Development of E-ice-COLD-PCR assay combined with HRM analysis for Nucleophosmin1 gene mutation detection in acute myelogenous leukemia

Abstract

Mutations of the nucleophosmin1 (NPM1) gene represent the most frequent molecular alteration in acute myelogenous leukemia (AML), especially in patients with AML who have a normal karyotype. These alterations have been shown to carry favorable prognostic significance in patients with AML. Several methods have been developed for detection of NPM1 gene mutations. However, their ability to detect low levels of mutations in a wild-type background is limited. In this study, the Enhance improved and complete enrichment Co-amplification at Lower Denaturation temperature Polymerase Chain Reaction (E-ice-COLD-PCR) assay combined with High Resolution Melting (HRM) analysis was developed and validated for highly specific and sensitive screening for NPM1 gene mutations. A total of 83 blood samples from patients with AML were collected, and their DNA was extracted. For mutational analysis, the E-ice-COLD-PCR assay for the detection of NPM1 gene mutations was developed. PCR products were analyzed by HRM analysis. All positive samples were confirmed by direct sequencing. This assay enabled detection specificity and sensitivity of NPM1 mutations in 9/83 patients with AML. Direct sequencing results were 100% concordant with this method. In addition, the limit of detection was 12.5% mutant in the final concentration of 5 ng genomic DNA. The E-ice-COLD-PCR assay with HRM analysis is a highly specific and sensitive screening method for enrichment of detecting NPM1 gene mutations. This method has both a short turn around time and easier interpretation compared to those of other methods.

Fig 1. Detecting NPM1 gene mutations in patient samples using a standard PCR assay.

The PCR products are visualized on 10% polyacrylamide gel. MT indicates NPM1 mutated control and WT indicates NPM1 wild-type control. A double band indicates a heterogeneous mutation.

Fig 2. HRM difference curves of Tc determinations.

(a) The height of the difference curve for mutants increased with increasing Tc from 80–90°C. (b) No signals of wild-type products were produced when increasing the Tc from 80–88°C, while the wild-type was amplified when increasing the Tc to more than 88°C and presented the signal in the HRM difference curve.

Fig 3. The E-ice-COLD-PCR assay combined with HRM analysis of four types of NPM1 mutations.

Following the E-ice-COLD-PCR assay, the PCR products were analyzed by HRM analysis. The HRM difference curves show the peaks of NPM1 mutation types A, B, D, and J, while no peak in the NPM1 wild-type indicated that the E-ice-COLD-PCR assay can inhibit wild-type amplification. Meanwhile, it can enhance the amplification of the four types NPM1 mutations.

Fig 4. LOD of the E-ice-COLD-PCR assay combined with HRM analysis.

Following the E-ice-COLD-PCR assay, the PCR products were analyzed by HRM analysis. The HRM difference curves show the height of the curves decreased with the decreasing percentage of mutants, and the peak disappeared with the decreasing percentage of mutants from 6.25% to 0%.

Fig 5. Detecting NPM1 gene mutations by HRM analysis.

Detecting NPM1 gene mutations using the E-ice-COLD-PCR assay and generating the data into HRM difference curves. Red line indicates mutant control. Green line indicates wild-type control. The wild-type samples present the peak at the same location as the wild-type control, and the mutant samples present the peak at the same location as the mutant control.

Fig 6. Direct sequencing in the reverse direction of mutated products.

(a) Mutated products from the standard PCR assay (b) Mutated products from the E-ice-COLD-PCR assay. The results showed that the NPM1 mutation type A is a 4 bp insertion.