Aplicació de la tecnologia de seqüenciació massiva a la determinació del genotip Kell Fetal a partir del plasma matern
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Knowledge
of
the
fetal
Kell
genotype
in
pregnancies
of
sensitized
women
with
anti-K
is
of
great
value
to
determine
if
the
fetus
is
at
risk
of
haemolytic
anaemia
and
allows
a
better
management
of
such,
otherwise,
high‐risk
pregnancies.
Several
non-invasive
approaches
for
fetal
Kell
genotyping
have
been
developed
ages
earlier
than
20
weeks.
The
next-generation
sequencing
(NGS)
Technology
offers
unprecedented
possibilities
of
massive
parallel
analysis
of
gene-targets
amplified
sequences
from
maternal
plasma
cell-free
(cf)
DNA.
Initial
proof
of
concept
studies
have
shown
promising
results.
Objectives:
1)
To
design
a
NGS
approach
for
the
fetal
Kell
genotype
analysis
in
plasma
cfDNA
and
2)
To
evaluate
its
sensitivity
and
the
reliability
of
the
fetus
in
a
validation
study
with
a
panel
of
clinical
samples
from
sensitized
pregnant
women.
Materials
and
Methods:
A
total
of
17
plasma
samples
from
pregnant
women
with
anti‐K
have
been
prospectively
collected
during
the
past
three
years.
Cell‐free
DNA
has
been
extracted
from
2
ml
of
these
plasma
samples
as
well
as
from
artificial
chimeric
mixtures
of
plasma
from
individuals
with
known
genotype
(major
KEL2/2,
minor
KEL1/2)
using
the
QIAsymphony
extractor.
A
125b
bp
KEL
gene
fragment
encompassing
the
KEL1/2
SNP
has
been
amplified
using
gene-specific
primers
with
NGS
suitable
adaptors.
The
resulting
amplification
products
have
been
pooled
with
other
amplicons
and
sequenced
in
a
MiSeq
Desktop
Sequencer.
The
NGS
pipeline
output
paired
sequence
files
have
been
used
as
input
for
the
analysis
with
CLC
Genomics
Workbench
software. Results:
Initial
tests
were
performed
with
plasma
DNA
from
the
artificial
chimeric
mixtures,
having
a
1
to
20%
minor
component
represented.
Parallel
amplification
of
the
AMEL
gene
and
the
SRY
locus
has
been
optimized
as
well
to
include
a
fetal
sex
marker.
Optimal
amplicon
size
and
input
plasma
DNA
have
been
assessed
with
a
restricted
panel
of
clinical
samples
representing
the
real
scenario
of
application.
Conclusion:
This
NGS
method
for
non-invasive
fetal
Kell
genotyping
has
shown
specific
and
sensitive
detection
of
the
fetal
KEL1
allele
in
clinical
samples
of
sensitized
pregnant
women.
It
is
also
compatible
with
the
simultaneous
sequence
analysis
of
multiple
amplicons
from
different
loci.
In
this
sense,
further
developments
may
include
other
loci
of
blood
group
polymorphisms,
potentially
involved
in
haemolytic
disease
of
the
newborn