Validating the Sensitivity of High-Resolution Melting Analysis for JAK2 V617F Mutation in the Clinical Setting

Abstract

Background: Janus kinase 2 (JAK2) plays an important role in normal hematopoietic growth factor signaling. The detection of the JAK2 V617F mutation (c.1849GNT, GTC → TTC) is crucial for the diagnosis of myeloproliferative neoplasm (MPN) and has become the essential criteria for diagnosis of MPN by the WHO. High-resolution melt (HRM) curve analysis is a nongel-based, closed-tube method, in which PCR amplification and subsequent analysis are sequentially performed in the well, making it more convenient than other scanning methodologies.

Methods: We evaluated JAK2 V617F mutation by HRM. Twenty-nine patients diagnosed with MPN were examined. We studied the analytical sensitivity of the HRM analysis using real-time polymerase chain reaction (PCR) for identifying the JAK2 V617F mutation. Additionally, the sensitivity of HRM analysis and allele-specific PCR (AS-PCR) assay was compared.

Results: The JAK2 V617F mutation was successfully discriminated at an abundance of 6% or above in HRM analysis. Both HRM analysis and AS-PCR showed 100% accuracy with detection limits of 6% and 2.5%, respectively.

Conclusion: HRM analysis is a fast, simple, reliable, and nonexpensive method for the detection of the JAK2 V617F mutation. However, more validation of the detection limits of HRM analysis should be performed before declaration of the analytic sensitivity of the method.

Keywords: JAK2; high-resolution melt curve; myeloproliferative neoplasm.

Figure 1

AS‐PCR assay. (A) Identification of the JAK2 V617F mutation. M, DNA 100‐bp ladder marker; PC, positive control; NC, negative control; B, reagent blank control; S1, sample 1 (mutant type); S2, sample 2 (wild type). (B) Sequence data (left, wild type; right, mutant type). (C) AS‐PCR analysis of mixtures wild type (G allele) and mutant type (T allele) (M, DNA ladder marker; 1: 0%; 2: 1%; 3:2.5%; 4: 5%; 5: 6%; 6: 7%; 7: 8%; 8: 10%; 9: 15%; 10: 20%; 11: 25%; 12: 50%; and 13: 100% mutant type).

Figure 2

HRM analysis. (A) LOD analysis mixed the wild‐type DNA (G allele) with the DNA of the mutant type (T allele) to attain different mutant concentrations (0%, 1%, 2.5%, 5%, 7%, 10%, 15%, 20%, 25%, 50%, and 100% mutant type). (B) LOD analysis mixed the wild‐type DNA (G allele) with the DNA of the mutant type (T allele) to attain different mutant concentrations (6%, 7%, and 8% mutant type). (C) Precision analysis of 6% JAK2 V617F mutation samples from three different patients (patients A, B, and C). (D) Sensitivity and specificity analysis for 24 MPD patients and 20 healthy individual patients.