Assay discrepancy in mild haemophilia A due to a factor VIII missense mutation (Asn694Ile) in a large Danish family

Abstract

Summary. Factor VIII gene analysis in a large consanguinous Danish family comprising 24 affected males and four homozygously affected females revealed an Asn694Ile mutation within the A2 domain. The factor VIII gene mutation led to a mild haemophilia A phenotype with factor VIII function displaying discordance between one-stage clotting and chromogenic two-stage assays. In one-stage assays, values ranged from 0´05 to 0´30 IU/ml (males) and from 0´19 to 0´29 IU/ml (homozygous affected females), whereas the chromogenic two-stage assay produced values of around only 50% of the one-stage result [0´02±0´12 IU/ ml (males); 0´06±0´10 IU/ml (females)]. The differences are suggested to be caused by the effect of the mutation on the active cleaved form of the factor (F)VIII protein. As the original amino acid (Asn) is conserved in all known FVIII A2 sequences, but not in ceruloplasmin, we suggest that Asn694 is involved in an A2-specific functional role. Examination of a homology model of the A domains predicts that the Asn694Ile mutation (i) results in the loss of two potential hydrogen±bonding interactions and (ii) hampers the integration of the bulky side-chain of Ile into the A2 domain core, probably causing an altered stability and/or folding of the protein. Interestingly, the disease in this Danish family was originally proposed to be von Willebrand±Ju È rgens disease. However, the current study rules out the co-existence of either von Willebrand's disease or the presence of the Normandy variant of von Willebrand factor (type 2N).

Molecular mechanisms of mild and moderate hemophilia A

“…Later on, biochemical analysis of recombinant FVIII carrying the substitution Arg531His indicated that, following activation by thrombin, the mutant FVIII had an accelerated rate of spontaneous inactivation due to an increased rate of dissociation of the A2 subunit [45,46]. This observation suggested a role for Arg531 in stabilizing the activated FVIII molecule.…”

Evaluation of three automated chromogenic FVIII kits for the diagnosis of mild discrepant haemophilia A

“…, 1996; Mazurier et al. , 1997) or Coamatic (Schwaab et al. , 2000) have been used, as well as from Dade (Sysmex, Milton Keynes; Keeling et al.…”

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

“…A family pedigree has been described in which all members have 2-st FVIII activities that are one half of that measured by 1-st assay. 36 This pedigree included affected males as well as homozygous females. Molecular analysis identified a missense mutation at residue 694 within the A2 subunit where ILE is substituted for ASN.…”