Distinctive Patterns of MicroRNA Expression Associated with Karyotype in Acute Myeloid Leukaemia Amanda Dixon-McIver 1 , Phil East 2 , Charles A. Mein 3 , Jean-Baptiste Cazier 1,2 , Gael Molloy 1 , Tracy Chaplin 1 , T. Andrew Lister 1 , Bryan D. Young 1 , Silvana Debernardi 1 * 1 Institute of Cancer, Medical Oncology Centre, Barts and The London, School Of Medicine, London, United Kingdom, 2 Cancer Research UK, Bioinformatics & Biostatistics Service, London, United Kingdom, 3 Genome Centre, Barts and The London, School Of Medicine, London, United Kingdom Abstract Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults; however, the genetic aetiology of the disease is not yet fully understood. A quantitative expression profile analysis of 157 mature miRNAs was performed on 100 AML patients representing the spectrum of known karyotypes common in AML. The principle observation reported here is that AMLs bearing a t(15;17) translocation had a distinctive signature throughout the whole set of genes, including the up regulation of a subset of miRNAs located in the human 14q32 imprinted domain. The set included miR-127, miR-154, miR- 154*, miR-299, miR-323, miR-368, and miR-370. Furthermore, specific subsets of miRNAs were identified that provided molecular signatures characteristic of the major translocation-mediated gene fusion events in AML. Analysis of variance showed the significant deregulation of 33 miRNAs across the leukaemic set with respect to bone marrow from healthy donors. Fluorescent in situ hybridisation analysis using miRNA-specific locked nucleic acid (LNA) probes on cryopreserved patient cells confirmed the results obtained by real-time PCR. This study, conducted on about a fifth of the miRNAs currently reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub- classify cancer and suggests a role in the aetiology of leukaemia. Citation: Dixon-McIver A, East P, Mein CA, Cazier J-B, Molloy G, et al. (2008) Distinctive Patterns of MicroRNA Expression Associated with Karyotype in Acute Myeloid Leukaemia. PLoS ONE 3(5): e2141. doi:10.1371/journal.pone.0002141 Editor: Cathal Seoighe, University of Cape Town, South Africa Received January 24, 2008; Accepted March 20, 2008; Published May 14, 2008 Copyright: ß 2008 Dixon-McIver et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funds from Cancer Research UK, Project Grant (C6277/A6789) to BDY and SD, Barts and The London, Research Advisory Board Studentship (ONA100P) to AD-M. Competing Interests: The authors have declared that no competing interests exist. * E-mail: silvana.debernardi@cancer.org.uk Introduction Acute myeloid leukaemia (AML) arises from the accumulation of myeloid precursor cells arrested at early stages of differentiation. Analysis of the karyotype of leukaemic cells has identified non- random somatically acquired translocations, inversions, and deletions, which are often associated with specific subtypes of AML [1]. The major gene fusion events are the t(8;21), t(15;17), inv(16), and the 11q23 rearrangements which together account for approximately 20% of all AMLs and result in the expression of chimeric proteins.[2]. Of the remaining AMLs, a substantial proportion, possibly as much as 40% [3], lacks any visible chromosomal abnormality and cannot be consistently associated with any known genetic lesion. Large scale clinical studies have demonstrated that cytogenetic abnormalities provide valuable information of prognostic relevance. Leukaemias fall into three broad cytogenetic prognostic risk groups, with the t(8;21), t(15;17), and inv(16) leukaemias having a more favourable outcome, whereas those with loss of chromosome 7, deletion of chromosome 5q and more complex karyotypes having an adverse outcome. All the other subtypes of AML, including those with rearrangement of 11q23 and normal karyotype, have an intermediate prognostic risk group [3]. Several studies have shown that genome-wide gene expression profiling can clearly distinguish the major cytogenetic groups, including normal karyotype samples, identifying specific sets of genes with expression patterns highly correlated with each karyotypic class [4–8] and so providing a better understanding of the underlying disease biology. A new class of small non-coding RNA molecules, designated as microRNAs (miRNAs) [9], has been shown to play key roles in a number of regulatory functions, including modulation of haema- topoiesis and cell differentiation in mammals [10]. MiRNAs are single stranded RNAs, typically 19–25 nucleotides in length, generated from endogenous transcripts and evolutionarily con- served. They modulate gene expression by complementarity- mediated binding to target mRNAs resulting in the repression of translation [11] or in the cleavage of the target transcript [12,13]. There are several indications that miRNAs might be a new class of genes involved in human tumourigenesis. A proportion of human miRNA genes is reported to be located in regions involved in cancer [14] and several examples of an association between disrupted expression of specific miRNAs and cancer have been shown in a variety of tissues [15–18]. Lu and collaborators [19] were the first to observe distinct patterns of miRNA expression across tumour types, and miRNA profiles reflecting the develop- mental lineage and the differentiation state of the tumour. The importance of miRNAs in AML has recently been emphasised by studies from this laboratory. Using a quantitative real-time PCR assay specific to the mature miRNA [20], we have demonstrated that the expression of a limited number of miRNAs in AML correlates with the AML global expression profile, and that miR-181a correlates with the morphological subtype and the PLoS ONE | www.plosone.org 1 May 2008 | Volume 3 | Issue 5 | e2141