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PGD in Italy: 1PB Testing
Mutation analysis
The diagnostic protocol involves a fluorescent multiplex PCR
analysis of highly polymorphic STR markers, linked to the
disease causing genes, to identify the haplotype associated
with the maternal mutation. In fact, any informative
polymorphism, which lies in close proximity to the disease
causing gene, can be used as a tool to ascertain the
presence or absence of the mutation, without its direct
detection, just evaluating the inheritance of the haplotypes,
obtained by segregation analysis of the STR alleles (Figure
2).
Because 1PB is the mirror image of the oocyte, its genotype
is indirectly derived from the opposite diagnosis of the
1PB.The presence of the haplotype associated with the
mutated allele in 1PB indicates that both copies of the
mutated gene have been extruded and the oocyte can be
predicted to be free of mutation. On the contrary, if 1PB
analysis reveals the presence of the haplotype associated
with the normal allele, the oocyte presumptively contains
the mutant gene. If crossing over does occur, the 1PB will
contain both the normal and the mutated haplotype, showing a
heterozygous genotype for the STR markers investigated
(Figure 1).
The implementation of an indirect diagnosis, by haplotype
analysis, precludes the need of detecting the specific
causative mutation, and therefore provides a unique protocol
applicable to most of the patients at risks of transmitting
a specific gene defect. This approach can be carried out for
any mutations combination, without the need to develop a
specific diagnostic experimental design for each couple. As
a consequence, a substantial shortening of the preclinical
work-up time necessary for each case can be achieved.
A panel of 6 closely linked highly polymorphic STR markers
is studied, to ensure a sufficient informativity in all
cases and also to avoid misdiagnosis in recombinant oocytes
due to possible allele drop-out (ADO) occurrences. The
co-amplification of several polymorphic markers reduces the
risk of amplification failure and increases the assay
accuracy by allowing the detection of potential ADO
occurring in multiple markers, which would lead to diagnose
a recombinant heterozygous oocyte as hemizygous, thus
reducing substantially the risk of misdiagnosis. In fact, in
such a case, misdiagnosis is only possible in the very
unlikely hypothesis that ADO of the wild-type allele occurs
in all amplified markers.
Figure 2. Example of electropherograms showing the
fluorescent PCR products obtained from 1PB after multiplex
amplification of 5 informative STR markers flanking the HBB
gene. The x-axis shows length of PCR products in bp and the
y-axis shows the fluorescence intensity in Relative
Fluorescence Units (RFU). On top of the electropherogram the
marker name is located above the corresponding alleles
(peaks). Numbers next to each peak represent the size of the
allele (in bp). The upper lane shows the haplotype
associated with the maternal mutation, therefore the
corresponding oocytes is diagnosed as mutation-free. The
haplotype associated with the normal allele is shown in the
middle lane, thus the corresponding oocyte contains the
mutant gene. The lower lane shows the profile of a carrier
female, presenting both the mutant and the normal haplotype.
The maternal haplotype was established by family studies.
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Limitations
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