Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematologic disorders, with distinct subtypes defined by cytogenetics, the number of affected lineages, severity of cytopenia, cellular dysplastic morphology, and blast counts.1 Extensive next-generation sequencing has recently been performed in adult MDS; these studies revealed that mutations most frequently occurred in genes involved in RNA splicing, the cohesin complex, chromatin modification, DNA methylation, transcriptional regulation, and signal transduction.102 In contrast, relatively little is known about recurrently affected genes and pathways in childhood MDS and their contribution to disease pathogenesis.1211
We performed for the first time within this disease group a systematic investigation of the importance of mutations and recurrently affected pathways found in a variety of hematologic diseases using deep sequencing, and compared our findings with published data from adult MDS patients. DNA samples from 24 primary and 14 secondary pediatric MDS cases were analyzed. Information on patients’ characteristics is provided in Online Supplementary Table S1. Institutional review board approval for these studies was obtained in the participating centers. Thirty-nine samples were sequenced using the TruSight Myeloid Sequencing Panel (Illumina, San Diego, CA, USA) on the MiSeq platform and prepared according to the TruSight DNA Amplicon Sequencing Panel guide (Illumina). Average gene coverage was 2805. The MDS sample from patient 5 was analyzed using whole exome sequencing (WES), as previously described,13 with an average gene coverage of 78. Further details on the patient cohort, on the bioinformatic analyses, and candidate mutation selection are provided in Online Supplementary Table S1 and Figure S1.
In total, we found 28 mutations in 18 genes (Figure 1). Details on the mutations, frequency, the variant coverage, and variant allele frequencies (VAFs) in healthy individuals as assessed by population-based sequencing efforts, as well as predicted effects of the alterations for each patient are shown in Online Supplementary Table S2. TP53, BCOR and RUNX1 mutations were present in 3 patients. ASXL1, GATA2, PTPN11 were mutated twice and mutations in WT1, DNMT3A, CUX1, STAG2, IKZF1, CSF3R, PHF6, ATRX, CBL, EZH2, IDH2, CDKN2A were found in single patients only. Twenty-one patients (55%) had none of these mutations, but 13 of them had a cytogenetic abnormality (Figure 1 and Online Supplementary Table S1), most commonly monosomy 7. Overall, at least one genetic or cytogenetic aberration was present in 30 of the 38 (79%) patients. This percentage is identical to that previously reported for adult MDS.7 There was no difference in frequencies or type of mutations between primary and secondary MDS samples. Unfortunately, no material of the primary diseases was available in the secondary MDS cases, thus, the possibility that these MDS cases are minimal residual diseases (MRDs) of the primary malignancies [e.g. of the acute myeloid leukemia (AML) patients] cannot be excluded. This represents an interesting question to be investigated in independent studies.
A previous report by Hirabayashi et al.11 suggests a lack of mutations in the splice factor-encoding genes in pediatric MDS, as evidenced by Sanger sequencing of mutational hotspots in SF3B1, U2AF35 and SRSF2. This finding contrasts strongly with results in adult MDS patients, in which mutations in genes involved in RNA splicing are the most common abnormality (Table 1), occuring as clonal mutations and early in disease evolution.974 Because the resolution of conventional Sanger sequencing is low, and given the previously reported impact of subclonal mutations in adult MDS on patient survival,147 we assessed whether subclonal aberrations in the splice factor-encoding genes SF3B1, SRSF2, ZRSR2 and U2AF1 could be detected in our cohort. None of the pediatric MDS cases in our study had clonal or subclonal mutations in these genes, corroborating the findings by Hirabayashi et al. Another mechanism that is recurrently affected in adult MDS is the formation of the cohesin complex (Table 1).10 We found only one mutation in STAG2 in our pediatric MDS cohort (Figure 1), while other genes of this complex, such as SMC1A, SMC3 and RAD21, were not mutated. On the other hand, we identified both clonal and subclonal mutations in genes involved in chromatin modification, DNA methylation, signaling and transcription (Figure 1 and Online Supplementary Table S2), in frequencies comparable to those reported for adult MDS (Table 1).7
In summary, our study shows that approximately 45% of pediatric MDS patients carry at least one mutation, primarily occurring in genes associated with chromatin modification, DNA methylation and transcription, but rarely in genes involved in RNA splicing and function of the cohesin complex. Because the latter mechanisms are most commonly affected in adult MDS, these data point towards a clear distinction between the pathogenesis of pediatric and adult patients, which may have implications for future therapy approaches in the different age groups.
References
- Niemeyer CM, Baumann I. Myelodysplastic syndrome in children and adolescents. Semin Hematol. 2008; 45(1):60-70. PubMedhttps://doi.org/10.1053/j.seminhematol.2007.10.006Google Scholar
- Boultwood J, Perry J, Pellagatti A. Frequent mutation of the polycomb-associated gene ASXL1 in the myelodysplastic syndromes and in acute myeloid leukemia. Leukemia. 2010; 24(5):1062-1065. PubMedhttps://doi.org/10.1038/leu.2010.20Google Scholar
- Grand FH, Hidalgo-Curtis CE, Ernst T. Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms. Blood. 2009; 113(24):6182-6192. PubMedhttps://doi.org/10.1182/blood-2008-12-194548Google Scholar
- Graubert TA, Shen D, Ding L. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 2012; 44(1):53-57. PubMedhttps://doi.org/10.1038/ng.1031Google Scholar
- Langemeijer SM, Kuiper RP, Berends M. Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat Genet. 2009; 41(7):838-842. PubMedhttps://doi.org/10.1038/ng.391Google Scholar
- Levine RL, Loriaux M, Huntly BJ. The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood. 2005; 106(10):3377-3379. PubMedhttps://doi.org/10.1182/blood-2005-05-1898Google Scholar
- Papaemmanuil E, Gerstung M, Malcovati L. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013; 122(22):3616-3627. PubMedhttps://doi.org/10.1182/blood-2013-08-518886Google Scholar
- Walter MJ, Ding L, Shen D. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia. 2011; 25(7):1153-1158. PubMedhttps://doi.org/10.1038/leu.2011.44Google Scholar
- Yoshida K, Sanada M, Shiraishi Y. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011; 478(7367):64-69. PubMedhttps://doi.org/10.1038/nature10496Google Scholar
- Kon A, Shih LY, Minamino M. Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms. Nat Genet. 2013; 45(10):1232-1237. PubMedhttps://doi.org/10.1038/ng.2731Google Scholar
- Hirabayashi S, Flotho C, Moetter J. Spliceosomal gene aberrations are rare, coexist with oncogenic mutations, and are unlikely to exert a driver effect in childhood MDS and JMML. Blood. 2012; 119(11):e96-99. PubMedhttps://doi.org/10.1182/blood-2011-12-395087Google Scholar
- Wlodarski MW, Hirabayashi S, Pastor V. Prevalence, clinical characteristics, and prognosis of GATA2-related myelodysplastic syndromes in children and adolescents. Blood. 2016; 127(11):1387-1397. PubMedhttps://doi.org/10.1182/blood-2015-09-669937Google Scholar
- Beekman R, Valkhof MG, Sanders MA. Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia. Blood. 2012; 119(22):5071-5077. PubMedhttps://doi.org/10.1182/blood-2012-01-406116Google Scholar
- Murphy DM, Bejar R, Stevenson K. NRAS mutations with low allele burden have independent prognostic significance for patients with lower risk myelodysplastic syndromes. Leukemia. 2013; 27(10):2077-2081. PubMedhttps://doi.org/10.1038/leu.2013.160Google Scholar