title: Homozygous type 2N R854W von Willebrand factor is poorly secreted and causes a severe von Willebrand disease phenotype.
abstract: BACKGROUND
  von Willebrand disease (VWD) type Normandy (VWD 2N) is caused by mutations at the factor (F)VIII-binding site of von Willebrand factor (VWF), located in the D'and D3 domains on the N-terminus of mature VWF. The R854Q mutation is the most frequent cause of this phenotype.
  
  
  OBJECTIVE
  We report the characterization of a homozygous VWD 2N mutation, R854W, detected in a patient with a severe VWD phenotype.
  
  
  METHODS
  The plasma VWF phenotype was studied, transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed, and the results were compared with those obtained with wild-type (WT) VWF. Furthermore, expression was also examined in HEK293 cells, which form Weibel-Palade body-like granules when transfected with WT VWF.
  
  
  RESULTS
  The multimer analysis of plasma VWF showed the lack of the typical triplet structure, with the presence of the central band only, and a relative decrease in the high molecular mass multimers. Homozygous expression of recombinant R854W VWF resulted in normal amounts of cellular VWF, but with a severe reduction in secretion into the medium. Severe reductions in FVIII binding to R854W VWF, glycoprotein Ib binding activity and collagen binding of secreted W854 VWF was observed, and reproduced the phenotypic parameters of plasma VWF. In HEK293 cells, homozygous R854W VWF failed to form Weibel-Palade body-like granules.
  
  
  CONCLUSIONS
  Our results demonstrate that a homozygous R854W mutation in the D' domain of VWF induces impaired secretion and activity of the protein, thereby explaining the severe phenotype of the patient.
authors.affiliation: Department of Cellular Therapy and Haematology, San Bortolo Hospital, Vicenza, Italy. castaman@hemato.ven.it
authors.forename: G
authors.initials: G
authors.lastname: Castaman
authors.forename: S H
authors.initials: SH
authors.lastname: Giacomelli
authors.forename: P
authors.initials: P
authors.lastname: Jacobi
authors.forename: T
authors.initials: T
authors.lastname: Obser
authors.forename: U
authors.initials: U
authors.lastname: Budde
authors.forename: F
authors.initials: F
authors.lastname: Rodeghiero
authors.forename: S L
authors.initials: SL
authors.lastname: Haberichter
authors.forename: R
authors.initials: R
authors.lastname: Schneppenheim
mesh_terms: D000328:Adult; D001777:Blood Coagulation; D002460:Cell Line; D004252:DNA Mutational Analysis; D005260:Female; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D010641:Phenotype; D011994:Recombinant Proteins; D014842:von Willebrand Diseases / Q000235:genetics*; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014841:von Willebrand Factor