title: Functional characterisation of the type 1 von Willebrand disease candidate VWF gene variants: p.M771I, p.L881R and p.P1413L.
abstract: BACKGROUND
  Abnormalities in the biosynthetic pathway or increased clearance of plasma von Willebrand factor (VWF) are likely to contribute to decreased plasma VWF levels in inherited type 1 von Willebrand disease (VWD). Recent studies demonstrated that 65% of type 1 VWD patients have candidate VWF mutations, the majority of which are missense variants. The purpose of this study was to explore the effects of three VWF missense mutations (p.M771I, p.L881R and p.P1413L) located in different functional domains of VWF, reported as candidate mutations in type 1 VWD patients in the course of the MCMDM-1VWD study.
  
  
  METHODS
  The focus of these studies was on the intracellular biosynthetic processing and localisation of VWF in a heterologous cell system. Molecular dynamic simulation for p.M771I and p.P1413L was also performed to analyse the conformational effects of the changes.
  
  
  RESULTS
  As determined by immunofluorescence antibody staining and confocal microscopy of HEK293 cells, the intracellular localisation of recombinant VWF with the p.M771I variation was impaired. Transient transfection studies and phorbol myristate acetate stimulation in COS-7 cells revealed significant intracellular retention. In addition, major loss of VWF multimers was observed for only the p.M771I mutation. Molecular dynamic simulations on p.M771I mutant VWF revealed distinct structural rearrangements including a large deviation in the E' domain, and significant loss of β-sheet secondary structure.
  
  
  CONCLUSIONS
  The pathogenic effects of candidate VWF gene mutations were explored in this study. In vitro expression studies in heterologous cell systems revealed impaired secretion of VWF and a dominant negative effect on the processing of the wild-type protein for only the p.M771I mutation and none of the mutations affected the regulated secretion.
authors.affiliation: Department of Molecular Biology and Genetics, Istanbul Arel University, Istanbul, Turkey.
authors.forename: Ergul
authors.initials: E
authors.lastname: Berber
authors.affiliation: Department of Molecular Biology and Genetics, Istanbul Arel University, Istanbul, Turkey.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Ozbil
authors.affiliation: Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada.
authors.forename: Christine
authors.initials: C
authors.lastname: Brown
authors.affiliation: Department of Internal Medicine, Cerrahpasa Medical Faculty, Istanbul University, Istanbul.
authors.forename: Zafer
authors.initials: Z
authors.lastname: Baslar
authors.affiliation: Department of Molecular Biology and Genetics, Boğaziçi University, Istanbul, Turkey.
authors.forename: S Hande
authors.initials: SH
authors.lastname: Caglayan
authors.affiliation: Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Lillicrap
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006801:Humans; D056004:Molecular Dynamics Simulation; D017354:Point Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D014162:Transfection; D056725:von Willebrand Disease, Type 1 / Q000235:genetics*; D014841:von Willebrand Factor / Q000032:analysis / Q000235:genetics*
chemical_list: D014841:von Willebrand Factor