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

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