title: Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping.
abstract: OBJECTIVE
  The factor VIII (FVIII) B domain shares very little amino acid homology with other known proteins and is not directly necessary for procoagulant activity. Despite this, missense mutations within the B domain have been reported in patients with hemophilia A. Given that the B domain is dispensable for secretion and function of FVIII, we hypothesized that these mutations should not be causative of hemophilia A in these patients.
  
  
  METHODS
  Plasmid vectors containing B domain missense mutations that were reported to be associated with moderate/severe hemophilia A (T751S, D826E, V993L, H1047Y, T1353A, N1441K, L1462P, E1579D, A1591S, P1641L and S1669L) were analyzed for their effect on synthesis and secretion compared with FVIII wild-type (WT) following transient transfection into COS-1 and CHO cells in vitro. Further, H1047Y, N1441K and E1579D mutants were expressed in vivo in a hemophilia A mouse model by hydrodynamic tail-vein injection.
  
  
  RESULTS
  FVIII activity and antigen levels for all mutants expressed into the conditioned media of COS-1 and CHO cells were similar to FVIII WT. Also, plasma expression of these mutants was similar to FVIII WT in hemophilia A mice. An in vivo tail clip bleeding assay also demonstrated that blood loss from hemophilia A mice expressing FVIII WT, H1047Y, N1441K and E1579D was similar.
  
  
  CONCLUSIONS
  We conclude that most missense mutations within the FVIII B domain would be unlikely to lead to severe hemophilia A and that the majority of such missense mutations represent polymorphisms or non-pathologic mutations.
authors.affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ogata
authors.forename: S R
authors.initials: SR
authors.lastname: Selvaraj
authors.forename: H Z
authors.initials: HZ
authors.lastname: Miao
authors.forename: S W
authors.initials: SW
authors.lastname: Pipe
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D005169:Factor VIII / Q000235:genetics*; D005838:Genotype; D006467:Hemophilia A / Q000209:etiology* / Q000235:genetics; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000235:genetics; D014162:Transfection
chemical_list: D005169:Factor VIII