title: Denaturing high-performance liquid chromatography: a valid approach for identifying NPM1 mutations in acute myeloid leukemia.
abstract: NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.
authors.affiliation: Laboratory of Cytogenetics and Molecular Genetics, University of Perugia, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Roti
authors.forename: Roberto
authors.initials: R
authors.lastname: Rosati
authors.forename: Rossella
authors.initials: R
authors.lastname: Bonasso
authors.forename: Paolo
authors.initials: P
authors.lastname: Gorello
authors.forename: Daniela
authors.initials: D
authors.lastname: Diverio
authors.forename: Massimo Fabrizio
authors.initials: MF
authors.lastname: Martelli
authors.forename: Brunangelo
authors.initials: B
authors.lastname: Falini
authors.forename: Cristina
authors.initials: C
authors.lastname: Mecucci
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003593:Cytoplasm / Q000235:genetics; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D000090243:Nucleophosmin
chemical_list: C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin