TET2Mutation/5-hmC Level In Myelodysplastic Syndromes And Its Clinical Significance For Prognostic Evaluation

Myelodysplastic syndrome (MDS) is a heterogeneous group of disorders of hematologic malignancies characterized by ineffective hematopoiesis and risk of progression to acute myeloid leukemia (AML). According to the2008WHO classification system for hematologic cancers, the primary MDS is one of five major categories of myeloid neoplasms. As a highly heterogeneous disease, the molecular pathogenes-is of MDS has not been well defined. Recent advances of sequencing studies suggest that multiple mutations are related to MDS pathogenesis and progression to AML. Particular advances are the recent recognition that genes involved in the regulation of histone modification and DNA methylation are recurrently mutated in MDS, providing an important link between genetic and epigenetic alterations in MDS.TET2is a tumor suppressor gene in myeloid malignancies, and is the most frequently mutated gene in MDS so far. As an alpha-ketoglutarate (α-KG) and Fe (Ⅱ)-dependent oxygenase, TET2catalyzes the conversion of5-methylcytosine (5-mC) to5-hydroxymethylcytosine (5-hmC) and the5-hmC leads to demethylation of cytosine. As a novel epigenetic marker, the discovery of5-hmC promotes demethylation research of human genome.In this study, to provide experimental evidence for the pathogenesis and prognosis of MDS, we examined the relationship between TET2mutations and5-hmC levels, the epigenetic effects of TET2and5-hmC on the methylation of P15and hMSH2, and explored the prognostic effects of TET2and5-hmC in MDS patients, following the prognostic categories of Revised International Prognostic Scoring System (IPSS-R).Firstly, screening of all coding exons of TET2was carried out by PCR amplification and directly sequencing. In addition, for IDH1and IDH2mutational analyses, exons4of IDH1and IDH2were amplified. It was showed that TET2mutated in16.39%(95%CI,7.10%-25.68%) of61MDS patients, mutations clustered in the conserved domain Boxl, and frameshift was more common than other mutation types. MDS patients with TET2mutations exhibited significantly higher neutrophil counts than TET2mutations-negative. We found no IDH1/IDH2mutation in61MDS patients, suggesting IDH1/IDH2mutation was not very common in MDS. Then, the5-hmC level in BMMNC was detected by Hydroxymethylated DNA Quantification Kit. It was showed that5-hmC levels of patients with MDS were significantly lower than those of controls and5-hmC levels in MDS with BM blast≥5%were obviously lower those with BM blast>5%. Then, the CD34+cells were acquired from BMMNC of MDS patients using CD34positive selection immunomagnetic beads. The5-hmC assay showed that hematopoietic stem/progenitor cells (CD34+) exhibited lower5-hmC level than their more differentiated counterparts (CD34-) both in controls and MDS patients, and more important,5-hmC in CD34+cells of MDS patients was lower than in CD34+cells of controls. This suggested that5-hmC might affect differentiation of hematopoietic cells. We also found that5-hmC levels of patients with TET2mutation were obviously lower than those without TET2mutation, which reflected that TET2mutations could affect its catalytic function.To assess whether TET2and5-hmC might be involved in the methylation or mRNA expression of other genes, we observed methylation status and mRNA expression of TET2, P15and hMSH2by methylation-specific PCR and real-time quantitative PCR, respectively. We found that all MDS patients had no methylation at TET2promoter region. P15and hMSH2methylation were all mostly identified in IPSS-R higher risk group patients. Patients with methylation P15or hMSH2had decreased5-hmC levels and TET2mRNA expression compared with patients without methylation. Correlation analysis showed that P15and hMSH2mRNA expression all had a positive correlation with5-hmC levels. These results suggested that there was certain intrinsic contact between TET2/5-hmC and P15/hMSH2methylation.Finally, to evaluate prognostic values of TET2mutations and5-hmC changes in MDS, the Kaplan-Meier method and multivariate Cox hazards model were performed, respectively. Overall survival (OS) was also analyzed according to the standard of IPSS-R for MDS. With the increase of IPSS-R risk, the2-year OS of this group of MDS patients was reduced. It is worth noting that a correlation between higher5-hmC and longer OS in patients with MDS is confirmed, suggesting5-hmC levels may be used as a new prognostic indicator of MDS. It is likely that5-hmC will ultimately be combined with IPSS-R to improve the prognostic categories of MDS.