From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
Hemophilia
By Mario
Pecorara,
Lucia
Angelo
Boeri,
Elio
A: Carrier
Detection
Casarino,
Claudio
and
Pier Giorgio
Molinari,
Raffaello
Angela
Loi,
Mori,
Massimo
De Biasi,
Lucia
Nicola
Perseu,
EMOPHILIA
A is the
disorder,
affecting
deficiency
or abnormality
inherited
as a X-linked
been assigned
years
ago,
ratio
of
Willebrand
80%
most
common
trait.’
to band
q28
carrier
detection
The
locus
of cases.36
for factor
Prenatal
analysis
diagnosis
of
has
blood
fetal
was
cloned9”
and
VIII-C
were
the factor
chromosome-specific
closely
restriction
VIII
a
is
has
a few
of the
have
prenatal
been
morphisms
were informative
families
investigated,
whereas
probes
showed
by
20
within
random
known
VIII
gene,
to carrier
of hemophilia
out
1 8 to
polymorphisms
polymorphisms
applied
at
In addition,
probes,
length
diagnosis
the Stl4
DNA
to the factor
linked
carried
a procedure
associated
Recently,
the factor VIII
detected.’2’4
DNA
fragment
discoveries
three”
been
obtained
DX 1 315
A.2122
The
detected
several
(RFLPs).
These
intragenic
only in a limited
the DXI 3 and,
recombination
X
and
detection’”7’#{176}
with
and
factor
VIII
In this article,
we describe
in combination
probes,
with
of hemophilia
Italian
the use of a factor
X
and STI4,
DX13
diagnosis
VIII
chromosome-specific
for carrier
detection
A in a large
series
and
After
informed
AND
consent
DNA
citrate,
Blood,
by a modification
analysis.
and
Vol
70,
From
the
the
Venous
DNA
was
No 2 (August).
of Laurell’s
blood
extracted
1987:
samples
from
pp 53 1-535
IV
of
blood
samples
the hemoClinic in
in Florcenter of
di
WBCs
Scrivano,
Barbujani,
ed
Address
coagulant
vWF:Ag
collected
standard
into
payment.
1987
Clinica
Cagliani,
Sardinia,
article
This
article
must
accordance
with
Ban;
sulle
istituto
di
Cagliani.
17, /987.
Ingegnenia
to Professor
ofthis
in
April
1986
costs
II.
di Ricerca
Cagliani;
P.F.
Ereditanie,
requests
Ravenna;
Medica
Istituto
accepted
CNR
Policlinico.
di
Universit#{224} Studi
dell’Et#{225} Evolutiva.
s/n, 1-09100
“advertisement”
©
Malattie
reprint
publication
charge
1986;
/9,
e Biologia
Jenner
di
dell’Et#{225} Evolutiva,
by a grantfnom
delle
Clinica
Istituto
Fi-
Palermo,
Emofilia.
Ospedale
Mediterranee-CNR
August
Molecolani
Centro
Universit#{226} di Ferrara;
e Biologia
Supported
Napoli;
Anemie
Gaslini,
Carreggi.
Universit#{227} Studi
Trasfusionale.
Coagulazione.
Giannina
Ospedale
Pediatrica,
Centro
di Zoologia.
indicate
by
Maria
Guido
Instituto
Ematologia;
Pellegrini,
Ban;
di
Talassemie
di
Clinica
Nuovo
Istituto
Pediatria,
Divisione
istituto
The
technique.25
were
Ripa,
Pirastu
Divisione
Genova-Quarto
Italy.
Submitted
obtained,
Angela
Teo
Chorionic
villous samples were obtained
by the transcervical
route under ultrasound
guidance
using a malleable
silver
cannula.27 The chorionic
villous samples were checked microscopically to exclude contamination
with maternal
decidua
before being
dissolved in 10 mmol/L
of Tris-HC1
(hydroxymethilaminomethane
hydrocloride)
pH 7.5, 1 mmol/L
of EDTA,
2% sodium
dodecyl
sulfate (SDS),
8 mol/L of urea, and 0.3 mol/L of NaCl. This was
followed by extraction
with phenol and then chloroform.
The DNA
was then precipitated
with ethanol and reconstituted
in TE buffer (1
mmol/L
of Tris-HCI
pH 7.5, 1 mmol/L
of EDTA);
10 zg of DNA
was digested
with the restriction
endonucleases
Bcll, BglII, or Taql
(Amersham,
UK), under conditions
recommended
by the manufacturers. Resulting
fragments
were separated
by electrophoresis
on a
0.8% agarose gel and transferred
to nitrocellulose
filters by Southern
blotting.28
Bound DNA was hybridized
with probes radiolabeled
with 32P by nick-translation.
Hybridization
was performed
overnight at 42 #{176}C
in a solution containing
50% formamide,
50 mmol/L
of Hepes, pH 8.0, 5 x Denhardts
[ I x - 0.2% bovine serum albumin
(BSA), 0.2% polyvinyl-pyrolidone,
0.2% Ficoll 400), 6 x I x - 0.15
mol/L ofsodium
chloride and 0.01 5 mol/L ofsodium
citrate (SSC),
Laboratorio
prenatal
of families
were collected
from members
of 61 families followed at
philia center of Gaslini Institute
in Genoa, the Pediatric
Palermo,
the hematology
department
of Carreggi
Hospital
ence, and the 2nd medical
clinic and the hemophilia
Pellegrini
Hospital in Naples.
Coagulation
and immunologic
assays.
Factor VIII
activity
(VIII-C)
was measured
by a one-stage
method24;
determined
Mario
Universit#{225} di
METHODS
was
Mancuso,
Bencivelli,
the carrier
status
in 39 of 45 female
relatives
of sporadic
patients.
The combination
of RFLP analysis
and biological
assay
of factor
VIII allowed
us to identify
a de novo
mutation
in the maternal
grandfather
in 7 of 1 2 of the
families
with sporadic
cases,
for which
members
of three
generations
were
available
for study.
Nine
of 1 0 couples
requesting
prenatal
diagnosis
provided
informative
RFLP
DNA
pattern.
Carrier
status
was excluded
in two women,
two
fetuses
were
shown
to be female,
and prenatal
diagnosis
was carried
out in five pregnancies
by DNA analysis.
Prenatal
testing
was
successful
in three
instances
and
failed
in two
because
a sufficient
amount
of chorionic
villous
DNA
was not obtained
for the analysis.
S 1987
by Grune
& Stratton,
Inc.
Clinica
MATERIALS
was
and
Ospedale
cDNA
random
descent.
Subjects.
Cao,
renze;
h7,19,22.23
probe
Giuseppe
Franco
Analysis
poly-
number
of the
less frequently,
the
Morfini,
by DNA
techniques.26
from
and
of chromosome
X.2 Until
was based on estimation
weeks
of gestation
by fetoscopy,”8
with a high risk of fetal mortality.
St14,’6
bleeding
It results
VIII-C
factor
VIII
coagulant
activity
(FVIII-C)
to von
factor
antigen
(vWF:Ag),
which
is accurate
in
conventional
gene
human
1 male in 10,000.
of clotting
factor
-
Diagnosis
Ciavarella,
Antonio
In this
study.
we used
DNA polymorphisms
for carrier
detection
and prenatal
diagnosis
of hemophilia
A in a large
group
of Italian
families.
The
restriction
fragment
length
polymorphisms
(RFLPs)
investigated
were
the intragenic
polymorphic
Bcfl
site
within
the factor
VIII gene;
the
extragenic
multiallelic
Taq I system
at the St14 locus;
and
the extragenic
BgI II site at the DX1 3 locus.
The factor
VIII
probe
was informative
in 30%. St14 in 82%,
and DX1 3 in
60% of obligate
carriers.
The combination
of factor
VIll-BcI
I and St14- Taq I showed
that 91 % of obligate
carriers
were
heterozygotes
for one or both; with all three
probes,
only
4% of obligate
carriers
were
noninformative.
In families
clearly
segregating
for hemophilia
A, RFLP analysis
allowed
us to define
the carrier
status
for the hemophilia
A gene in
all 27 women
tested.
RFLP analysis
allowed
us to exclude
H
Prenatal
Genetica
(U.O.
No
Antonio
e Basi
58) AC.
Cao,
Universit#{225} degli
Istituto
di
Studi.
Via
Italy.
were
defrayed
therefore
/8
U.S.C.
be
in part
hereby
§1734
by page
marked
solely
to
this fact.
by Grune
& Stratton,
Inc.
0006-4971/87/7002-0033$3.OO/O
531
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
532
PECORARA
of salmon sperm DNA,
10% dextran
sulfate and 4 x 106
of heat-denatured
probe. After hybridization,
excess probe
was washed off in 2 x SSC for 30 minutes
at room temperature,
followed
by 1 /2 hours at 50 #{176}C
in 0. 1 x SSC and 0.1% sodium
dodecyl
sulfate
(SDS).
DNA bands that hybridized
to the 32Plabeled
probe were detected
by autoradiography
with x-ray film
(X-Omat,
AR; Kodak) on intensifying
screens (Cronex,
Du Pont,
UK).
DNA probes used were the I .3-kilobase
(kb) fragment
of factor
VIII eDNA
covering
exons 14 through
19 (kindly
provided
by
Chiron
Corporation,
Emeryville,
CA)”;
DXI3,
a cloned sequence
localized
to band Xq28 by somatic
cell hybrids30 and by in situ
hybridization3’
(kindly provided
by Dr K. E. Davies);
and 5114, a
3-kb DNA
fragment
originating
from the q26-qter
region of the X
chromosome’6
(kindly
provided by Dr J. L. Mandel).
200
Table
zg
Restriction
No.
We
unrelated
families
for the presence
tives
the
factor
VIII
referred
350
ofa
gene
shown
in Fig
I . The
BclI
Stl4
Taq
I reveals
probe
fragments
with
a probe
probe
hybridized
for
to fragments
to human
DNA
for
linkage
between
phase
hemophilia
phism).
(30%),
was
The
St14
informative
heterozygosity
at
with
children
the
detectable
factor
VIII
probe
informative
the
allelic
was
was
in
DNA
and
156
5.8/43
5.3/4.6
2.8/57
1.8
3.4/ 14.5
B.
no.
of
I8
unrelated
females
59
65
48
was
informative
in 19 of 23 (82%),
combined,
St14
summa-
from
famisibships
who
Total
no.
No.
for
21/35
47/73
31/48
of
unrelated
obligate
27
23
23
9/30
19/82
14/60
studied
of
heterozygotes
for
were
were heterozygous
the three
probes
carriers
were
BclI
the
informative
the
factor
and
the
observed
The
markers.
Bc/I
at
BglI
site
linkage
polymorphism
investigated
2). The
The
lod score
recombination
gene
limits
ned
gene
of
revealed
in 20 and
number
is <6.4
from
between
in 9 of 27
FVJIt-Bcl
was
probe
limits
and
subjects
showed
When
the factor
to other
to the
(Table
VIII
factor
confidence
DXI3
locus
VIII
homozygous
for the intragenic
was this polymorphic
by
19 families,
of meiotic
events
ana-
lyzed
was 36 for the St14
locus
and
31 for DX13.
No
recombination
was observed
between
the Stl4
and factor
VIII loci. The lod score for linkage
of factor
VIII to St14 is
4.8 at a recombination
fraction
of 0.0. This indicates
that
the
polymor-
VIII
DX13
respectively
women,
analyzed
of them
not shown).
offactor
and
St14
Eighteen
also
In none
were
(data
Linkage
for one or both polymorphisms.
were
combined,
23 of 24 (96%)
informative.
site,
polymorphism.’3
in whom
22 of 24 (9 1 %) obligate
-TaqI
heterozygotes
C.
allelic
size,’6’8
The
with Bgl II
are
of
carriers
When
with
ten
fragments
(informative
in 14 of 23 (60%).
Some
for more
than one RFLP.
were
1 73
6.6/0.6
RFLPs/%
exon
separate
proportion
polymorphism
A locus
V I I I and St I 4 probe
DX13-BglH
one
1 53
1.1/35
3.9/18
14
fragment
is absent.
at least
of constant
DNA digested
to define
least
exons
digested
the three
polymorphic
systems
investigated
rized in Table
I . Twenty-seven
obligate
carriers
heterozygotes
studied
in kb/%
carriers
DNA
fragments
of 5.8 and 2.8
of the allelic
DNA
fragments
affected
BgIlI/DX13
4.0/15
rela-
to the factor
and RFLPs
at
lies with at least two
were
also investigated
TaqI/St14
4.5/3
within
3’ to the
polymorphism
patterns
detects
a RFLP
with allelic
kb in length.
The frequencies
Bc/l/FVIII
Gene
4.8/15
X chromosome
DNA
sequences
linked
analysis
to human
polymorphic
VIII
RFLPs/%
from
their
a 879-base
pair (bp)
1, I 85 bp when the site
hybridized
in addition
samples
from
polymorphism
DX13
and St14,
closely
Typical
Southern
blotting
The
DXI3
DNA
Bcll
detected
VIII
gene yields
the site is present
and
Factor
0.8/65
polymor-
samples
common
factor
when
DNA
to as
gene.
are
of
extragenic
19, as well as for two common
which
are detected
by anonymous
through
RFLPs,
VIII
and
61 hemophiliac
and
at Three
and Extra
studied
intragenic
analyzed
the
Enzyme/Probe
Fragments
(Bdi/FVIII)
BgIll/DX13)
of X chromosomes
alleles
No.
phisms.
DNA
Intra
A.
Total
of
of Allelic
Systems
(TaqI/St14.
RESULTS
Frequency
1 . Frequencies
Polymorphic
cpm/mL
ET AL
recombination
Stl4
the
DXI3
and
for linkage
locus.
factor
fraction
Carrier
detection.
out
in 27 families
VIII
of0.045,
Carrier
two
was
loci
that
with
detection
at least
with
95%
(Fig
2).
to DX I 3 is 2.1 at a
indicating
units,
with
recombinant
VIII/St14
of factor
is <17.6
recombination
from DX I 3 locus.
units
One
factor
VIII
confidence
95%
(Table
3) was
hemophiliac
car-
males
in
I
Fig
1
Autoradiogram
.
hybridized
to
DX13
(left),
probes
(right) following
and BcIl, respectively.
ment
length
were
investigated
of
St14
digestion
and
3.9.
with
Arrows:
polymorphisms
DX13; 4.8. 4.5. 4.0.
kb for FVIII.
leukocyte
and
BgIll.
FVIII
TaqI.
restriction
(RFLPs)
were:
DNA
(middle).
5.8
at each
and
3.4 kb for St14;
2.8
fraglocus
kb
for
0.8 and 1.1
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
HEMOPHILIA
A BY
Table
2.
DNA
533
ANALYSIS
Linkage
of Factor
VIII L ocus to Other
No. of Recombinants
Pairwise
Cross
Total
x St14
x DX13
FVlll
FVIII
sibships
different
hemophiliacs
in which
inheritance
at risk
29 women
The
criteria.
2.1
probe
with
(group
was
established,
site
in 82%,
34
one sibship
clearly
revealed
and
the TaqI
A gene.
of
investigated.
Definition
analysis
agreed
with
factor VIII-C/vWF:
informative
in
informative
probe
was
informative
Stl4
and
of
34
in I 5 of 30
VIII
families
(86%).
By adding
the number
of informative
In
RFLPs
were
carrier
cases.
two
found.
of
34
RFLP
RFLP
on the
analysis
basis
of the
allowed
investigated
86
33
14
47
by (%)
50
BcII/FVllI-TaqI/St14
91
86
revealed
possible
was
by the
probe
in
1 1 (33%)
of the
carriers
tested
the ratio of factor
in 16 of 19 females
status
the factor
VIII
probe
was
the
St14
was
(47%),
and
by RFLP
of conventional
in whom both
the
DX I 3 probe
combination
informative
of the
in 29 of
34
of the DX13
probe,
increased
to 32 of 34
(6%)
no
factor
the carrier
The
informative
exclusion
female
relatives
the mother
was
risk.
67
41
conventional
at
Defined
(%)
(%)
60
allowed
us to establish
females
identified
45
BgIII/DX13
families
analysis
excluded
status
34
29
probe
the analysis
families
was
status in 39 of 45 (86%)
In those families
in which
a carrier
four
only
was
status
27
VIII
The
(50%).
probes
Carriers’
Carriers’
investigated
factor
in 20 of 30 (66%),
factor
(94%).
II),
carriers
82
system
families
of possible
With
Cases
30
the results of the ratio
Ag in 1 2 of I 3 individuals
16
group
of families
No.
biologic
of the
of sporadic
classified
as
VIII
status
assay
II
Families
Sporadic
TagI/St14
of the carrier
assays
were carried
out.
In the sporodic
cases (group
more
No.
Group
With
Cases
BcIl/FVIII
by the DXI3
the
Factor
we investigated
escaped
identification.
In obligate
carriers,
VIII-C/vWF:Ag
was in the carrier
range
(84%)
or
Families
Inh&ited
of genetic
revealed
None
by RF LP Analysis
basis
60%.
RFLP
analysis
allowed
classification
of
women
as carriers
and
18 (67%)
as non-carriers
hemophilia
D etection
on the
by the
BgIII
one
In the first
II).
carriers
in 30% ofcases,
informative
St14
4.2
0.045
in
A Carrier
Groupi
0
and
Hemophilia
Lod Score
1/31
I)
3.
Table
0/36
of being
Bc/I
Recombination
Fraction
Meiosis
(group
in only
Markers
assay,
in all
and
RFLP
analysis
gated.
In
generations
biologic
were
of
7
concordant
12
in 17 of 25 carriers
families,
in
which
were available
for study,
assay
with
RFLP
analysis
investi-
members
of
the combination
revealed
a
three
of the
de novo
occurrence
of hemophilia
A mutation
in the maternal
father.
An example
of this type of de novo mutation
grandis shown
in Fig 3.
Prenatal
ten
diagnosis.
pregnant
At least
tive
Prenatal
women
1 of the
in all but
from
3 polymorphisms
investigated
in 2 and
the
remaining
not
Prenatal
fetal
blood
unsuccessful
2 cases
yield
with
analysis
and
DXI3
male
probe
fetuses,
informaand
Carrier
status
In the remaining
was
in
7
1. DNA
in I case;
of
parents
fetus
decided
villous
DNA
was
in the other
analysis
and the diagnoses
analysis.
In the
chorionic
amount
of an affected
the
4).
determined
to be female
and no
out. In the remaining
5, prenatal
by RFLP
analysis
in 3, using the
a sufficient
diagnosis
by
(Table
was male
predicted
a normal
and 2 affected
fetuses,
were
confirmed
by fetal
or cord
blood
did
requested
was
the fetus
terminated.
in 2 women.
electively
analysis
pregnancies,
2 fetuses
were
further
analysis
was carried
diagnosis
was accomplished
probe
was
A families
In this family,
I family.
the pregnancy
was
excluded
by RFLP
St14
diagnosis
hemophilia
biopsy
for
analysis.
accomplished
case,
fetoscopy
to continue
the
by
was
preg-
nancy.
I
H
II
I
a94
3
3
4.5
3.9-4
4
3.9-4
34
N
lii
m
LI
!
4.5-3.9
N
fl5-8
4
:,,
Uoe
oxta-
egi
S?14.
T.qI
II
FVIN-ScII
Fig 2.
Pedigree.
illustrating
a crossing-over
between
the
DX13 and the St14/FVIII
loci. Bars: three chromosome
haplotypes
and the length
of the restriction
fragment
length
polymorphisms
(RFLPs)
of these
three
loci (bottom).
In 111-5, a recombination
occurred
between
DX13
and
VWF
St14/FVIII.
4
4
3.9-3.4
N
FVIH
VWF
Fig 3.
Pedigree
illustrating
a de novo occurrence
of hemophilia A mutation.
Numbers
below the symbols
denote
the alleles
of the St14 locus. Factor VIII biologic activity
was measured
by the
FVlll/von
Willebrand
factor
(vWf)
ratio in the available
females.
These
ratios were
normal
(N) in all except
in Il-I. in which
it was
decreased.
Thus. the X chromosome
with
the 4-kb St14 allele that
she passed to the affected
son (111-2) already
carried
the hemophiliac mutation.
Because
111-3 did not have hemophilia
and 11-3 is not a
carrier.
the
mutation
must
have
occurred
in the
grandfather
(I-i).
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
PECORARA
534
Table
4.
Prenatal
Pregnancies
of He mophilia
Diagnosis
by DNA
Informative
RFLP
Analysis
a useful
9
noninformative,
aborted)
probes,
applied.
analysis
1 (Male.
Noninformative
Exclusion
of carrier
RFLP
status
tion
2
determination
Sex
by
analysis
addition
2
DXI3
loci. Previous
studies
DXI3
and the factor
have
VIII
allow
of the
status in the most
hemophilia
A families
in
two subjects
are affected
in separate
sibships
carrier
at least
(group
I in Table
probes
alone
that
St14,
3). Use
carriers.
hemophilia
86%
of
families
informative
analysis
female
analysis
mother
factor
allowed
relatives
on the
basis
VIII,
with
family
exclusion
(Table
can be used for carrier
of the sporadic
patients
carrier
of three
DNA
permit
detection
combination
In
A, when
RFLP
ofthe
or in various
of the
able,
a combination
and DXI3-will
and
was
shows
VIII,
St14,
and
allowed
detection
of
sporadic
patients
of
members
of the
3).
were
factor
avail-
carrier
Despite
identification
has been
of the conventional
DXI3
state
this,
in 7 of I 2 (58%)
members
of three
This
indicates
a high
the
hemophilia
A
and
parents
phisms
and,
within
for carrier
because
VIII
RFLP
the
factor
the
three
loci
DX13/St14/factor
St14
in
gene
and
are
DX13
be
one recombinant
and
the
factor
clearly
require
and
useful
the
it did
since
the
morphisms
caused
and
site
Bc/I
the
hemophilia
intragenic
not
factor
diagnosis
increase
VIII
polymorphism
gene
BglI
detection
brother,
grand-
was
VIII/St14
the
gene,
within
In
not
obtained
of other
particularly
intron
this
was
information
Investigation
A
is negligible.
polymorphism
the
22,’
risk
age
of
analysis
random
carrier
with
the
with
in 70%
to 95%
VIII
extragenic
assay.
be
investigated.
of chorionic
predict
that
of error,
definite
which
of cases.3
sites
may
iac
male
or
father
the
the
factor
VIII
ofwomen
criteria
track
gene
of
by linkdetected
by
conventional
be used
to calcu-
of pedigree
information,
assay.
Prenatal
diagnosis
successfully
in 7 ofthe
10
the data
reported,
diagnosis
the
instances,
conventional
factor
still be required
in the future,
within
as
in
were
caused
by insufficient
DNA
for analysis
in two cases
is available.
to
in
DNA
Because
detection
with
analysis
prenatal
A proband
either
this,
advantages
is accurate
produced
in
we confi-
of hemophilia
linkage
analysis
with intragenic
or extragenic
sites will be accomplished
in -95%
of families
A by
polymorphic
in which
According
at risk for being carriers
have no living hemophilmutant
gene.34
In
VIII assay
of fetal blood
even as more polymorphic
and
a
to recently
or new
random
probes
these
will
sites
are
identified.35
ACKNOWLEDGMENT
polythank
We
be
probe
Despite
uninformative
RFLPs
in one. From
study,
as well as in those
already
dently
recombination
site
the
polymorphic
Failures
villous
95%
6.4%
combined
Computer
as
the
was
carrier
however,
must
study,
a
risk
has
assay,
results
locus and the DXI3
probes,
particularly
diagnosis.
detection
biologic
misdiagnosis,
probes
this
the St14
a small
late the genetic
risk on the basis
RFLP
segregation,
and biological
ofhemophilia
A was accomplished
recently
should
carries
These
X chromosome
of
and
or prenatal
identification
the
locus
published
studies,36
-30%
of hemophilia
A on genetic
primarily
between
thus
hemophilia
polymor-
of hemophilia
by recombination
polymorphism.
in the
XbaI
prenatal
of
esti-
for
carrier
and
this
analysis.
as maternal
analysis
compared
In
the factor
VIII
of these
random
detection
cases
amount
in which
previous
that carrier
of the mother,
as well
occurgrand-
mutation
with
indicates
testing
siblings,
misdiagnosis
experience,
of spontaneous
possible,
the father.
Bc/I
VIII gene should
be used
whenever
the factor
detection
for
DX I 3 probe
gene.’7
the
probability
VIII
that between
17.4%.
Use
carrier
for
factor
VIII
along
VIII.
interval
the
factor
assay.
cases
available
in agreement
nonaffected
polymorphic
of sporadic
were
frequency
gene,
mates.32’33
This study
and prenatal
diagnosis
affected
families
generations
in
only when the
identified
as a
RFLP
analysis
enabled
us to establish
a de novo
rence
of the hemophilia
A mutation
in the maternal
discovered
families
shown
two
crossovers
between
gene’822
and one between
the
and
between
This
study
probes-factor
using
whereas
loci
confidence
DISCUSSION
our
VIII
with
locus
us to order
follows:
2
Failures
father
gene,
the
DX13/Stl4
3 (2 by TaqI-St14
by RFLP analysis
1 byBg/ll-DX13)
96%
In
8
M
Diagnosis
which
markers.
the factor
to the hemophilia
A locus,
can
the St14 locus showed
no recombina-
VIII
between
within
analysis
linked
study,
the factor
with
available
sites
linkage
closely
In this
observed
of the fetus
F
to the
the polymorphic
which
10
monitored
ET AL
Gampell
R. Loi and P. Mameli
for editorial
for typing
the manuscript
and J.
review.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1987 70: 531-535
Hemophilia A: carrier detection and prenatal diagnosis by DNA analysis
M Pecorara, L Casarino, PG Mori, M Morfini, G Mancuso, AM Scrivano, E Boeri, AC Molinari, R De Biasi
and N Ciavarella
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