Rapid screening and sensitive detection of NPM1 (nucleophosmin) exon 12 mutations in acute myeloid leukaemia

Scholl, Sebastian; Mügge, Lars-Olof; Landt, Olfert; Lončarević, Ivan F.; Kunert, Christa; Clement, Joachim H.; Höffken, K.

doi:10.1016/j.leukres.2006.12.011

Cited by 32 publications

(16 citation statements)

References 21 publications

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“…Alleles A and B have been reported to account for 78% and 12% of all NPM1 mutations, respectively 36. (2) Deletion of 4 or 5 nucleotides and insertion of 9 new nucleotides in that position, like alleles E and F. (3) Deletion of 9 nucleotides and insertion of 14 new nucleotides.…”

Section: Discussionmentioning

confidence: 99%

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia

Pazhakh

Zaker

Atashrazm

2011

Annals of Saudi Medicine

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BACKGROUND AND OBJECTIVES:Nucleophosmin gene mutations are frequently reported in acute myeloid leukemia (AML) patients with normal karyotype, which is also frequently associated with internal tandem duplication mutations in the FMS-like tyrosine kinase-3 gene. We sought to detect the nucleophosmin and FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) mutations among Iranian patients with AML and to assess the relationship between these mutations and the subtypes of the disease.DESIGN AND SETTING:Cross-sectional study of patients referred during 2007 through 2009.PATIENTS AND METHODS:Bone marrow and peripheral blood samples of 131 AML patients were randomly collected at the time of diagnosis and prior to treatment and the DNA extracted. After amplifying the nucleophosmin and FLT3 gene regions, positive cases were screened by conformation-sensitive gel electrophoresis and agarose gel electrophoresis techniques.RESULTS:Of 131 patients, 23 (17.5%) (0.95% CI=0.107-0.244) had nucleophosmin gene mutations. The highest frequency of such mutations was found among the subtypes of M4 (30.4%), M3 (21.7%) and M5 (17.4%). There was a high frequency of these mutations in the M3 subtype as well as a high frequency of allele D in all subtypes. Also, 21 (16.0%) samples (0.95% CI=0.092-0.229) had FLT3/ITD mutation, of which 8 samples had mutant nucleophosmin (8 of 23, 35%), and another 13 samples had wild-type nucleophosmin gene (13 of 108, 12%). There was a high degree of association between the occurrence of nucleophosmin and FLT3/ITD mutations (P=.012).CONCLUSION:Our data showed a high frequency of NPM1 mutations in the monocytic subtypes of AML, as well as a high degree of association between the occurrence of NPM1 and FLT3/ITD mutations.

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Section: Discussionmentioning

confidence: 99%

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia

Pazhakh

Zaker

Atashrazm

2011

Annals of Saudi Medicine

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“…The fragment analysis method is one of the most commonly used for detection of NPM1 gene mutations because the mutations almost always involve a net insertion of four nucleotides. 24,34,35 In the heteroduplex method, after amplification using the PCR, Figure 1. Graph of Ϫd(F1)/dT versus temperature showing that the use of a LNA PCR clamp enhances the sensitivity of the NBQ assay.…”

Section: Resultsmentioning

confidence: 99%

Rapid Method for Detection of Mutations in the Nucleophosmin Gene in Acute Myeloid Leukemia

Laughlin

Becker

Liesveld

et al. 2008

The Journal of Molecular Diagnostics

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Mutations in exon 12 of the nucleophosmin gene (NPM1) that cause the encoded protein to abnormally relocate to the cytoplasm are found at diagnosis in about 50% of karyotypically normal acute myeloid leukemias and are associated with a more favorable outcome. We have devised a PCR-based assay for NPM1 exon 12 mutations using differential melting of an oligo probe labeled with a fluorescent dye. The nucleobase quenching (NBQ) phenomenon was used to detect probe hybridization, and an oligonucleotide containing locked nucleic acid (LNA) nucleotides was used as a PCR clamp to suppress amplification of the normal sequence and enhance the analytical sensitivity of the assay. After the NBQ assay, the specimens with a mutation were removed from the capillary and sequenced to identify the mutation. The use of the LNA clamp facilitates interpretation of the mutant sequence because of the lower intensity of the overlapping normal sequence. Analysis of a series of 70 patient specimens revealed 17 positive for an NPM1 mutation and 53 negatives. All of the NBQ results (positives and negatives) were confirmed with other methods. The analytical sensitivity of the NBQ assay is variable depending on the concentration of the PCR clamp and other parameters. Using a 100 nmol/L concentration of the LNA clamp, NPM1 mutations were detectable in a 10-fold excess of wild-type DNA. This assay may be valuable for screening disease specimens. Nucleophosmin is a member of a family of chaperone proteins and is normally located in the nucleolus, where it appears to have a role in the maturation of ribosomes. The nucleophosmin gene (official symbol: NPM1; also known as nucleolar phosphoprotein B23 and numatrin) has been found mutated in about 50% of newly diagnosed adult acute myeloid leukemia (AML) patients with a normal karyotype.2 It has also been shown to be a partner gene in several translocations including t(2; 5)(p23;q35) in anaplastic large-cell non-Hodgkin's lymphoma, 3 t(5;17)(q32;q12) in rare cases of acute promyelocytic leukemia, 4 and t(3;5)(q25.1;q34) in AML and myelodysplastic syndrome. 5 The mutations found in AML almost always involve a 4-bp insertion in a limited region of exon 12. The insertions cause a change in the reading frame, the destruction of the nucleolar localization signal, and the creation of a carboxy terminus that completes a nuclear export signal.6 As a result, the NPM1 protein relocalizes to the cytoplasm (NPMc ϩ ).6,7 Exceptions have been reported, including two AMLs with NPMc ϩ having an 8-nucleotide insertion in exon 11. 8,9 The most common mutation, called type A, is a duplication of 4 nucleotides after position 863 in the cDNA (c.860_863dupTCTG) and accounts for about 75% of the mutations in adults, but a lower fraction of the pediatric cases.10,11 NPM1 with AML-type mutations has been shown to gain the ability to transform primary mouse embryonic fibroblasts, probably due to inactivation of Arf. 12NPM1 mutations are associated with normal karyotype, internal tandem duplication in the juxtamembrane region...

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“…In addition, the sensitivity of this method is low, with the lower limit of detection beginning at ~20% (10). Therefore, polymerase chain reaction (PCR) methods such as electrophoresis (11), melting curve analysis (12), high-resolution melting (HRM) analysis which detects the different melting temperatures of the PCR products (13), locked nucleic acid clamp-mediated PCR (10,14), and capillary electrophoresis (15,16) have been investigated as potentially more sensitive methods for detection of this 4 bp mutation.…”

Section: Introductionmentioning

confidence: 99%

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells

et al. 2016

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Abstract. Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61˚C and 68˚C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68˚C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.

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Rapid screening and sensitive detection of NPM1 (nucleophosmin) exon 12 mutations in acute myeloid leukaemia

Cited by 32 publications

References 21 publications

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia

Rapid Method for Detection of Mutations in the Nucleophosmin Gene in Acute Myeloid Leukemia

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells

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