Comparative study of different methodologies to detect the JAK2 V617F mutation in chronic BCR-ABL1 negative myeloproliferative neoplasms

Abstract

ObjectivesA mutation in the JAK2 gene, V617F, has been identified in several BCR-ABL1 negative myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Defining the presence or absence of this mutation is an essential part of clinical diagnostic algorithms and patient management. Here, we aimed to evaluate the performance of three PCR-based assays: Amplification Refractory Mutation System (ARMS), High-Resolution Melting analysis (HRM), and Sanger direct sequencing, and compare their results with those obtained by a PCR restriction fragment polymorphism assay (PCR-RFLP).Design and methodsWe used blood samples from 136 patients (PV=20; PMF=20; ET=28, and other MPN suspected cases=68).ResultsComparable results were observed among the four assays in patients with PV, PMF, and MPN suspected cases. In patients with a diagnosis of ET, the JAK2 V617F mutation was detected in 67.8% of them by the PCR-ARMS and PCR-HRM assay and in 64% of them by the conventional Sanger sequence approach. The PCR-ARMS and PCR-HRM assays were 100% concordant. With these tests, only one of the 20 patients with ET and one of the three patients with clinically suspected MPN gave different results compared with those obtained by the PCR-RFLP.ConclusionsOur results have demonstrated that the PCR-ARMS and PCR-HRM assays could detect the JAK2 V617F mutation effectively in MPN patients, but PCR-HRM assays are rapid and the most cost-effective procedures.

Systematization of analytical studies of polycythemia vera, essential thrombocythemia and primary myelofibrosis, and a meta-analysis of the frequency of JAK2, CALR and MPL mutations: 2000–2018

“…In their combined measurement, an OR of 3.5 was obtained (95% CI = 1.6–7.7). Eight studies used the WHO 2008 criteria [41, 48–51, 54, 62, 66], for which an OR of 4.7 (95% CI = 3.1–7.5) was found. Three studies used WHO 2008 criteria and AS-PCR, and their combined measurement was an OR of 3.8 (95% CI = 1.6–8.3).…”

Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study

“…Applied to ddPCR, LoD could be defined as the smallest number of target nucleic acid molecules that is statistically distinguishable from the background or negative control. One approach for estimating LoD and LoB for a ddPCR assay has been experimentally demonstrated in a study to determine lower limits of detection for cancer-related mutations [33,34]. As is well-known, accurate quantification of nucleic acid molecules by ddPCR is based on the correct sorting of single droplets into negative and positive populations, since the proportion of positive partitions is an integral component of the equation used to determine copy number concentration.…”

Sequence analysis of theN-acetyltransferase 2 gene (NAT2) among Jordanian volunteers

“…Previous studies on NAT2 gene among Jordanians genotyped major NAT2 genetic polymorphisms using the PCR-RFLP method [11,28]. It has been reported that DNA sequencing is the golden method for genotyping [29], while the PCR-RFLP technique has large errors with a higher probability of false-positive results [30]. Some studies have found that the PCR-RFLP technique is less accurate than other genotyping assays, such as direct DNA sequencing and pyrosequencing [31–33].…”