Development of COLD-PCR assay combined with HRM analysis for nucleophosmin1 gene mutation detection in acute myelogenous leukemia

Abstract

Mutations of nucleophosmin1 (NPM1) gene represent the most frequent molecular alteration in acute myelogenous leukemia (AML), especially in the AML patients with normal karyotype. These alterations have been shown to carry favorable prognostic significance in AML because their clinical importance in terms of risk assessment and treatment decision ns in AML patients. Several methods have been developed for detection of NPM gene mutations. However, most of which are expensive, technically challenging, complicated, and require additional laboratory equipment that is not available in some laboratories with limited resources. More importantly, their inability to detect low levels of mutations in a wild-type background is limited. In this study, E-ice-COLD-PCR assay combined with HRM analysis for NPMI gene mutations detection were developed and validated for highly specific, short time-to-result, and sensitive screening for detection of NPMI gene mutations with easier result interpretations. Blood samples from 23 patients who were diagnosed with AML at Maharaj Nakorn Chiang Mai hospital were collected and then DNAs were extracted. For mutational analysis, E-ice-COLD-PCR assay for detection of NPMI gene mutations was developed that cover 4 mutation types (type A, B, D, and J). These four types are the most prevalent mutations found in Thailand. PCR products were then analyzed by HRM analysis and detected NPMI gene mutations using 2% agarose gel electrophoresis. All of positive samples were confirmed by direct sequencing in both directions. The E-ice-COLD-PCR assay with HRM analysis enabled detection specificity and sensitivity of NPMI mutations in 2/23 AML patients. The specificity was confirmed in the positive samples by direct sequencing analysis and the results were 100 % concordant with this method. In addition, the limit of detection (LOD) was 12.5% mutant in final concentration of 5 ng genomic DNA. This study concluded that E-ice COLD-PCR assay with HRM analysis is highly specific and sensitive screening method for enrichment of detecting NPMI gene mutations. This method is short time-to-results and easier interpretation of the results. Moreover, performing E-ice-COLD-PCR prior to direct sequencing allows substantially increasing the limit of detection of direct sequencing. In addition, E-ice-COLD PCR protocol can easily be integrated in the routine molecular diagnosis of AML using standard laboratory equipment.