title: Characterisation of five factor XI mutations.
abstract: A large scale factor XI (FXI) mutation screening program identified a number of novel candidate mutations and previously reported mutations and polymorphisms. Five potential missense mutations were selected for further study; these included two novel missense mutations - Met-18Ile (p.Met1Ile) and Met102Thr (p.Met120Thr), two previously reported missense mutations - Tyr133Ser (Tyr151Ser) and Thr575Met (Thr593Met), and one amino acid substitution previously reported as a polymorphism - Arg378Cys (Arg396Cys). The substitutions were recreated by the site-directed mutagenesis of a FXI cDNA and stably expressed in a BHK-570 cell line. Subsequent analysis of both the conditioned media and cell lysates showed that three of the substitutions, Met-18Ile, Met102Thr andTyr133Ser, prevented secretion of the mutated protein from the transfected cell line, resulting in a cross-reactive material negative (CRM-) phenotype. The remaining two mutants, Thr575Met and Arg378Cys, secreted significant levels of FXI into the conditioned media; however, these mutant FXIs were shown to have negligible factor IX activation activity in an APTTbased assay. These results confirmed all five of the missense mutations as being causative of factor XI deficiency, despite one having been previously reported as a polymorphism (Arg378Cys) and one (Tyr133Ser) as a mild mutation - FXI:C 38 U/dl in a homozygous patient.
authors.affiliation: Centre for Haemostasis and Thrombosis, Haemophilia Reference Centre, 1st Floor North Wing, St. Thomas' Hospital, London SE1 7EH, United Kingdom. michael.mitchell@kcl.ac.uk
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Mitchell
authors.forename: Letian
authors.initials: L
authors.lastname: Dai
authors.forename: John B
authors.initials: JB
authors.lastname: Clarke
authors.forename: Paula H B
authors.initials: PH
authors.lastname: Bolton-Maggs
authors.forename: Geoffrey F
authors.initials: GF
authors.lastname: Savidge
authors.forename: Anwar
authors.initials: A
authors.lastname: Alhaq
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001777:Blood Coagulation / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D019281:Dimerization; D015949:Factor IXa / Q000378:metabolism; D005172:Factor XI / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005173:Factor XI Deficiency / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006801:Humans; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010314:Partial Thromboplastin Time; D010641:Phenotype; D014162:Transfection
chemical_list: D005172:Factor XI; D015949:Factor IXa