Mutation analysis of DICER1 gene in hematologic tumors

MicroRNAs (miRNAs) regulate gene expression by pairing to mRNA of the target genes [1]. Dicer, an endonuclease (RNase III), is a protein involved in miRNA biogenesis [1]. During biogenesis, precursor miRNAs are processed by Dicer to generate a short RNA duplex [1]. Mounting evidence indicates that not only miRNAs themselves, but also the genes involved in miRNA biogenesis, play important roles in cancer pathogenesis [1]. Heravi-Moussavi et al . [2] recently identifi ed frequent somatic mutations of DICER1 , the gene encoding Dicer protein, in Sertoli – Leydig cell tumors (60%), and less frequent mutations in juvenile granulosa cell tumors, yolk sac tumors, mature teratomas, testicular non-seminomatous germ cell tumors and embryonal rhabdomyosarcomas. Th e mutations were recurrent in exons 25 and 26 of DICER1 , and most of the mutations were missense mutations occurring at Asp 1709 and Glu 1813 residues [2]. Because miRNA biogenesis and regulation are ubiquitous to all cell types, it is important to know whether any other types of human tumors carry DICER1 recurrent mutations. However, to date, the mutation status of the DICER1 gene in hematologic tumors remains unknown. In the present study, we attempted to determine whether the DICER1 gene is somatically mutated in acute leukemias and other common hematologic tumors. For this, we analyzed somatic mutations of DICER1 gene in fresh bone marrow aspirates of 798 hematologic tumors (acute myelogenous leukemia [AML], acute lymphoblastic leukemia [ALL], multiple myeloma and myelodysplastic syndrome) (Table I) by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. Also, we analyzed the gene in paraffi n-embedded tissues of 132 non-Hodgkin lymphomas (NHLs). Approval was obtained from Th e Catholic University of Korea, College of Medicine ’ s institutional review board for this study. To date, all of the recurrent DICER1 somatic mutations in Sertoli – Leydig cell tumors have been detected in exons 25 and 26 [2]. Genomic DNA each from tumor cells and normal cells of the same patients were amplifi ed by PCR with specifi c primer pairs for exon 25 (forward and reverse, respectively: 5 ’ -ATGTGGGGATAGTGTAAATGC-3 ’ and 5 ’ -GGGTCTTCATAAAGGTGCTT-3 ’ ) and exon 26 (5 ’ -GGCCTTTTTGCTTACAAGTCA-3 ’ and 5 ’ -GGGATAGTACACCTGCCAGAC-3 ’ ). Radioisotope ([ 32 P]dCTP) was incorporated into the PCR products for detection by autoradiography. Other procedures of the PCR-SSCP and DNA sequencing were described in our previous studies [3]. On the SSCP autoradiogram, all of the PCR products were clearly seen. SSCP analysis of aberrantly migrating bands led to the identifi cation of one DICER1 somatic mutation in an NHL (a diff use large B-cell lymphoma), but no DICER1 mutation was detected in the other tumors. Th e mutation was a missense mutation that substituted Glu 1813 residue (c.5438A C [p.Glu1813Ala]) (Figure 1). Importantly, this mutation was the same mutation as previously reported in Sertoli – Leydig cell tumors [2]. We repeated the experiments twice, including PCR-SSCP and direct DNA sequencing analysis to ensure the results, and found that the data were consistent. A main concern in cancer research is to identify whether any mutation found in a cancer type is common in other cancer types as well. In the present study, we analyzed DICER1 somatic mutation in various hematologic tumors. However, the prevalence of the DICER1 mutation in exons 25 and 26 was very low (0.1% of samples). Taken together, our results suggest that DICER1 exons 25 and 26 mutations may be very