scholarly journals Targeted Resequencing of MLL-PTD Positive AML Patients Reveals a High Prevalence of Co-Ocurring Mutations in Epigenetic Regulator Genes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1035-1035
Author(s):  
Sylvia Herold ◽  
Thoralf Stange ◽  
Matthias Kuhn ◽  
Ingo Roeder ◽  
Christoph Röllig ◽  
...  
Keyword(s):  
Data Analysis ◽  
Hot Spots ◽  
Final Analysis ◽  
Coding Region ◽  
Research Support ◽  
Targeted Resequencing ◽  
Whole Exome ◽  
High Prevalence ◽  
Epigenetic Regulator ◽  
Regulator Genes

Abstract Background Partial tandem duplication mutations of the Mixed Lineage Leukemia gene (MLL-PTD) can be found in about 10% of patients with AML, especially in patients with normal karyotype AML. The mutation generates a self-fusion within the N-terminal part of MLL and has been shown to be leukemogenic in mouse models. In patients, the presence of the mutation is associated with poor prognosis. Little is known on the molecular profile of patients with MLL-PTD and on the cooperating mutations. In order to identify accompanying molecular alterations, we performed whole exome sequencing (WES) of eight AML patients harbouring MLL-PTD mutations. Based on the observed alterations we then designed a custom amplicon panel and performed targeted resequencing in a cohort of 90 MLL-PTD mutated AML patients. Materials and Methods All patients included in this analysis were treated in prospective treatment protocols of the Study Alliance Leukemia (SAL). To enrich for malignant cells and to obtain germline reference material (T-cells), FACS sorting was performed on viable cells banked at diagnosis. After whole genome amplification of the primary DNA, whole exomes were enriched (TruSeq chemistry; Illumina), and paired-end sequenced using Illumina HiSeq2000 2x100 bp runs. Resulting data were mapped against human genome (Hg19). Only somatic single nucleotide variants (SNVs) were included in the final analysis. Based on the SNVs identified by whole exome sequencing (WES), a custom amplicon panel (TruSeq Custom Amplicon, TSCA, Illumina) for targeted resequencing was designed. The assay included either the entire coding region or mutational hot spots of 56 genes (Fig.1). In total, 700 targets were amplified in a single reaction for each patient and paired end sequenced on a MiSeq NGS system (Illumina). Paired end reads were BWA mapped against targeted regions and data analysis was done using the Sequence Pilot software package (JSI Medical Systems) with a 20% variant allele frequency (VAF) mutation calling cutoff. Only non-synonymous variants not specified as SNP in the db137 database and predicted as deleterious (Provean) were included in the final analysis. All variations were confirmed by Sanger sequencing. Results WES of eight MLL-PTD (7/8 FLT3-ITD negativ) patients revealed a total 490 SNVs (range 13-254 per patient). Most frequently mutated genes were DNMT3A, IDH1/2 and TET2. Somatic mutations were also found in genes rarely mutated in AML, such as ATM, GNAS, TET1 and EP300. Based on the WES-data, 90 MLL-PTD patients were screend for a panel of 56 genes using the TSCA assay, which revealed in total of 169 mutations. 18 genes were not found to be mutated and in 8 patients, no co-occurring mutations were identified. Due bad assay performance EP300, EZH1, JAK3, MLL2, MLL3 and NOTCH1 were excluded from the data analysis. Here again, the most frequently mutated genes were DNMT3A (34.4%), IDH1 (20.0%), IDH2R140 (18.9%), IDH2R172 (7.9%), TET2 (16.7%) and FLT3 (11.3%). Mutations were less frequently found in RUNX1 (8.9%) and ASXL1, SMC1A, U2AF1 (5.6% each) (Fig. 1). In addition to these known genes, most prevalent mutations were found in ATM (8.9%) as well as DNMT3B and TET1 (4.4% each). Overall, we oberserved a low frequency of mutations in typical class 1 genes such as NRAS, KRAS and FLT3, which was lower than reported in the TCGA data set. Conclusions This analysis in a large set of patients with MLL-PTD mutations did not reveal any new and specific individual mutation present in patients with this alteration. Instead, our finding of a very high prevalence of alterations in epigenetic regulator genes, found in more than 85% of patients with MLL-PTD, strongly argues for a particular disease biology in these patients. These findings might also implicate that treatment based on demethylating agents or histone-deacetylase inhibitors might be especially attractive in patients with MLL-PTD. Figure 1: Figure 1:. Distribution of mutations in MLL-PTD patients The assay included either the entire coding region or mutational hot spots of the following 56 genes; ASXL1, ATM, BCOR, BRAF, CBL, DDR1, DNMT1, DNMT3A, DNMT3B, EIF4A2, EP300, ETV6, EZH1, EZH2, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KDM4A, KDM5A, KDM5C, KDM6A, KIT, KRAS, MET, MLL, MLL2, MLL3, NOTCH1, NOTCH4, NPM1, NRAS, PDGFRA, PDGFRB, PHF6, PTEN, PTPN11, RAD21, RUNX1, SF3A1, SF3B4, SMC1A, SMC3, SMC4, TET1, TET2, TP53, U2AF1 and WT1. Disclosures Thiede: AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

Analysis of Molecular Predictors of Response to 5-Azacitine Treatment in AML and MDS Patients Preemptively Treated for Molecular Relapse of Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2384-2384
Author(s):  
Meredith Lilly ◽  
Beatrice Haack ◽  
Anne Otto ◽  
Katja Sockel ◽  
Jan Moritz Middeke ◽  
...  
Keyword(s):  
Clinical Response ◽  
Recurrent Disease ◽  
Molecular Evidence ◽  
Loss Of Function ◽  
Research Support ◽  
Free Survival ◽  
Predictors Of Response ◽  
Epigenetic Regulator ◽  
Regulator Genes

Abstract Background: Relapse of disease remains the major cause of treatment failure in patients with acute myeloid leukemia (AML) or advanced myelodysplastic syndrome (MDS), even after allogeneic hematopoietic stem cell transplantation (HSCT). Treatment of relapsed AML or MDS is difficult, especially after HSCT, and long-term prognosis of patients suffering from relapse is dismal. One approach to overcome this problem is to use sensitive molecular diagnostic strategies to detect recurring disease already at the level of minimal residual disease (MRD), thus avoiding the development of overt hematologic relapse by treatment of patients at the stage of molecular relapse. We have recently implemented preemptive treatment with the demethylating drug 5-Azacitidine (AZA) in patients with molecular evidence of recurrent disease in a prospective Phase II study (RELAZA). In this study, 80% of the patients showed responses, with reduction of MRD and prolonged leukemia free survival, 20% of patients even showed molecular clearance of their leukemia and long-term disease free survival. More recently, results from several groups studying demethylating agents in MDS or AML suggested that patients with mutations in genes involved in epigenetic DNA-modification, such as TET2, DNMT3A or IDH1 or IDH2 might be more responsive to treatment with these drugs. Since we observed varying clinical response in the patients treated preemptively with AZA for molecular evidence of recurrent disease, we correlated the clinical response in these patients with the presence of mutations in epigenetic regulator genes in order to identify potential predictors of response. Patients and Methods: A cohort of 44 patients (23 f/21 m), median age 55.6 years (range 21-75 years), in hematological remission with AML (N=40) or MDS (N=4) were given AZA to treat molecular relapse defined by mutant NPM1 (N=23) or CD34+ chimerism (N=21). Patients were monitored post allogeneic HSCT (N=26) or standard chemotherapy (N=18). The cohort received a median of 5 cycles of AZA (ranging from 1-18 cycles). DNA taken at first diagnosis was analyzed using amplicon based resequencing on a MiSeq next generation sequencing system for the following genes, either analyzing the complete coding region (EZH1, EZH2, DNMT3A, TET1 and TET2) or hot-spot regions (ASXL1, ASXL2, IDH1, IDH2). First diagnosis samples were unavailable for 4 patients. In these, DNA from sorted CD34+ cells taken at the time of molecular relapse was used as a substitute. Results: Amplicon sequencing revealed mutations in one or more genes in 25/44 patients (56.8%). With 15 mutations (34%), DNMT3A was the most frequently mutated gene, the majority of the alterations (9; 60%) were located in exon 23. Mutations in TET2 were found in 8 patients, IDH1 was mutated twice, ASXL2, EZH2 and TET1 were mutated once each. In 20 of the 44 patients (45.5%), no mutations in the investigated genes were found. A comparison of primary response to AZA-treatment (defined as stabilization or decrease of the MRD-marker) between patients with and without mutations revealed no significant difference (79.2 vs 66.6%; P=.48). Likewise, the rate of hematologic relapse was comparable in both cohorts (54% vs. 56%). However, a more detailed look at the patients with mutations revealed differences. The highest initial response rate was observed in patients with DNMT3A mutations (87%), whereas patients with isolated TET2 mutations were less likely to respond (50%). Also, the rate of hematologic relapse was highest in patients with TET2-mutations (75%) compared to patients with DNMT3A-mutations alone (41.6%). In support of a role of TET2-mutations in mediating resistance, an analysis of matched diagnosis and relapse samples in three patients indicated persistence of TET2-loss of function mutations in one patient as well as an acquisition of a second mutant TET2- allele or a switch to a loss-of function-mutation in two patients, indicating that a clonal evolution favoring a subclone with an inactivating TET2-allele under treatment with AZA occurred. Conclusions: Our data confirm that mutations in epigenetic regulator genes are common in patients with AML. Although based on small numbers, these preliminary data do not support that mutations in these genes are associated per se with an improved response to treatment with AZA, but might indicate a differential effect of certain alterations, i.e. DNMT3A-mutations or mutations of TET2. Disclosures Middeke: Genzyme: Speakers Bureau. Thiede:AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

Epigenetic Regulator Genes Direct the Fate of Multipotent Progenitor Cell of Origin in Lineage Switched MLL-AF4 Leukaemia

2019 ◽  
Author(s):  
Ricky Tirtakusuma ◽  
Paul Milne ◽  
Anetta Ptasinska ◽  
Claus Meyer ◽  
Sirintra Nakjang ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Cell Of Origin ◽  
Epigenetic Regulator ◽  
Regulator Genes

scholarly journals Prevalence of MDR organism (MDRO) carriage in children and their household members in Siem Reap Province, Cambodia

2020 ◽  
Vol 2 (4) ◽  
Author(s):  
Shweta R Singh ◽  
Bunsoth Mao ◽  
Konstantin Evdokimov ◽  
Pisey Tan ◽  
Phana Leab ◽  
...  
Keyword(s):  
Final Analysis ◽  
Cross Sectional Study ◽  
Cross Sectional ◽  
Middle Income ◽  
E Coli ◽  
Middle Income Countries ◽  
Siem Reap ◽  
High Prevalence ◽  
Significant Public Health ◽  
History Of

Abstract Background The rising incidence of infections caused by MDR organisms (MDROs) poses a significant public health threat. However, little has been reported regarding community MDRO carriage in low- and middle-income countries. Methods We conducted a cross-sectional study in Siem Reap, Cambodia comparing hospital-associated households, in which an index child (age: 2–14 years) had been hospitalized for at least 48 h in the preceding 2–4 weeks, with matched community households on the same street, in which no other child had a recent history of hospitalization. Participants were interviewed using a survey questionnaire and tested for carriage of MRSA, ESBL-producing Enterobacterales (ESBL-E) and carbapenemase-producing Enterobacterales (CPE) by culture followed by antibiotic susceptibility testing. We used logistic regression analysis to analyse associations between collected variables and MDRO carriage. Results Forty-two pairs of households including 376 participants with 376 nasal swabs and 290 stool specimens were included in final analysis. MRSA was isolated from 26 specimens (6.9%). ESBL-producing Escherichia coli was detected in 269 specimens (92.8%) whereas ESBL-producing Klebsiella pneumoniae was isolated from 128 specimens (44.1%), of which 123 (42.4%) were co-colonized with ESBL-producing E. coli. Six (2.1%) specimens tested positive for CPE (4 E. coli and 2 K. pneumoniae). The prevalence ratios for MRSA, ESBL-producing E. coli and ESBL-producing K. pneumoniae carriage did not differ significantly in hospital-associated households and hospitalized children compared with their counterparts. Conclusions The high prevalence of ESBL-E across both household types suggests that MDRO reservoirs are common in the community. Ongoing genomic analyses will help to understand the epidemiology and course of MDRO spread.

scholarly journals Differences in physical symptoms between those with and without kidney disease: a comparative study across disease stages in a UK population

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Thomas J. Wilkinson ◽  
Daniel G. D. Nixon ◽  
Jared Palmer ◽  
Courtney J. Lightfoot ◽  
Alice C. Smith
Keyword(s):  
Kidney Disease ◽  
Early Stage ◽  
Symptom Burden ◽  
Final Analysis ◽  
Physical Symptoms ◽  
Disease Trajectory ◽  
High Prevalence ◽  
Comparative Group ◽  
The Difference ◽  
Disease Stages

Abstract Background Those living with kidney disease (KD) report extensive symptom burden. However, research into how symptoms change across stages is limited. The aims of this study were to 1) describe symptom burden across disease trajectory, and 2) to explore whether symptom burden is unique to KD when compared to a non-KD population. Methods Participants aged > 18 years with a known diagnosis of KD (including haemodialysis (HD) and peritoneal dialysis (PD)) and with a kidney transplant) completed the Leicester Kidney Symptom Questionnaire (KSQ). A non-KD group was recruited as a comparative group. Multinominal logistic regression modelling was used to test the difference in likelihood of those with KD reporting each symptom. Results In total, 2279 participants were included in the final analysis (age 56.0 (17.8) years, 48% male). The main findings can be summarised as: 1) the number of symptoms increases as KD severity progresses; 2) those with early stage KD have a comparable number of symptoms to those without KD; 3) apart from those receiving PD, the most frequently reported symptom across every other group, including the non-KD group, was ‘feeling tired’; and 4) being female independently increased the likelihood of reporting more symptoms. Conclusions Our findings have important implications for patients with KD. We have shown that high symptom burden is prevalent across the spectrum of disease, and present novel data on symptoms experienced in those without KD. Symptoms requiring the most immediate attention given their high prevalence may include pain and fatigue. Trial registration The study was registered prospectively as ISRCTN11596292.

scholarly journals Gravity Probe B data analysis: II. Science data and their handling prior to the final analysis

2015 ◽  
Vol 32 (22) ◽  
pp. 224019 ◽  
Author(s):  
A S Silbergleit ◽  
J W Conklin ◽  
M I Heifetz ◽  
T Holmes ◽  
J Li ◽  
...  
Keyword(s):  
Data Analysis ◽  
Final Analysis ◽  
Science Data ◽  
Gravity Probe B ◽  
Gravity Probe

Data Analysis and Presentation

2018 ◽  
pp. 177-192
Author(s):  
John R. B. Lighton
Keyword(s):  
Data Analysis ◽  
Temperature Effects ◽  
Final Analysis ◽  
Analysis Of Covariance ◽  
Neutral Zone ◽  
Metabolic Rates ◽  
Treatment Groups ◽  
Metabolic Data ◽  
Interspecific Comparisons ◽  
Selection Of

This chapter discusses ways of analyzing and presenting metabolic data while avoiding common mistakes. Topics covered include vital information often omitted from manuscripts; how to analyze the allometry of metabolic rate on mass; the mistake of reporting mass-specific or “mass-independent” metabolic rates; methods for quantifying differences between treatment groups by analysis of covariance; the importance of phylogeny in interspecific comparisons; the importance of the temperature at which measurements are made, including mammals (the thermal neutral zone); the necessity of leaving an “audit trail” from raw data through to final analysis; analyzing temperature effects such as Q10 correctly; and the proper selection of metabolic data.

scholarly journals Familial Intracranial Aneurysm in Newfoundland: Clinical and Genetic Analysis

2019 ◽  
Vol 46 (5) ◽  
pp. 518-526
Author(s):  
Amy E. Powell ◽  
Bridget A. Fernandez ◽  
Falah Maroun ◽  
Barbara Noble ◽  
Michael O. Woods
Keyword(s):  
Intracranial Aneurysm ◽  
Sanger Sequencing ◽  
Variant Prioritization ◽  
Future Studies ◽  
Whole Exome ◽  
High Prevalence ◽  
History Of ◽  
Multiplex Families ◽  
Genetic And Environmental Factors ◽  
Population Controls

ABSTRACT:Objective:Intracranial aneurysm (IA) is an expansion of the weakened arterial wall that is often asymptomatic until rupture, resulting in subarachnoid hemorrhage. Here we describe the high prevalence of familial IA in a cohort of Newfoundland ancestry. We began to investigate the genetic etiology of IA in affected family members, as the inheritance of this disease is poorly understood.Methods:Whole exome sequencing was completed for a cohort of 12 affected individuals from two multiplex families with a strong family history of IA. A filtering strategy was implemented to identify rare, shared variants. Filtered variants were prioritized based on validation by Sanger sequencing and segregation within the families.Results:In family R1352, six variants passed filtering; while in family R1256, 68 variants remained, so further filtering was pursued. Following validation by Sanger sequencing, top candidates were investigated in a set of population controls, namely,C4orf6c.A1G (p.M1V) andSPDYE4c.C103T (p.P35S). Neither was detected in 100 Newfoundland control samples.Conclusion:Rare and potentially deleterious variants were identified in both families, though incomplete segregation was identified for all filtered variants. Alternate methods of variant prioritization and broader considerations regarding the interplay of genetic and environmental factors are necessary in future studies of this disease.

Somatic Mutations of Epigenetic Regulator Genes in Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1756-1756 ◽  
Author(s):  
Yao-hui Huang ◽  
Weili Zhao
Keyword(s):  
B Cell ◽  
Somatic Mutations ◽  
Cell Lymphoma ◽  
Univariate Analysis ◽  
B Cell Lymphoma ◽  
Genome Database ◽  
Large B Cell Lymphoma ◽  
Epigenetic Regulator ◽  
Regulator Genes ◽  
Large B Cell

Abstract Background. Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive types of B-cell lymphoma with high heterogeneity, accounting for 30-40% of newly diagnosed non-Hodgkin lymphoma (NHL), and dysfunction of epigenetic regulation has been found as a common and important feature of B cell lymphomas. To identify epigenetic associated genes mutations in DLBCL, including KMT2D, CREBBP, EP300, EZH2 and MEF2B, we sequenced tumour DNA from 226 Chinese DLBCL cases by applying next generation sequencing technology (NGS). A total of 679 consecutive Chinese patients with previously untreated DLBCL at our institution from December 2006 and January 2016 were enrolled in this study, and we assessed the predictive value of clinical and mutational pattern of epigenetic associated genes in a large single-institution cohort of these patients. Methods. Genomic DNA was extracted from 226 subjects with DLBCL formalin-fixed paraffin-embedded tumor tissue, using a QIAamp DNA FFPE Tissue Kit (Qiagen). Specific primers, producing amplicons about 200 bp at the coding regions of the genes of interest , were designed at the UCSC website (http://genome.ucsc.edu/cgi-bin/hgGateway ). Microfluidic PCR reactions ran in a 48 ¡Á 48 Access array system (Fluidigm) with FastStart High Fidelity PCR system (Roche) and high-throughput DNA sequencing was performed on Illumina Genome Analyzer IIx (GAIIx) and HiSeq2000 systems, according to the manufacturer's instructions. SAMtools version 0.1.19 was used to generate chromosomal coordinate-sorted bam files and to remove PCR duplications. Sequences for epigenetic associated genes were obtained from the UCSC Human Genome database, using the corresponding mRNA accession number as a reference, and those containing splice-site, nonsense or coding-region indel mutations, were selected for Gene Ontology analysis. All of the results were also confirmed by Sanger sequencing. Baseline characteristics of patients were analysed using two-sided c2 test. Overall survival (OS) was estimated using the Kaplan-Meier method and compared by log-rank test. Univariate hazard estimates were generated with unadjusted Cox proportional hazards models. Covariates demonstrating significance with P<0.100 on univariate analysis were included in the multivariate model. Statistically significance was defined as P<0.05. All statistical analyses were carried out using Statistical Package for the Social Sciences (SPSS) 20.0 software (SPSS Inc., Chicago, IL, USA). Results. Overall, 105 of 226 Chinese DLBCL cases were identified to have at least one mutation in epigenetic regulator genes. Somatic mutations in KMT2D were most frequently observed (24.3%), followed by CREBBP, EP300, EZH2 and MEF2B (15.5%,10.6%,4.4% and 2.2%, respectively)(Figure1,A,B). Association of mutated genes according to the conceptual classification. Circos plot of mutated genes according to the function is shown, and overlap mutations between epigenetic regulator genes mutations were frequently observed (Figure1, C). Clinically, mutation-positive DLBCL patients presented shorter OS than patients those without mutations (P=0.0286, Figure 1,D) among 226 DLBCL cases. A total of 679 Chinese DLBCL cases were enrolled in univariate analysis, and R-IPI, Complete Remission (CR), epigenetic related mutations were significant prognostic factors for OS. In further multivariate analysis, R-IPI (RR=2.72,95%CI=1.619-4.567,P<0.000), CR (RR=0.129,95%CI=0.076-0.219,P<0.000), epigenetic related mutations (RR=1.605,95%CI=1.007-2.557,P=0.046) are independent prognostic factor for OS. Conclusion. Our study provided the mutational spectrum of epigenetic regulator genes in DLBCL, and the relationships between mutations and clinic suggested some therapeutic efficiency. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Whole Exome Sequencing for Mutation Screening in Hemophagocytic Lymphohistiocytosis

2020 ◽  
Author(s):  
Edris Sharif Rahmani ◽  
Majid Fathi ◽  
Mohammad Foad Abazari ◽  
Hojat Shahraki ◽  
Vahid Ziaee Fellow ◽  
...  
Keyword(s):  
Immune System ◽  
Data Analysis ◽  
Exome Sequencing ◽  
Molecular Characterization ◽  
Whole Exome Sequencing ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Mutation Screening ◽  
Positive Family History ◽  
Whole Exome

Background: Hemophagocytic lymphohistiocytosis (HLH) is an immune system disorder characterized by uncontrolled hyper-inflammation owing to hypercytokinemia from the activated but ineffective cytotoxic cells. Establishing a correct diagnosis for HLH patients due to the similarity of this disease with other conditions like malignant lymphoma and leukemia and similarity among its two forms is difficult and not always a successful procedure. Besides, the molecular characterization of HLH due to the locus and allelic heterogeneity is a challenging issue. Materials and Methods: In this experimental study, whole exome sequencing (WES) was used for mutation detection in a four-member Iranian family with children suffering from signs and symptoms of HLH disease. Data analysis was performed by using a multi-step in-house WES approach on Linux OS. Result: In this study, a homozygous nucleotide substitution mutation (c.551G>A:p.W184*) was detected in exon number six of the UNC13D gene. W184* drives to a premature stop codon, so produce a truncated protein. This mutation inherited from parents to a four-month female infant with an autosomal recessive pattern. Parents were carrying out the heterozygous form of W184* without any symptoms. The patient showed clinical signs such as fever, diarrhea, hepatosplenomegaly, high level of ferritin, and a positive family history of HLH disease. W184* has a damaging effect on cytotoxic T lymphocytes, and natural killer cells. These two types of immune system cells without a healthy product of the UNC13D gene will be unable to discharge toxic granules into the synaptic space, so the inflammation in the immune response does not disappear. Conclusion: According to this study, WES can be a reliable, fast, and cost-effective approach for the molecular characterization of HLH patients. Plus, WES specific data analysis platform introduced by this study potentially offers a high-speed analysis step. This cost-free platform doesn't require online data submission.

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