Characterization of the microRNAome in a patient with acute myeloid leukemia.

Abstract

10501 Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have been implicated in the pathogenesis of human cancer. Herein, we use next generation sequencing approaches to comprehensively assess miRNA expression, discover novel miRNAs and identify genetic variants of all known miRNA genes and miRNA binding sites in a patient with AML. Methods: The patient (AML1) was a female in her 50s with FAB M1 AML with normal 46XY karyotype. Massively-parallel sequencing of small RNAs isolated from the myeloblasts of AML1 was performed using the ABI SOLiD sequencing platform. Pooled RNA isolated from CD34+ bone marrow cells of 4 healthy volunteers (CD34) was used as control. To detect genetic variants of miRNA genes, we used 454-based sequencing of all 695 miRNA genes in the Sanger miR database (version 12.0). Finally, we analyzed the previously generated whole genome sequence for AML1 for genetic variants in the 3′- untranslated regions (3′-UTR) of all coding genes. Results: 28×106 and 20×106 small RNA sequence reads were obtained from AML1 and CD34 respectively. 8 novel miRNAs were identified from sequences that mapped to unannotated regions of human genome. Expression of 498 known miRNAs were detected with miR-233 being the most highly expressed miRNA in both AML1 and CD34; remarkably, it represented 47.3% of all miRNA reads in AML1. MiRNA gene sequencing of AML1 leukemic blast identified several single nucleotide variants. The whole genome sequence of AML1 skin DNA was used to differentiate germline polymorphism (SNPs) from somatic mutations. 13 novel SNPs and no somatic mutation were detected. Analysis of the 3′UTR of all coding genes in leukemic blasts and skin of AML1 revealed a single somatic mutation in the 3′-UTR of TNFAIP2. This mutation results in suppression of TNFAIP2 protein expression possibly by creating one or more new miRNA binding site. However, no recurrent mutations in the 3′-UTR of TNFAIP2 were detected in an additional 180 patients with AML. Conclusions: These data demonstrate the feasibility of next generation sequencing technologies to identify novel miRNAs, accurately measure mature miRNA expression, and identify both somatic and germline genetic variants of miRNA genes in primary cancer. No significant financial relationships to disclose.