Abstract
Mutations of nucleophosmin (NPM1) gene represent the most frequent molecular alteration in acute myelogenous leukemia (AML), especially in the AML patients with normal karyotype. Moreover, NPM1 gene mutations have been shown to carry favorable prognostic significance in AML. Therefore, Screening of NPM1 mutations show valuable clinical importance in terms of risk assessment and treatment decisions of physician in AML patients. In this study, we developed a in house test kit for detection of NPM1 gene mutation cover 4 types (type A, B, D and type J) in 1 reaction based on Enhanced-improved and complete enrichment-co amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) combined with High-resolution melting (HRM) analysis. Method: For E-ice-COLD-PCR, blocker probe is very importance to block wild-type amplification. Blocker probe hybridized complete match with wild-type sequence while incomplete match with mutant sequence. After critical temperature step in each cycle, blocker probe allows completely denature mutant-blocker probe heteroduplex while wild-type-blocker probe heteroduplex remain strongly hybridized, which only allow the amplification of mutant. The PCR reaction was performed using 1x NPM1 HRM master mix, 400 nM of NPM-LNA probe, 5 ng of genomic DNA and sterile water with a total reaction volume of 25 μL. The thermocycling program consisted of an initial denaturation at 95°C for 3 min followed by 40 cycles of denaturation at 95°C for 5 s, probe hybridization at 70°C for 30 s, critical denaturation at 88°C for 40 s, primer annealing at 60°C for 10 s and extension at 72°C for 30 s, with a final extension at 72°C for 3 min. The final step was melting curve with continuous increase in temperature from 65°C to 85°C (0.2°C per acquisition, 5 s hold before each acquisition) by using CFX96 Touch Real-Time PCR or Mini MJ Thermal Cycler (conventional PCR machine) A total of 83 AML blood samples from Maharaj Nakorn Chiang Mai Hospital were evaluated by NPM1 HRM master mix, standard PCR and direct sequencing. Results: we show herein 2x NPM1 HRM master mix exhibited similar sensitivity and specificity with direct sequencing. NPM1 mutations were detected in 9 of 83 AML patients with 2x NPM1 HRM master mix. Moreover, The PCR products from E-ice-COLD PCR were confirmed by direct sequencing. The result show that 100% concordance with E-ice-COLD-PCR assay and direct sequencing with type A mutation and easily to interpretation. Interestingly, The PCR products from standard PCR show that 8 of 83 patients were positive. One sample showed inconsistent result between standard PCR assay and direct sequencing. Perhaps, the low level of mutations in wild-type background interferes the direct sequencing led to verification of the mutation sample as wild-type. Therefore, performing E-ice-COLD-PCR prior to direct sequencing allow substantial increasing the limit of detection of direct sequencing. Conclusions: Compared to standard PCR and direct sequencing, a new test kit, 2x NPM1 HRM master mix was shown to detect mutations in the NPM1 gene cover 4 types in 1 reaction. Moreover, E-ice-COLD- PCR is a PCR-based method that can be use for enrichment low level of mutated in mixtures of wild-type and mutant allele. E-ice-COLD PCR protocol can easily be integrated in the routine molecular diagnosis of AML using Real-time PCR or conventional PCR machine.
