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Intracellular accumulation of factor VIII induced by missense mutations Arg 593 →Cys and Asn 618 →Ser explains cross‐reacting material‐reduced haemophilia A
Author(s) -
Joost Roelse,
R. T. M. De Laaf,
Steven Timmermans,
Marjolein Peters,
Jan A. van Mourik,
Jan Voorberg
Publication year - 2000
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1046/j.1365-2141.2000.01834.x
Subject(s) - haemophilia a , missense mutation , intracellular , antigen , microbiology and biotechnology , transfection , mutation , chemistry , biology , haemophilia , biochemistry , gene , immunology , genetics
Patients with cross‐reacting material (CRM)‐reduced haemophilia A exhibit reduced levels of factor VIII antigen. In this study, we determined the molecular basis of the genetic defect in the factor VIII gene induced by either the Arg 593 →Cys or the Asn 618 →Ser missense mutation, identified in two CRM‐reduced haemophilia A patients. We introduced either the Arg 593 →Cys or the Asn 618 →Ser mutation into a B‐domain‐deleted factor VIII cDNA and expressed the modified cDNAs in C127 cells. Reduced levels of factor VIII activity and factor VIII antigen in conditioned medium of transfected cells indicated that the secretion of both factor VIII variants was impaired. The ratio of factor VIII antigen present in cell extract to that in conditioned medium was 1.9 and 2.4 times higher for rFVIII‐R593C and rFVIII‐N618S, respectively, than for rFVIII. Metabolic labelling and immunoprecipitation of transfected cells revealed that rFVIII‐R593C and rFVIII‐N618S persisted somewhat longer inside the cell than factor rFVIII. Intracellular accumulation and subsequent degradation of factor VIII‐R593C and factor VIII‐N618S may explain the reduced levels of both factor VIII activity and antigen in plasma of mild haemophilia A patients with corresponding genetic defects.