Figure 1.
Identification of JAK2 exon 12 mutations.
A: Sequence of F537-I540delinsLV mutation of PV4 revealing a 10 base-pair deletion and a four base-pair insertion. B: Difference plot of high resolution melting (HRM) analysis detecting N542-E543del, F537-I540delinsLV and K539L JAK2 exon 12 mutations in PV1, PV3, PV4, PV5 and PV6. C: Phase-contrast microphotograph showing Epo-independent growth of endogenous erythroid colonies (EEC) at day 14 of culture. Scalebar: 200 µm. HRM, high resolution melting.
Figure 2.
Amplification plots of dilution series and standard curve of qPCR assay.
A: Representative amplification plots of plasmid containing JAK2 exon 12 mutation diluted into wildtype genomic DNA detected by the mutation specific assay. The ten fold dilution series starting at 15,000 copies is continued as two-fold dilutions after 15 copies down to 1.9 copies as indicated. B: Representative standard curve for the mutation and wildtype assays producing correlation coefficients >0.990. The average slope of the standard curves was −3.4.
Figure 3.
Reproducibility of mutant allele burden determination by the qPCR assays.
Histogram plots showing reproducibility of percentage mutant allele burden in six separate qPCR runs. The mutant allele burden was determined for 11 different DNA samples from four different patients as indicated. The JAK2 exon 12 mutations include K539L (PV6), V536-I546dup11 (PV4), and N542-E543del (PV1 and PV2) in high, intermediate and low levels of mutant allele burden. The data is presented as percentage mean values ± standard deviation (SD).
Figure 4.
Time-course of JAK2 exon 12 allele burden development.
A and B: Graphs describing the development of the N542-E543del mutant allele burdens of PV1 and PV2 during a 420 and 735 day period respectively. C: Plot of V536-I546dup11 mutant allele burden of PV3 during a 203 day period.
Figure 5.
Mutant JAK2 exon 12 allele burden in bone marrow and peripheral blood cell lineages.
Histogram plot displaying the JAK2 exon 12 mutation burden in bone marrow, peripheral blood, CD16+ granulocytes, CD14+ monocytes, CD3+ T-lymphocytes, and CD19+ B-lymphocytes in patients PV1-PV6. PV2 had very low level of JAK2 exon 12 mutant allele burden except for the bone marrow sample. PV1-PV3 and PV5 appeared to be heterozygous, whereas PV4 and PV6 were homozygous. Note that DNA from bone marrow samples could not be obtained from patients PV5 and PV6. BM, bone marrow and PB, peripheral blood.
Table 1.
Primer and probe sequences for JAK2 exon 12.