A Simple, Rapid, and Sensitive Method for the Detection of the JAK2 V617F Mutation

Angela Y.C. Tan, PhD; David A. Westerman, MBBS, FRACP, FRCPA; Alexander Dobrovic, PhD

Disclosures

Am J Clin Pathol. 2007;127(6):977-981. 

In This Article

Discussion

The ACB-PCR (first described by Orou et al[19]) is based on the amplification refractory mutation system method[20] in which relevant alleles are targeted using specific primers. Allele-specific assays such as the amplification refractory mutation system are efficient techniques for the detection of monomorphic mutations because they are simple and easy to perform. However, standard allele-specific assays can be prone to mispriming.[12,21,22] Therefore, we chose to adopt an ACB-PCR assay to detect the V617F JAK2 mutation. In an ACB-PCR assay, 3 primers are combined in a single tube, a forward primer with a modified 3' end, a second forward primer that targets the mutant allele, and a reverse primer. Under optimized conditions, only the mutant allele (the allele of interest) is amplified, and owing to the 3' modification of the normal primer, amplification of the normal allele is inhibited.

Theoretically, any blocking group can be incorporated at the 3' end of the normal primer to prevent amplification, ie, a dideoxynucleotide. In this study, a phosphate group was selected because it was an inexpensive modification that was widely available from commercial oligonucleotide companies.

Similar to any PCR test, some optimization of the ACB-PCR assay was required but was minimal owing to the attachment of the 3' phosphate group on the normal primer. Under optimal routine conditions, the ACB-PCR assay was able to detect the V617F mutation at the level of 1% or approximately 1 mutant cell per 100 normal cells. This high level of detection sensitivity was equivalent to that of previous allele-specific assays.[18]

The advantage of such a high detection level of the ACB-PCR assay was made apparent when studying a cohort of ET cases. In 3 ET cases, the V617F mutation would not have been detected by sequencing because the mutant peak was at the level of background on the chromatograms. Low levels of V617F in patients with ET may be due to heterogeneity in the granulocyte population, and the potential to miss the V617F mutation in some patients with ET using less sensitive techniques has been reported.[23,24]

We detected the JAK2 V617F mutation in 6 of 8 patients with PRV, 10 of 15 with ET, and 1 of 2 with IMF. The pattern of incidence in each of the disease states is consistent with previous reports.[2,3,4,5,7] The mutation was not present in the patient with postpolycythemic myelofibrosis and myeloid metaplasia.

The ACB-PCR assay described in this article has subsequently been implemented in the routine diagnostic laboratory of the Pathology Department, the Peter MacCallum Cancer Centre. Currently, the V617F mutation has been identified in 15 (83%) of 18 patients with PRV, 14 (56%) of 25 with ET, and 5 (71%) of 7 with IMF. The ACB-PCR assay is rapid and highly sensitive. It is a suitable technique for V617F mutation detection with wider appeal for routine diagnostic laboratories because it uses standard equipment, has few sample manipulation steps, and can be performed directly on DNA extracted from peripheral whole blood samples.

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