title: The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes.
abstract: 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.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.|Department of Medicine, McMaster University, Hamilton, ON, Canada.|Hamilton Regional Laboratory Medicine Program, Hamilton, ON, Canada.
authors.forename: Catherine P M
authors.identifier: http://orcid.org/0000-0002-2843-0817
authors.initials: CP
authors.lastname: Hayward
authors.affiliation: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada.|Molecular Genetics, University of Toronto, Toronto, ON, Canada.
authors.forename: Minggao
authors.initials: M
authors.lastname: Liang
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.
authors.forename: Subia
authors.initials: S
authors.lastname: Tasneem
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.
authors.forename: Asim
authors.initials: A
authors.lastname: Soomro
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.|Hamilton Regional Laboratory Medicine Program, Hamilton, ON, Canada.
authors.forename: John S
authors.initials: JS
authors.lastname: Waye
authors.affiliation: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada.|The Dalla Lana School of Public Health and Institute of Medical Sciences, University of Toronto, ON, Canada.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Paterson
authors.affiliation: Hematology/ Oncology, Centre Hospitalier Universitaire Sainte-Justine, Montreal, QC, Canada.
authors.forename: Georges E
authors.initials: GE
authors.lastname: Rivard
authors.affiliation: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada.|Molecular Genetics, University of Toronto, Toronto, ON, Canada.|Heart & Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, Toronto, Canada.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Wilson
mesh_terms: D005166:Factor V Deficiency / Q000235:genetics*; D020440:Gene Duplication; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism*; D009154:Mutation; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D014568:Urokinase-Type Plasminogen Activator