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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Factor XI deficiency in French Basques is caused predominantly by an ancestral
Cys38Arg mutation in the factor XI gene
Ariella Zivelin, Frederic Bauduer, Louis Ducout, Hava Peretz, Nurit Rosenberg, Rivka Yatuv, and Uri Seligsohn
Inherited factor XI deficiency is an injuryrelated bleeding disorder that is rare in
most populations except for Jews, in
whom 2 mutations, a stop mutation in
exon 5 (type II) and a missense mutation
in exon 9 (type III), predominate. Recently,
a cluster of 39 factor XI–deficient patients
was described in the Basque population
of Southwestern France. In this study, we
determined the molecular basis of factor
XI deficiency in 16 patients belonging to
12 unrelated families of French Basque
origin. In 8 families, a nucleotide 209T>C
transition in exon 3 was detected that
predicts a Cys38Arg substitution. Four
additional novel mutations in the factor XI
gene, Cys237Tyr, Tyr493His, codon
285delG, and IVS6 ⴙ 3A>G, were identified in 4 families. Expression studies
showed that Cys38Arg and Cys237Tyr
factor XI were produced in transfected
baby hamster kidney cells, but their secretion was impaired. Cells transfected with
Tyr493His contained reduced amounts of
factor XI and displayed decreased secretion. A survey of 206 French Basque
controls for Cys38Arg revealed that the
prevalence of the mutant allele was 0.005.
Haplotype analysis based on the study of
10 intragenic polymorphisms was consistent with a common ancestry (a founder
effect) for the Cys38Arg mutation. (Blood.
2002;99:2448-2454)
© 2002 by The American Society of Hematology
Introduction
Factor XI is a zymogen that, upon activation to factor XIa,
promotes coagulation by activating factor IX.1 It is currently
postulated that factor XI does not play an important role in the
initiation of blood coagulation but is essential for its propagation
after small amounts of thrombin are generated.2 Factor XI is
activated by thrombin preferentially on the surface of platelets,
which leads to further thrombin generation even after the clot has
formed.3-5 The additional amount of thrombin then activates
thrombin-activatable fibrinolysis inhibitor, which results in clot
stabilization owing to resistance to fibrinolysis.5
Factor XI circulates as a homodimer of identical 80-kd subunits
linked by a disulfide bond. Upon activation by thrombin, factor XI
is cleaved at Arg369–Ile370, yielding a heavy chain and a light
chain, with the latter harboring the active catalytic site. The heavy
chain is composed of 4 “apple domains,” each composed of 90 to
91 amino acids and stabilized by 3 disulfide bridges.6,7 Apple
domain 1 contains the binding site for high–molecular weight
kininogen, thrombin, and prothrombin8-10; apple domain 3 contains
the binding site for factor IX and platelets11,12; and apple domain 4
harbors the binding sites for factor XIIa and for dimerization of the
80-kd subunits.13,14
The human factor XI gene consists of 15 exons and is localized
on the long arm of chromosome 4 (4q35).15,16 The gene is
approximately 50 kilobases (kb) in length,17 and not 23 kb as
initially reported.15 So far, 26 mutations causing factor XI deficiency have been reported.18,19 Of these, 4 are nonsense mutations,
5 are splice-site mutations, 3 are small insertion/deletion mutations,
and the remaining 14 are missense mutations. Six of the missense
mutations were localized to the apple 4 domain, and expression of 5
of them revealed impaired secretion of factor XI from transfected
cells.20,21 For one of the mutations, Phe283Leu (also designated
type III mutation), the impaired secretion was attributed to a defect
in dimerization of the factor XI monomers.21
Inherited factor XI deficiency is a rare autosomal recessive
injury-related bleeding disorder.22 The disorder is common in Jews
of mainly Ashkenazi (European) origin, among whom 2 mutations,
a stop mutation in exon 5 (type II) and a missense mutation in exon
9 (type III), predominate.23,24 A survey of 125 unrelated Ashkenazi
Jewish patients with severe factor XI deficiency disclosed that the
type II and type III mutations each accounted for 49.2% of the
mutant alleles.25 Another cluster of 39 cases with factor XI
deficiency was recently identified among Basques residing in
Southern France.26 In this study, we determined the molecular basis
of factor XI deficiency in 16 French Basque patients and addressed
the question of whether one or more mutations account for the
relatively high frequency of the disorder in this population.
From the Institute of Thrombosis and Hemostasis, the Chaim Sheba Medical
Center, Tel-Hashomer, Israel, and the Department of Clinical Hematology,
Centre Hospitalier de la Cote Basque, Bayonne, France.
Sheba Medical Center, Tel-Hashomer 52621, Israel; e-mail: seligson@sheba.
health.gov.il.
Submitted September 26, 2001; accepted November 20, 2001.
Reprints: Uri Seligsohn, Institute of Thrombosis and Hemostasis, The Chaim
2448
Patients, materials, and methods
Patients
The diagnosis of factor XI deficiency (factor XI activity below 50 U/dL)
was established in 12 probands and 4 immediate relatives who reside in the
Basque region of Southwestern France following a finding of prolonged
activated partial thromboplastin time (aPTT). Only 1 of the 16 patients was
tested because of hematoma following herniotomy; the remaining patients
were routinely tested for aPTT. All patients were questioned about previous spontaneous and injury-related bleeding manifestations. The ethnic
origin of the patients was defined according to the place of birth of the
individual’s grandparents or parents. Blood samples were obtained from
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
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BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
factor XI–deficient patients who gave their consent to participate in this
study, and from control French Basque subjects referred for routine
complete blood count.
DNA purification
DNA was extracted from whole blood of patient’s leukocytes by a standard
procedure.27 Drops of blood remaining after completion of the complete
blood count in controls were mounted on Blood Collection Cards (Gentra
Systems, Minneapolis, MN) and shipped to the laboratory in Israel. Small
disks were punched out from the cards and treated with DNA purification
solution (Gentra Systems) according to the manufacturer’s instructions. By
this procedure, the DNA remains on the disks and can be used directly for
polymerase chain reaction (PCR).
Mutation identification
PCR amplifications of exons 3 through 15 of the factor XI gene were
performed with the use of genomic DNA as template, with primers and
conditions as described by Imanaka et al.28 Direct sequencing of amplified
fragments was carried out by an automatic sequencer (ABI Prism 377,
Perkin Elmer, Foster City, CA).
Analysis of polymorphisms
The CA repeat polymorphism in intron B and the AT repeat polymorphism
in intron M were determined as previously reported.25,29 The polymorphisms 231C⬎T and 138C⬎A in intron A19 were analyzed by amplification
of intron A and digestion with BseDI (Fermentas, Vilnius, Lithuania) and
MslI (New England Biolabs, Beverly, MA), respectively. The 3 previously
described polymorphisms 472C⬎T in exon 5, 844A⬎G in exon 8, and
1234T⬎C in exon 11, which are in strong linkage disequilibrium, were
analyzed by PCR amplification of the corresponding exons and digestion
with the restriction enzymes AatlI (New England Biolabs), Bsh1236I
(Fermentas), and HphI (New England Biolabs), respectively.19,30 Intron E
dimorphism, comprising a 150–base pair (bp) fragment (allele 1) or a
140-bp fragment (allele 2), was detected as described.31 The recently
reported polymorphisms 1855G⬎T and 1882G⬎A in exon 15 were
analyzed by amplification of exon 15 and simultaneous digestion with
BsrBI and RsaI (New England Biolabs).32 All digestions were carried out
according to the manufacturer’s instructions and analyzed on either 3%
agarose gels (Amresco, Solon, OH) or 4% agarose-SFR (super-fine resolution)
gels (Amresco) containing ethidium bromide for UV visualization.
Construction of expression vectors
Human factor XI complementary DNA (cDNA) in pBR322 vector was
kindly provided by Dr Dominic Chung from the University of Washington,
Seattle, and was subcloned by BamHI (New England Biolabs) digestion
into pGEM7 (Promega, Madison, WI) for mutagenesis. The sequence
alterations 209T⬎C, 807G⬎A, 1059G⬎T, and 1574T⬎C were introduced
into factor XI cDNA by PCR site-directed mutagenesis by means of, in each
instance, 2 overlapping oligonucleotide primers that incorporated the
corresponding single base pair substitutions.33 The created mutants were
confirmed by sequencing. PCR products containing each of the 4 alterations
were cut by specific restriction enzymes for replacement of the corresponding normal cDNA in pGEM7-factor XI. The corresponding 5⬘ and 3⬘
restriction enzymes for 209T⬎C, 807G⬎A, 1059G⬎T, and 1574T⬎C PCR
fragments were as follows: BbsI and BsaMI; Tth111I and PstI; PstI and
BstEII; and BclI and PpuMI, respectively (New England Biolabs).
Wild-type (WT) and mutated cDNAs were subcloned into the BamH I
site of pcDNA3 (Invitrogen, Carlsbad, CA) mammalian expression vector
carrying the geneticine resistance gene as a selection marker. Plasmids were
transformed in Escherichia coli DH5␣ and grown in Luria-Bertani medium
(DIFCO, Detroit, MI) supplemented with 100 ␮g/mL ampicillin (Sigma, St
Louis, MO) for large-scale preparation of DNA by means of Concert
nucleic acid purification system (Gibco BRL, Paisley, United Kingdom).
Cell culture and transfection
Baby hamster kidney cells (BHKs) were grown in Dulbecco modified Eagle
medium (DMEM) supplemented by 2 mg/mL L-glutamine and 5% fetal
MOLECULAR BASIS OF FACTOR XI DEFICIENCY IN BASQUES
2449
calf serum (FCS). Medium and FCS were purchased from Biological
Industries, Beit-Haemek, Israel. The cells were transfected by means of
LipofectAMINE reagent (Gibco BRL) with 1 ␮g either WT factor XI or
mutant factor XI pcDNA3s. Mock cells were produced by transfecting
BHK cells with 1 ␮g native pcDNA3 vector. Selection for stably transfected
cells was carried out by growing the cells in medium containing 0.7 ␮g/mL
G418 (Gibco BRL) for 2 weeks. Medium was collected for factor XI assays
from confluent plates containing about 106 cells grown for 24 hours with 7
mL FCS-free medium (Optimem 1) (Gibco BRL).
Factor XI activity and antigen measurements
Factor XI activity was measured by an aPTT-based assay with severe factor
XI–deficient plasma used as substrate (factor XI activity below 1 U/dL) and
1/10 to 1/640 dilutions of normal reference plasma used for construction of
a standard curve. The medium of cultured BHK cells was concentrated ⫻
20 by filtration on a centricon PM-30 (Millipore, Bedford, MA) and assayed
directly without the usual 1/5 to 1/10 dilution prior to testing. The
sensitivity of factor XI activity measurements was thus as low as 0.01 U/dL.
Factor XI antigen was determined by an enzyme-linked immunosorbent
assay (ELISA) using 4 ␮g/mL anti–factor XI monoclonal antibodies for
capturing (Enzyme Research, South Bend, IN), followed by incubation with
samples containing factor XI antigen and binding of 1:1000 dilution of
polyclonal goat anti–factor XI immunoglobulin (Ig)–G (Enzyme Research).
Amplification and detection were achieved with biotinylated antigoat IgG
and avidin peroxidase by means of the Vectastain ABC kit (Vector
Laboratories, Burlingame, CA). Standard curves were constructed with
reference plasma diluted 1/25 to 1/800 in Tris-buffered saline (TBS) (50
mM Tris, 150 mM NaCl), pH 7.5. When no factor XI antigen was detected
in the medium of transfected cells, ELISA measurements were repeated
with 2 additional capture monoclonal antibodies (mAbs), kindly provided
by Dr Joost Meijers (University of Amsterdam, The Netherlands). One
antibody, mAb-3, was directed against an epitope on apple 2, and the other,
mAb-5, was directed against an epitope on apple 1. This precaution was
taken to confirm that the absence of factor XI antigen was not due to a loss
of a recognition site. Measurement of factor XI antigen was performed
either in the medium or in ⫻ 10 concentration of the medium. The lower
limit of factor XI antigen detection was 0.5 ng/mL.
Western blot analysis of factor XI antigen
Proteins were analyzed by sodium dodecyl sulfate–polyacrylamide gels (SDSPAGE) and immunoblotting. Factor XI antigen was detected on the gel by means
of 1:1000 dilution of polyclonal goat anti–factor XI IgG (Enzyme Research)
followed by 1:5000 dilution of peroxidase-labeled antigoat IgG (Sigma) and
chemiluminescence (ECL Western blotting detection reagent) (Amersham Pharmacia Biotech, Buckinghamshire, England).
Immunoprecipitation
The medium of BHK cells was filtered through a 0.22-␮ membrane
(Millipore) and centrifuged for 15 minutes at 14 000g to remove cell debris.
One milliliter of medium was incubated for 1 hour at 4°C with 3 ␮g
nonimmune mouse IgG (Sigma) followed by 60 ␮L 10% Pansorbin cells for
1 hour at 4°C (Calbiochem-Novabiochem, La Jolla, CA). After centrifugation, the supernatant was transferred to a tube containing 3 ␮g monoclonal
antibody against factor XI (Enzyme Research) and incubated on ice for 1
hour. Immune complexes were removed by addition of 60 ␮L goat
antimouse IgG coupled to agarose (Sigma), incubation for 2 hours with
mixing at 4°C, and centrifugation. The agarose beads were washed twice
with 50 mM Tris (pH 7.5), 0.5 M NaCl, and 0.1% NP-40, followed by a
wash with TBS, pH 7.5. Proteins were eluted in sample buffer (0.125 M
Tris-HCl, pH 6.8; 5% SDS; 25% glycerol; and bromophenolblue as dye)
and incubated for 15 minutes at 60°C before SDS-PAGE.
Metabolic labeling of factor XI in transfected cells
Transfected cells in 6-well plates, each containing 105 cells, were exposed
to methionine-free DMEM for 1 hour; then, 150 ␮Ci (5.55 MBq) [35S]
protein mix (New England Biolabs) was added for a 30-minute pulse. The
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2450
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
ZIVELIN et al
cells were chased with cold methionine (2 mM) for 1, 3, 6, and 20 hours and
then washed twice with phosphate-buffered saline (PBS) and lysed in 1 mL
cold lysis buffer (10 mM Hepes, pH 7.5; 50 mM NaCl; 5 mM EDTA; 0.3 M
sucrose; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS) for 10
minutes. The lysates were centrifuged at 14 000g for 15 minutes at 4°C and
kept frozen. Cell lysates were immunoprecipitated by monoclonal antibodies as described, eluted into reduced sample buffer, and separated by
SDS-PAGE. NuPage 4% to 12% Bis Tris gels in 2-N-morpholinoethanesulfonic acid running buffer were used according to the manufacturer’s
instructions (Invitrogen). The gels were fixed in isopropanol/acetic acid/
water (25:10:65), vacuum-dried, and exposed to Biomax MS film with the
use of Biomax Transcreen-LE (Kodak, Rochester, NY) at ⫺70°C.
Immunostaining of intracellular factor XI
Transfected cells were grown on glass slides to subconfluence and then
washed in PBS, dried for 24 hours, and fixed in cold acetone for 15 minutes.
The cells were then incubated in TBS (pH 7.6) containing mAb-3 or mAb-5
(10 to 20 ␮g/mL) and stained with an alkaline phosphatase–antialkaline
phosphatase kit (APAAP) (Dako, Glostrup, Denmark). Microscopy and
photography were done by Duet (BioView, Rehovot, Israel).
Results
Bleeding manifestations
None of the 16 patients with severe or mild factor XI deficiency
exhibited spontaneous bleeding. Hemostatic challenges occurred in
10 patients, 2 of whom with mild factor XI deficiency displayed (1)
a hematoma following cervical disc surgery and (2) mild postpartum bleeding, respectively (Table 1). Two siblings with severe
factor XI deficiency underwent uneventful surgical procedures
with plasma replacement therapy.
Identification of candidate mutations
Table 1 displays the origin of the 12 families studied, the
individuals’ factor XI activities, and the mutations that were
identified. In 8 of the 12 families, a nucleotide (nt) 209T⬎C
transition in exon 3 was detected, which predicts a Cys38Arg
substitution. Patient 1 was homozygous for Cys38Arg, and 8
patients from 6 families were heterozygous for Cys38Arg. The
twins of family VIII who had severe factor XI deficiency were
compound heterozygotes for the Cys38Arg and a novel mutation,
nt 1574T⬎C transition in exon 13, which predicts a Tyr493His
substitution.
The 2 siblings of family IX were of mixed Basque and Jewish
origin and had factor XI activity of less than 1 U/dL. Both were
compound heterozygotes for the type II nonsense mutation observed in Jews (Glu117Stop) and for a novel nt 807G⬎A change in
exon 8 predicting Cys237Tyr substitution. Both siblings also
harbored a rare 1059G⬎T polymorphism in exon 9, predicting
Cys321Phe substitution,32 which was phased to the Cys237Tyrbearing allele (data not shown).
Patient 14 was heterozygous for a novel mutation, a deletion of
nt G in codon 285, which causes a frameshift, leads to a stop codon
at 5 codons downstream, and predicts a truncated protein. Since the
patient was of mixed Basque and non-Basque origin and other
family members were unavailable for examination, it is uncertain
whether the mutation originated in the Basque side of the family.
In patient 15, the only sequence alteration found was a substitution of
the third nucleotide of intron 6 (IVS6 ⫹ 3A⬎G). Conceivably, this
change affects the acceptor splice site of intron 6 because the calculated
splice site consensus score (reflecting similarity of an observed splice
site to the consensus sequence) decreased from 0.98 in the normal
sequence to 0.56 in the altered sequence.34 Unfortunately, messenger
RNA was not available from this patient to demonstrate alternative
splicing. This sequence alteration is probably not a polymorphism since
it was not found in 114 alleles of a mixed population surveyed for
single-nucleotide polymorphisms of the factor XI gene.32
In patient 16, who was of mixed Basque and non-Basque origin,
no candidate mutation was found. Collectively, 5 novel alterations in the factor XI gene were identified in 12 families with factor XI deficiency; 1 of these alterations, Cys38Arg, is common in
French Basques.
Expression of mutated recombinant factor XI in BHK cells
To evaluate the correlation of the identified candidate missense
mutations with the factor XI phenotype, mutated and normal
recombinant factor XI was expressed in BHK cells, and the
secretion and intracellular contents of factor XI were assessed. In
Table 1. Mutations identified in patients with factor XI deficiency in Southwestern France
Patient
Gender/Age
Factor XI
activity,
U/dL
I
1
F/50
⬍1
Breast surgery
None
None
Basque
Cys38Arg/Cys38Arg
II
2
M/22
40
Herniorrhaphy
None
None
Basque
Cys38Arg/N
3
M/28
40
None
—
—
—
Cys38Arg/N
4
F/53
45
None
—
—
—
Cys38Arg/N
III
5
M/40
36
Cervical disc surgery
None
Hematoma
Basque
Cys38Arg/N
IV
6
F/35
30
None
—
—
Basque
Cys38Arg/N
V
7
M/61
34
None
—
—
Basque
Cys38Arg/N
VI
8
F/51
40
None
—
—
Basque
Cys38Arg/N
VII
9
F/34
18
Delivery
None
Mild
Basque
Cys38Arg/N
VIII
10
F/22
2
Open fracture
None
None
Basque
Cys38Arg/Tyr493His
11
F/22
1
Dental extraction
None
None
—
Cys38Arg/Tyr493His
12
M/13
⬍1
Tonsillectomy
Plasma
None
Basque/Jewish
Cys237Tyr/Glu117Stop
Family
IX
Bleeding following hemostatic challenge
Challenge
Coverage
Bleeding
Origin
Genotype
13
M/10
⬍1
Testicular surgery
Plasma
None
—
Cys237Tyr/Glu117Stop
X
14
M/17
48
Appendectomy
None
None
Basque/non-Basque
Codon 285delG/N
XI
15
M/5
40
Adenoidectomy
Tranexamic
None
Basque
IVS6 ⫹ 3A ⬎ G/N
—
Basque/non-Basque
Not detected
acid
XII
16
F/39
N indicates normal genotype.
35
None
—
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BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
MOLECULAR BASIS OF FACTOR XI DEFICIENCY IN BASQUES
2451
Table 2. Factor XI produced by baby hamster kidney cells transfected with normal and mutated recombinant complementary DNAs
Recombinant cDNA
Factor XI secreted in medium
No.
transfections
Activity, U/dL
Antigen, ng/mL
Specific activity,
U/mg
Intracellular factor XI
in lysate
Antigen, ng/mL
WT
6
1.45 ⫾ 0.80
99 ⫾ 47
146
187 ⫾ 74
Cys38Arg
3
⬍ 0.01
⬍1
—
138 ⫾ 60
Cys237Tyr
4
⬍ 0.01
3.1 ⫾ 2.2
—
172 ⫾ 46
Tyr493His
3
0.18 ⫾ 0.08
11.5 ⫾ 3.0
157
58 ⫾ 8
Cys321Phe*
3
1.32 ⫾ 0.16
80 ⫾ 8.7
165
178 ⫾ 21
Results are given as mean ⫾ SD.
cDNA indicates complementary DNA; WT, wild type.
*Rare polymorphism.
comparison with the medium of cells transfected with normal
recombinant factor XI, the medium of cells transfected with factor
XI Cys38Arg contained neither factor XI antigen, which was
separately assayed by 3 monoclonal antibodies, nor factor XI
activity (Table 2). The medium of cells transfected with recombinant Cys237Tyr contained 3% of factor XI antigen secreted by
normal cells but no demonstrable factor XI activity. The medium of
cells transfected with Tyr493His contained approximately 12% of
factor XI antigen and activity secreted by normal cells. Immunoprecipitation of the media from cells containing the 3 mutations and
immunoblotting with a polyclonal antibody revealed no factor XI
for Cys38Arg, a small amount for Tyr493His (Figure 1), and trace
amounts for Cys237Tyr after long exposure (data not shown).
The Cys321Phe alteration did not contribute to factor XI
deficiency; the recombinant Cys321Phe protein had activity similar
to that of WT although it migrated as a monomer on nonreduced
SDS-PAGE (Table 2; Figure 1).
Since no factor XI antigen was detected in the medium of BHK
cells transfected with Cys38Arg and only trace amounts were
present in the medium of cells transfected with Cys237Tyr factor
XI, we addressed the question of whether the protein was normally
produced but not secreted by the cells. As shown in Table 2,
comparable amounts of factor XI antigen were observed in lysates
of cells transfected with either WT, Cys38Arg, or Cys237Tyr factor
XI. In contrast, measurements of Tyr493His cell lysates revealed
only one third of the amount of factor XI produced by WT cells.
Figure 1. Western blot analysis of immunoprecipitated factor XI secreted by BHK cells transfected with
mutant factor XI constructs. Twenty-microliter samples
of WT, mutants, and mock (M) were size-fractionated in 2
experiments (panels A and B) under nonreducing conditions on a 7.5% SDS-PAGE and reacted with polyclonal
rabbit antihuman factor XI antibodies. Factor XI dimer
was detected only in WT and Tyr493His samples, and
factor XI monomer was observed only in the Cys321Phe
mutant. A fast-migrating nonspecific band was observed
in all samples, including the 2 mocks. A nonspecific
slow-migrating band present in panel A most probably
corresponds to mouse IgG, which is recognized by the
secondary peroxidase-conjugated antibody. This band is
absent from samples shown in panel B, probably because the secondary antibody used in this experiment
was preadsorbed with mouse IgG, thereby eliminating
cross-reactivity.
The presence of Cys38Arg factor XI in BHK transfected cells was
evaluated by immunostaining of cells grown on glass slides with 2
different monoclonal antibodies. The mAb-3 directed against an epitope
on apple 2 clearly detected Cys38Arg factor XI in the cells (Figure 2). In
contrast, mAb-5 directed against an epitope on apple 1 did not recognize
the Cys38Arg factor XI, suggesting that the mutation, which is localized
in apple 1, probably interfered with recognition of the epitope. Both
mAb-3 and mAb-5 stained Cys237Tyr factor XI in transfected cells,
indicating that the production of the protein was not impaired by this
mutation (data not shown).
The intracellular degradation of factor XI in transfected BHK
cells was assessed in pulse-chase experiments. Figure 3 demonstrates that similar amounts of factor XI were produced by WT and
Cys38Arg-transfected cells. Moreover, the preservation of the
factor XI band of the Cys38Arg cells at 1 to 6 hours of chase
indicated that there was no accelerated degradation of factor XI.
Similar results were obtained in Cys237Tyr- and Tyr493Histransfected cells (data not shown).
Prevalence of Cys38Arg mutation in the French
Basque population
An allele-specific restriction analysis was devised for identification
of the nt 209T⬎C substitution, which is responsible for the
Cys38Arg mutation. The mutant allele 209C was cleaved by the
enzyme TaiI (New England Biolabs), whereas the normal 209T
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2452
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
ZIVELIN et al
Figure 2. Immunostaining of BHK cells transfected
with WT or Cys38Arg factor XI. Cells grown on glass
slides were reacted with 2 different anti–factor XI monoclonal antibodies, and immune complexes were detected
by antimouse IgG coupled to alkaline phosphatase. The
mAb-3 directed against an epitope on apple 2 detected
Cys38Arg factor XI in the cells, whereas the mAb-5
directed against an epitope on apple 1 (the site of the
mutation) failed to detect Cys38Arg factor XI. Original
magnification ⫻ 200.
allele was resistant to this enzyme (Figure 4). A survey for the
mutation in 206 French Basque subjects detected 2 heterozygotes,
predicting an allele frequency of 0.005 in this population (95%
confidence interval, 0.0006-0.0174).
Identification of a possible founder haplotype for Cys38Arg
To discern whether the Cys38Arg substitution observed in 10
unrelated Basque individuals (8 patients and 2 subjects from the
general Basque population) stemmed from a single mutational
event (a common founder), we examined a series of 10 polymorphisms spanning the factor XI gene. This analysis enabled us to
make a distinction between alleles bearing the mutation and those
not bearing the mutation among carriers and controls, respectively.
Table 3 demonstrates that all subjects bearing the Cys38Arg
mutation carried a unique intron B polymorphism (12 CA repeats),
which was not detected in French Basque controls (P ⬍ .0001).
Interestingly, the prevalence of this polymorphic allele was 2% in
Ashkenazi Jews.25 The frequency of intron A ⫺138A allele in
carriers of Cys38Arg was also significantly different from controls
(P ⫽ .002). For the remaining 8 polymorphisms, the frequency in
controls and carriers of the Cys38Arg mutation was not statistically
different. These data define a unique haplotype bearing the
Cys38Arg mutation and suggest that the mutation occurred in a
founder individual.
Discussion
The presented data established the molecular basis for factor XI
deficiency in 11 of 12 unrelated families residing in the Basque area
of France. In 8 Basque families, we identified a novel 209T⬎C
nucleotide alteration, which predicts a Cys38Arg substitution. A
survey among 206 French Basques revealed that the Cys38Arg
allele frequency is 0.005. Haplotype analysis based on examination
of 10 intragenic polymorphic sites in 10 carriers of the Cys38Arg
mutation (only 1 being a homozygote) was consistent with a
founder effect and indicated that the mutation probably occurred in
an individual who had an haplotype that is extremely rare among
Basques (Table 3).
The French Basques are thus the third population in which an
ancestral mutation causing severe factor XI deficiency is prevalent.
In previous studies, we identified the type II mutation (a stop in
exon 5) in Ashkenazi Jews, Iraqi Jews, and Palestinian Arabs23,25,35
and estimated that it occurred in a founder approximately 2500
years ago.36 The type II mutation was also reported in 2 Portuguese
families, and haplotype analysis demonstrated that it was of the
same ancestry.19 Type III missense mutation, also shown to have a
distinct founder, was estimated to have evolved more recently and
is confined to Ashkenazi Jews.25,36
Figure 3. Pulse-chase experiments of Cys38Arg and
WT factor XI. Transfected cells were radiolabeled by
35S-methionine and then chased by cold methionine for 1
to 20 hours. Cell lysates were precipitated by monoclonal
antibodies against factor XI, electrophoresed on gradient
4% to 12% SDS-PAGE gels under reducing conditions,
and exposed to autoradiography. Note that Cys38Arg
was produced similarly to WT and did not undergo
accelerated degradation.
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BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
MOLECULAR BASIS OF FACTOR XI DEFICIENCY IN BASQUES
2453
Figure 4. Detection of factor XI Cys38Arg mutation by
allele-specific restriction analysis. Depicted are a
normal genotype (N), a homozygote for Cys38Arg (Hom),
and a heterozygote (Het). Numbers on the left of the gel
indicate the size in base pairs of the bands.
Patients with severe factor XI deficiency are at significant risk
of injury-related bleeding, particularly during operations involving
tissues containing fibrinolytic activity such as the urinary tract, oral
cavity, and nasal mucosa.35,37 In view of the high prevalence of
factor XI deficiency in Israel, it has become mandatory to screen
for factor XI deficiency prior to surgery by testing the aPTT. The
prevalence of Cys38Arg heterozygotes is 1:100 in French Basques,
which predicts a prevalence of severe factor XI deficiency of
1:40 000. From the limited clinical information presented in Table
1, it is difficult to determine whether homozygotes or heterozygotes
for the Cys38Arg mutation are at significant risk of bleeding; 1
homozygote had no bleeding following breast surgery and 2
heterozygotes had mild bleeding following cervical disc surgery
and delivery, respectively.
In 2 of the 3 mutations described in this study, highly conserved
cysteine residues in the apple domain region (heavy chain) were
substituted. The cysteine residues in the 4 apple domains are
characteristic for the PAN module superfamily, which includes
prekallikrein, a number of Caenorhabditis elegans proteins, and
the N-terminal domains of the plasminogen/hepatocyte growth
factor family.38 The Cys38Arg mutation disrupts the C32-C38 bond
in apple domain 1, and the Cys237Tyr mutation disrupts the
C208-C237 bond in apple domain 3. Our expression studies
showed that although both mutant proteins were synthesized in
BHK transfected cells, they were not secreted (Table 2, Figures
1-2) and also did not rapidly degrade intracellularly (Figure 3).
Interestingly, in 6 of 7 previously described missense mutations, all
localized in the apple domains, expression studies also demonstrated significantly impaired secretion.20,21 For the type III mutation (Phe283Leu), the impaired secretion was attributed to defective factor XI dimerization, but for the remaining mutations the
mechanism has not been established.
In the 2 siblings who carried the Cys237Tyr mutation, a rare
Cys321Phe polymorphism was also detected. The unpaired C321
was previously shown to play a role in stabilization of factor XI
dimers by formation of an intermonomer disulfide bond.14 However, a Cys321Ser substitution did not impair factor XI dimer
formation, secretion, or activity.14 In this study, we expressed the
naturally occurring Cys321Phe polymorphism in BHK cells and
also found normal factor XI secretion and specific activity (Table
2); however, on nonreducing denaturing SDS-PAGE, the protein
appeared as a monomer rather than dimer (Figure 1). Interestingly,
rabbit factor XI contains a histidine instead of cysteine at the
equivalent position and displays similar features: a monomer on
SDS-PAGE, but a dimer by size-exclusion chromatography.39
The third Tyr493His mutation is located in the catalytic domain
of factor XI. The tyrosine residue is conserved in human, bovine,
and rabbit factor XI and human prekallikrein. Interestingly, in
mouse factor XI, this residue is replaced by histidine similarly to
the mutation detected. The Tyr493His mutation caused a 90%
reduction of factor XI measured in the medium of transfected BHK
cells. This reduced amount probably stems from decreased synthesis and secretion of factor XI since (1) the amount of factor XI in
cell lysates was approximately a third of the amount in WT cells
Table 3. Allele frequency of 10 factor XI intragenic polymorphisms in French Basque controls and carriers of Cys38Arg mutation
Controls
Carriers of Cys38Arg*
No.
alleles
Frequency of bold-faced
allele, %
⫺231C ⬎ T
82
77
20
⫺138C ⬎ A
56
39
20
Intron B, 1, 2, 3, 4
88
0
20
55
⬍.0001
1
Exon 5, 472C ⬎ T
84
93
20
100
NS
C
Polymorphism†
No.
alleles
Frequency of bold-faced
allele, %
P
Founder
haplotype
75
NS
C
80
.002
A
Intron A
Intron E, ⫺431 1, 2
62
65
18
78
NS
1
Exon 8, 844A ⬎ G
90
92
20
100
NS
A
Exon 11, 1234T ⬎ C
90
90
20
95
NS
T
Intron M, 1, 2, 3, 4
90
75
20
80
NS
3
1855G ⬎ T
80
91
20
95
NS
G
1882G ⬎ A
80
91
20
95
NS
G
Exon 15
NS indicates not statistically significant.
*One homozygote, 1 compound heterozygote, and 8 heterozygotes.
†The allele appearing in bold in each polymorphism in this column is the allele whose frequency is measured in 2 later columns.
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
2454
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
ZIVELIN et al
(Table 2); (2) there was no accelerated intracellular degradation;
and (3) the secreted amount of factor XI was disproportionally low
(Table 2).
The Basque population is one of the oldest populations in
Europe.40 Several unique genetic features have been observed in
Basques. Examples are the following: (1) The Rh-negative blood
group was found in about 20% of Basques; this frequency
surpasses the frequency of most populations.40 (2) The frequency
of delta 508 mutation among cystic fibrosis–bearing alleles in
Basques is 87%, which by far exceeds the 53% frequency observed
in Spaniards.41,42 (3) Factor V Leiden, which is confined to whites,
appears to be absent in Basques.43 The present study identified a
new characteristic genetic feature: the Cys38Arg mutation in the
factor XI gene among French Basques. However, since a recent
study demonstrated significant differences in genetic markers
among autochthonous Basque subgroups,44 it remains to be established whether the Cys38Arg mutation is evenly distributed among
all Basque subgroups.
Acknowledgments
We thank Nurit Kornbrot, Aviya Ifrah, and Sali Usher for expert
technical assistance.
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From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
2002 99: 2448-2454
doi:10.1182/blood.V99.7.2448
Factor XI deficiency in French Basques is caused predominantly by an
ancestral Cys38Arg mutation in the factor XI gene
Ariella Zivelin, Frederic Bauduer, Louis Ducout, Hava Peretz, Nurit Rosenberg, Rivka Yatuv and Uri Seligsohn
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