{'fraction_non_alphanumeric': 0.028721957851801497, 'fraction_numerical': 0.022660321776569226, 'mean_word_length': 5.40065264684554, 'pattern_counts': {'":': 0, '<': 22, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Expression Profiles of DNA Methylation and Demethylation Machinery Components in Pediatric Myelodysplastic Syndrome: Clinical Implications

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.


Introduction
Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal hematopoietic stem cell diseases. MDS is characterised by ineffective hematopoiesis, presence of dysplasias, peripheral blood cytopenia and an increased risk of evolution to acute myeloid leukemia (AML). 1,2 MDS has a higher incidence in adults and elderly individuals. By contrast, MDS is a rare disease in children, occurring in approximately 2-7% of the childhood hematological malignancies. [3][4][5][6] Pediatric patients with MDS have some distinct genetic, epigenetic and clinical characteristics when compared to adult patients. [4][5][6] Thus, pediatric MDS has a specific classification proposed by Hasle and colleagues in 2003 5 and revised in 2016. 6 These patients can be classified as refractory cytopenia of childhood (RCC), refractory anemia with excess of blasts (RAEB) and refractory anemia with excess of blasts in transformation (RAEB-t). 5,6 Recently, Locatelli and Strahm (2018) used for the advanced pediatric MDS the WHO classification of myeloid neoplasms. So, MDS with ≥2% of blasts in peripheral blood (PB) or 5-19% of blasts in bone marrow (BM) is classified as myelodysplastic syndrome with excess of blasts (MDS-EB). 7 However, in pediatric MDS classification, the subtype MDS-EB in transformation (MDS-EB-t) is maintained. This subtype is characterized by 20-29% of blasts in the BM. 6,7 Due to the heterogeneity and rarity of pediatric MDS, molecular alterations involved in initial and subsequent stages during its development and progression to AML are not well defined. Several studies in adult patients indicate the important role of epigenetic changes in the pathogenesis of MDS. [6][7][8][9] This epigenetic nature can be explained as follows: (1) among the most frequently mutated genes in MDS, approximately 45% are responsible for the regulation of epigenetic mechanisms and (2) MDS is considered the most responsive disease to treatment with DNA methyltransferase inhibitors. 9,10 However, there are few studies focused on the genes that act on the epigenetic mechanisms in pediatric patients with MDS, and these studies have shown that mutations in these genes are rare. 11 Nevertheless, epigenetic mediators could be dysregulated by other mechanisms, such as transcriptional alterations and primarily involve components of the DNA methylation and demethylation machinery to result in aberrant methylation profiles. 12 Aberrant promoter hypermethylation involving genes encoding cell adhesion molecules, cell cycle regulators and tumor suppressor genes have been associated with hematopoiesis dysregulation. In MDS, a high frequency of hypermethylation of p15 INK4B , cadherin 1 (CDH1), death-associated protein kinase (DAPK) and suppressor of cytokine signalling (SOCS-1) has been observed. 13 In pediatric MDS, p15 INK4B methylation has been associated with disease pathogenesis and poor prognosis. 14 DNA methylation is considered a guardian of hematopoietic stem cell fate because it acts to maintain the balance of these cells, their self-renewal capacity, and differentiation in specific hematopoietic cell populations. 12 DNA methyltransferases (DNMTs) are enzymes responsible for catalysing the insertion of a methyl group on carbon 5 of a cytosine in the CpG context. DNMT1 is associated with the maintenance of DNA methylation patterns, while DNMT3A and DNMT3B mediate de novo methylation. 15 By contrast, DNA demethylation can occur passively during replication through the inhibition of the methylation maintenance process or actively and independent of DNA replication. 16 Active demethylation is initiated by two independent pathways. The first involves the progressive oxidation of 5-methylcytosine (5mC) and is catalysed by the teneleven-translocation (TET) family of enzymes; the second is driven by the apolipoprotein B mRNA editing enzyme (APOBECs) family, which deaminates 5mC and 5-hydroxylmethyl cytosine (5hmC). [17][18][19] In both cases, mispairing takes place and the base excision repair machinery replaces the modified base by an unmethylated cytosine. 18 The balance between the enzymes that act on DNA methylation and demethylation is essential for the maintenance of genomic stability and is referred to as the DNA methylation and demethylation machinery. 18 It has been suggested that increased expression of de novo or maintenance DNMTs contributes to the development of leukemia by inducing aberrant hypermethylation of important regions in the genome. 20 Up to date, to the best of our knowledge, there are no studies focusing on DNMTs expression in pediatric MDS, and only one study evaluated TET2 expression in pediatric patients. 21 Even in adult patients, few studies have been performed to analyse the expression of DNA methylation and demethylation machinery components. 17,22 Although the APOBEC family is an important component in the demethylation machinery 18,19 and APOBEC3B has been described as a driving mutagenic agent during cancer development and progression, 23 no studies involving the APOBEC family have been performed in MDS. Thus, the aim of this study was to analyse the expression of DNMT1, DNMT3A, DNMT3B (components of the DNA methylation machinery), TET2 and APOBEC3B (components of the DNA demethylation machinery) in pediatric patients with MDS and investigate their associations with MDS subtypes, cytogenetics, evolution to AML and the p15 INK4B methylation levels of gene to verify the role of epigenetic alterations during pediatric MDS pathogenesis.

Statistical Analyses
Comparisons of the DNMTs, TET2, and APOBEC3B expression between patients and donors, MDS subtypes, karyotypes and evolution from MDS to AML were performed using the Mann-Whitney U-test. Correlations between the expression levels of each pair of genes considered in this study were evaluated by the Spearman rank correlation coefficient. The analyses of p15 INK4B MtL and its association with the expression of DNA methylation and demethylation machinery components were performed using the Mann-Whitney U-test. Statistical analyses were performed using GraphPad Prism (Graphpad Software, USA). A p-value < 0.05 was considered statistically significant in all analyses.

Associations of DNMTs, TET2, and APOBEC3B Genes Expression with the MDS Subtypes, Cytogenetics and Disease Evolution
When considering MDS subtypes, we observed a higher DNMT1 expression (p<0.01) at initial stage (RCC) ( Figure 3A) and a higher expression of DNMT3A (p< 0.02) ( Figure 3B) and DNMT3B (p<0.007) ( Figure 3C) in advanced stages (MDS-EB/MDS-EB-t) compared to controls. TET2 and APOBEC3B expression did not significantly differ between MDS subtypes and controls ( Figure 3D and E).
We also analysed the association between DNMTs, TET2 and APOBEC3B expression and the evolution from MDS to AML. The evolution of disease was observed in 28% of the patients (11/39). Patients who progressed to AML had higher expression levels of DNMT1, DNMT3A, and DNMT3B when compared to donors (p<0.04, p<0.02 and p<0.005, respectively) ( Figure 5A-C). TET2 and APOBEC3B expression did not differ between patients who showed evolution from MDS to AML and controls ( Figure 5D and E). Table 2  according to MDS subtypes, karyotypes and evolution from MDS to AML.

Analysis of p15 INK4B Promoter Methylation and Its Association with the Expression of DNA Methylation/ Demethylation Machinery Components
The analysis of p15 INK4B promoter MtL in 20 patients showed that three had no methylation (15%), 11 had methylation (55%) and six were hypermethylated (30%). When comparing the patients who presented methylation with the donors, we observed a statistically significant increase in the first group (p<0.03) ( Figure 6A) and when subdividing MDS cases in methylated and hypermethylated, increased MtL were observed in the latter group ( Figure 6B). Next, we compared DNMTs expression in the different groups defined by p15 INK4B methylation status and showed increased DNMT1 expression in patients with methylation (p<0.02) and hypermethylation (p<0.009) when compared to donors ( Figure 6C). For the other genes (DNMT3A, DNMT3B, TET2, and APOBEC3B) no statistically significant differences were observed (data not shown).

Discussion
In the present study, we described the expression levels of the DNA methylation and demethylation machinery components in pediatric patients with MDS. DNMT1, DNMT3A, and DNMT3B were overexpressed in pediatric MDS when compared with healthy pediatric donors. The initial stage of MDS showed a higher expression of DNMT1, while in advanced stages an increased expression was observed for DNMT3A and DNMT3B. DNMTs overexpression in MDS was previously demonstrated in adult patients. 25,26 Langer et al observed DNMT1, DNMT3A and DNMT3B overexpression in initial stage and in advanced stage when compared to control group. 25 Hopfer et al showed a higher expression of DNMT3A and DNMT3B in advanced stages relative to initial stage. 26 DNMTs activity is essential for normal hematopoiesis as well as in the process of blood cell differentiation. 20,26 DNMT1 has been shown to be important for HSC selfrenewal, niche retention, and multilineage hematopoietic differentiation. 27 DNMT1 overexpression has already been associated with aberrant methylation in tumour suppressor genes. 12 DNMT3A controls the balance between selfrenewal and differentiation in normal hematopoiesis 28 and alterations in this enzyme result in HSC expansion, impaired differentiation and seems to be associated with  29 Recently, it has been demonstrated that DNMT3B acts as a master regulator of transcription in the pediatric AML genome. 30 As DNMT1 acts on maintenance methylation while DNMT3A and DNMT3B mediate de novo methylation, 15 our results suggest that the three enzymes act in the development of the disease and mainly in its evolution to AML. However, it is noteworthy that the increased expression of DNMT3A and DNMT3B suggesting that new patterns of methylation occur, especially in patients with disease evolution. In fact, some studies have identified aberrant methylomes in adult patients with MDS, as well as hypermethylation of specific genes, particularly during the course of AML. 10,12,17,26,31 In our results, we observed an association between p15 INK4B promoter methylation and higher DNMT1 expression levels. In this way, it is possible that DNMT3A and DNMT3B produce new methylation patterns during the development of the MDS and that DNMT1 acts by maintaining these patterns.
Some studies demonstrated that there is a coordinate action of the DNMTs. 32,33 In accordance, a significant correlation between the expression of these enzymes was observed in the present study, suggesting that there is a cooperative action of the DNMTs expression in pediatric MDS pathogenesis.
TET2 and APOBEC3B act as components of the DNA demethylation machinery and they have relevant roles in cells homeostasis. TET2 has been shown to regulate myeloid differentiation and clonal cell expansion during hematopoiesis. Its decreased expression has been previously reported in patients with MDS/AML, and it has also been associated with a poor prognosis. 34,35 APOBEC3B, in addition to its potential role in DNA demethylation, has also been shown to be an important source of mutations in human cancer. 23 Through its deamination activity, APOBEC3B can induce C to T and C to G transitions, leaving specific footprints in tumour DNA. Although this APOBEC-mediated mutational signature has been reported in several solid tumours, it is still poorly explored in the context of hematological malignancies. 19,36 In adult MDS patients, C to T transitions are the most frequent point mutations. 11,31,37 Furthermore, a more recent study with MDS pediatric patients showed that its mutational load is also characterised by C to T substitutions. 19

Donors
Methylated Hypermethylated In our study, TET2 and APOBEC3B expression levels did not differ between pediatric patients and donors nor according to MDS subtypes. On the other hand, APOBEC3B expression levels were higher in patients with normal karyotypes when compared with patients with abnormal karyotypes and with healthy donors. In pediatric MDS, most patients with RCC show normal karyotypes. 38 However, some of these patients will manifest rapid disease evolution, suggesting the contribution of molecular alterations. 9 Our results suggest a possible role of APOBEC3B at initial stage of disease.
We also analysed the correlation between the expression of DNMTs, TET2, and APOBEC3B in pediatric patients with MDS, showing that DNMTs and TET2 expression are not significantly correlated. However, it was possible to observe a correlation between DNMTs and APOBEC3B expression. Alterations in DNMTs also cause DNA imbalances and/or histone modifications, which may lead to chromatin remodelling, genomic instability, and gene expression pattern changes. 39 So, our results reinforce the relevant role of DNMTs and also suggests the participation of APOBEC3B, possibly contributing to genomic instability in the pathogenesis of pediatric MDS.
Taken together, our results suggest the presence of an imbalance between the DNA methylation and demethylation machinery components during pediatric MDS pathogenesis, with an overexpression of DNMT1, DNMT3A, and DNMT3B (Figure 7). Our results have clinical implications and indicate the importance of hypomethylating agents for preventing or delaying the progression to leukemia, especially for children undergoing hematopoietic stem cell transplantation (HSCT). Waespe et al demonstrated that azacytidine treatment prior to hematopoietic stem cell transplantation was well tolerated in pediatric patients with advanced MDS and was associated with superior event-free survival. 40 Therefore, laboratory tests for detecting the level of DNMTs expression may aid the prediction of disease evolution and may select the patients with MDS for the use of DNA methyltransferases inhibitors.
Since this study was the first to address the alterations in DNA methylation and demethylation machinery components in pediatric patients with MDS, it is necessary to confirm our results in other cohorts to provide a better understanding of the aberrant expression of these genes in the pathogenesis of pediatric MDS.

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.

Data Sharing Statement
The data used to support the findings of this study are included within the article and the original data used to support the findings of this study are available from the corresponding author upon request.