Allelic Expression Imbalance of <em>JAK2</em> V617F Mutation in <em>BCR-ABL</em> Negative Myeloproliferative Neoplasms

<div><p>The discovery of a single point mutation in the <em>JAK</em>2 gene in patients with <em>BCR/ABL</em>-negative myeloproliferative neoplasms (MPNs) has not only brought new insights and pathogenesis, but also has made the diagnosis of MPNs much easier. Although, to date, several mechanisms for the contribution of single <em>JAK</em>2V617F point mutation to phenotypic diversity of MPNs have been suggested in multiple studies, but it is not clear how a unique mutation can cause the phenotypic diversity of MPNs. In this study, our results show that allelic expression imbalance of <em>JAK</em>2 V617F mutant frequently occurs and contributes to phenotypic diversity of <em>BCR-ABL</em>-negative MPNs. The proportion of <em>JAK</em>2 V617F mutant allele was significantly augmented in RNA levels as compared with genomic DNA differently by distinct MPNs subtypes. In detail, preferential expression of <em>JAK2</em> mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with essential thrombocythemia and twofold increase in polycythemia vera. In conclusion, allelic expression imbalance of <em>JAK</em>2 V617F mutant proposes another plausible mechanism for the contribution of single <em>JAK</em>2 point mutation to phenotypic diversity of MPNs.</p> </div

Ratio of the <i>JAK</i>2 mutant allelic load of gDNA and cDNA.

<p>Amplification of <i>JAK</i>2 mutant allele was observed in all MPN patients. The ratio of <i>JAK</i>2 mutant from genomic and cDNA disclosed markedly difference from MPN patients (n = 57) with the <i>JAK</i>2 V617F mutation. Allelic expression imbalance of <i>JAK</i>2 V617F mutation is associated with MPN phenotypes.</p

Proportion of the <i>JAK</i>2 V617F mutant according to treatment response in 15 PV patients with the <i>JAK</i>2 V617F mutation.

<p><i>JAK</i>2 mutant load predicted treatment response and strategy at diagnosis.</p

Demographic and hematologic characteristics of 78 patients with MPNs.

<p>PV, polycythemia vera; ET, essential thrombocythemia; PMF, primary myelofibrosis; UC, unclassifiable MPN; WBC, white blood cell count; PLT, platelet count.</p

Quantitation of <i>JAK</i>2 mutant by real time PCR and pyrosequencing from the patients with JAK2 V617F mutation.

1<p>Formula for proportion of <i>JAK</i>2 mutant: 1/(1+1/2ΔCt), ΔCt = Ct wild type – Ct mutant.</p>2<p>Statistically significant difference among each MPNs by Kruskall-Wallis test (<i>P = 0.006</i>).</p><p>qPCR, real-time PCR; other Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052518#pone-0052518-t001" target="_blank">Table 1</a>.</p

MICs of AMB and triazoles of 31 <i>A</i>. <i>terreus</i> isolates from respiratory and ear specimens.

<p>MICs of AMB and triazoles of 31 <i>A</i>. <i>terreus</i> isolates from respiratory and ear specimens.</p

Genetic relatedness by microsatellite analysis and virulence in the <i>G</i>. <i>mellonella</i> model of 15 respiratory and 16 ear isolates of <i>A</i>. <i>terreus</i> from six hospitals.

<p>Genetic relatedness by microsatellite analysis and virulence in the <i>G</i>. <i>mellonella</i> model of 15 respiratory and 16 ear isolates of <i>A</i>. <i>terreus</i> from six hospitals.</p

Diversity of clinical isolates of <i>Aspergillus terreus</i> in antifungal susceptibilities, genotypes and virulence in <i>Galleria mellonella</i> model: Comparison between respiratory and ear isolates

<div><p>We analyzed the antifungal susceptibility profiles, genotypes, and virulence of clinical <i>Aspergillus terreus</i> isolates from six university hospitals in South Korea. Thirty one isolates of <i>A</i>. <i>terreus</i>, comprising 15 respiratory and 16 ear isolates were assessed. Microsatellite genotyping was performed, and genetic similarity was assessed by calculating the Jaccard index. Virulence was evaluated by <i>Galleria mellonella</i> survival assay. All 31 isolates were susceptible to itraconazole, posaconazole, and voriconazole, while 23 (74.2%) and 6 (19.4%) showed amphotericin B (AMB) minimum inhibitory concentrations (MICs) of ≤ 1 mg/L and > 4 mg/L, respectively. Notably, respiratory isolates showed significantly higher geometric mean MICs than ear isolates to AMB (2.41 <i>vs</i>. 0.48 mg/L), itraconazole (0.40 <i>vs</i>. 0.19 mg/L), posaconazole (0.16 <i>vs</i>. 0.08 mg/L), and voriconazole (0.76 <i>vs</i>. 0.31 mg/L) (all, <i>P</i> <0.05). Microsatellite genotyping separated the 31 isolates into 27 types, but the dendrogram demonstrated a closer genotypic relatedness among isolates from the same body site (ear or respiratory tract); in particular, the majority of ear isolates clustered together. Individual isolates varied markedly in their ability to kill infected <i>G</i>. <i>mellonella</i> after 72 h, but virulence did not show significant differences according to source (ear or respiratory tract), genotype, or antifungal susceptibility. The current study shows the marked diversity of clinical isolates of <i>A</i>. <i>terreus</i> in terms of antifungal susceptibilities, genotypes and virulence in the <i>G</i>. <i>mellonella</i> model, and ear isolates from Korean hospitals may have lower AMB or triazole MICs than respiratory isolates.</p></div

Antifungal susceptibilities, microsatellite genotypes and virulence of 31 <i>A</i>. <i>terreus</i> isolates from six hospitals in South Korea.

<p>Antifungal susceptibilities, microsatellite genotypes and virulence of 31 <i>A</i>. <i>terreus</i> isolates from six hospitals in South Korea.</p

Genetic relationships of 31 <i>A</i>. <i>terreus</i> isolates according to source.

<p>The dendrogram is based on a categorical analysis of seven microsatellite markers in combination with unweighted pairgroup method using the arithmetic average (UPGMA) clustering. The number on the tree indicates the branch length, showing the difference along a branch. All 31 isolates (R1 to R15 and E1 to E16) comprised 27 distinct genotypes (GT 1 to GT 27) by 7 microsatellite markers. However, when a cluster is defined as the isolation of two or more strains with a branch length distance of < 0.63, ear isolates comprise clusters II and V, and the respiratory isolates comprise clusters I, III, and IV, suggesting a closer genetic relatedness among isolates from the same body site (ear or respiratory tract). Five isolates (R7, R15, E1, E4, and R12) were unique to a single isolate, which did not cluster with other isolate as a branch length distance of < 0.63. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186086#pone.0186086.t002" target="_blank">Table 2</a> for detailed information on each isolate.</p