scholarly journals TET2 mutations in acute myeloid leukemia: a comprehensive study in patients of Sindh, Pakistan

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10678
Author(s):  
Abdul Rehman Khalil Shaikh ◽  
Ikram Ujjan ◽  
Muhammad Irfan ◽  
Arshi Naz ◽  
Tahir Shamsi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Exome Sequencing ◽  
Genetic Variants ◽  
Myeloid Leukemia ◽  
Sanger Sequencing ◽  
Normal Karyotype ◽  
Marrow Transplant ◽  
Sequencing Analysis ◽  
Frameshift Deletion ◽  
Acute Myeloid

Background The tet oncogene family member 2 (TET2) gene has been reported to be involved in DNA methylation and epigenetic regulation in acute myeloid leukemia (AML). Various studies have proven functional role of TET2 mutations in AML. We herein studied the frequency and genotype-phenotype correlation of TET2 gene in AML patients in Sindh, Pakistan. Patients and methods The current study was carried out at Liaquat University of Medical & Health Sciences, Jamshoro, Pakistan, in collaboration with National Institute of Blood Disease & Bone Marrow Transplant, Karachi, Pakistan, during the period from June 2019 to June 2020. A total of 130 patients diagnosed with AML were screened for TET2 mutations. Whole exome sequencing of 14 individuals was carried out to find the genetic variants in TET2 gene. The pathogenicity of the variants was predicted by SIFT, PolyPhen2, Mutation Taster and CADD Phred scores. The allele frequency of the variants was compared with global population using 1000 genomes project and Exome Aggregation Consortium (ExAC). Furthermore, exon 3 and exon 5 of the TET2 gene were sequenced by using Sanger sequencing. The findings were correlated with subtypes of AML and corresponding karyotypes. Results Through the exome sequencing, 17 genetic variants (13 SNPs and four indels) were identified in 14 individuals. Of these, four variants that is, one frameshift deletion, one frameshift insertion and two nonsense variants were novel and not present in dbSNP151 database. Three novel variants were found in exon 3 including two frameshift variants that is, p.T395fs and G494fs, predicted as deleterious by CADD Phred scores, and one stop-gain variant (p.G898X) predicted as deleterious by Mutation Taster and CADD Phred scores. One novel non sense variant (p.Q1191X) was found in the exon 5 predicted as deleterious by SIFT, Mutation Taster and CADD Phred scores. Sanger sequencing analysis revealed one novel deletion at g105233851: del.TAGATAGA, and one novel SNP g;105233861 T>G identified in the TET2 gene. Majority of the exon 3 mutations were seen in the patients diagnosed with AML with maturation, and had a normal karyotype. Conclusion TET2 mutations were identified in around 16% of the total patients of our study indicating other mechanisms being involved in pathophysiology of AML in this cohort. The TET2 mutations provide a prognostic value in determining AML classification.

Whole-Exome Sequencing Identifies Recurrent Mutations of BCOR in Acute Myeloid Leukemia with Normal Karyotype

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 71-71 ◽  
Author(s):  
Brunangelo Falini ◽  
Vera Grossmann ◽  
Enrico Tiacci ◽  
Antony Holmes ◽  
Alexander Kohlmann ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Exome Sequencing ◽  
Myeloid Leukemia ◽  
Normal Karyotype ◽  
Employment Equity ◽  
Normal Cells ◽  
Genetic Syndrome ◽  
Whole Exome ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Abstract 71 Acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) represents about half of all adult AML. NPM1 and CEBPA mutations define WHO provisional entities accounting for ∼60% of CN-AML, but the remaining cases (∼40%) remain poorly characterized. To address this issue, we carried out whole-exome-sequencing (WES) of leukemic and normal cells from one patient with CN-AML that lacked mutations in NPM1, CEBPA, FLT3-ITD, and MLL-PTD. Using this approach, we identified a clonal somatic mutation of BCOR, a gene located on chromosome Xp11.4, that was present in the leukemic but not normal cells of the index AML case. The BCOR (BCL6 co-repressor) gene encodes for an ubiquitously expressed nuclear protein that is involved in repressing the activity of BCL6 and other transcriptional factors. BCOR is a key transcriptional regulator of early embryonic development, mesenchymal stem cell function and hemopoiesis. Germline mutations of BCOR are responsible for the oculo-facio-cardio-dental (OFCD) genetic syndrome that is inherited in an X-linked pattern and comprises microphtalmia, dysmorphic appearance, dental abnormalities (radiculomegaly), hammer-toe deformity and cardiac defects. WES findings in the index case were subsequently validated and further studied in a total cohort of 514 AML patients. We first performed deep-sequencing analyses of all exons of the BCOR gene in an initial set of 82 AML cases that were selected because they showed the same genetic characteristics of our index patient (i.e. normal karyotype without NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Disruptive BCOR mutations (i.e., nonsense mutations, out-of-frame small indels, and consensus splice-site mutations) were detected in 14/82 (17.1%) of these cases. We next assessed the frequency of BCOR mutations in a series of unselected CN-AML patients (n=262) and found that they occurred in 4.2% of cases, mostly showing the typical features of BCOR-mutated cases (absence of NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Almost mutual exclusion of BCOR and NPM1 mutations was further confirmed in a separate series of 71 NPM1-mutated only AML patients. No BCOR mutations were observed in the 89 AML cases with recurrent cytogenetic abnormalities investigated, including t(8;21)(q22;q22) (n= 29), inv(16)(p13q22) (n=40), t(15;17)(q22;q12) (n=10), and t(11q23)/MLL (n=10), and in the 10 patients with double CEBPA-mutated AML studied. BCOR mutations were: i) scattered across the whole length of the coding sequence with no hotspots identified; ii) somatic in origin and disruptive molecular events similar to germline BCOR mutations causing the OFCD genetic syndrome; iii) associated with markedly decreased BCOR mRNA levels, absence of full-length BCOR and absent or low expression of a truncated BCOR protein; iv) almost mutually exclusive with NPM1 (only 1.5% of the 197 NPM1-mutated AML investigated carried BCOR mutations); v) rarely associated with FLT3-ITD; and vi) frequently associated with DNMT3A and RUNX1 mutations, suggesting cooperation with the respective mutated pathways. Clinically, BCOR mutations correlated with poor outcome among the cohort of 160 CN-AML patients evaluated (28.0% versus 66.3% overall survival at 2 years, P=0.024). We also searched for BCOR mutations in the human AML cell lines OCI-AML2, OCI-AML3, KG1a, U937, HL-60, HL-60R, HB4, AML193, and MVP-11. Only HL-60 and HL-60R (a ATRA-resistant derivative of HL-60) carried a BCOR mutation that consisted of a hemizygous G to T transition at position 4616 in exon 10, leading to the Glu1442X nonsense mutation. Western blot analysis of HL-60 cells resulted in the absence of the full-length BCOR protein (predicted MW: 192 kDa) and presence of a low intensity 156 kDa band likely corresponding to a truncated BCOR protein. In conclusion, our results implicate for the first time BCOR in the pathogenesis of CN-AML and suggest it may act as tumor suppressor gene. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

High Frequency of GATA2 Mutations in Cytogenetically Normal Acute Myeloid Leukemia with Biallelic CEBPA Mutations Identified by Exome Sequencing

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 72-72 ◽  
Author(s):  
Annika Dufour ◽  
Nikola Konstandin ◽  
Bianka Ksienzyk ◽  
Evelyn Zellmeier ◽  
Tobias Benthaus ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Exome Sequencing ◽  
Zinc Finger ◽  
Myeloid Leukemia ◽  
Sanger Sequencing ◽  
Haematopoietic Stem Cell ◽  
Missense Mutations ◽  
Cebpa Mutations ◽  
Exome Sequence ◽  
Acute Myeloid

Abstract Abstract 72 Cytogenetically normal acute myeloid leukemia (CN-AML) with biallelic CEBPA gene mutations (biCEPBA) represents a distinct genetic entity associated with a favorable clinical outcome (Dufour et al, JCO, 2010; Green et al, JCO, 2010; Pabst et al, Br J Cancer, 2009; Wouters et al, Blood, 2009). Furthermore, biCEBPA mutations are seldomly associated with other known prognostic mutations, like mutated NPM1 or FLT3-ITD. So far, it is not known if other alterations cooperate with the biCEBPA mutations in the process of leukemogenesis. To identify collaborating mutations, we performed whole exome sequencing in five biCEBPA mutated CN-AML patients. We generated at least 5 Gbp of exome sequence for each of the biCEBPA AML samples and for the corresponding remission samples. This allowed us to cover at least 80% of RefSeq coding exon positions with a minimum read depth of 10. Comparison of the AML exome sequence with the remission exome sequence and exclusion of annotated polymorphisms led to the identification of leukemia-specific variants. So far, we were able to confirm between 2 to 10 non-synonymous coding somatic mutations per patient in addition to the previously known biCEBPA mutations using Sanger sequencing. Thus, we detected tumor-specific mutations (nonsense and missense) in a total of 22 genes. Two genes were found recurrently mutated in 2 of the 5 biCEBPA samples: DNMT3A (2/5) and GATA2 (2/5). GATA2 is a zinc finger transcription factor important for haematopoietic stem cell proliferation and normal megakaryocytic development. GATA2 mutations have recently been associated with familial monocytopenia and familial myelodysplastic syndrome (Hsu et al, Blood, 2011; Scott et al, ASH abstract 2010). In the M5 subtype of AML, GATA2 mutations were found at a low frequency of 3.6% (Yan et al, Nature Genetics, 2011). Interestingly, GATA2 is a direct protein interactor and negative regulator of CEBPA. (Huang et al., MCB, 2009; Tong et al, MCB, 2005). Therefore, we determined the frequency of GATA2 mutations in 32 patients with biCEBPA mutant AML by screening all coding exons of GATA2 using high resolution melting curve analysis. Aberrant melting curves were subsequently confirmed by Sanger sequencing. Interestingly, 13 out of 32 (40.6%) biCEBPA patients carried heterozygous missense mutations in GATA2 and strikingly these mutations were all located in the highly conserved N-terminal zinc finger domain of GATA2. The missense mutations A318T and G320D surrounding the C319 which coordiates the zinc atom were recurrently detected in 6 out of 13 biCEBPA patients (3 with A318T and 3 with G320D). Two patients were found to carry each two different mutations in GATA2. 4 out of 13 biCEBPA patients with GATA2 mutations who could be analyzed during molecular remission had lost the GATA2 mutation at remisssion. Furthermore, no GATA2 mutations were found in 38 patients with a monoallelic CEBPA mutation and in 90 CN-AML patients with wildtype CEBPA. We are currently analyzing the functional consequences of these GATA2 mutations. In summary, we describe for the first time the specific association of mutations within the N-terminal zinc finger of GATA2 with biallelic CEBPA mutations in cytogenetically normal AML. Although high throughput sequencing so far has mainly revealed an increasing genetic heterogeneity in AML, our results suggest that there is an association of distinct mutations in defined genetic subgroups of AML. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype

Blood ◽  
2011 ◽  
Vol 118 (23) ◽  
pp. 6153-6163 ◽  
Author(s):  
Vera Grossmann ◽  
Enrico Tiacci ◽  
Antony B. Holmes ◽  
Alexander Kohlmann ◽  
Maria Paola Martelli ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
Normal Karyotype ◽  
Genetic Alterations ◽  
World Health ◽  
Whole Exome ◽  
Acute Myeloid

Abstract Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.

scholarly journals Nanopore Targeted Sequencing for Rapid Gene Mutations Detection in Acute Myeloid Leukemia

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1026 ◽  
Author(s):  
Cumbo ◽  
Minervini ◽  
Orsini ◽  
Anelli ◽  
Zagaria ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Low Cost ◽  
Gene Mutations ◽  
Predictive Biomarkers ◽  
Molecular Testing ◽  
Sequencing Analysis ◽  
Cebpa Mutations ◽  
Targeted Gene Sequencing ◽  
Acute Myeloid

Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.

FLT3 Internal Tandem Duplication in Acute Myeloid Leukemia with Normal Karyotype

2007 ◽  
Vol 42 (3) ◽  
pp. 250 ◽  
Author(s):  
Sang-Ho Kim ◽  
Yeo-Kyeoung Kim ◽  
Il-Kwon Lee ◽  
Deog-Yeon Jo ◽  
Jong-Ho Won ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Normal Karyotype ◽  
Internal Tandem Duplication ◽  
Flt3 Internal Tandem Duplication ◽  
Acute Myeloid

Normal karyotype acute myeloid leukemia with the CD7+ CD15+ CD34+ HLA-DR + immunophenotype is a clinically distinct entity with a favorable outcome

2014 ◽  
Vol 93 (6) ◽  
pp. 957-963 ◽  
Author(s):  
Noriyoshi Iriyama ◽  
Norio Asou ◽  
Yasushi Miyazaki ◽  
Shunichiro Yamaguchi ◽  
Shinya Sato ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Normal Karyotype ◽  
Favorable Outcome ◽  
Distinct Entity ◽  
Hla Dr ◽  
Acute Myeloid

FLT3 mutations have no prognostic impact in elderly patients with acute myeloid leukemia and normal karyotype

2009 ◽  
Vol 84 (8) ◽  
pp. 532-534 ◽  
Author(s):  
Felicetto Ferrara ◽  
Clelia Criscuolo ◽  
Cira Riccardi ◽  
Tiziana Izzo ◽  
Mariangela Pedata ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Elderly Patients ◽  
Myeloid Leukemia ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Flt3 Mutations ◽  
Acute Myeloid

scholarly journals BCOR Mutations in Acute Myeloid Leukemia: Clonal Evolution and Prognosis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Ashley Zhang ◽  
Yuntao Liu ◽  
Shuning Wei ◽  
Benfa Gong ◽  
Chunlin Zhou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Risk Stratification ◽  
Myeloid Leukemia ◽  
Statistical Difference ◽  
Tp53 Mutation ◽  
Clonal Evolution ◽  
Normal Karyotype ◽  
Abnormal Karyotype ◽  
Acute Myeloid

Background BCOR gene is a transcription repressor that may influence normal hematopoiesis and is associated with poor prognosis in acute myeloid leukemia (AML) with normal karyotype. However, due to the rare mutation frequency in AML (3.8%-5%), clinical characteristics and prognosis of AML patients with BCOR mutation including abnormal karyotype are still unknown. In addition, the clonal evolution of AML patients with BCOR mutation has not been fully investigated. Methods By means of next generation of sequencing, we performed sequencing of 114 genes related to hematological diseases including BCOR on 509 newly diagnosed AML patients (except for acute promyelocytic leukemia) from March 2017 to April 2019. The 2017 European Leukemia Net (ELN) genetic risk stratification was used to evaluate prognosis. Overall survival (OS) was defined as the time from diagnosis to death or last follow-up. Relapse-free survival (RFS) was measured from remission to relapse or death. Clonal evolution was investigated through analyzing bone marrow samples at diagnosis, complete remission (CR) and relapse from the same patient. Result Among 509 AML patients, we found BCOR mutations in 23 patients (4.5%). BCOR mutations were enriched in patients with mutations of RUNX1 (p = 0.008) and BCORL1 (p = 0.0003). Patients with BCOR mutation were more at adverse ELN risk category compared to patients without BCOR mutation (p = 0.007). Besides, there was a larger proportion of patients with normal karyotype in BCOR mutation group but it had not reached statistical difference (62.5% vs 45.5%, p = 0.064). The abnormal karyotype in patients with BCOR mutations included trisomy 8, t(9;11), inv(3), -7 and complex karyotype.There were no significant differences in age, sex, white blood cell count, hemoglobin or platelet count between the two groups. More patients died during induction (13.0% vs 3.5%, p = 0.56) and fewer patients achieved CR after 2 cycles of chemotherapy when patients had BCOR mutations (69.6% vs 82.5%, p = 0.115) but the difference had not reached statistical difference . Patients with BCOR mutations had inferior 2-year OS (52.1% vs 70.7%, p = 0.0094) and 2-year RFS (29.8% vs 61.1%, p = 0.0090). After adjustment for ELN risk stratification, BCOR mutation was still remain a poor prognostic factor. However, the adverse prognostic impact of BCOR mutation is overcome by hematopoietic stem cell transplantation (HSCT), in which there was no difference between BCOR mutation group and wild type group (p = 0.474) (Figure 1). Through analysis of paired bone marrow sample at diagnosis, remission and relapse, we revealed the clonal evolution that BCOR mutation was only detected at diagnosis sample as a subclone and diminished at CR and relapse while TP53 mutation was only detected at relapse with a variant allele frequency (VAF) of 25.5%. We also found BCOR mutation at another patient's diagnosis and relapse sample while TP53 mutation was detected at relapse with VAF of 11.8%. Conclusion BCOR is associated with RUNX1 mutation and higher ELN risk. AML patients with BCOR mutation including normal and abnormal karyotype conferred a worse impact on OS that can be overcome by HSCT. BCOR mutation is a subclone at diagnosis or relapse in some patients, in which TP53 mutation clone occurred at relapse. Disclosures No relevant conflicts of interest to declare.

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