Portada Simposios - Supplements - Haematologica

128 Haematologica (ed. esp.), volumen 85, supl. 2, octubre 2000
A
B
Figure 3. A) PCR strategy for detection of intron 22 inversion. Recombination between F8A gene located inside intron 22 of FVIII gene
and either of two distal telomeric homologous genes (F8A’ and F8A˝) results in distinguishable PCR products sizes. B) Agarose
gel electrophoresis of PCR products amplified from 8 inversion-negative individuals, 2 inversion-positive patients and a carrier woman.
M: size marker.
Laboratory techniques for the genetic
diagnosis of hemophilia
The molecular diagnosis of hemophilia has been
achieved by a number of techniques that are continuously
improved with better knowledge of the responsible
genes. In severe hemophilia A, the routine
protocol begins with characterization of the most
frequent mutation (intron 22 inversion) by means of
Southern Blot, a tedious and time-consuming technique
that commonly requires the use of radioactive
isotopes. However, recent description of a simple,
single-tube PCR technique allows determination of
the intron 22 gene inversion in patients and carriers
in less time and at lower cost (fig. 3). Moreover,
when applied to prenatal diagnosis, using amniofiltrated
fluid of 11 weeks’ gestation, we found this
technique to be highly sensitive and reliable. If the inversion
is ruled out, the genetic study can be performed
in several ways depending on the laboratory
facilities, human resources and the diagnostic accuracy
required. Techniques for the molecular diagnosis
of Hemophilia A and B can be classified into three
groups according to the degree of sensitivity and
simplicity, and the information provided:
1. Linkage analysis techniques: these are indirect
techniques based on the peculiarity that a number
of polymorphic markers are present along the human
genome. These markers, which can be intragenic or
extragenic, allow identification and follow-up of the
chromosome that carries the gene with the genetic
defect, though they are not related with the pathology.
Intragenic polymorphisms are most suitable
for diagnosis since the probability of recombination
between the marker and the gene defect is lower. The
polymorphic markers used in the linkage analysis of
hemophilia include the Restriction Fragment Length Polymorphism
(RFLP), when the presence or absence of a
restriction site is analyzed, or Short Tandem Repeats
(STR), comprised of short sequences repeated in a
variable number, with a characteristic pattern for