The Missense Mutation Arg593 → Cys Is Related to Antibody Formation in a Patient With Mild Hemophilia A

Abstract

The development of inhibitory antibodies to factor VIII in patients affected by a mild form of hemophilia A (factor VIII < 0.05 IU/mL) is considered a rare event. In this study, we evaluated the relationship between genotype and anti-factor VIII antibody formation in a patient with mild hemophilia A. Mutation analysis showed that a missense mutation in the factor VIII gene leading to replacement of Arg593 by Cys in the A2 domain of factor VIII was associated with hemophilia A in this patient. The anti-factor VIII antibodies present in the patient's plasma were characterized using metabolically labeled factor VIII fragments expressed in insect cells. The anti-factor VIII antibodies, composed of subclasses IgG2 and IgG4, reacted with both the fragment corresponding to the factor VIII heavy chain and the A2 domain. The Arg593 → Cys substitution was introduced into the cDNA encoding the A2 domain of factor VIII and the resulting construct was expressed in insect cells. Strikingly, the metabolically labeled A2 domain carrying the Arg593 → Cys mutation was not recognized by the anti-factor VIII antibodies present in the plasma of the patient. These data indicate that the anti-factor VIII antibodies are exclusively directed against exogenous factor VIII. This strongly suggests that the Arg593 → Cys substitution results in recognition of wild-type factor VIII as nonself and is thereby related to the formation of anti-factor VIII antibodies after factor VIII replacement therapy in this particular patient.

Intracellular accumulation of factor VIII induced by missense mutations Arg593→Cys and Asn618→Ser explains cross‐reacting material‐reduced haemophilia A

“…Similar levels of factor VIII activity and antigen were observed for both rFVIII-R593C and FVIII-R593C present in the plasma of patient AMC-64 (Table I). In previous studies, identical levels of factor VIII antigen and activity levels were reported in an unrelated patient (AMC92) with the same genetic defect (Pieneman et al, 1995;Fijnvandraat et al, 1997). We do not have an explanation for the deviations between the different reports.…”

“…Both the Arg 593 !Cys and the Asn 618 !Ser mutations may be associated with inhibitor development (Fijnvandraat et al, 1997;Thompson et al, 1997;Hay et al, 1998;Vlot et al, 1998;van den Brink et al, 1999). Intracellular accumulation of rFVIII-R593C and rFVIII-N618S is most probably related to suboptimal folding of these variants in subcellular compartments.…”

“…Previously, the missense mutations Arg 593 !Cys and Asn 618 !Ser have been identi®ed in patients with mild haemophilia A, who developed inhibitory antibodies directed against factor VIII (Fijnvandraat et al, 1997;Thompson et al, 1997;Vlot et al, 1998;van den Brink et al, 1999). Inhibitor formation in these patients may be linked to structural alterations in the A2 domain of factor VIII induced by the missense mutations Arg 593 !Cys and Asn 618 !Ser.…”

HLA‐DR‐restricted T‐cell responses to factor VIII epitopes in a mild haemophilia A family with missense substitution A2201P

“…Neutralizing antibodies to FVIII, clinically termed ÔinhibitorsÕ, are a serious complication of FVIII infusion that develop in approximately one-third of severe patients, and they generally occur within the first five years of FVIII treatment [2][3][4]. FVIII inhibitors develop less frequently in mild/moderately severe patients [5][6][7], in whom they may occur later in life. Thus, the highest risk for inhibitor development is associated with mutations resulting in the absence or severe truncation of the FVIII protein [8,9].…”

Analysis of factor VIII inhibitors in a haemophilia A patient with an Arg593→Cys mutation using phage display

“…Peripheral blood mononuclear cells were used from a mild haemophilia A patient with a factor VIII inhibitor. Approximately half of the anti-factor VIII antibodies in plasma of this patient were of subclass IgG4 (Fijnvandraat et al, 1997). RNA was isolated from the peripheral blood cells and used for the construction of a phage display library as described previously (van den Brink et al, 2000).…”