Profile of genetic mutations and aberrant methylation.

<p>(A) Mutation status of RAS pathway genes and secondary genes (<i>SETBP1</i> and <i>JAK3</i>) identified as gene targets. Aberrant methylation scores (AMS) in a cohort of 92 patients with juvenile myelomonocytic leukemia are summarized. A rhombus denotes a patient with Noonan syndrome-associated myeloproliferative disorder. (B) Mutations in <i>SETBP1</i> and <i>JAK3</i> were associated with a higher AMS. The mean AMS of patients with <i>SETBP1</i> and/or <i>JAK3</i> mutations was higher than that of patients without secondary mutations (p = 0.03).</p

Summary of DNA methylation in candidate genes.

<p>(A) The dot plot represents the frequencies of methylated CpG sites for each candidate gene in the 92 patients with juvenile myelomonocytic leukemia. Aberrant hypermethylation was defined as >3 standard deviations above the mean methylation level of the healthy control population. The threshold values of each gene are shown as red broken lines. (B) Kaplan–Meier plots of the patient groups, defined by aberrant methylation of the indicated genes, are shown for <i>BMP4</i>, <i>CALCA</i>, <i>CDKN2A</i>, <i>CDKN2B</i>, <i>H19</i>, and <i>RARB</i>.</p

Hypermethylation status and clinical outcome in patients with juvenile myelomonocytic leukemia (JMML).

<p>(A) Kaplan–Meier curves represent the probability of transplantation-free survival (TFS) in the 92 patients with JMML. TFS was defined as the probability of being alive and transplantation free. Both death and transplantation were considered events. The probability of 5-year TFS in the aberrant methylation score (AMS) 0 cohort (solid line) was significantly higher than that in the AMS 1–2 (long dashed line) and AMS 3–4 cohorts (dashed line), p < 0.001. (B) Kaplan–Meier curves represent the probability of overall survival (OS) in the 92 patients with JMML. Death was considered an event. The probability of OS in both the AMS 0 (solid line) and 1–2 cohorts (long dashed line) was significantly higher than that in the AMS 3–4 cohort (dashed line), p < 0.001.</p

Patient characteristics.

<p>Patient characteristics.</p

Multivariate model for transplantation-free and overall survival.

<p>Multivariate model for transplantation-free and overall survival.</p

Single Nucleotide Polymorphism Array Lesions, <em>TET2, DNMT3A</em>, <em>ASXL1</em> and <em>CBL</em> Mutations Are Present in Systemic Mastocytosis

<div><p>We hypothesized that analysis of single nucleotide polymorphism arrays (SNP-A) and new molecular defects may provide new insight in the pathogenesis of systemic mastocytosis (SM). SNP-A karyotyping was applied to identify recurrent areas of loss of heterozygosity and bidirectional sequencing was performed to evaluate the mutational status of <em>TET2, DNMT3A, ASXL1, EZH2, IDH1/IDH2</em> and the <em>CBL</em> gene family. Overall survival (OS) was analyzed using the Kaplan-Meier method. We studied a total of 26 patients with SM. In 67% of SM patients, SNP-A karyotyping showed new chromosomal abnormalities including uniparental disomy of 4q and 2p spanning <em>TET2</em>/<em>KIT</em> and <em>DNMT3A.</em> Mutations in <em>TET2, DNMT3A, ASXL1</em> and <em>CBL</em> were found in 23%, 12%, 12%, and 4% of SM patients, respectively. No mutations were observed in <em>EZH2</em> and <em>IDH1/IDH2</em>. Significant differences in OS were observed for SM mutated patients grouped based on the presence of combined <em>TET2/DNMT3A/ASXL1</em> mutations independent of <em>KIT</em> (<em>P</em> = 0.04) and sole <em>TET2</em> mutations (<em>P</em><0.001). In conclusion, <em>TET2, DNMT3A</em> and <em>ASXL1</em> mutations are also present in mastocytosis and these mutations may affect prognosis, as demonstrated by worse OS in mutated patients.</p> </div

Kaplan-Meier survival curves estimated according to presence of specific mutations or accumulation of several mutations in patients with systemic mastocytosis.

<p>Differences in OS for SM patients are shown (A-B). For each group number of analyzed cases and <i>P</i> value are presented, respectively.</p

Characteristics of patients carrying <i>TET2</i>, <i>DNMT3A</i>, <i>ASXL1</i> and <i>CBL</i> family mutation.

<p>WHO, World Health Organization; SM, systemic mastocytosis; ISM, indolent SM; SM-AHNMD, SM with associated non-mast cell lineage disease; ASM, aggressive SM; MSC, mast cell sarcoma; M, male; F, female; dx, diagnosis; Age, y, years; Neg., negative; Pos., positive.</p>*<p>UPD2pterp13.1 (homozygous mutation).</p>†<p>UPD4q12qter (homozygous mutation).</p>‡<p>Germ-line confirmation.</p>§<p>Mutations reported at <a href="http://www.sanger.ac.uk" target="_blank">http://www.sanger.ac.uk</a>.</p>II<p>Associated hematological non mast cell disease: chronic myelomonocytic leukemia.</p>¶<p>Associated hematological non mast cell disease: chronic myelomonocytic leukemia and chronic lymphocytic leukemia.</p

Single nucleotide polymorphism array-based karyotyping (SNP-A) of mastocytosis patients.

<p>(A) Overview of all genetic aberrations found by SNP-A analysis in patients with systemic mastocytosis. Green represents gain, red represents loss, black represents somatic uniparental disomy (UPD). UPD involving the <i>KIT</i> and <i>TET2</i> genes on chromosome 4q and UPD involving the <i>DNMT3A</i> gene on chromosome 2p were noted in one patient each, as indicated. (B) Representative SNP-A analysis of loss of heterozygosity (LOH), UPD, and gain by Genotyping Console v3.0. The top track of each panel shows LOH. The second track shows raw copy number for each SNP along the chromosome, while the third track shows allele calls (AA, AB, BB). Each region of genomic change is indicated by vertical black bars.</p

Localization of mutations identified in systemic mastocytosis.

<p>In a cohort of 26 patients with systemic mastocytosis, 14 mutations were identified. Genomic sequencing of protein-coding regions and splice sites revealed missense (black), nonsense (orange), and frameshift mutations (blue) in <i>TET2</i>, <i>DNMT3A</i>, <i>ASXL1,</i> and <i>CBL</i>. Most mutations were found in conserved domains and specific known conserved motifs and domains are shown for each protein: cysteine-rich region (C-rich-), double strand â helix (DSBH), PWWP domain (characterized by the presence of a highly conserved proline–tryptophan–tryptophan–proline motif), ADD (<i>ATRX, DNMT3,</i> and <i>DNMT3L</i>)-type zinc finger (ZNF) domain, methyltransferase (MTase) domain, amino-terminal ASX homology (ASXN) region, ASXM domain, nuclear receptor coregulator binding (NR box) motifs, carboxyterminal plant homeodomain (PHD) domain, tyrosine kinase binding (TKB) domain, linker sequence (L), RF domain (RF), proline-rich region (PPP), and leucine zipper LZ/ubiquitin-associated domain (UBA). Two changes occurred in a homozygous state, as indicated by the symbol # and the others in heterozygous state.</p