSP) was carried out to assay 5-mC of p15, CDH1, WWOX, PARK2. Quantification of 5-mC and 5-hmC (except PARK2) was carried out by methylation sensitive restriction enzyme assay (MSRE) with glucosylation and Q-PCR. Total DNA 5-mC and 5-hmC were analyzed by ELISA. The mRNA expression levels of p15, CDH1, FHIT, WWOX, PARK2, DNMT1, 3A, TET2 were quantified by Q-PCR.

Results: MSP revealed that p15, CDH1, WWOX and PARK2 were methylated in 43.1%, 94.3%, 35.7% and 36.9% of AML, respectively. PARK2 methylation was not found in t(15;17) APL, but in 32% of normal karyotype AML (NK-AML), in 67% of t(8;21) CBF-AML. In contrast, the p15 methylation was found in 83.3% of APL, 45.5% of NK-AML, 50% of CBF-AML. WWOX methylation was found in 42.9% of APL, in 16% of NK-AML and 66.7% of CBF-AML. Adverse karyotype AML (adv-AML) tended to show lower % of WWOX, PARK2 and p15 methylation with 15.8%, 21.1% and 18.8% compare to good risk karyotype. The frequency of the methylation of PARK2 and WWOX were varied among karyotypes and the methylation was mutually exclusive. ELISA demonstrated that mean % of total 5-mC DNA was 1.08% and ratio of 5-hmC in 5-mC was 0.95% in AML. Interestingly, 5-hmC was 0% in adv-AML although 5-mC existed (mean: 1.05%). Locus specific MSRE-QPCR demonstrated that mean % of 5-mC of p15, CDH1, WWOX and FHIT were 6.62%, 1.25%, 8.33%, 2.88%, respectively., In adv-AML, 5-hmC of CDH1, WWOX and FHIT were not detected, although 5-mC of these genes were detected (0.41%, 9.0%, 2.14%) in accordance with whole DNA analysis. In good and intermediate AML, 5-hmC of these genes was 3.44%, 1.07%, 2.69% ,respectively. RQ-PCR demonstrated that CDH1, p15, WWOX, PARK2 and epigenetic modifier DNMT1, DNMT3A and TET2 expression were not different among various karyotype risks, but only FHIT expression significantly higher in good risk group (p=0.047). The expression levels of the genes were not significantly different between mentylated and unmethylated. The ratio of 5-hmC/5-mC of the TSGs tended to be associated with the expression levels of the corresponding genes, but the association did not reach statistical significance. DNMT3A expression in AML with 5-mC PARK2 was higher than in other AML (p=0.016). Contrary to the intuition, DNMT3A expression was positively correlated with FHIT, PARK2 expression (r=0.776, p<0.001, r=0.689, p<0.001). CDH1 expression was positively correlated with DNMT1 and negatively correlated with TET2 expression (r=0.447, p=0.009, r=-0.349, p=0.022). OS and EFS were not different among the methylation status of these genes.

Conclusion: CFS genes are selectively methylated in AML. MSRE-QPCR can distinguish 5-mC and 5-hmC and quantify the ratio of them with locus specific manner. The relationship between gene expression and 5-hmC, 5-mC should be pursued.