Childhood myelodysplastic syndromes (MDS) are a rare group of disorders characterized by clonal defects of stem and progenitor cells, resulting in ineffective hematopoiesis, which manifests throughout childhood and adolescence.1. In contrast to adult MDS, the knowledge of genetic alterations in the pediatric population is elementary. Recently, our group identified germline GATA2 mutations accounting for 15% of advanced primary MDS (MDS with excess of blast, MDS-EB) and one third of cases with monosomy 7,2 the most frequent cytogenetic aberration in childhood MDS.3. However, with the exception of our previous finding implicating the lack of spliceosomal gene aberrations,4 there is no systematic data elucidating the mutational landscape in primary childhood MDS. Next-generation sequencing (NGS) studies in adult MDS and myeloproliferative neoplasms (MPN) indicate a manageable number of recurrently mutated genes.5, 6, 7, 8, 9 We utilized these results for a focused mutational discovery in childhood MDS. We studied a cohort of 50 children and adolescents (28 males and 22 females) diagnosed with primary MDS10 at a median age of 9.0 (1.1-17.4) years. On the basis of paucity of gene mutations in low-risk, in contrast to high-risk MDS in adults,5, 6, 7 we purposely biased our study cohort towards more advanced disease in order to increase the odds of identifying mutations. The study cohort was enriched for cases with monosomy 7 (-7) (48%) and MDS-EB (38%, Table 1). Germline predisposition, namely GATA2 and RUNX1 deficiency, had previously been established in one-third of our cohort (18 cases, Table 1). Addnl. familial MDS with unresolved genetic cause had been documented in five patients of four pedigrees. Patients with known inherited bone marrow failure syndromes had been excluded from this study. We used DNA from purified bone marrow granulocytes collected at diagnosis to perform capture-based next-generation sequencing (NGS) of 104 genes associated with myeloid neoplasia (Supplementary Table 1). In addition, we employed a sensitive allele-specific PCR to screen for SETBP1 mutations reported in myeloid malignancies (Supplementary Figure 1). All samples were run in duplicates on HiSeq2000 sequencer (Illumina, San Diego, CA, USA) as previously described.9 Genetic variants with an allelic frequency (VAF)≥5% detected in both independent runs were evaluated further and confirmed using Sanger sequencing. The known polymorphisms present at >1% frequency in population databases were not considered in this study. Anal. of germline status was performed in specimens of non-myeloid origin (Supplementary Figure 2). Further details on the aforementioned methods are provided in Supplementary Methods. Overall, 72% (36/50) of patients carried 64 mutations in 25 of 105 analyzed genes (Figure 1a). Germline validation confirmed 24 somatic mutations in 17 patients (34%), and 35 germline variants in 25 patients (50%), while for 4 instances (8%) the status of 4 missense substitutions could not be resolved (Supplementary Tables 2-5). Somatic mutations were significantly overrepresented in MDS-EB vs RCC (68 vs 13%, P<0.001) and in patients with -7 vs other karyotypes (56 vs 18%, P<0.001, Table 1). Addnl., we studied the impact of marrow fibrosis found in 42% of the patient cohort. Surprisingly, the presence of fibrosis was not associated with higher mutational ratios (Table 1). Mutations frequently encountered in adult MDS and in age-related clonal hematopoiesis such as TET2, DNMT3A, TP53 and the spliceosome complex were not detected. In detail, we found recurrent somatic mutations in SETBP1 (18%, 9/50), ASXL1 (8%, 4/50), RUNX1 (6%, 3/50), PTPN11 (6%, 3/50) and NRAS (4%, 2/50), while KRAS, EZH2, NF1 and NPM1 were mutated in only one patient each (Figure 1a). The RAS pathway genes PTPN11, NRAS and KRAS were altogether altered in 12% of cases. Notably, the top three mutated genes found in this study, i.e., SETBP1, ASXL1 and RUNX1 emerged only in the context of -7 karyotype, and were affected in 36, 16, and 12% of -7 cases, resp. Conversely, PTPN11 and NRAS mutations were not unique to -7 but also associated with normal karyotype (Figure 1a). Next, we addressed the question whether GATA2 or RUNX1 deficiency is associated with specific somatic mutations. In germline GATA2-mutated (GATA2mut) patients, most common somatic hits were SETBP1, RUNX1 and ASXL1 (Supplementary Table 3, Figure 1a). This was likely related to the underlying -7, as within this cytogenetic category also GATA2-wild-type cases displayed a comparable mutational pattern (Table 1, Figure 1b). Of the three patients with RUNX1 deficiency, two had normal karyotypes and no addnl. alterations, while in one patient, -7 and somatic mutations in NRAS and KRAS were found. We did not test for somatic mutations in CDC25C gene, previously implicated in one study as a biomarker predicting leukemic progression in patients with germline RUNX1 mutations.11. This association, however, could not be reproduced in another cohort of 13 RUNX1-mutated patients.12. The recurrence of coexisting clonal events may well reveal a cooperation of various genetic mechanisms in the evolution of childhood MDS. The interconnection between recurrent somatic mutations (>1), karyotypes and germline GATA2 and RUNX1 predisposition is illustrated in Figure 1b. Concomitant somatic mutations observed in 35% of the mutated cohort mainly involved SETBP1 and ASXL1, and were associated with -7 (Figures 1a and b). A previous study described the association of SETBP1-ASXL1 mutations as a cooperative mechanism towards leukemic transformation.13. Of note, within the -7 subgroup in our study, only 33% of patients without increase in blasts (RCC) carried somatic mutations, while the mutational load increased to 77% in MDS-EB (P<0.05, not shown). It is currently unknown, to what extent germline mutations or rare variants predispose to MDS. Thus, we finally explored germline variants of unknown significance (gVUS) and found a total of 17 gVUS in 13 patients (Figure 1a). These gVUS affect highly conserved amino acids and are suggested to be pathogenic based on in silico scoring (Supplementary Table 4). Unexpectedly, 6 of 13 patients with germline gVUS also had a known disease-causing germline GATA2 mutation (46%). It is reasonable to speculate that these gVUS modify the phenotype of GATA2 deficiency, possibly 'facilitating' an earlier onset of MDS in a GATA2-deficient background. Similarly, Hind et al.14 postulated that certain germline variants might exert a predisposing effect in individuals with MPN. Of the remaining seven patients with gVUS, four cases of three pedigrees had familial MDS. Patient D493 and D494 were siblings and both carried a novel heterozygous missense substitution in SH2B3 (p.Pro507Arg). Mutations in SH2B3 were previously recounted in MPN, acute lymphoblastic leukemia and juvenile myelomonocytic leukemia (JMML) as both germline and somatic events.15. Two other index cases with familial MDS had gVUS in PTEN (p.Tyr188Cys) and IRF8 (p.Tyr242His). PTEN is a known cancer-predisposition gene associated with Cowden disease; furthermore, PTEN downregulation was reported in JMML and other myeloid disorders.16. IRF8 is a transcription factor involved in lineage commitment and in myeloid cell maturation, but to date, no association with MDS has been described. Another germline missense variant in PTPN11 (p.Glu97Gln) was detected in patient D225 who was diagnosed at 5 years of age with hypercellular RCC, having a normal karyotype and unremarkable family history. Missense PTPN11 mutations underlie 50% of Noonan syndrome and the vast majority affects the N-SH2 and PTP protein domains.17. The missense alteration p.Glu97Gln is located in the N-SH2 domain. However, the patient did not exhibit Noonan-like features, although he had mild developmental delay and a marginally enlarged spleen. The predisposing effect of the other gVUS in PIK3C3, WT1 and EPOR genes is uncertain and awaits further evaluation in larger patient cohorts. In this study, we assessed the mutational profiles of a selection of patients with primary childhood MDS. Altogether, our results indicate that genes known to be commonly mutated in adult MDS, such as TET2, DNMT3A, TP53 and the spliceosome complex, are not involved in disease pathogenesis in children. Instead, somatic driver mutations in SETBP1, ASXL1, RUNX1 and the RAS oncogenes define the genomic landscape of the pediatric counterpart. This reinforces the notion that primary pediatric MDS, as an early onset disease, requires direct leukemogenic hits as opposed to mutations with delayed oncogenic potential like those encountered in adult patients. Due to the design of our study, the patient cohort was biased towards monosomy 7 and MDS-EB, whereas normal karyotypes and less advanced disease were underrepresented. This, however, certainly does not weaken our finding, exposing the association between -7 karyotype and a high mutational burden that even continues to increase with higher blast counts. We also show that various somatic mutations are found in the -7 background. These findings lead to the assumption that monosomy 7 arises as an ancestral clone, which then rapidly acquires driver mutations, resulting in disease progress. Future studies characterizing the clonal architecture of pediatric MDS at single-cell level are required to address this hypothesis. Finally, the variety of known and novel germline mutations portrayed in our cohort points to the significant role of genetic predisposition as a hallmark of pediatric MDS. Collectively, our results help define the mutational landscape in MDS in children and adolescents.