title: Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.
abstract: The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.
authors.affiliation: Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Stockley
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.
authors.forename: Shaista P
authors.initials: SP
authors.lastname: Nisar
authors.affiliation: Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Vincenzo C
authors.initials: VC
authors.lastname: Leo
authors.affiliation: Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Essa
authors.initials: E
authors.lastname: Sabi
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Cunningham
authors.affiliation: Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jeroen C
authors.initials: JC
authors.lastname: Eikenboom
authors.affiliation: National Haemophilia Center, University Hospital, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Stefan
authors.initials: S
authors.lastname: Lethagen
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schneppenheim
authors.affiliation: Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Anne C
authors.initials: AC
authors.lastname: Goodeve
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Mundell
authors.affiliation: Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Martina E
authors.initials: ME
authors.lastname: Daly
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D057809:HEK293 Cells; D006470:Hemorrhage / Q000503:physiopathology; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014842:von Willebrand Diseases / Q000235:genetics*; D014841:von Willebrand Factor / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D043562:Receptors, G-Protein-Coupled; D014841:von Willebrand Factor