The duplication mutation of Quebec platelet disorder dysregulates <i>PLAU</i>, but not <i>C10orf55</i>, selectively increasing production of normal <i>PLAU</i> transcripts by megakaryocytes but not granulocytes

<div><p>Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing <i>PLAU</i> (the uPA gene) and <i>C10orf55</i>, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that <i>PLAU</i> duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique <i>PLAU</i> transcripts; 2) increased uPA in leukocytes; 3) altered levels of <i>C10orf55</i> mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in <i>PLAU</i> transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed <i>PLAU</i> alleles in proportions consistent with an extra copy of <i>PLAU</i> on the disease chromosome, unlike QPD megakaryocytes. QPD <i>PLAU</i> transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for <i>C10orf55</i>, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.</p></div

<i>PLAU</i> transcript levels in QPD and control leukocytes.

<p>Bars indicate standard errors and the p values compare data for QPD to control samples. A) T/C allele ratios for <i>PLAU</i> rs4065 were assessed for affinity purified granulocytes and monocytes from heterozygous QPD (n = 5) and control (n = 4) subjects, with simultaneous evaluation of QPD megakaryocytes as a known abnormal control (* indicates significantly higher values, p<0.0005, for QPD megakaryocytes compared to affinity isolated QPD leukocytes). B) <i>PLAU</i> mRNA expression levels (shown as NRQ) in affinity purified granulocytes and monocytes, and in total blood leukocytes that had been rapidly isolated using leukodepletion filters.</p

Global expression differences in QPD megakaryocytes.

<p>A) Differential expression (DE) analysis of genes, comparing QPD to control megakaryocytes. Log transformed p-values and fold-changes are shown on the vertical and horizontal axes, respectively. Horizontal dashed lines delineate the Bonferroni corrected FDR threshold of 0.05. Vertical dashed lines delineate a fold change of +/- 2 fold. Genes are shaded based on their mean expression level. Only genes exceeding the FDR and fold change cutoffs are labeled. Genes annotated as involved in type-1 interferon signaling (GO:0034340) are outlined in yellow. B) Manhattan plot of DE analysis for megakaryocytes. Genes are plotted based based on chromosomal position and significance. Genes in red are down-regulated in QPD and those in blue are up-regulated in QPD. Dashed red lines delineate the Bonferroni corrected FDR threshold of 0.05. Arrows mark the positions of <i>PLAU</i>, <i>EGR1 and TFPI2</i>. C) Unsupervised clustering of log transformed FPKM (fragments per kilobase of transcript per million mapped reads) values for a panel of literature-reported megakaryocyte marker genes for both megakaryocyte and granulocyte samples from individual QPD (Q) and control (C) subjects. Genes and samples were clustered based on Euclidean distance using the hclust function in R with default parameters. Markers were selected as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173991#pone.0173991.ref031" target="_blank">31</a>].</p

C10orf55 protein levels in QPD and control cells.

<p>ELISA data (medians, bars indicate ranges) comparing data for platelet, cultured megakaryocyte, granulocyte and monocyte lysates from QPD (Q, n = 6) and control (C; n = 6) subjects.</p

Exon and splice junction usage for <i>PLAU</i> in QPD versus control megakaryocytes and granulocytes, evaluated using JunctionSeq.

<p>The plots for megakaryocytes (A) and granulocytes (B) show the relative expression levels for each tested exon and splice junction, normalized to compensate for differences in gene expression, which are shown to the right as gene-level expression values. Vertical axis for the top panels are log-transformed except for the area between 0–1 which is plotted on a linear scale. The horizontal axis indicates the alpha numerical designations for exons (E) and junction sequences (J) for all annotated exons and splice variants in the Gencode v19 reference model. Only exons and junctions with coverage above the automatic detectable count threshold are plotted. The diagrams below indicate the positions of these elements on the full gene model that shows all annotated exons (boxes) and splice junctions (arcs). Exons and splice junctions which did not pass the automatic detectable count threshold are colored white and gray, respectively. The bottom panel compares the findings to all annotated <i>PLAU</i> isoforms, annotated as in the middle panel. The transcript model that corresponds to <i>PLAU</i> variant 1 transcript is indicated. Features showing significantly differential usage between QPD and control (CTL) samples, based on a criteria of p <0.05, are not colored-coded as none were detected.</p

<i>TFPI2</i> mRNA and protein levels in QPD and control megakaryocytes and platelets.

<p>A) <i>TFPI2</i> ddPCR findings for cultured megakaryocytes from QPD (Q, n = 6) and control (C; n = 6) subjects. B) TFPI2 ELISA findings (medians, bars indicate ranges) for platelets and cultured megakaryocytes from QPD (Q, n = 6) and control (C; n = 6) subjects. p values shown compare QPD to control findings.</p

Comparison of gene expression at the <i>PLAU</i> locus in granulocytes and CD34+ derived megakaryocytes obtained using RNA-seq.

<p>A) UCSC genome browser screenshot showing normalized RNA-seq signal (tags per million) at the <i>PLAU</i> locus (merged for 3 individuals, for each cell and subject type; the signal tracks for individual samples are shown for reference in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173991#pone.0173991.s001" target="_blank">S1 Fig</a>). Numbers indicate highest value (log scaled) within viewing range per track. Colors indicate reads that mapped to sense (pink) and antisense (blue) strands. Thick bars in the gene model represent exons, thin bars represent untranslated regions. <i>PLAU</i> variant 1 is shown. Chromosome position and genome assembly are displayed in the top track. B) Expression of SNPs in QPD megakaryocyte (top) and granulocytes (bottom). The proportion of reads mapping to control (red) and QPD (blue) alleles for cells from 2 QPD individuals with two alleles for each of the SNP shown, and n values indicate the total read coverage at each SNP. Only SNPs with > 10 mapped reads in each sample are shown. Purple lines delineate the position of individual SNP. * indicates SNP also evaluated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173991#pone.0173991.g002" target="_blank">Fig 2</a>. ^‡ indicates allele ratios for rs2227574 after correction for alignment bias. CTL, control; Mk, megakaryocyte; G, granulocyte.</p

uPA in QPD and control leukocytes.

<p>A) Median uPA levels in granulocytes and monocytes compared to platelets (Plt) from QPD (n = 6) and control (n = 9) subjects (bars indicate ranges). B-D) Western blot images showing the forms of uPA visualized in QPD and control (respectively labeled Q and C) monocyte (B) and granulocyte (C, D) lysates, after separation by non-reduced (B, C) or reduced (D) SDS-PAGE, with comparison to two chain (tc), single chain (sc) and low molecular weight (LMW) uPA and uPA in QPD platelet lysates (Plt). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173991#pone.0173991.s004" target="_blank">S1 Supporting Information</a> shows for reference the scans of original Western blots that were used to prepare panels B-D of Fig 1.</p