Methylation status of DDIT3 gene in chronic myeloid leukemia

Abstract

Background: DNA-damage-inducible transcript 3 (DDIT3), a candidate tumor suppressor gene (TSG), has been found involved in the regulation of cellular growth and differentiation. The epigenetic changes of TSGs are recently recognized as an abnormal mechanism contributing to the development of chronic myeloid leukemia (CML). The aim of this study was to investigate the methylation status of DDIT3 gene in CML patients.

Methods: The methylation status of DDIT3 promoter was detected in the bone marrow mononuclear cells from 53 patients with CML using methylation-specific PCR (MSP). The expression levels of DDIT3 and bcr/abl transcript were determined by real-time quantitative PCR (RQ-PCR). Clinical data of these patients were collected and analyzed.

Results: The aberrant methylation of DDIT3 gene promoter was found in 35 of 53 (66%) CML cases. Correlation was not found between DDIT3 promoter hypermethylation and the age, sex, hemoglobin concentration, platelet counts, chromosomal abnormalities, bcr/abl transcript, and staging of CML patients (P > 0.05), but found between DDIT3 promoter hypermethylation and WBC counts of CML cases (R = 0.781, P < 0.001). The level of DDIT3 transcript in CML patients was significantly lower than that in controls (median 3.28 vs 19.69, P < 0.001), however, there was no difference in the level of DDIT3 transcript between methylation-positive CML cases (0.05-65.32, median 2.13) and methylation- negative CML cases (0.12-126.04, median 3.92) (P > 0.05).

Conclusion: Our results demonstrate that aberrant methylation of DDIT3 occurs in CML frequently.

Figure 1

MSP results of DDIT3 gene in CML. U and M represent PCR results by using primer sets for methylated and unmethylated DDIT3 gene, respectively. 1: positive control (positive controls of methylation and unmethylation are genomic DNA of placenta which is modified with or without M.SssI); 2: sample of one BM donor; 3,4: samples of two cases at CP; 5: sample of one case at AP; 6: sample of one case at BC; 7: ddH2O; Mark: Gene Ruler™ 100 bp DNA Ladder.

Figure 2

The sequencing results of MSP products in one patient with CML. I: The sequencing result of methylated product, CG was not changed after bisulfite treatment; II: The sequencing result of unmethylated product, T was replaced by C after bisulfite treatment.