scholarly journals Postnatally Acquired Mutations Underlie the Progression of Transient Leukemia to Myeloid Leukemia of Down Syndrome

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 442-442
Author(s):  
Jian Chen ◽  
Yue Li ◽  
Fouad Yousif ◽  
Sagi Abelson ◽  
Sanaz Manteghi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Gene Mutations ◽  
Cohesin Complex ◽  
Loss Of Function ◽  
Digital Droplet Pcr ◽  
Whole Exome ◽  
Transient Leukemia ◽  
Droplet Pcr ◽  
A Minor

Abstract INTRODUCTION . Transient Leukemia (TL; also termed Transient Myeloproliferative disorder, TMD, and Transient Abnormal Myelopoiesis, TAM) occurs in 10-30% of newborns with Down syndrome (DS). Approx. 20% of infants with TL go on to develop acute myeloid leukemia of DS (ML-DS), typically within the first four years of life. Somatic, clone-specific mutations of GATA1 are found both in the blasts of TL and ML-DS, are concordant within the same individual and thought to function as initiating event in the development of ML-DS. In contrast, additional mutations of cohesin complex and related genes (e.g. RAD21, STAG2, CTCF), epigenetic regulators (e. g. EZH2) and signal transducers (e.g. within RAS, JAK signaling pathways) have been identified only in ML-DS blasts and are thought to cooperate with mutant GATA1 in the progression from TL to ML-DS. It is not known whether these cooperating mutations already mark a minor subclone of TL blasts at birth - allowing, at least in principle, a genetic risk stratification of TL - or are acquired postnatally during the first four years of life. OBJECTIVES . We tested the functional impact of impaired function of cohesin complex genes, CTCF and EZH2 on the progression of TL to ML-DS. We asked if mutations representing putative genetic progression events were already detectable at birth in a minor clone of TL blasts or were acquired postnatally (during the first four years of life). METHODS. The spectrum of GATA1 and cooperating mutations was determined by whole exome sequencing in fractions of TL and ML-DS blasts sorted from blood and bone marrow samples of five patients who had successively developed both disorders including one with a relapse of ML-DS. Corresponding normal T lymphocyte fractions of each patient at the stage of TL and ML-DS served as controls. Numbers of blasts harboring specific mutations were quantified by digital droplet PCR (BioRad, Inc.). Primary TL cells were transduced with lentivirus encoding shRNA (pLVX-shRNA, Clontech, Inc.) to suppress expression of cohesin complex genes, CTCF and EZH2 and intrafemurally injected into 8 week old NSG recipient mice. Engraftment in the bone marrow was assessed 8 weeks later by flow cytometry and GATA1 mutational analysis and compared to TL cells transduced with control vector. RESULTS. TL blasts harbored fewer mutations than those of ML-DS. GATA1 mutations were concordant in TL and ML-DS blasts in the same patient, consistent with development of ML-DS from subclone of TL. Knockdown of RAD21 expression in primary TL blasts, mimicking loss of function mutation of a cohesin complex gene, resulted in significantly increased engraftment of transduced cells in xenograft recipients compared to controls. This finding is consistent with RAD21 loss of function mutations playing the role of a progression event. Mutations of cohesin complex genes (SMC1A, STAG2, RAD21), NRAS and other putative cooperating mutations (with mutant GATA1) were not detectable in any sample of primary TL blasts by either whole exome sequencing or digital droplet PCR. The same result was obtained with control T lymphocytes sorted from TL samples. ML-DS blasts in one case were oligo-clonal with regard to cohesin complex gene mutations. Relapse in this patient arose from a minor clone as defined by cohesin complex gene mutations; mutations of NRAS, KNASL1 and SMC1A were present in ML-DS blasts but absent at relapse. CONCLUSIONS . Increased engraftment of TL cells with suppressed RAD21 expression is consistent with a model in which RAD21 loss of function mutations function as a progression event in the development of ML-DS. Absence of detectable cohesin complex gene mutations and other putative cooperating events in TL blasts suggests these mutations are acquired during the first four years of life and do not mark a minor clone of TL blasts present at birth. Genomic screening of TL blasts at birth therefore is unlikely to predict the risk for development of ML-DS. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Genetic Control of Stoichiometry Underlying Autism

2020 ◽  
Vol 43 (1) ◽  
pp. 509-533 ◽  
Author(s):  
Robert B. Darnell
Keyword(s):  
Large Scale ◽  
Biochemical Analysis ◽  
Critical Role ◽  
Gene Mutations ◽  
Loss Of Function ◽  
Case Control Studies ◽  
Neurologic Disorder ◽  
Regulatory Variants ◽  
Whole Exome ◽  
Number Variation

Autism is a common and complex neurologic disorder whose scientific underpinnings have begun to be established in the past decade. The essence of this breakthrough has been a focus on families, where genetic analyses are strongest, versus large-scale, case-control studies. Autism genetics has progressed in parallel with technology, from analyses of copy number variation to whole-exome sequencing (WES) and whole-genome sequencing (WGS). Gene mutations causing complete loss of function account for perhaps one-third of cases, largely detected through WES. This limitation has increased interest in understanding the regulatory variants of genes that contribute in more subtle ways to the disorder. Strategies combining biochemical analysis of gene regulation, WGS analysis of the noncoding genome, and machine learning have begun to succeed. The emerging picture is that careful control of the amounts of transcription, mRNA, and proteins made by key brain genes—stoichiometry—plays a critical role in defining the clinical features of autism.

scholarly journals EZH2 in Myeloid Malignancies

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1639 ◽  
Author(s):  
Jenny Rinke ◽  
Andrew Chase ◽  
Nicholas C. P. Cross ◽  
Andreas Hochhaus ◽  
Thomas Ernst
Keyword(s):  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Myeloproliferative Neoplasms ◽  
Gene Mutations ◽  
Loss Of Function ◽  
Disease Pathogenesis ◽  
Catalytic Core ◽  
Myeloid Malignancies ◽  
Treatment Free Remission

Our understanding of the significance of epigenetic dysregulation in the pathogenesis of myeloid malignancies has greatly advanced in the past decade. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic core component of the Polycomb Repressive Complex 2 (PRC2), which is responsible for gene silencing through trimethylation of H3K27. EZH2 dysregulation is highly tumorigenic and has been observed in various cancers, with EZH2 acting as an oncogene or a tumor-suppressor depending on cellular context. While loss-of-function mutations of EZH2 frequently affect patients with myelodysplastic/myeloproliferative neoplasms, myelodysplastic syndrome and myelofibrosis, cases of chronic myeloid leukemia (CML) seem to be largely characterized by EZH2 overexpression. A variety of other factors frequently aberrant in myeloid leukemia can affect PRC2 function and disease pathogenesis, including Additional Sex Combs Like 1 (ASXL1) and splicing gene mutations. As the genetic background of myeloid malignancies is largely heterogeneous, it is not surprising that EZH2 mutations act in conjunction with other aberrations. Since EZH2 mutations are considered to be early events in disease pathogenesis, they are of therapeutic interest to researchers, though targeting of EZH2 loss-of-function does present unique challenges. Preliminary research indicates that combined tyrosine kinase inhibitor (TKI) and EZH2 inhibitor therapy may provide a strategy to eliminate the residual disease burden in CML to allow patients to remain in treatment-free remission.

scholarly journals Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status

2020 ◽  
Vol 4 (5) ◽  
pp. 845-854 ◽  
Author(s):  
Raksha Shrestha ◽  
Mamiko Sakata-Yanagimoto ◽  
Koichiro Maie ◽  
Motohiko Oshima ◽  
Masatomo Ishihara ◽  
...  
Keyword(s):  
Median Survival ◽  
Myeloid Leukemia ◽  
Recurrent Events ◽  
Loss Of Function ◽  
Genetic Event ◽  
Hematopoietic Stem ◽  
Age Related ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
The Impact

Abstract Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.

Detection of Nucleophosmin Gene Mutations in Plasma from Patients with Acute Myeloid Leukemia: Clinical Significance and Implications.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2855-2855
Author(s):  
Wanlong Ma ◽  
Xi Zhang ◽  
Iman Jilani ◽  
Farhad Ravandi ◽  
Elihu Estey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Gene Mutations ◽  
Leukemic Cells ◽  
Striking Difference ◽  
Plasma Dna ◽  
Npm1 Mutation ◽  
Acute Myeloid

Abstract Nucleotides insertion in the nucleophosphamin (NPM1) gene has been reported in about one third of patients with acute myeloid leukemia (AML). Multiple studies showed that the presence of NPM1 mutations associated with better outcome in patients with AML. Studies reported to date have analyzed leukemic cells obtained from bone marrow or peripheral blood. We tested for mutations in the NPM1 gene using peripheral blood plasma and compared results with clinical outcome from a single institution. Analyzing plasma from 98 newly diagnosed patient with AML showed NPM1 mutation in 24 (23%) of patient while only one (4%) of 28 previously untreated patients with myelodysplastic syndrome (MDS) showed NPM1 mutation. Compared with peripheral blood cells, 2 (8%) of the 24 positive patients were negative by cells; none were positive by cells and negative by plasma. Most of the mutations detected (45%) were in patients with FAB classification M2, M4 and M5. In addition to the reported 4 bp insertion, we also detected 4 bp deletion in one patient in cells and plasma. Patients with NPM1 mutation had a significantly higher white blood cell count (P = 0.0009) and a higher blast count in peripheral blood (P = 0.002) and in bone marrow (P = 0.002). Blasts in patients with NPM1 mutant expressed lower levels of HLA-DR (P = 0.005), CD13 (P = 0.02) and CD34 (P < 0.0001), but higher CD33 levels (P = 0.0004). Patients with NPM1 mutation appear to have better chance of responding to standard therapy (P = 0.06). Event free survival of patients with NPM1 mutation was longer (P = 0.056) than in patients with intermediate cytogenetic abnormalities. The most striking difference in survival was in patients who required >35 days to respond to therapy (Figure). Survival was significantly longer in patients with NPM1 mutation requiring >35 days to respond (P = 0.027). This data not only support that NPM1 plays a significant role in the biology and clinical behavior of AML, but also show that plasma DNA is enriched with leukemia-specific DNA and is a reliable source for testing. Figure Figure

DNA-Based Methods for Measurable Residual Disease Detection in NPM1-Mutated Acute Myeloid Leukemia; Establishment of Cut-Offs for qPCR, Digital Droplet PCR and Targeted Deep Sequencing

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-6
Author(s):  
Louise Pettersson ◽  
Sofie Johansson Alm ◽  
Alvar Almstedt ◽  
Vladimir Lazarevic ◽  
Gustav Orrsjö ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Gold Standard ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Npm1 Mutation ◽  
Digital Droplet Pcr ◽  
Droplet Pcr ◽  
Targeted Deep Sequencing ◽  
Measurable Residual Disease

For detection of measurable residual disease (MRD) in acute myeloid leukemia with NPM1 mutations, RT-qPCR with quantification of leukemic transcripts is currently considered the method of choice; however, MRD can also be determined with DNA-based methods, offering certain advantages. For example, digital droplet PCR (ddPCR) and targeted deep sequencing (deep seq) do neither require standard curves nor reference genes and are thus less labor intense than RT-qPCR. Also, deep seq allows for quantification independently of type of NPM1 mutation. In addition, DNA-based techniques enable MRD assessment of other mutations, beyond the reach of RT-qPCR, which is limited to analyses of highly expressed genes or fusion transcripts (e.g. core binding factor leukemias). With the rapid development of highly sensitive DNA-based techniques for MRD detection, there is a need to establish clinically relevant cut-offs for accurate interpretation of MRD results and risk stratification. Here, we compare and provide MRD cut-offs for three different DNA-based MRD methods for NPM1 mutations: quantitative PCR (qPCR), ddPCR and deep seq. To compare the DNA-based methods with RT-qPCR, we analyzed 110 follow-up peripheral blood (PB) or bone marrow (BM) samples from 32 AML patients harboring NPM1 mutation type A. First, we compared the mere detectability of leukemic signals (without reference to specific MRD cut-off points). We found a high correlation between results from RT-qPCR and the three DNA-based methods (Rs=0.936 for RT-qPCR vs qPCR, Rs=0.774 for RT-qPCR vs ddPCR and Rs=0.743 for RT-qPCR vs deep seq, p&lt;0.001). As expected, RT-qPCR was the most sensitive method. Among the DNA-based methods, qPCR was the most sensitive, detecting leukemic DNA in 95% (55/58) of the RT-qPCR positive samples, compared to 72% (42/58) and 62% (36/58) for ddPCR and deep seq, respectively. Interestingly, the transcript level for a given amount of measurable leukemic DNA (RNA copy number per leukemic DNA molecule) fluctuated considerably between different follow-up samples for certain patients. In some cases, the RNA/DNA ratio exceeded a hundredfold difference between different follow-up time points in both PB and BM. Hence, transcript analysis may be more complex than just a simple measurement of leukemic cell burden, which in turn may influence accurate risk stratification and treatment decisions, if relying on RT-qPCR measurements alone. To select adequate DNA MRD cut-offs, we performed ROC curve analyses for each method at various DNA cut-offs, comparing them with the gold standard RT-qPCR cut-off. In BM, this cut-off can be defined as a less than 3 log reduction of mutated NPM1 transcripts vs diagnosis, separating MRDhigh from MRDlow/undetectable (sometimes inaccurately termed "MRD-positivity" and "MRD-negativity", respectively). In PB, the mere detectability of mutated NPM1 transcripts is considered the relevant cut-off. DNA cut-offs were chosen based on the area under the curve (AUC) for the ROC analyses (Table 1), and influenced by available literature including recommendations of prognostically relevant MRD levels. For qPCR, a cut-off at 0.1% leukemic DNA was judged relevant in BM. For ddPCR and deep seq, 0.05% was chosen to adjust for measuring allelic ratio (variant allele frequency (VAF)) rather than mutant DNA alone. In PB, the selected cut-off was detectable leukemic signal irrespective of DNA method. We next determined the accuracy of the selected cut-offs, for identification of samples with clinically relevant MRD, by comparing them with the gold standard RT-qPCR. In general, the selected DNA cut-off values generated high specificity as well as high positive and negative predictive values (Table 1). The vast majority of all MRD analyses (93% (368/395)) showed concordant results irrespective of MRD method. In BM samples, MRD assessment by the DNA based methods agreed with MRD status as determined by RT-qPCR (MRDhigh high vs MRDlow/undetectable) in 93% (62/67) of the analyses for qPCR, 96% (64/67) for ddPCR, and 97% (65/67) for deep seq. In PB, the agreement was 95% (41/43), 88% (38/43) and 86% (37/43), respectively. In summary, we found strong agreement between different MRD methods and based on this could provide clinically relevant cut-offs for risk stratification. Thus, in BM follow-up samples from AML patients with NPM1 mutation, we propose 0.1% leukemic DNA as cut-off for qPCR and 0.05% VAF for ddPCR and deep seq. Disclosures No relevant conflicts of interest to declare.

TET2, ANPM1 and FLT3-ITD Gene Mutations in Normal Karyotype Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1413-1413
Author(s):  
Aining Sun ◽  
Xiaopeng Tian ◽  
Jia Yin ◽  
Weiyang Li ◽  
Suning Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Gene Mutations ◽  
Normal Karyotype ◽  
Genetic Mutation ◽  
Wild Type ◽  
Cell Percentage ◽  
High Bone ◽  
Acute Myeloid

Abstract Abstract 1413 Objective: Analyze the molecular genetics characteristics of acute myeloid leukemia with normal karyotype and explore the relationship between different genetic mutation patterns and prognosis. Methods: A total of 373 acute myeloid leukemia (AML) with normal karyotype diagnosed and treated in the First Affiliated Hospital of Soochow University from 2005 to 2010 were recruited in this research to assess the genetic mutation patterns. The target genes which was extracted from bone marrow cell were amplified by PCR and analyzed by massively DNA sequencing. All of the TET2, DNMT3A, IDH1, IDH2, EZH2, CBL, ASXL1, MLL-PTD, NPM1, WT1, RUNX1, c-KIT, FLT3-ITD, FLT3-TKD, N-RAS and JAK2V617F gene mutations were detected in our study. Results: (1). A total of 16.1% of patients had TET2 mutations, 31.6% had FLT3 internal tandem duplications (ITDs), 6.2% had FLT3 tyrosine kinase domain mutations, 2.4% had c-KIT mutations, 37.8% had NPM1 mutations, 11.3% had WT1 mutations, 5.9% had RUNX1 mutations, 11.5% had ASXL1 mutations, 3.8% had MLL partial tandem duplications (PTDs), 7.8% had IDH1 mutations, 7.8% had NRAS mutations, 12.3% had IDH2 mutations, 1.6% had EZH2 mutations, 14.7% had DNMT3A mutations and no mutations were found of CBL and JAK2V617F. In conclusion, there are 77% (287/373) gene mutations hide in normal karyotype AML patients.(2). We found that the TET2 gene mutations were associated with DNMT3A (P = 0.041) and RUNX1 (P <0.001) mutations, but mutually exclusive with IDH2 (P = 0.021), or IDH1/2 (P = 0.006) gene mutations. NPM1 gene mutations were highly correlated with DNMT3A mutations (P <0.0001), IDH1 mutations (P <0.0001) and IDH2 mutations (P = 0.001), but mutually exclusive with RUNX1 mutations (P=0.003). IDH2 mutations and WT1 mutations were mutually exclusive (P = 0.01); DNMT3A mutations were associated with NRAS mutations (P = 0.01). In addition, study have shown that the number of gene mutations was closely associated with older age, high white blood cell and high bone marrow blast cell percentage, but wasn't correlated with gender, hemoglobin and platelet levels.(3). In the NPM1m+ patients, TET2 mutations were associated with shorter median OS in contrast to TET2 wild type (9.9 vs. 27.0 months, P= 0.023). Surprisingly, in NPM1m+/FLT3-ITDm- group, TET2 mutations was also an unfavorable prognostic factor, which was closely associated with shorter median OS compared to TET2 wild type (9.5 vs. 32.2 months, P=0.013). Conclusion: Gene mutations incidence was high in normal karyotype AML patients. TET2 mutations was an unfavorable prognostic factor which was closely associated with shorter median OS in contrast to TET2 wild type in NPM1m+/FLT3-ITDm-group. In addition, The number of gene mutation was closely associated with older age, high white blood cell levels and high bone marrow blast cell percentage. Disclosures: No relevant conflicts of interest to declare.

Recurrent Mutations of Multiple Components of Cohesin Complex in Myeloid Neoplasms

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 782-782
Author(s):  
Ayana Kon ◽  
Lee-Yung Shih ◽  
Masashi Minamino ◽  
Masashi Sanada ◽  
Yuichi Shiraishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Gene Mutations ◽  
Cohesin Complex ◽  
Wild Type ◽  
Myeloid Neoplasms ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Equity Ownership

Abstract Abstract 782 Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in both acute and chronic myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. In the previous study, we analyzed 29 paired tumor-normal samples with chronic myeloid neoplasms with myelodysplastic features using whole exome sequencing (Yoshida et al., Nature 2011). Although the major discovery was frequent spliceosome mutations tightly associated with myelodysplasia phenotypes, hundreds of unreported gene mutations were also identified, among which we identified recurrent mutations involving STAG2, a core cohesin component, and also two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex conserved across species and is composed of four core subunits, i.e., SMC1, SMC3, RAD21 and STAG proteins, together with several regulatory proteins. Forming a ring-like structure, cohesin is engaged in cohesion of sister chromatids in mitosis, post-replicative DNA repair and regulation of gene expression. To investigate a possible role of cohesin mutations in myeloid leukemogenesis, an additional 534 primary specimens of various myeloid neoplasms was examined for mutations in a total of 9 components of the cohesin and related complexes, using high-throughput sequencing. Copy number alterations in cohesin loci were also interrogated by SNP arrays. In total, 58 mutations and 19 deletions were confirmed by Sanger sequencing in 73 out of 563 primary myeloid neoplasms (13%). Mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205) and CML (8/65), with much lower mutation frequencies in MPN (2/76), largely in a mutually exclusive manner. In MDS, mutations were more frequent in RCMD and RAEB (19.5%) but rare in RA, RARS, RCMD-RS and 5q- syndrome (3.4%). Cohesin mutations were significantly associated with poor prognosis in CMML, but not in MDS cases. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles was performed in 19 cases with cohesin mutations. Majority of the cohesin mutations (16/19) existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we investigated a possible impact of mutations on cohesin functions, where 17 myeloid leukemia cell lines with or without cohesin mutations were examined for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of one or more components of cohesin was substantially reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we examined the effect of forced expression of wild-type cohesin on cell proliferation of cohesin-defective cells. Introduction of the wild-type RAD21 and STAG2 suppressed the cell growth of RAD21- (Kasumi-1 and MOLM13) and STAG2-defective (MOLM13) cell lines, respectively, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, 23 cohesin-mutated cases of our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Alternatively, a growing body of evidence suggests that cohesin regulate gene expression, arguing for the possibility that cohesin mutations might participate in leukemogenesis through deregulated gene expression. Of additional note, the number of non-silent mutations determined by our whole exome analysis was significantly higher in 6 cohesin-mutated cases compared to non-mutated cases. Since cohesin also participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, we report a new class of common genetic targets in myeloid malignancies, the cohesin complex. Our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Whole Exome Sequencing Shows a Paucity Of Somatic Gene Mutations In Pediatric Idiopathic Bone Marrow Failure Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3708-3708
Author(s):  
Atsushi Narita ◽  
Hideki Muramatsu ◽  
Kenichi Yoshida ◽  
Yusuke Okuno ◽  
Asahito Hama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Somatic Mutations ◽  
Bone Marrow Failure ◽  
Gene Mutations ◽  
Bone Marrow Failure Syndrome ◽  
Cell Lineages ◽  
Bone Marrow Failure Syndromes ◽  
Whole Exome

Abstract Introduction Pancytopenia accompanied by a severe decrease in bone marrow (BM) cellularity in children can be due to a broad variety of underlying disorders. Appropriate classification of bone marrow failure syndrome in children is challenging, particularly in relation to the morphological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study was to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 patients with these disorders through the use of exome sequencing. Patient and Methods Diagnosis of AA, RCC, or RCMD was made on basis of the 2008 World Health Organization (WHO) classification criteria. AA patients exhibited no morphologically dysplastic changes in any of their hematopoietic cell lineages, while RCC patients had<10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients classified as RCMD exhibited >10% of the dysplastic changes in two or more cell lineages. Blood and BM samples were obtained from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). Exome capture from paired DNA (non-T cells/CD3+ lymphocyte) was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate somatic nucleotide changes were validated by Sanger sequencing. Results Exome sequencing pipeline identified a total of 193 non-synonymous somatic mutations or indels candidates among the 29 patients (range, 2–15 per patient). After validation by Sanger sequencing, one nonsense, 11 missense, and two frame-shift mutations were confirmed as non-silent somatic mutations. The average numbers of mutations per sample were not significantly different when comparing morphological diagnostic groups (0.50 in AA, 0.36 in RCC, 0.60 in RCMD). Of these validated genes, BCOR (n = 2) and CSK (n = 2) mutations were recurrent genetic events. BCOR is a frequent mutational target in myelodysplastic syndrome, whereas CSK somatic mutations were not reported in human cancers. BCOR mutations were found both in AA (c.472delA:p.S158fs; patient 13) and in RCMD (c.G3856T:p.E1286X; patient 39). Both patients with CSK mutations were classified as RCC (c.G994A:p.D332N; patient 23 and 27). When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0% - 0%) vs. 6% (0% - 26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100% - 100%) vs. 95% (70% - 99%)] were not significantly different. Conclusion Whole exome sequencing analysis was used for gene mutational profiling of patients with idiopathic bone marrow failure syndromes; i.e., AA, RCC, and RCMD. Although BCOR and CSK somatic mutations were recurrently identified, idiopathic bone marrow failure syndromes in children are characterized by a paucity of gene mutations, irrespective of morphological diagnosis. These findings suggest that morphological diagnosis based on WHO classification system does not discriminate the mutational profile and pathogenesis of bone marrow failure in children. Disclosures: No relevant conflicts of interest to declare.

Whole-Exome Resequencing Identifies Somatic Mutations Of BCOR and BCORL1 Transcriptional Corepressor Genes and Major Cohesin Complex Component Genes In Pediatric Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 834-834
Author(s):  
Norio Shiba ◽  
Kenichi Yoshida ◽  
Yasunobu Nagata ◽  
Ayana Kon ◽  
Yusuke Okuno ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Transcriptional Corepressor ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Pediatric Aml ◽  
Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Currently, targeted sequencing efforts have identified several mutations that carry diagnostic and prognostic information such as RAS, KIT, and FLT3 in both adult and pediatric AML, and NPM1 and TET2 in adult AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be enabled the discovery of recurrent mutations in IDH1/2, and DNMT3A in adult AML. However, these mutations are extremely rare in pediatric AML. Methods To reveal a complete registry of gene mutations and other genetic lesions, whole-exome resequencing of paired tumor-normal DNA from 19 cases were analyzed with a mean coverage of approximately x100, and 82 % of the target sequences were analyzed at more than x20 depth on average. We selected various cases in age, FAB classification and karyotypes, including 5 cases with core-binding-factor AML, 6 cases with MLL-rearrangement and 2 acute megakaryoblastic leukemia cases. Results and Discussion A total of 80 somatic mutations or 4.2 mutations per sample were identified. As the mean number of somatic mutations reported in adult AML was about ten, somatic mutations in pediatric AML might be fewer than in adult AML. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BRAF, BCORL1, DAZAP1, CUL2, ASXL2, MLL2, MLL3, SMC3 and RAD21. Among these, what immediately drew our attention were SMC3 and RAD21, because they belong to the major cohesin components. Cohesin is a multimeric protein complex conserved across species and composed of four core subunits, i.e., SMC1, SMC3, RAD21, and STAG proteins, forming a ring-like structure. Cohesin is engaged in cohesion of sister chromatids during cell division, post-replicative DNA repair, and regulation of global gene expression through long-range cis-interactions. Furthermore, we also drew our attention to BCORL1, because it is a transcriptional corepressor, and can bind to class II histone deacetyllases (HDAC4, HDAC5, HDAC7), to interact with the CTBP1 corepressor, and to affect the repression of E-cadherin. BCOR is also a transcriptional corepressor and play a key role in the regulation of early embryonic development, mesenchymal stem cell function and hematopoiesis. To confirm and extend the initial findings in the whole-exome sequencing, we studied mutations of the above 8 genes, in pediatric AML (N = 190) using a high-throughput mutation screen of pooled DNA followed by confirmation/ identification of candidate mutations. In total, 32 mutations were identified in 31 of the 190 specimens of pediatric AML [BCOR (N = 7), BCORL1 (N = 7), RAD21 (N = 7), SMC3 (N = 5), SMC1A (N = 1), and STAG2 (N = 3)]. The mutually exclusive pattern of the mutations in these BCOR, BCORL1 and cohesin components genes was confirmed in this large case series, suggesting a common impact of these mutations on the pathogenesis of pediatric AML. The 4-year overall survival of these cases with major cohesin components gene mutations was relatively favorable (12/16 or 75.0%), but the outcome of cases with BCOR or BCORL1 cases was unfavorable (8/14 or 57.1%). Conclusion Whole exome resequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document