Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including IntroductionChildren with Down syndrome (DS), characterized by constitutional trisomy 21, have a 50-fold increased risk of developing acute leukemia in the first few years of life. 1,2 This comprises the normally extremely rare subtype acute megakaryoblastic leukemia (AMKL) as well as the common variety (B-cell precursor) of acute lymphoblastic leukemia (ALL). 2-4 Both DS-AMKL and the transient myeloproliferative disorder (TMD) that often precedes it are consistently associated with acquired mutations in the GATA1 gene. 5 Concordant GATA1 mutations in the blast cells of identical twins with TMD 6 and mutations in the neonatal blood spots of DS newborns 7 indicate that this is an early/prenatal event in DS leukemogenesis.Much less is known about the genetic events predisposing to DS-ALL. Candidate genes for activating mutations in DS-ALL include FLT3 and RAS, both mutated in high hyperdiploid ALL (with acquired trisomy 21), 8,9 PTPN11 and BRAF, mutated in B-cell precursor ALL. 10,11 An additional candidate is the JAK2 pseudokinase domain mutation JAK2⌬IREED, reported in a single case of DS-ALL. 12 A different JAK2 pseudokinase domain mutation, JAK2V617F, is frequently found in the myeloproliferative disorders (MPDs) and believed to be an initiating event. [13][14][15] Submicroscopic deletions that involve genes linked functionally to deregulation of cell cycling or B-cell differentiation were also implicated in the molecular pathogenesis of ALL. [16][17][18][19] In this study we undertook both sequencing of candidate genes and high-resolution singlenucleotide polymorphism (SNP) array analysis to identify genetic events associated with ALL in DS. Methods PatientsPatients' samples (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article) were collected, informed consent was obtained in accordance with the Declaration of Helsinki, and the study was carried out with approval of the ethical review committee from all participating institutions. DNA was extracted from archival (frozen cell or cytogenetic-fixed pellet) leukemic samples. SequencingPrimers were designed to amplify the following candidate genes: FLT3 ITD, FLT3 kinase domain, KIT kinase domain, PTPN11, BRAF, NRAS, and KRAS (Table S2A). Exons 12 to 23 of the JAK2 gene were polymerase chain reaction (PCR)-amplified and sequenced using previously described primers. 13 All samples found to be mutated were PCR-amplified and sequenced in a second, independent experiment. Pyrosequencing was carried out in accordance to the manufacturer's instructions (Biotage AB, Uppsala, Sweden) using the primers shown in Table S2B. Ba/F3 prolifer...

IntroductionThe t(12;21)(p13;q22) is one of the most common chromosome translocations in childhood acute lymphoblastic leukemia (ALL). [1][2][3] This translocation is associated with rearrangement of the TEL (ETV6) gene on chromosome 12 and the AML1(CBFA2) gene on chromosome 21 resulting in TEL-AML1 fusion. Although many studies on the TEL-AML1 fusion gene focused on the biologic significance of leukemogenesis, little is known about the mechanisms involved in the cleavage of TEL and AML1 and subsequent fusion of the TEL-AML1 gene. Chemotherapy-related secondary leukemia is frequently associated with rearrangement of the MLL(ALL1) gene. Several clinicoepidemiologic studies have indicated that topoisomerase-II (topo-II) inhibitors cause rearrangement of the MLL gene. 4 These findings are supported by in vitro studies that have demonstrated that MLL rearrangement could be induced by topo-II inhibitors in human hematopoietic cells. 5 Site-specific cleavage within the MLL gene can also be induced by nongenotoxic stimuli involved in apoptotic cell death, suggesting that this cleavage is not specific to topo-II inhibitors but is rather part of a chromatin fragmentation similar to the generalized cellular response to apoptotic stimuli. 6 Based on these early findings, we speculated that general apoptotic stimuli on hematopoietic cells could play a key role in chromosomal translocation involving not only MLL but also TEL, another gene frequently altered in hematopoietic malignancies. To investigate whether apoptotic stimuli could trigger a cascade of events that ultimately leads to the formation of the TEL-AML1 fusion gene, we studied the in vitro conditions inducing the formation of DNA double-strand break (DSB) ends within TEL and AML1 genes. Our study indicates that DSBs of TEL and AML1 can be induced by apoptotic stimuli and this DNA cleavage appears to be the first step in the generation of TEL-AML1 fusion in immature B lymphocytes, which is considered to be an early or initiating event in childhood ALL. 7,8 Materials and methods Cell linesRS4;11 is an ALL cell line and has MLL-AF4 fusion gene as result of t(4;11)(q21;q23). 9 The TS-2 cell line was established from childhood pre-B ALL 10 and carries t(1;19)(q23;p13) without the E2A gene rearrangement. REH, a t(12;21)-positive ALL cell line, 11 was used as a positive control for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for TEL-AML1 fusion transcript. Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines (EB-LCLs) were established from peripheral blood lymphocytes infected with the EBV strain B95-8, as described previously. 12 Cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 in air. Peripheral blood and cord blood samplesPeripheral blood samples of nonleukemic individuals were obtained from outpatients consulting several hospitals during December 1997 to April 1998 with informed consent. These individuals had no history of hematopoietic malignancy or administration of an...