Simple, Gel-Based Determination of Nucleophosmin Mutations in Acute Myeloid Leukemias.

Mutations in the Nucleophosmin, member 1 (NPM1) gene, have been shown to be significant for prognosis and treatment of cytogenetically normal (CN) patients with acute myeloid leukemia (AML). To increase sensitivity for minimal residual disease (MRD) detection, especially in AMLs without a distinct phenotype or genetic marker, some assays have specifically targeted the individual mutation, which has led to an increased complexity of these tests. Other assays use expensive equipment such as capillary electrophoresis to differentiate between the usual 4bp size difference in the mutated gene and the wildtype(wt) gene. We developed a simple PCR-based procedure that amplifies the exon 12 region of the NPM1 gene and produces a small product (156bp for the wt and 160 for the mutant genes) using RNA that can be separated on a 4% metaphor agarose gel. This approach is clinically relevant since it can easily distinguish greater than 95% of the mutations. Eight of 24 AMLs of various types were positive for the mutation using our technique and those that were cloned and sequenced yielded the most common insertion TCTG, confirming its accuracy.