A denaturing high-performance liquid chromatography (DHPLC) assay could be used to identify NPM1 mutations in leukemia

11-14-2006

According to recent research from Italy, "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+)," explained G. Roti and colleagues, University of Perugia. "Here, we designed and tested a 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."

The researchers concluded, "Our results suggest that DHPLC detects NPM1 mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML."

Roti and colleagues published their study in the Journal of Molecular Diagnostics (Denaturing high-performance liquid chromatography - A valid approach for identifying NPM1 mutations in acute myeloid leukemia. J Mol Diagn, 2006;8(2):254-259).

For additional information, contact C. Mecucci, University of Perugia, Laboratory of Cytogenetics and Molecular Genetics, I-06123 Perugia, Italy.