Hayward Catherine P M, Liang Minggao, Tasneem Subia, Soomro Asim, Waye John S, Paterson Andrew D, Rivard Georges E, Wilson Michael D
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.
Department of Medicine, McMaster University, Hamilton, ON, Canada.
PLoS One. 2017 Mar 16;12(3):e0173991. doi: 10.1371/journal.pone.0173991. eCollection 2017.
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 PLAU (the uPA gene) and C10orf55, 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 PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 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 PLAU 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 PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU 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 C10orf55, 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.
魁北克血小板紊乱症（QPD）是一种独特的出血性疾病，它会使巨核细胞和血小板中的尿激酶型纤溶酶原激活剂（uPA）显著增加，但血浆或尿液中的uPA却不会增加。其病因是10号染色体上一个包含PLAU（uPA基因）和功能未知基因C10orf55的78 kb区域发生串联重复。QPD使血小板和巨核细胞中的uPA增加100倍以上，远远超过基因重复所预期的增加幅度。为了研究PLAU重复对QPD中基因表达和转录本结构的组织特异性影响，我们测试了QPD是否会导致：1）正常或独特PLAU转录本的过表达；2）白细胞中uPA增加；3）巨核细胞和白细胞中C10orf55 mRNA和/或蛋白质水平改变；4）巨核细胞基因表达的整体变化。对来自供体的原代细胞和培养的巨核细胞进行定量逆转录聚合酶链反应分析、RNA测序和蛋白质表达分析。从QPD受试者快速分离的血液白细胞显示PLAU转录本水平仅增加3.9倍，这与亲和纯化的QPD白细胞中uPA正常至轻微增加一致。所有受试者的粒细胞中的uPA比单核细胞多，淋巴细胞中的uPA极少。与QPD巨核细胞不同，QPD白细胞表达PLAU等位基因的比例与疾病染色体上额外的PLAU拷贝一致。QPD的PLAU转录本与参考基因模型一致，巨核细胞中来自疾病染色体的读数比例远高于粒细胞。QPD和对照巨核细胞中C10orf55的读数极少，且QPD巨核细胞或血小板中的C10orf55蛋白未增加。最后，我们对QPD巨核细胞的转录组分析揭示了1型干扰素途径的整体下调。我们认为血液中uPA的内源性低水平受到积极调控，并且在QPD中调控机制以巨核细胞特异性方式被破坏。
