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

Abstract

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...

A monoclonal antibody against mutated nucleophosmin 1 for the molecular diagnosis of acute myeloid leukemias

“…7 Currently available methods to detect NPM1 mutations include DNA sequencing, real-time quantitative polymerase chain reaction (PCR), denaturing high-performance liquid chromatography, capillary electrophoresis, locked nucleic acid-mediated PCR clamping, and allele-specific reverse-transcribed PCR assays. [10][11][12][13][14][15][16][17][18] Although highly specific, these methods are expensive and laborious. Alternatively, NPM1 mutations can be identified indirectly, using antibodies that recognize epitopes that are common for both the wild-type (wt) and mutant NPM1 proteins and allow recognition of the cytoplasmic dislocation of NPM1.…”

Genetic Tests To Evaluate Prognosis and Predict Therapeutic Response in Acute Myeloid Leukemia

“…Alternative molecular methods are feasible. 12,71,72 At least 40 molecular variants exist, most of which result in a 4-bp enlargement, although rarely an amplicon up to 11 bp larger than normal is seen. Frameshift mutations interfere with the nucleolar localization motif in the C-terminal end of the protein.…”

Detection of Exon 12 Mutations in the JAK2 Gene

“…24,25 We previously used such a strategy to improve the analytical sensitivity of an assay for NPM1 mutations in acute myeloid leukemia. 26 For the JAK2 exon 12 mutations, we aimed for and achieved a higher level of analytical sensitivity mostly by increasing the concentration of the clamp. Using the clamp at a concentration of 1 mol/L, we were able to detect the JAK2 exon 12 mutations N542-E543del and F537-K539delinsL when the heterozygous patient specimen DNA was diluted in a 1000-fold excess of normal DNA (Figures 1 and 2; data not shown).…”