title: Identification and characterization of the elusive mutation causing the historical von Willebrand Disease type IIC Miami.
abstract: Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation.
  
  
  
  Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo.
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Obser
authors.affiliation: CLOT-ED, Inc., Islamorada, FL, USA.
authors.forename: M
authors.initials: M
authors.lastname: Ledford-Kraemer
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Oyen
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: M A
authors.initials: MA
authors.lastname: Brehm
authors.affiliation: INSERM UMR_S 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicetre, France.
authors.forename: C V
authors.initials: CV
authors.lastname: Denis
authors.affiliation: Institute of Pharmaceutical Biology/ZAFES/DCAL, Biocenter, Johann-Wolfgang-Goethe-University Frankfurt, Frankfurt/Main, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Marschalek
authors.affiliation: Blood Research Institute of Blood Center of Wisconsin and Medical College of Wisconsin, Milwaukee, WI, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Montgomery
authors.affiliation: Departments of Medicine and Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Sadler
authors.affiliation: Medilys Central Laboratory Coagulation, Asklepios Clinic Altona, Hamburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Schneppenheim
authors.affiliation: Medilys Central Laboratory Coagulation, Asklepios Clinic Altona, Hamburg, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Budde
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. schneppenheim@uke.de.
authors.forename: R
authors.initials: R
authors.lastname: Schneppenheim
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D003894:Deamino Arginine Vasopressin / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D005799:Genes, Dominant; D005808:Genes, Recessive; D005838:Genotype; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D009154:Mutation; D010641:Phenotype; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D056728:von Willebrand Disease, Type 2 / Q000235:genetics* / Q000378:metabolism; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014841:von Willebrand Factor; D003894:Deamino Arginine Vasopressin
keywords: ADAMTS-13, mouse; genetics; physiopathology; von Willebrand disease, type 2A; von Willebrand factor