Novel splicing-factor mutations in juvenile myelomonocytic leukemia

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are heterogeneous groups of chronic myeloid neoplasms characterized by clonal hematopoiesis, varying degrees of cytopenia or myeloproliferative features with evidence of myelodysplasia and a propensity to acute myeloid leukemia (AML).1 In recent years, a number of novel gene mutations, involving TET2, ASXL1, DNMT3A, EZH2, IDH1/2, and c-CBL, have been identified in adult cases of chronic myeloid neoplasms, which have contributed to our understanding of disease pathogenesis.2, 3, 4, 5, 6, 7 However, these mutations are rare in pediatric cases, with the exception of germline or somatic c-CBL mutations found in 10–15% of chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML),8 highlighting the distinct pathogenesis of adult and pediatric neoplasms.9 Recently, we reported high frequencies of mutations, involving the RNA splicing machinery, that are largely specific to myeloid neoplasms, showing evidence of myeloid dysplasia in adult.10 Affecting a total of eight components of the RNA splicing machinery (U2AF35, U2AF65, SF3A1, SF3B1, SRSF2, ZRSR2, SF1 and PRPF40B) commonly involved in the 3′ splice-site (3′SS) recognition, these pathway mutations are now implicated in the pathogenesis of myelodysplasia.10 To investigate the role of the splicing-pathway mutations in the pathogenesis of pediatric myeloid malignancies, we have examined 165 pediatric cases with AML, MDS, chronic myeloid leukemia (CML) and JMML for mutations in the four major splicing factors, U2AF35, ZRSR2, SRSF2, and SF3B1, commonly mutated in adult cases. Bone marrow or peripheral blood tumor specimens were obtained from 165 pediatric patients with various myeloid malignancies, including de novo AML (n=93), MDS (n=28), CML (n=17) and JMML (n=27), and the genomic DNA (gDNA) was subjected to mutation analysis (Supplementary Table 1). The status of the RAS pathway mutations for the current JMML series has been reported previously (Supplementary Table 2).11, 12 Nineteen leukemia cell lines derived from AML (YNH-1, ML-1, KASUMI-3, KG-1, HL60, inv-3, SN-1, NB4 and HEL), acute monocytic leukemia (THP-1, SCC-3, J-111, CTS, P31/FUJ, MOLM-13, IMS/MI and KOCL-48) and acute megakaryoblastic leukemia (CMS and CMY) were also analyzed for mutations. Peripheral blood gDNA from 60 healthy adult volunteers was used as controls. Informed consent was obtained from the patients and/or their parents and from the healthy volunteers. We previously showed that for U2AF35, SRSF2 and SF3B1, most of the mutations in adult cases were observed in exons 2 and 7, exon 1, and exons 14 and 15, respectively.10 Therefore, we confirmed mutation screening to these ‘hot-spot' exons. In contrast, all the coding exons were examined for ZRSR2, because no mutational hot spots have been detected. Briefly, the relevant exons were amplified using PCR and mutations were examined by Sanger sequencing, as previously described.10 The Fisher's exact test was used to evaluate the statistical significance of frequencies of mutations for U2AF35, SF3B1, ZRSR2 or SRSF2 in adult cases and pediatric cases. This study was approved by the Ethics Committee of the University of Tokyo (Approval number 948-7). No mutations were identified in the 28 cases with pediatric MDS, which included 13 cases with refractory anemia with excess blasts, 5 with refractory cytopenia of childhood, 2 with Down syndrome-related MDS, 2 with Fanconi anemia-related MDS, 2 with secondary MDS and 4 with unclassified MDS. Similarly, no mutations were detected in 93 cases with de novo AML or in 17 with CML, as well as 19 leukemia-derived cell lines. Our previous study in adult patients showed the frequency of mutations in U2AF35, SF3B1, ZRSR2 or SRSF2 to be 60/155 cases with MDS without increased ring sideroblasts and 8/151 de novo AML patients, emphasizing the rarity of these mutations in pediatric MDS (P