Abstract

Haemophilia A (HA) and B (HB) are the most common inherited bleeding diseases. HA and HB are X-linked recessive disorders caused by mutation in the factor VIII gene which maps to Xq28 and factor IX located at Xq27, respectively; resulting in absence or deficiency of these proteins. Several mutations have been reported as responsible for the disturbance of these genes; therefore, the use of direct molecular techniques to analyze the carrier status of women and their affected fetuses in not easy to perform. Thus, gene linked polymorphisms analysis is the most convenient molecular test since it is independent from the nature of the mutation, allowing the identification of the mutant X chromosome by following its segregation along the pedigree. The main objective of this research was to perform the molecular diagnosis of HA or HB carrier status in pregnant women and male fetuses affected or not, who were referred to the Medical Genetic Unit of the University of Zulia (UGM-LUZ), Maracaibo, Venezuela. Molecular analysis for HA and HB was performed in 32 DNA samples from 8 pregnant women, 8 fetuses, 8 affected and 8 healthy males. Using the Polymerase Chain Reaction (PCR), a 142 bp (bases pairs) fragment, which corresponds to intron 18 of the Factor VIII gene, was amplified. This fragment has a restriction polymorphism for the enzyme Bcl I. Additionally, a Duplex PCR was performed for the STRs (short tandem repeat) of introns 13 and 22 of the same gene. On the other hand, Hinf I, Xmn I y Taq I polymorphism in the factor IX gene were also amplified, so, we were able to build the haplotypes for each one of the key members in the families affected. The latter, allowed us to identify, in five of the eight cases, the mutant X chromosome responsible of HA and HB, thus, prenatal diagnosis was possible with the following results: three healthy males fetuses, two affected males fetuses ith HA and three females fetuses.