NATURE MEDICINE VOLUME 16 | NUMBER 1 | JANUARY 2010 49 ARTICLES Myelodysplastic syndrome (MDS), one of the most common hemato- poietic malignancies, arises in primitive CD34 + hematopoietic stem/ progenitor cells 1–4 . MDS is characterized by ineffective hematopoiesis and dysplasia in one or more lineages of the bone marrow 5 . The para- doxical finding of normal or increased cellularity in the marrow in the context of peripheral blood cytopenias has been attributed to increased proliferation of hematopoietic cells that is counterbalanced by a simultaneous increase in apoptosis 6 . The majority of affected individuals either succumb to the consequences of marrow failure or progress to acute myeloid leukemia (AML) 5 . One of the most common subtypes of MDS, 5q– syndrome, is defined by an isolated interstitial deletion of chromosome 5q, refrac- tory anemia, variable neutropenia and normal or high platelet counts associated with hypolobulated megakaryocytes 7 . Although the com- mon deleted region (CDR) on chromosome 5q has been mapped to a 1.5-megabase region on band q33.1, distal breakpoints at band q33-q35 are more common, and, less often, deletions of the 5q arm are limited to a region distal to the CDR 8–10 . The CDR in 5q– syndrome contains several genes that are implicated in hematopoiesis 7 . Recently, the erythroid differentiation defect observed in 5q– syndrome has been attributed to the RPS14 gene located within the CDR of chromosome 5q 11 . However, haploinsufficiency of RPS14, which encodes ribosomal protein S14, does not explain several of the other features of 5q– syndrome, namely, thrombocytosis associated with megakaryocytic dysplasia, neutropenia and clonal dominance (that is, replacement of the marrow by the malignant cells). Thus, loss of other genes probably contributes to the full manifestation of 5q– syndrome. We postulated that loss of noncoding transcripts encoding miRNAs within the CDR may result in haploinsufficiency by loss of inhibition of their targets. miRNAs are small 21- to 25-nucleotide noncoding RNAs that post-transcriptionally repress specific messenger RNA targets through interaction with the 3′ untranslated region (UTR) 12 . We evaluated the expression of miRNAs located on chromosome 5q in MDS and found lower expression of miR-145 (5q33.1) and miR-146a (5q33.3) in individuals with MDS with deletion of the long arm of chromosome 5 (del(5q)). Concurrent loss of both miR-145 and miR-146a resulted in activation of innate immune signaling through elevated expression of their respective miRNA targets, TIRAP and TRAF6. Knockdown of miR-145 and miR-146a together or overexpression of TRAF6 in mouse HSPC recapitulated features of 5q– syndrome, such as increased platelet counts associated with dys- plastic megakaryopoiesis and neutropenia, through cell-autonomous and cell-nonautonomous mechanisms. RESULTS Expression of miRNAs on chromosome 5q in MDS bone marrow We performed massively parallel sequencing of small RNA librar- ies from five cell lines, with or without deletion of chromosome 5q (Supplementary Fig. 1), to a minimum depth of 4 million tags, to identify potential miRNAs on chromosome 5q. We found 25 Identification of miR-145 and miR-146a as mediators of the 5q– syndrome phenotype Daniel T Starczynowski 1,2 , Florian Kuchenbauer 1 , Bob Argiropoulos 1 , Sandy Sung 1 , Ryan Morin 1 , Andrew Muranyi 1 , Martin Hirst 1 , Donna Hogge 1 , Marco Marra 1 , Richard A Wells 3 , Rena Buckstein 3 , Wan Lam 1,2 , R Keith Humphries 1,4 & Aly Karsan 1,2 5q– syndrome is a subtype of myelodysplastic syndrome characterized by severe anemia and variable neutropenia but normal or high platelet counts with dysplastic megakaryocytes. We examined expression of microRNAs (miRNAs) encoded on chromosome 5q as a possible cause of haploinsufficiency. We show that deletion of chromosome 5q correlates with loss of two miRNAs that are abundant in hematopoietic stem/progenitor cells (HSPCs), miR-145 and miR-146a, and we identify Toll–interleukin-1 receptor domain–containing adaptor protein (TIRAP) and tumor necrosis factor receptor–associated factor-6 (TRAF6) as respective targets of these miRNAs. TIRAP is known to lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6 in mouse HSPCs resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow progressed either to marrow failure or acute myeloid leukemia. Thus, inappropriate activation of innate immune signals in HSPCs phenocopies several clinical features of 5q– syndrome. 1 British Columbia Cancer Agency Research Centre, Vancouver, British Columbia, Canada. 2 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada. 3 J.D. Crashley Myelodysplastic Syndrome Laboratory, Molecular and Cellular Biology, Sunnybrook Research Institute, Toronto, Ontario, Canada. 4 Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Correspondence should be addressed to A.K. (akarsan@bccrc.ca). Received 20 May; accepted 30 September; published online 8 November 2009; doi:10.1038/nm.2054 © 2010 Nature America, Inc. All rights reserved.