Quebec Platelet Disorder Is Caused by a Cis-Acting Mutation near the Plasminogen Activator Gene (PLAU) That Increases PLAU Transcription by Megakaryocytes.
Authors: Diamandis Maria , Paterson Andrew D , Rommens Johanna M , Veljkovic D. Kika , Blavignac Jessica , Waye John S , Derome Francine , Rivard Georges E. , Hayward Catherine PM 
Published: October 2019

Abstract
Quebec platelet disorder (QPD) is an autosomal dominant disorder, with high penetrance, that is associated with increased risks for bleeding and a gain-of-function defect in fibrinolysis. Its hallmark feature is a markedly increased platelet content of urokinase plasminogen activator (uPA), without systemic fibrinolysis or increased uPA in plasma or urine. Recent studies of 28 affected and 110 unaffected individuals from the QPD pedigree linked the disorder to a 2 megabase region on chromosome 10q containing PLAU, the uPA gene (J Thromb Haemost2007;5: Suppl.2: O-S-021). This led us to investigate if QPD was caused by a mutation within or near PLAU, and if the disorder increased transcription of one or both PLAU alleles in megakaryocytes. Single nucleotide polymorphism (SNP) arrays (Illumina 1M BeadChip), Southern blotting, and sequencing were used to investigate genetic changes within the linked region (n= 1–3 QPD and 1–3 control individuals). To assess expression of PLAU alleles, heterozygous individuals (n= 4 QPD, 5 controls) were evaluated by a quantitative Taqman® 5’ nuclease assay for SNP rs4065 alleles (T/C; T allele inherited by all affected individuals) using reverse transcribed mRNA from platelets, CD 34+ hematopoietic progenitors, and saliva. Quantitative RTPCR of QPD platelet mRNA was used to determine if the disorder increased expression of the genes flanking PLAU on chromosome 10 which encode vinculin (VCL) and calmodulin-dependent protein kinase IIγ (CAMK2G). No abnormalities near PLAU were detected in QPD subjects evaluated by Southern blotting. No insertions or duplications were detected by SNP array analysis, which provided data on 80 markers over the region of chromosome 10 nearest to PLAU. Analysis of a rare SNP, 13.7 kb upstream of PLAU, that was present in 28/28 with QPD and 4/110 unaffected family members, confirmed linkage of the region near PLAU to QPD (LOD score +11.8). Sequence analyses confirmed a common QPD haplotype for the linked region and excluded the possibility that QPD resulted from mutations in the promoter or in the 11 exons and 10 introns of PLAU. No mutations were found by extended sequencing of the regions extending 20 kb upstream and 1 kb downstream of PLAU which includes all of its known regulatory elements and the 3’ message instability region. Analysis of SNP rs4065 in PLAU indicated that there was a marked increased expression of the linked T allele in QPD platelets (>150-fold increase; p = <0.016 compared to controls; confirmed by sequencing) but minimal (2–4 fold) increased expression of this allele in QPD CD 34+ cells and saliva cells. Unlike PLAU, VCL and CAMK2G were not overexpressed in QPD platelets. Our data indicate that inheritance of QPD results from a cis-regulatory defect, outside the known regulatory elements of PLAU, that generates profibrinolytic platelets by markedly increasing transcription of the linked PLAU allele during megakaryopoiesis, without increasing PLAU transcription by non-hematopoietic saliva cells.