Analysis of Methylation Status of ZAR1, GATA4, CDH22, SOX3, SLC16A5, PFN2, EHD3 and TBPL1 in Hematological Malignancies

DNA cytosine methylation in CpG dinucleotides is an important epigenetic event and critical for the control of gene expression, and appears to play a crucial role in tumorgenesis in hematologic malignancies. To identify novel tumor-specific differentially methylated regions in human leukemia/lymphoma, we performed methylation analysis of new target lesions in hematological malignancies.

The aberrant methylation at 8 new candidate human homologous genomic regions (ZAR1, GATA4, CDH22, SOX3, SLC16A5, PFN2, EHD3 and TBPL1), where hypermethyloation status was identified through a mouse-skin cancer model study by the RLSG method, were analysed with quantitative DNA methylation analysis using the Sequenom MassARRAY system. 8 human leukemia/lymphoma cell lines (HL-60, KG-1, Jurkat, MOLT-4, NALM-6, K562, Ramos, and Raji), and bone marrow or peripheral blood samples from 20 patients with leukemia before treatment were obtained for samples (10 in AML, 4 in Ph- ALL, 2 in Ph+ ALL, 2 in CML-CP, 1 in CML-BC, and 1 in CLL). Normal lymphocyte cells from four healthy individuals were used as normal controls. Each data were assigned as hypermethylaion when the average methylation levels of the entire target regions were more than 50%.

In ZAR1, SLC16A5 and EHD3, hypermethylation level was seen in all leukemia/lymphoma cell lines. GATA4, CDH22 and SOX3 showed hypermethylaion in all tumor cell lines except for AML cell line KG-1. PFN2 showed hypermethylation in all cell lines except for KG-1, T-ALL cell line Jurkat and MOLT-4. No aberrant methylation among tumor and normal cells was observed in TBPL1. Interestingly, DNA methylation patterns of ZAR1, GATA4, CDH22, and SOX3 were different between the clinical specimens of AML and ALL. Hypermethylation of those genes were frequently observed in ALL samples but less in AML. The Mann-Whitney U-test showed that the differences were significant in all ZAR1, GATA4, CDH22, and SOX3 (p < 0.01, respectively). DNA samples from CML-CP patients showed no abnormal methylation patterns in all genes. DNA from T-CLL patient showed hypermethylation only in EHD3. No abnormal methylation pattern was observed in SLC16A5 and PFN2 in clinical specimens. None of the normal control samples showed aberrant methylation in any genes.

We identified regions aberrantly methylated with high frequency among the new candidate genes in leukemia and lymphoma, and demonstrated a distinct methylation pattern between tumors and normal lymphocytes. Aberrant DNA methylation of ZAR1, GATA4, CDH22 and SOX3 may be associated to differentiation to lymphoid populations of leukemia.

No relevant conflicts of interest to declare.