Mutations in a Subgroup of Patients With Mild Haemophilia a and a Familial Discrepancy Between the One‐stage and Two‐stage Factor Viii:c Methods

Abstract

A subgroup of patients with haemophilia A who have a familial discrepancy between the one-stage and two-stage factor VIII:C results has previously been described. These patients show factor VIII:C levels by one-stage assay that are 2-7-fold higher than their two-stage results. We have studied 10 such families and identified six different mutations in the factor VIII gene in this group. The chemical cleavage method and DNA sequencing was used to identify mutations in factor VIII gene fragments generated by reverse transcription and PCR. All available family members were tested to confirm the presence of the mutation in affected individuals. These patients were found to have one of six single point substitutions causing a missense mutation and alteration to one codon in exons 7, 11, 14 or 18. The mutations comprise three that have not previously been described (Ala284Glu. Arg698Leu. Leu1932Phe) and three that have been previously described (Ser289Leu, Arg531His, Arg698Trp). Alterations to the amino acid composition of the A1, A2 and A3 domains of factor VIII are predicted by these molecular studies. In contrast, a control group of 23 mild haemophilia families with equivalent factor VIII:C results by one-stage and two-stage assays did not have any of the above mutations. Detailed studies in seven of these latter families identified four mutations affecting the A3, C1 and C2 domains of factor VIII. These findings suggest a genetic basis to the unusual factor VIII phenotype but do not explain the mechanism of the discrepant factor VIII activity.

Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa

“…27 The slightly higher 2-st activity results obtained with the recombinant-derived mutant proteins are consistent with those obtained from the few reported patient plasmas where a chromogenic assay rather than the classical 2-st assay was used. 18 Following immunoaffinity purification of FVIII WT and mutant proteins from the conditioned medium, similar discrepancy in 1-st/2-st activities were obtained (data not shown).…”

“…[14][15][16][17][18] Initially, the molecular mechanism for one of these mutations, a missense mutation, ARG 531 HIS , was characterized to result from increased rate of inactivation of FVIIIa by increased rate of A2 subunit dissociation. 19 On thrombin activation, the ARG 531 HIS A2 subunit exhibited a 4-fold increased rate of dissociation from the A1/A3-C1-C2 heterodimer.…”

New developments in laboratory diagnosis and monitoring

“…These patients have bleeding histories compatible with the lower levels obtained in a two-stage clotting or chromogenic assay. In many cases the genetic defect has been identified, so there is no doubt that these subjects do indeed have haemophilia [4,5]. In our experience about 5-10% of mild haemophilia A patients have a normal one-stage assay result.…”

A1 Subunit-mediated Regulation of Thrombin-activated Factor VIII A2 Subunit Dissociation

“…These patients originally were identified based on having higher fVIII levels in a one-stage coagulation assay compared with a two-stage coagulation or two-stage chromogenic substrate assay (22,(45)(46)(47)(48). In the one-stage assay, fVIII is activated by thrombin or factor Xa that are formed endogenously during the ϳ45-90 s period between addition of calcium and the formation of the fibrin clot.…”