ABSTRACT
This study was aimed to establish the PCR methods to detect nucleophosmin (NPM) gene and its mutation. 2 leukemia cell lines and 23 specimens from patients with acute myelogenous leukemia (AML) were investigated. The level of NPM mRNA was detected by RT-PCR. The exon-12 of NPM gene in leukemia cell lines was amplified by PCR and sequenced. Using the plasmid containing cDNA of NPM mutation A as a positive template, the PCR procedure to detect mutation A was established and evaluated. Then, the mutation of NPM was analyzed in 23 AML specimens. The results indicated that the expression level of NPM in leukemia cell lines was higher than that in normal cells. Different overexpression levels of NPM mRNA were found in all 23 AML specimens. PCR indicated that mutation had been not occurred at NPM exon-12 in THP1 and K562 cells, but a T base was deleted at 3' untranslated region of NPM gene in K562 cells. The PCR used for directly detecting NPM mutation A can specially amplify the NPM mutation gene. The method was reproducible, whose coefficient of variability was 1.6% and 3.1% in intra-and inter-assays respectively. The lowest detectable limit was 100 pg cDNA. Using the PCR methods, NPM mutation A could be detected in 2 out of 23 AML specimens, but NPM mutation A was not found in THP1 and K562 cells. It is concluded that the RT-PCT method detecting NPM mRNA level and the PCR method detecting directly NPM mutation are established. NPM mRNA is overexpressed in leukemia cells; NPM mutation A occurs in some AML patients.