Detection of carriers of haemophilia A: use of bioassays and restriction fragment length polymorphisms (RFLP)

Abstract Background: Haemophilia A is a sex-linked bleeding disorder carried by unaffected females. Currently, the two main methods used for the determination of carrier status in women from families with haemophilia A are bioassays and DNA-based assays using restriction fragment length polymorphisms (RFLP).
Aim: The aim of this paper was to assess the current usefulness of these two methods.
Methods: Bioassays measured factor VIII coagulation activity by a two-stage coagulation assay and von Willebrand antigen by immunoelectrophoresis. RFLP were determined with two intragenic probes (pi 14 and p486) and two linked probes (Stl4 and DX13). Data were analysed using a Bayesian analysis to allow for all possible recombination events. We also incorporated an estimate for the risk of mosaicism into calculations in isolated haemophilia families. Both bioassays and RFLP were used to determine carrier status in 63 women, 31 from known haemophilia families and 32 from families of isolated cases. The techniques were assessed for their ability to classify the patients as normal (p<0.2) or carrier (p>0.7). Where applicable, intron 22 inversion was also tested.
Results: In the known families, six women could not be classified after bioassay, but all could be classified by RFLP. Of the 32 women from families of isolated cases, eight were unclassified by bioassay and 12 were not definitely classified using RFLP. However, RFLP was useful in determining that a recent mutation had occurred in six of the eight families in which DNA from the grandparents was available.
Conclusion: For diagnosis of carriers of haemophilia, RFLP is the preferred method in familial haemophilia, but is less useful in isolated haemophilia.