TET2 Promoter DNA Methylation and Expression Analysis in Pediatric B-cell Acute Lymphoblastic Leukemia

Abstract

TET2 is a novel tumor suppressor gene involved in several hematological malignancies of myeloid and lymphoid origin. Besides loss-of-function mutations and deletions, hypermethylation of the CpG island at the TET2 promoter was found in human cancer. Previous analysis revealed no TET2 mutations in acute lymphoblastic leukemia (ALL). Since the TET2 promoter methylation status in pediatric ALL has not been reported, the aim of the present study was to determine if promoter hypermethylation may be a mechanism of TET2 inactivation in a group of pediatric ALL cases. Methylation of TET2 promoter region in one (1/45) ALL B-common patient was detected by methylation specific polymerase chain reaction (PCR) and subsequently analyzed by bisulfite sequencing. We found no correlation between promoter methylation and gene expression, measured by quantitative reverse transcriptase-PCR, however the level of TET2 expression in ALL group was significantly decreased compared to children's normal peripheral blood mononuclear cells and isolated B-cells. TET2 promoter hypermethylation seems to have limited clinical relevance in childhood B-cell ALL due to its low frequency.

Keywords: B-cell acute lymphoblastic leukemia; DNA methylation; TET2.

Figure 1.

(A) An example of methylation-specific PCR result of TET2 promoter methylation analysis. (1-3) Acute lymphoblastic leukemia (ALL) patients, patient number 3 is the one with TET2 promoter methylation: M - product amplified with primers specific for methylated TET2 sequence, U - product amplified with primers specific for unmethylated TET2 sequence; meth – methylated control DNA (normal human DNA methylated in vitro with SssI DNA methyltransferase), unmeth – unmethylated control DNA (commercially available universal unmethylated human DNA); NTC – no template control; M – marker. (B-C) DNA sequencing analysis of methylation specific PCR product in ALL patient number 3 (B) and methylation positive control (C). Each sequenced clone is represented by a row. Black circles correspond to methylated Cs, white circles correspond to unmethylated Cs, and small vertical lines without a circle correspond to missing values (e.g. caused by sequencing errors). (D) The comparison of TET2 expression level in ALL patients, normal peripheral blood mononuclear cells (PBMCs) and isolated normal CD19+ cells. Patient number 3 (TET2 methylation positive) is marked with a black dot. (E) The comparison of TET2 expression levels between ALL patients stratified according to blasts percentage.