title: Hypermethylation and down-regulation of DLEU2 in paediatric acute myeloid leukaemia independent of embedded tumour suppressor miR-15a/16-1.
abstract: BACKGROUND
  Acute Myeloid Leukaemia (AML) is a highly heterogeneous disease. Studies in adult AML have identified epigenetic changes, specifically DNA methylation, associated with leukaemia subtype, age of onset and patient survival which highlights this heterogeneity. However, only limited DNA methylation studies have elucidated any associations in paediatric AML.
  
  
  METHODS
  We interrogated DNA methylation on a cohort of paediatric AML FAB subtype M5 patients using the Illumina HumanMethylation450 (HM450) BeadChip, identifying a number of target genes with p <0.01 and Δβ >0.4 between leukaemic and matched remission (n = 20 primary leukaemic, n = 13 matched remission). Amongst those genes identified, we interrogate DLEU2 methylation using locus-specific SEQUENOM MassARRAY® EpiTYPER® and an increased validation cohort (n = 28 primary leukaemic, n = 14 matched remission, n = 17 additional non-leukaemic and cell lines). Following methylation analysis, expression studies were undertaken utilising the same patient samples for singleplex TaqMan gene and miRNA assays and relative expression comparisons.
  
  
  RESULTS
  We identified differential DNA methylation at the DLEU2 locus, encompassing the tumour suppressor microRNA miR-15a/16-1 cluster. A number of HM450 probes spanning the DLEU2/Alt1 Transcriptional Start Site showed increased levels of methylation in leukaemia (average over all probes >60%) compared to disease-free haematopoietic cells and patient remission samples (<24%) (p < 0.001). Interestingly, DLEU2 mRNA down-regulation in leukaemic patients (p < 0.05) was independent of the embedded mature miR-15a/16-1 expression. To assess prognostic significance of DLEU2 DNA methylation, we stratified paediatric AML patients by their methylation status. A subset of patients recorded methylation values for DLEU2 akin to non-leukaemic specimens, specifically patients with sole trisomy 8 and/or chromosome 11 abnormalities. These patients also showed similar miR-15a/16-1 expression to non-leukaemic samples, and potential improved disease prognosis.
  
  
  CONCLUSIONS
  The DLEU2 locus and embedded miRNA cluster miR-15a/16-1 is commonly deleted in adult cancers and shown to induce leukaemogenesis, however in paediatric AML we found the region to be transcriptionally repressed. In combination, our data highlights the utility of interrogating DNA methylation and microRNA in combination with underlying genetic status to provide novel insights into AML biology.
authors.affiliation: Cancer & Disease Epigenetics, Murdoch Childrens Research Institute, Melbourne, Victoria, Australia. leah.morenos@mcri.edu.au.
authors.forename: Leah
authors.initials: L
authors.lastname: Morenos
authors.forename: Zac
authors.initials: Z
authors.lastname: Chatterton
authors.forename: Jane L
authors.initials: JL
authors.lastname: Ng
authors.forename: Minhee S
authors.initials: MS
authors.lastname: Halemba
authors.forename: Mandy
authors.initials: M
authors.lastname: Parkinson-Bates
authors.forename: Francoise
authors.initials: F
authors.lastname: Mechinaud
authors.forename: Ngaire
authors.initials: N
authors.lastname: Elwood
authors.forename: Richard
authors.initials: R
authors.lastname: Saffery
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Wong
mesh_terms: D000293:Adolescent; D000970:Antineoplastic Agents / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002648:Child; D002675:Child, Preschool; D002869:Chromosome Aberrations; D002880:Chromosomes, Human, Pair 11; D002898:Chromosomes, Human, Pair 8; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015973:Gene Expression Regulation, Leukemic; D056426:Genetic Loci; D006801:Humans; D007223:Infant; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D062085:RNA, Long Noncoding; D012074:Remission Induction; D015398:Signal Transduction; D014166:Transferases; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; C109420:DLEU2 lncRNA, human; C505361:MIRN15 microRNA, human; C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D062085:RNA, Long Noncoding; D025521:Tumor Suppressor Proteins; D014166:Transferases