Findings

Introduction

New sequencing technologies have enabled the identification of several mutations in epigenetic regulator genes, such as Ten-eleven-translocation 2 (TET2), which encodes an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC). Mutations in the coding regions of TET2 lead to loss of TET2 protein function, reduce 5-hmc levels and cause a significant shift towards monocyte/granulocyte differentiation in detriment of erythroid/lymphoid differentiation [1, 2]. TET2 mutations confer a worse prognosis in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) [3, 4]. We have recently reported that TET2 mRNA expression is downregulated in MDS and AML, and predicts poor overall survival in MDS patients [5]. However, the correlation between the presence of TET2 mutations and TET2 transcript levels has rarely been addressed [4, 6]. We, herein, expanded our previous observations and aimed to investigate TET2 mutational status and the impact of this mutational status on TET2 expression in a cohort of MDS and AML patients. For this purpose, TET2 mutation analysis and TET2 gene expression were tested by Sanger sequencing and quantitative PCR, respectively, in bone marrow samples from healthy donors and myeloid neoplasm patients.

Materials and methods

Patients’ characteristics

Bone marrow samples were obtained from a total of 22 healthy donors, and 19 patients with myeloid neoplasms (MDS = 10 and AML = 9) followed at the outpatient clinics of the University of Campinas, who had also been included in a previous study [5]. The present study was approved by the Institutional and National Review Board in accordance with the Helsinki Declaration. Written informed consent was obtained from all patients who participated in this study. Patients were diagnosed according to the 2008 Word Health Organization criteria [7]. Patients’ characteristics are described in Table 1.

Table 1 Patients’ characteristics
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Polymerase chain reaction (PCR) and DNA sequencing

The screening of TET2 mutations was performed on coding exons 3, 4, 5, 6, 7, 8, 9, 10 and 11 (GenBank reference NM_0175628.4). Primer sequences and PCR conditions were previously described [8]. Amplicons were sequenced with an ABI 3500 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) using the Big Dye terminator V1.1 cycle sequencing kit and analyzed using CLC Main Workblench v.7.6.2 software (Qiagen, Aarhus, Denmark). All alterations found were searched in SNP (dbSNP; http://www.ncbi.nlm.nih.gov/projects/SNP), Ensembl Genome Browser databases (http://asia.ensembl.org/index.html), and in the Catalogue of Somatic Mutations in Cancer (COSMIC; http://cancer.sanger.ac.uk/cosmic).

Quantitative PCR (qPCR)

Total RNA was extracted from cells using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The reverse transcription reaction was performed using RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). TET2 mRNA level was detected by Maxima Sybr green qPCR master mix (MBI Fermentas, St. Leon-Rot, Germany) in the ABI 7500 Sequence Detection System (Life Technologies) using specific primers: forward 5′- ACGCAAGCCAGGCTAAACA -3′, reverse 5′- GCTGGGACTGCTGCATGA -3′; HPRT1 (hypoxanthine phosphoribosyltransferase 1) was used as the endogenous control: forward 5′-GAACGTCTTGCTCGAGATGTGA-3′, reverse 5′-TCCAGCAGGTCAGCAAAGAAT-3′. The relative quantification value was calculated using the equation, 2-ΔΔCT [9]. A negative ‘No Template Control’ was included for each primer pair. The dissociation protocol was performed at the end of each run to check for non-specific amplifications. Three replicas were run on the same plate for each sample.

Statistical analyses

Statistical analyses were performed using GraphPad Instat 5 (GraphPad Software, Inc., San. Diego, CA, USA). For comparisons, Mann-Whitney test was used for measured factors with two levels. A p value <0.05 was considered as statistically significant.

Results and discussion

We analyzed the mutation status of TET2 exons and the impact of this status on TET2 expression in bone marrow cells from myeloid neoplasm patients. In total, 17 TET2 variants were detected. After excluding confirmed SNPs (P29R [rs12498609], V218M [rs6843141], P363L [rs17253672], G355D [rs61744960], H1778R [rs62621450], I1762V [rs2454206] and L1721W [rs34402524]), TET2 mutations were observed in 8/19 (42 %) patients with myeloid neoplasms [4/10 (40 %) MDS and 4/9 (44 %) AML], including six missense (E709K, Y867H, H924R, S1109P, P1723S and H1868L) and three stop codon (E1073X, S1516X and S1518X) mutations (Fig. 1). A total of nine TET2 mutations were found in exon 3 (n = 5) or exon 11 (n = 3) in eight patients; one patient had two mutations. Four TET2 mutations (E1073X, S1109P, S1518X and H1868L) found in our study had not been previously described in the COSMIC database. Among the six missense mutations, three mutations presented a high probably of damage to protein function, according to Polyphen2 analysis. The PolyPhen2 scores for predicted damage of TET2 Y867H, S1109P and H1868L mutations were 0.999, 0.995 and 1, respectively (http://genetics.bwh.harvard.edu/pph2/). TET2 mutations in exons have been implicated in protein loss-of-function and myeloid neoplasm mechanism of disease [1, 2], which supports our focus on sequencing TET2 coding regions only. The relevance of mutations in the TET2 promoter region has not been explored yet in myeloid neoplasms.

Fig. 1
figure 1

TET2 mutations identified in myelodysplastic syndromes and acute myeloid leukemia patients. In a cohort of 19 patients, nine TET2 mutations were identified in eight patients. Genomic sequencing of protein-coding regions revealed missense (black arrows), and stop codon (red arrows) mutations in TET2; Sanger sequencing analysis is illustrated in the figure. TET2 protein primary structure indicating the domains and specific known conserved motifs are shown: cysteine-rich region (C-rich), double strand beta helix (DSBH). The aminoacid position is indicated

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TET2 expression was reduced in MDS/AML patients (median 0.8 [minimum 0.01- maximum 6.41]), compared with healthy donors (2.72 [0.43–31.49]); p <0.005 (Fig. 2a). The standard deviation of TET2 expression for the healthy donors was high in our cohort of patients, which is in agreement with reports from other authors [4, 10]. TET2 expression was similar in TET2 wild type (0.8 [0.01–3.79]) and in mutated patients (1.82 [0.14–6.41]), for the entire cohort, and among patients with MDS and AML only, all p >0.05 (Fig. 2b–d). These findings are in agreement with Jankowska and colleagues [4] and Coutinho and colleagues [6], who reported TET2 downregulation in 16 patients with MDS/myeloproliferative neoplasms and in 12 patients with pediatric MDS, respectively, regardless of TET2 mutational status. Reduced TET2 levels in MDS patients have also been previously reported by our group [5], Li and collaborators [10], and Zhang and collaborators [11]. However, the TET2 mutation was not evaluated in these studies.

Fig. 2
figure 2

TET2 expression, according to TET2 mutational status, in myelodysplastic syndromes and acute myeloid leukemia patients. a qPCR analysis of Ten-eleven-translocation 2 (TET2) mRNA levels in total bone marrow cells from healthy donors and from patients with the diagnosis of myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML). TET2 expression in patients stratified according to TET2 mutational status for the entire cohort of MDS/AML patients (b), for patients with MDS (c) or AML diagnosis (d). The “y” axis represents the relative TET2 mRNA expression. The numbers of subjects studied and the p values (Mann-Whitney) are indicated in the graph. Abbreviations: WT wild type, MUT mutated

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Somatic mutations in TET2 have been described in healthy elderly individuals with clonal hematopoiesis and have been associated with alterations in DNA methylation [12]. TET2 mutations have been indicated as early events in clonal evolution and potently lead to the development of preleukemic hematopoietic stem cells [13, 14]. For this reason, growing attention has been given to the hypothesis that TET2 is a gate keeper in hematological malignancies. The occurrence of TET2 mutations may indirectly lead to the disruption of other important genes by inducing epigenetic changes, as recently described for JAK2 [15]. TET2 mutations alter the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevent JAK2 V617F from up-regulating a proliferative program [15]. In addition, Kameda et al. [16] and Rasmussen and collaborators [17] demonstrated, using mouse models, that the loss of Tet2 contributes towards the progression of myeloid neoplasms. Thus, reduced TET2 function, due to loss-of-function mutations, reduced mRNA expression due to epigenetic silencing or not yet elucidated mechanisms may have a great contribution to the malignant phenotype of hematological cancers.

Taken together, our findings suggest that decreased TET2 expression is observed in myeloid neoplasms and does not correlate with TET2 mutation status. Other molecular mechanisms, including mutations of additional genes and/or epigenetic regulation may affect TET2 expression in myeloid neoplasms [18]. TET2 mRNA expression levels, in addition to TET2 mutation status, may also play a role in myeloid neoplasm physiopathology. Future studies to further investigate the mechanisms that lead to reduced TET2 expression in hematological malignancies will be of importance.