1Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Medical University of Warsaw, Warsaw, Poland.
2Department of Pediatric Haematology & Oncology, Medical University of Warsaw, Warsaw, Poland.
Precursor B-cell acute lymphoblastic leukemia (B-cell ALL) is the most common neoplasm in children and is characterized by genetic and epigenetic aberrations in hematopoietic transcription factor (TF) genes. This study evaluated promoter DNA methylation and aberrant expression levels of early- and late-acting hematopoietic TF genes homeobox A4 and A5 (
Blood samples of 38 ALL patients and 20 controls were obtained. DNA was treated with sodium bisulfite and DNA methylation level of
Aberrant methylation of
Aberrant methylation and expression of the selected hematopoietic genes were correlated with demographic/clinical prognostic factors of pediatric ALL, such as age, WBC count, and NCI risk classification.
Precursor B-cell acute lymphoblastic leukemia (B-cell ALL) is the most common neoplasm in children and is characterized by excessive proliferation of abnormal and undifferentiated B-cells. Chromosomal translocations that frequently involve genes encoding hematopoietic transcription factors (TFs), such as ets variant 6-runt-related transcription factor 1 (
Besides genetic aberrations, epigenetic mechanisms are involved in the development of leukemia . The best-described epigenetic mechanism is the methylation of CpG islands in promoter regions that regulates gene expression. Several microarray-based studies have shown that pediatric patients with ALL show aberrant DNA methylation on a genome-wide scale, indicating a distinct DNA methylation pattern between patients with ALL and healthy individuals and among different B-cell ALL subtypes. Gene ontology analyses have shown that genes encoding TFs and other proteins involved in lymphocyte development are the most aberrantly methylated genes in pediatric patients with ALL [4, 5].
Previous studies on DNA methylation in pediatric patients with ALL have provided some insights on the promoter methylation status of genes encoding hematopoietic TFs. These studies have highlighted that early B-cell factor 1 and paired box 5 genes (
In this study we comprehensively analyzed promoter methylation and expression of selected hematopoietic TF genes homeobox A4 and A5 (
Blood samples were collected from 38 pediatric patients (age, 1-17 years) with ALL at the Medical University of Warsaw, Poland. The study was approved by the local ethics committee. Patient characteristics are listed in Table 1. Of the 38 patients, 35 had ALL B-common, 1 had ALL-proB, and 2 had ALL B-common/pre-B. Peripheral blood mono-nuclear cells (PBMCs) obtained from 20 anonymous healthy pediatric donors aged between 8 months and 17 years were used as controls.
DNA was extracted from 27 Ficoll-isolated PBMCs and 11 whole blood samples using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), was quantified using NanoDrop (Thermo Scientific, Wilmington, DE, USA), and was treated with sodium bisulfite by using EpiTect Kit (Qiagen), according to the manufacturer's recommendations. Promoter methylation of
Standard curves of known template concentration were used to quantify the PCR products. Samples were prepared using serial dilutions of plasmid DNA containing methylated inserts of the genes of interest. These recombinant plasmids were constructed by amplifying standard methylated genomic DNA (Qiagen) with primers for each selected gene and by cloning the PCR products with StrataClone Kit (Agilent Technologies, La Jolla, CA, USA). The recombinant plasmids were amplified in bacteria, purified using Plasmid Mini Kit (A&A Biotechnology, Gdynia, Poland), and quantified using Quant-iT PicoGreen (Invitrogen, Paisley, UK).
Percentage DNA methylation (percentage of methylated reference [PMR]) was calculated by dividing the gene of interest:
Gene expression and percentage DNA methylation (PMR value) were treated as continuous variables and were analyzed using 2-sided Mann-Whitney
To assess the possible prognostic value of promoter methylation and expression of the selected genes, we analyzed their relationship with clinical prognostic indicators such as age of patients, white blood cell (WBC) count at the time of diagnosis, blast count in the peripheral blood (PB) on day 8 or in the bone marrow (BM) on day 15, and relapse during follow-up. Data collection was terminated on September 10, 2014.
Significance threshold was set at α=0.05. All statistical analyses were performed using GraphPad Prism (La Jolla, CA, USA).
Promoter methylation level of
These 2 genes are located in the same chromosomal region. Interestingly, we observed an inverse correlation between promoter methylation levels of these genes in samples from ALL patients (Spearman correlation r=0.4415,
Gene expression analysis showed significantly decreased expression of
Comparison of expression level of the genes of interest showed correlation among genes encoding TAL1, homeobox proteins, and interferon regulatory factors. Inverse correlation was observed between
We assessed the correlation among DNA methylation and expression levels of the analyzed genes and demographic and clinical features of pediatric patients with leukemia. According to the National Cancer Institute (NCI) risk classification, patients with ALL who are aged 1-9 years and who have a WBC count of <50,000/µL are classified as standard risk patients while those aged ≥10 years and who have a WBC count of >50,000/µL have a high-risk and show poor prognosis. In the present study, we observed slightly elevated
No relationship was observed between promoter methylation and gene expression and blast count in PB on day 8 or blast count in BM on day 15. Interestingly, comparison of leukemic patients who experienced and did not experience relapse showed very slight but significant increase in
In leukemic patients, genes encoding hematopoietic TFs frequently show both genetic and epigenetic aberrations [1, 2, 4]. Such abnormalities result in impaired hematopoietic differentiation and may contribute to leukemogenic transformation. In the present study, we evaluated promoter DNA methylation and expression levels of 6 genes encoding earlyand late-acting hematopoietic TFs in samples from pediatric patients with B-cell ALL and healthy controls.
Genome-wide analysis  and analysis of candidate genes [6, 16] showed that promoters of
We did not observe a direct correlation between promoter methylation level of
Previous studies indicate that
IRF4 and IRF8 play a key role in B-cell development, particularly during pre-B cell transition to immature B-cells, class-switch recombination and plasma cell differentiation [12, 24]. Epigenetic silencing of
Only 1 patient with ALL showed a slight increase in
TAL1 (SCL) is a hematopoietic TF predominantly expressed in HSCs, early progenitor cells, and erythroid and megakaryocytic precursor cells .
From the viewpoint of development-hematopoiesis, an interesting relationship between the expressions of different genes can be observed. Decrease in the expression of the late leukemogenesis related gene
Irrespective of the dysregulation of leukemic blasts, some correlations were observed between the expressions levels of the studied genes that resembled their relationship in normal leukemogenesis.
In conclusion, the proportion of pediatric patients with B-cell ALL included in this study showed aberrant promoter methylation in key hematopoietic genes. Methylation was predominantly observed for the promoters of
Analysis of promoter methylation and expression of genes encoding hematopoietic TFs in pediatric patients with B-cell ALL. (