Genome-wide profiling of methylation identifies novel targets with aberrant hyper-methylation and reduced expression in low-risk myelodysplastic syndromes

of an unedited peer-reviewed manuscript that publication. NPG are providing this early version of the manuscript as a to our The manuscript typesetting and a proof review before it is published in its ﬁnal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply. Abstract 29 Gene expression profiling signatures may be used to classify the subtypes of MDS 30 patients. However, there are few reports on the global methylation status in MDS. The 31 integration of genome-wide epigenetic regulatory marks with gene expression levels 32 would provide additional information regarding the biological differences between MDS 33 and healthy controls. Gene expression and methylation status were measured using 34 high-density microarrays. A total of 552 differentially methylated CpG loci were 35 identified as being present in low-risk MDS; hyper-methylated genes were more 36 frequent than hypo-methylated genes. In addition, mRNA expression profiling identified 37 1005 genes that significantly differed between low-risk MDS and the control group. 38 Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of 39 the hyper-methylated genes were under-expressed in low-risk MDS cases. Gene 40 network analysis revealed molecular mechanisms associated with the low-risk MDS 41 group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and 42 ETS1 were identified as silenced genes. In addition, the immune response and miRNA 43 biogenesis were affected by the hyper-methylation and under-expression of IL27RA 44 and DICER1 . Our integrative analysis revealed that aberrant epigenetic regulation is a 45 hallmark of low-risk MDS patients and could play a central role in these diseases. 46 using the FDR threshold indicated. All the calculations described here were done using R and Bioconductor. suggested that the increased apoptosis of the bone marrow myeloid precursors is an important factor in the ineffective hematopoiesis of MDS patients. These studies also showed that the increased programmed cell death a pathophysiological mechanism rather than a compensatory process to counteract increased cell growth (31;33;34). Members of the BCL2 family are major regulators of these apoptotic pathways. The present study shows that BCL2 expression was significantly weaker in mononuclear low-risk MDS cells than in normal individuals. These results are in accordance with previous studies that showed reduced BCL2 expression in CD34+ cells of patients with early MDS subtypes (35). Furthermore, our study showed that BCL2 had significantly higher methylation levels in low-risk MDS samples. Consistent with the increasing evidence for a fundamental role of epigenetic silencing of apoptotic pathways in cancer (36-38), the hyper-methylation and the inverse correlation of mRNA expression of BCL2 would be expected to promote apoptosis in MDS patients. The under-expression of BCL2 in low-risk MDS due to aberrant methylation deserves further investigation as a low-risk MDS biomarker and supports a role for apoptosis-targeted therapy in these patients in the future. Our study found hyper-methylation and under-expression of the ETS1 gene in the same group of low-risk MDS patients compared with the control group. Several studies have indicated that the level of ETS expression is reduced during tumorigenesis. These analyses show that ETS1 suppresses tumorigenicity and the cases with a high level of 411 ETS1 expression had better outcomes for disease-free survival than those with a low level (39). These findings suggest that under-expression of ETS1 could have a crucial role in tumor promotion in MDS patients, especially during their early phases. ETS1 is a nuclear phosphoprotein that functions as a transcription factor by binding the target DNA sequences containing impairs with These suggest that the disruption of DICER1 by methylation or mutation may cause in mice resembling important features of human MDS. Our findings also showed that DICER1 had direct genetic interactions with ATXN1 , NFE2L3 and deregulated genes, which might affect the normal relationship of these genes with DICER1 and consequently the deregulation of the functions in which they are involved. Furthermore, our data showed an overall slight down-regulation of miRNAs in low-risk MDS (p-value = 0.039) which could be related to the deregulation of DICER1 . However, no significant differences were found for two miRNAs (miRNA- 145 and miRNA-196) that were examined individually.