Molecular diagnostics for the detection and characterization of genetic variants is based in the majority of cases on PCR technology. Since the primers and probes are designed according to the nucleotide sequence of the DNA region for the specific mutation, PCR technology can only be applied if the mutation is well known. In cases where the mutated region of a gene may have variable sequences, different between patients, a set of primers for each sequence variant is necessary, which involves a large volume of work. The nucleophosmin (NPM1) gene mutation is also included in this category where more than 24 variants of the gene are known of which 6 are more common.
The aim of our study was to establish a qPCR assay and a molecular kit (a set of primers) for detection and quantification of NPM1 gene mutations regardless of its sequence by using RNase H-dependent PCR (rhPCR). Several sets of rh-primers that show point mismatches in relation to some mutations have been designated and tested. The rh-primers carried a removable PCR blocker at the 3′ end. Only after the removal of this blocker by the RNaseH2 enzyme, the primers are expandable by DNA polymerase. qPCR was used to determine the extent to which the presence of these sequence mismatches could affect the H2-endonuclease activity of ribose digestion and PCR activation. The evaluation was carried out in a preclinical context, using synthetic fragments of NPM1 carrying mutations (AML mutation type A, B, D, I, K).
After establishing the optimal combination for the primer sets as well as the reaction conditions, the sensitivity limit was tested in samples with the number of target copies that varied decreasingly: 1000000, 100000, 10000, 1000, 100, 10. It was observed that regardless of the nature of the mutation, the same set of primers can detect 10 copies of mutant NPM1.
The main advantage of this assay is that different sequence-variable NPM1 mutations can be detected and quantified with high specificity through a single rhPCR reaction. It is no longer necessary to design, synthesize and optimize primers and probes for each individual mutation. Our study demonstrates the potential use of rhPCR for monitoring patients with AML for minimum residual disease and recurrence risk.
Victor Babes Institute of Pathology.
National Program 1N/2019/PN19.29.01.03.
All authors have declared no conflicts of interest.