PCR artifacts can explain the reported biallelic JAK2 mutations
Authors: R Gao, A H Zhao, Y Du, W T Ho, X Fu, Z J Zhao
Journal: Blood Cancer Journal (2012)

JAK2V617F, an activation mutant form of tyrosine kinase JAK2, is found in the majority of patients with myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis.Overwhelming studies have demonstrated the pathogenicity of JAK2V617F. 1 However, some still doubt that JAK2V617F is a primary molecular defect in causing the malignant disease.This uncertainty partly comes from the finding of biallelic JAK2V617F mutations. 2 --4 By using allele-specific PCR amplification of genomic DNAs and subcloning, Olcaydu et al. 2 demonstrated an infrequent occurrence of biallelic JAK2V617F mutations in o5% of MPN patients.However, a subsequent study by Lambert et al. 3 employed cDNA and identified biallelic JAK2V617F mutations in the majority of patients with ET.Based on this finding, it was concluded that independent JAK2V617F mutation events may occur in ET patients on a polyclonal background, and therefore, the presence of a JAK2 mutation in ET patients should not be equated with a malignant disease.A recent study by Beer et al. 4 essentially confirmed the results using each individual method but cautioned that PCR artifacts may affect the outcome, and they concluded that the true prevalence of biallelic JAK2 mutations in ET is approximately 5 --10%.Although the previous studies were performed with patient DNA samples of unknown compositions, we believe it is important to clarify them by using DNAs with absolute allelic sequence information.For this purpose, we amplified a 3707-bp DNA fragment from blood genomic DNAs of a heterozygous JAK2V617F-positive PV patient with primers P1 5 0 -CAGGCTGGAAT GCAGAAATACTAT-3 0 and P2 5 0 -ATCTAAGAAGCACAATAAAGCAAG GT-3 0 by using the high-fidelity Phusion polymerase.The fragment spans intron 12 --14 of the JAK2 gene and contains singlenucleotide polymorphism (SNP) rs10974944, the V617F mutation site, and SNP rs12343867.Upon cloning into the pBluescript KS vector, two clones, representing two distinct JAK2 alleles, were verified by complete DNA sequencing and were designated C --G --T for wild-type JAK2 with two non-risk SNPs and G --T --C for mutant JAK2V617F DNA with two risk SNPs as illustrated in Figure 1a.The DNAs were purified and mixed in equal proportions for further PCR amplification.PCR was conducted with primers derived from the T3 and T7 promoter regions of the vector, giving rise to a 3883-bp product.We first ran the PCR under the standard conditions recommended by the manufacturer of the Phusion polymerase, except that the extension time was 3.5 min instead of the suggested 15 s/kb.A typical reaction with a total volume of 20 ml contained 10 ng mixed DNA templates, 0.4 U of Phusion DNA polymerase, 0.2 mM dNTP (each) and 0.2 mM primers (each).The PCR was run for 25 or 40 cycles.Reactions were also conducted with three times the amount of Phusion polymerase of the standard procedure and a longer extension time of 7 min.The PCR products were digested with EcoRI and XhoI, and then ligated into the pBluescript KS vector opened with the same enzymes.The ligation products were used to transform Escherichia coli cells.At least 50 plasmid DNA clones were sequenced for each set of PCR conditions.The percentages of different clones are summarized in Figure 1b.In control experiments without PCR amplification, the sequencing results revealed approximately equal