Detection of Exon 12 Mutations in the JAK2 Gene

Abstract

JAK2 V617F is the most frequently found somatic mutation in polycythemia vera (PV). Among the cases negative for V617F , a significant fraction have a mutation in exon 12 of the JAK2 gene. Several groups have reported that the exon 12 mutations are present in only a small fraction of the blood cells in some patients. We have developed an assay to detect these mutations with an analytical sensitivity of 0.1% by using a "PCR clamp" to inhibit amplification of the normal sequence and enhance amplification of DNA containing a mutation in the clamp target sequence. The products of this reaction were analyzed by capillary electrophoresis to detect deletions , which are the most frequent type of exon 12 mutations , or by nucleotide sequencing to detect all of the mutations. In a survey of 34 specimens from patients with PV or idiopathic erythrocytosis who did not have a JAK2 V617F mutation , we found four with a mutation in exon 12 , 3 of 10 with PV , and 1 of 24 with idiopathic erythrocytosis. In two cases the mutation was present in a small fraction of the cells and difficult to detect without the use of the clamp. The use of an assay with increased analytical sensitivity enhances the ability to identify patients with mutations in exon 12

Requirements for Efficient PCR Clamping by Locked Nucleic Acid Oligonucleoties for Simple and Sensitive Detection of Somatic Mutations

“…Different studies use LNA-based PCR clamps either in 2-step PCR with 60-65°C annealing/ extension temperature [2,3,10] or in classic 3-step PCR protocol with 56-64°C annealing and 72°C extension temperature [1,4,9,11]. We speculated Figure 3.…”

“…A limitation of Sanger sequencing after PCR clamping were occasional mutation artifacts on wild-type DNA. The problem was previously reported [4,10] and is believed to result from mistakes of Taq DNA polymerase. The problem can be solved by the use of DNA polymerase with proofreading activity instead of Taq DNA polymerase [10].…”

“…The problem was previously reported [4,10] and is believed to result from mistakes of Taq DNA polymerase. The problem can be solved by the use of DNA polymerase with proofreading activity instead of Taq DNA polymerase [10]. Other potential options are a hi-fidelity version of Taq DNA polymerase [16], or use of less PCR cycles with PCR clamp.…”

“…It was proposed that clamping oligonucleotide can be degraded by the 5'-exonuclease activity of Taq DNA polymerase, and for efficient PCR clamping, the Stoffel fragment of DNA polymerase without 5'exonuclease activity is required [1,8]. Alternatively, some studies used Taq DNA polymerase and LNA oligonucleotides protected at the 5'-end with phosphorothioate modifications or employed Pfu DNA polymerase and its variants without 5'-exonuclease activity [9][10][11]. However, successful PCR clamping was also reported with regular Taq DNA polymerase and unprotected LNA oligonucleotides [2][3][4].…”

Myeloproliferative neoplasms and the JAK/STAT signaling pathway: an overview

“… 12 Besides the g.1849 G > T (p.V617F) mutation, 5 , 6 other mutations were found in exon 12 in 2007 namely: p.N542-543del, p.E543-D544del, p.K539L, p.F537-K539delinsL, p.H538-p.K539delinsL, p.H538QK539L, p.V536-1546dup11, p.F537-1546dup10+F547L, p.R541 E543delinsK-and-p.I540 E543delinsMK. 21 , 22 , 23 These were found to show low frequencies of about 3–4% in PV patients. There are valid techniques that detect these mutations and their active search is justified not only due to the absence of the exon 14 mutation, but also in patients that have erythrocytosis and in those with low levels of erythropoietin.…”