A multiplex snapback primer system for the enrichment and detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia-negative myeloproliferative neoplasms

Abstract

A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.

Figure 1

Detection of JAK2 V617F, MPL W515L, and MPL W515K mutations with a multiplex snapback primer system. Negative first-derivative (dF/dT) plot of melting curve consists of two melting regions. The stem-loop hairpin melting region for mutation discrimination (55–75°C) and the double-strand amplicon for DNA template amplification control (75–95°C). (a) Samples with the JAK2 V617F mutation (purple) showed a melting peak at 59.3°C, while the melting peaks at 63.2°C and 67.6°C indicated the presence of MPL W515K (ginger) and MPL W515L (blue) mutations, respectively. The amplification controls of JAK2 and MPL were presented by the melting peak at 81.1°C and 91.2°C, respectively. The mixture of three mutant allele-positive standards (red) generated all the three mutation melting peaks. In wild-type control sample (pink), only the double-strand control was amplified. (b) The amplicon melting curves of DNA from patients with JAK2 V617F, MPL W515L, MPL W515K, and concurrent JAK2 V617F and MPL W515K mutation.

Figure 2

Analytical sensitivity of the multiplex snapback primer system for mutant allele discrimination. (a) Serial diluted standards of 0.1% load MPL W515L (blue), MPL W515K (purple), and JAK2 V617F (ginger) specifically generated the corresponding melting peaks, which could be easily discriminated from the wild-type control (lake blue). (b) Serial JAK2 V617F dilution of 0.01% (pink), 0.1% (purple), 1% (blue), and 10% (ginger) mutation load. After the robust mutation enrichment, snapback primer system generated a melting curve with 0.1% mutation load that can be distinguished from the wild-type control.

Figure 3

ARMS for JAK2 V617F mutation. Amplified DNA products from the ARMS assay were subjected to 2% agarose electrophoresis for mutation identification. The 469 bp product served as the amplification control. Bands of 229 bp suggested the presence of the wild-type allele, while the mutant allele was indicated by the bands of 279 bp. M: 1,000 bp DNA ladder; BLK: no template amplification control; 1: HEL cell line DNA; 2: RPMI 8226 cell line DNA; 3: patient sample identified as JAK2 V617F-homozyous positive; 4: patient sample with heterozygous JAK2 V617F mutation.

Figure 4

ARMS for MPL W515L and MPL W515K mutation. The MPL W515L/K ARMS products after 2% agarose electrophoresis. The 246 bp band represented the amplification control. Bands of 98 bp were the wild-type allele product. Presence of the 188 bp amplicon suggested the mutant allele in DNA sample. MPL W515L ARMS system was in tracks of 1, 3, 5, 7, and 9; MPL W515K ARMS system was in tracks of 2, 4, 6, 8, and 10. M: 1,000 bp DNA ladder; BLK: no template amplification control; 1, 2: artificial plasmid with MPL W515L allele; 3, 4: artificial plasmid with MPL W515K allele; 5, 6: RPMI 8226 cell line DNA; 7, 8: patient sample with heterozygous MPL W515L mutation; 9, 10: patient sample with heterozygous MPL W515K mutation.

Figure 5

TaqMan probe qPCR assay for JAK2 V617F and MPL W515L/K mutation. (a) TaqMan system for JAK2 V617F detection. A sigmoidal shaped amplification curve in the fluorescent signal versus cycle number plot of FAM fluorescence channel (465 nm–510 nm) indicated the presence of JAK2 V617F mutant allele in the DNA sample, while the amplification curve in VIC channel (540 nm–580 nm) represents the wild-type JAK2 copies in the sample. JAK2 V617F and JAK2 wild-type homozygotes specifically produced the amplification curve in the FAM and VIC channel, respectively. The amplification of mutation heterozygote could be observed both in the FAM and VIC channel. (b) TaqMan system for MPL W515L/K mutations. The system was consisted of three allele-specific channels. The amplification curve of homozygous mutant and wild-type samples was only generated in the corresponding channel. The amplification curves of heterozygote with MPL W515L or MPL W515K mutation would be observed in both the wild-type channel and the mutant allele channel.

Figure 6

Snapback primer assay identifying a patient DNA with MPL W515L mutation. (a) Peripheral blood DNA from a patient with PMF (J247) was detected as weak MPL W515L mutation positive (C _t = 38.623). (b) Sanger sequencing of MPL W515L/K mutation after amplicon T-A cloning identified purified MPL W515L mutation clone. (c) Snapback primer assay discriminated the MPL W515L mutation from the wild-type control.