Abstract • 142

Carrier detection and prenatal diagnosis of severe Hemophilia A is requested by 100% of the Italian families involved in this disease. The improvement of the techniques employed and their accuracy is the topic of this abstract. At the Hemophilia Study Center of the G. Gaslini Children's Hospital 370 Italian families have been examined so far. Among them 1401 possible carriers were examined, 817 of whom proved to be carriers while 584 did not. In 78% of the cases restriction fragment length polymorphism (RFLPs) were employed as intragenic (BclI/Intron 18 or Xbal-Kpnl/Intron 22, M13/VNTR and 122/VNTR) or as extragenic markers (TaqI/ST14 and BglI/Dx13).

The main limits of this method are:

  • the misdiagnosis of the carrier status in cases of sporadic hemophilia when the probable carrier shares the same haplotype as the hemophilic

  • cases of homozigosity

  • when the hemophilic is not available for testing

  • the possibility of crossing over when extragenic RFLP are used (5% in our cases)

On the other hand RFLP maintain their diagnostic value in sporadic hemophilia when the probable carrier and the hemophiliac do not have the same haplotype, or when a female is a carrier due to the fact that she is the daughter of a hemophiliac or because she is a member of a family with familiar hemophilia.

The direct method of diagnosis we used is the detection of FVIII gene inversion (49% of our cases were positive) and the direct search of the FVIII gene mutation by DGGE or CSGE techniques (this technique was employed in 17% of our cases). Even this latter method has a pitfall when the hemophiliac mother in sporadic hemophilia has a Factor VIII gene normal (5 in our cases). In fact in this case a germinal mosaicism can not be excluded. Only in 5% of the cases carrier diagnosis was non possible.