ABSTRACT
PURPOSE: The aim of this study was to analyse the expression profiles of DNMT1, DNMT3A, DNMT3B (components of DNA methylation machinery), TET2 and APOBEC3B (components of DNA demethylation machinery) in pediatric MDS patients and investigate their associations with MDS subtypes, cytogenetics, evolution to acute myeloid leukemia (AML) and p15INK4B methylation level. PATIENTS AND METHODS: The expressions of DNMT1, DNMT3A, DNMT3B, TET2, and APOBEC3B were evaluated in 39 pediatric MDS patients by real-time quantitative PCR (qPCR). The quantification of p15INK4B methylation levels (MtL) was performed in 20 pediatric MDS patients by pyrosequencing. Mann-Whitney test was used to evaluate possible differences between the expression levels of selected in patients and donors, according to MDS subtypes, karyotypes, evolution to AML and p15INK4B MtL. The correlations between the expression levels of the different genes were assessed by Spearman rank correlation coefficient. RESULTS: We found that DNMTs expression levels were higher in pediatric MDS compared to donors [DNMT1 (p<0.03), DNMT3A (p<0.03), DNMT3B (p<0.02)]. TET2 and APOBEC3B expression levels did not show a statistically significant difference between pediatric patients and donors. Considering MDS subtypes, patients at initial stage presented DNMT1 overexpression (p<0.01), while DNMT3A (p<0.02) and DNMT3B (p<0.007) were overexpressed in advanced subtypes. TET2 and APOBEC3B expression did not differ in MDS subtypes. DNMT1 (p<0.03), DNMT3B (p<0.03), and APOBEC3B (p<0.04) expression was higher in patients with normal karyotypes, while patients with abnormal karyotypes showed higher DNMT3A expression (p<0.03). Karyotypes had no association with TET2 expression. DNMTs overexpression was observed in patients who showed disease evolution. A positive correlation was found between DNMTs expression and between APOBEC3B and DNMT3A/DNMT3B. However, TET2 expression was not correlated with DNMTs or APOBEC3B. p15INK4B MtL was higher in pediatric MDS patients compared with donors (p<0.03) and its hypermethylation was associated with increased DNMT1 expression (p<0.009). CONCLUSION: Our results suggest that the overexpression of DNMTs and an imbalance between the expressions of the DNA methylation/demethylation machinery components play an important role in MDS development and evolution to AML. These results have clinical implications indicating the importance of DNMTs inhibitors for preventing or delaying the progression to leukemia in pediatric MDS patients.
ABSTRACT
Pediatric myelodysplastic syndrome (MDS) is an uncommon disease and little is known about the molecular alterations of its development and evolution to acute myeloid leukemia (AML). The Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PCR2). It is a histone methyltransferase, that targets lysine 27 of histone 3. This methylated H3-K27 is usually associated with the silencing of genes that are involved in fundamental cellular processes, such as cell proliferation and differentiation. There are only few studies showing the status of EZH2 expression in patients with MDS and they were performed in adult MDS patients. The aim of this study was to analyze the EZH2 expression in pediatric patients with MDS and its association with karyotypes and evolution to acute myeloid leukemia (AML). We conducted the first study of EZH2 expression in pediatric patients with MDS. Considering the EZH2 expression levels in 42 patients and 17 healthy pediatric donors, it was possible to define three groups of expression in patients: low, intermediate, and high. The intermediate level encompassed patients with normal karyotypes, low level included patients with monosomy 7 and del(7q) and high level included patients with trisomy 8 and del(11q) (p < 0.0001). Comparing the leukemic evolution, the low expression group presented disease evolution in 100% (8/8) of the cases, the intermediate expression group showed disease evolution in 4.34% (1/23) and in the high expression group, 63.63% (7/11) patients showed evolution from MDS to AML (p < 0.0001). It is important to note that low and high EZH2 expression are associated with leukemic evolution, however low expression showed a stronger association with evolution from MDS to AML than the high expression. Our results suggest a scale of measure for EZH2 expression in pediatric MDS, where aberrant EZH2 expression may be a potential biomarker of disease evolution.
