Amplification plots of dilution series and standard curve of qPCR assay.

<p>A: Representative amplification plots of plasmid containing <i>JAK2</i> 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.</p

Reproducibility of mutant allele burden determination by the qPCR assays.

<p>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 <i>JAK2</i> 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).</p

Identification of <i>JAK2</i> exon 12 mutations.

<p>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 <i>JAK2</i> 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.</p

Mutant <i>JAK2</i> exon 12 allele burden in bone marrow and peripheral blood cell lineages.

<p>Histogram plot displaying the <i>JAK2</i> 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 <i>JAK2</i> 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.</p

Primer and probe sequences for <i>JAK2</i> exon 12.

<p>Primers used for qPCR screening and quantitative determination consisting of a common forward primer and probe in addition to a mutation specific reverse primer. The primers listed for quantitative determination were only used exclusively for determination of mutant allele burden. Lowercase letters in sequences indicate intended mismatches.</p

Fold changes for the 5 genes in ET, PV, and PMF compared to control subjects.

<p>Patient groups and genes are shown on the x-axis and fold changes on the Y-axis. NS: non-significant; S: significant. All genes FDR<0.05.</p

Flowchart illustrating the background for analytical approaches.

<p>Flowchart illustrating the background for analytical approaches.</p

Baseline subject characteristics.

<p>Values are number (column-%) or means (SD). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099388#pone.0099388-Hemminki1" target="_blank">[1]</a> Lymphoproliferative cancer defined as Hodgkin's lymphoma, non-Hodgkin lymphoma and chronic lymphocytic leukemia, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099388#pone.0099388-Chakravarty1" target="_blank">[2]</a> Calculated on previous hospital contacts (<3 years), <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099388#pone.0099388-Khurana1" target="_blank">[3]</a>>10 mg/L, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099388#pone.0099388-Mercer1" target="_blank">[4]</a>>0.5•10⁹/L.</p

Hierarchical Cluster analysis with euclidean distance in ET, PV and PMF patients.

<p>Rows in the heat map represent the five genes DEFA4, ELA2, CTSG, OLFM4, and AZU1, and columns represent patients. The color key ranges from green to red representing standardized expression values of −3.0 to 3.0. Green indicates low expression, black intermediate expression, and red high expression. Five major clusters can be identified. Cluster 1 (green, low expression), cluster 2 (green-black, low-intermediate expression), cluster 3 (black-red, intermediate expression), cluster 4 (red-black, intermediate-high expression), and cluster 5 (red, high expression).The dendogram shows the degree of similarity between patients.</p

Clinical and Biochemical Data in Cluster 1–5 with low, low-intermediate, intermediate, intermediate-high, and high expression values of the 5 genes, respectively.

<p>Median and range are shown. Abbreviations: ET =  Essential Thrombocythemia; PV =  Polycythemia Vera; PMF =  Primary Myelofibrosis; *  =  blood tests at the time of blood sampling for gene expression profiling; disease duration at the time of blood sampling; **:from diagnosis as assessed January 2013.</p