title: Establishment of a quenching probe method for detection ofNPM1mutations in acute myeloid leukemia cells.
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.
authors.affiliation: Department of Laboratory Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan; Department of Health Sciences, Saitama Prefectural University, Koshigaya, Saitama 343-8540, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Kawaguchi-Ihara
authors.affiliation: Department of Laboratory Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Itoh
authors.affiliation: Department of Health Sciences, Saitama Prefectural University, Koshigaya, Saitama 343-8540, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Murohashi
authors.affiliation: Department of Laboratory Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Tohda
keywords: NPM1; PCR; acute myeloid leukemia; melting curve analysis; quenching probe