Portada Simposios - Supplements - Haematologica

XLII Reunión Nacional de la AEHH y XVI Congreso de la SETH. Simposios
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Figure 4. Schematic representation of our direct-sequencing procedure for molecular diagnosis of hemophilia.
each individual. STR markers are commonly more informative,
since they are multi-allelic and thus the heterozygosity
in the population is higher.
2. Mutation screening techniques: This group includes
several methods that allow, on a physical or
chemical basis, the differentiation between a control
amplified DNA fragment and an identical fragment
carrying a mutation. Many screening techniques
have been designed and used for molecular diagnosis
of hemophilia, such as Single Strand Conformational
Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis
(DGGE), Conformational Sensitive Gel Electrophoresis
(CSGE), Amplification and Mismatch Detection
(AMD), Universal Heteroduplex Generator (UHG), etc.
In all cases the first step is identification of the fragment
carrying the mutation, which often involves
two or more candidates. Further DNA sequencing of
these fragments must be performed to confirm the
presence of a mutation and to determine if it is responsible
for the pathology.
3. Direct DNA sequencing: This is conceptually
the simplest approach since the gene associated to
the pathology is analyzed by determining the nucleotide
sequence. This methodology analyzes the regulatory
elements and sequences coding for all functional
protein domains. Because of the significant
recent advances in DNA sequencing technology, direct
sequencing of genes is increasing as the preferred
technique for the molecular diagnosis of a number
of hereditary diseases.
Optimized nucleotide sequencing
in hemophilia diagnosis
One of the main goals in our laboratory was the
design and implementation of a new protocol for rapid,
reliable and sensitive molecular diagnosis of Hemophilia
A and B, based on accurate identification
of the mutations responsible for these bleeding disorders.
Because of the high mutational variability
associated with both genes, it was essential to develop
a relatively simple and cost-effective procedure
with minimal hands-on time so that all the essential
regions of the gene could be studied efficiently. For
this purpose, we modified several previously described
protocols and designed new primers that amplify
a collection of fragments covering all gene essential
regions (23 amplimers for factor VIII and
7 for factor IX) under identical thermocycling parameters.
The complete procedure from blood sample
collection to mutation identification in hemophilia
A, including analysis of the intron 22 inversion,
can be done in less than 4 days. For hemophilia B,
we have been able to sequence the promoter, all
exons and the corresponding flanking intronic regions
in less than 15 hours thanks to the smaller size
of the FIX gene (fig. 4). We obtained similar results