Characterisation of five factor XI mutations

Michael J. Mitchell; Letian Dai; John B. Clarke; Paula H. B. Bolton-Maggs; Geoffrey F. Savidge; Anwar Alhaq

doi:10.1160/TH06-12-0704

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 97(06): 884-889
DOI: 10.1160/TH06-12-0704

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Characterisation of five factor XI mutations

Authors

Michael J. Mitchell

¹Centre for Haemostasis and Thrombosis, Haemophilia Reference Centre, St. Thomas Hospital, London, UK
Letian Dai

¹Centre for Haemostasis and Thrombosis, Haemophilia Reference Centre, St. Thomas Hospital, London, UK
John B. Clarke

¹Centre for Haemostasis and Thrombosis, Haemophilia Reference Centre, St. Thomas Hospital, London, UK
Paula H. B. Bolton-Maggs

²Clinical Haematology, Manchester Royal Infirmary, Manchester, UK
Geoffrey F. Savidge

¹Centre for Haemostasis and Thrombosis, Haemophilia Reference Centre, St. Thomas Hospital, London, UK
Anwar Alhaq

¹Centre for Haemostasis and Thrombosis, Haemophilia Reference Centre, St. Thomas Hospital, London, UK

Abstract

Summary

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 APTT based 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.

Keywords

Factor XI - site-directed mutagenesis - expression studies

Full Text

References

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