The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes
The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes
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DOI：
10.1371/journal.pone.0173991
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发表时间：
2017-03-16
期刊：
影响因子：
3.7
通讯作者：
Wilson, Michael D.
中科院分区：
文献类型：
--
作者：
Hayward, Catherine P. M.;Liang, Minggao;Wilson, Michael D.
魁北克血小板疾病（QPD）是一种独特的出血性疾病，其显著增加巨核细胞和血小板中的尿激酶纤溶酶原激活物（uPA），但不增加血浆或尿液中的uPA。其原因是10号染色体上含有PLAU（uPA基因）和C10 orf 55（一个功能未知的基因）的78 kb区域的串联重复。QPD使血小板和巨核细胞中的uPA增加>100倍，远远超过基因复制的预期。为了研究PLAU复制对QPD中基因表达和转录物结构的组织特异性影响，我们测试了QPD是否导致：1）正常或独特PLAU转录物的过表达; 2）白细胞中uPA的增加; 3）巨核细胞和白细胞中C10 orf 55 mRNA和/或蛋白水平的改变;以及4）巨核细胞基因表达的整体变化。制备来自供体的原代细胞和培养的巨核细胞用于定量反向聚合酶链反应分析、RNA-seq和蛋白质表达分析。从QPD受试者中快速分离的血液白细胞显示PLAU转录水平仅增加3.9倍，与亲和纯化的QPD白细胞中正常至最低限度增加的uPA一致。所有受试者的粒细胞中uPA含量高于单核细胞，淋巴细胞中uPA含量最低。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的PLAU额外拷贝一致。QPD PLAU转录物与参考基因模型一致，其中巨核细胞中源自疾病染色体的读段比例比粒细胞高得多。QPD和对照巨核细胞含有C10 orf 55的最小读数，并且C10 orf 55蛋白在QPD巨核细胞或血小板中没有增加。最后，我们的QPD巨核细胞转录组分析揭示了1型干扰素通路的整体下调。我们认为，低内源性水平的uPA在血液中的积极调节，并在QPD的巨核细胞特异性的方式中断的监管机制。
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.