- Identification of three F5 gene mutations associated with inherited coagulation factor V deficiency in two Chinese pedigrees
- 作者:刘立根|发布时间:2013-06-21|浏览量:504次
identification of three f5 gene mutations associated
with inherited coagulation factor v deficiency in two chinese
pedigrees
q.-h. fu,*1 r.-f. zhou,*1 l.-g. liu, w.-b. wang,* w.-m. wu,* q.-l. ding,* y.-q. hu,*
x.-f. wang,* z.-y. wang* and h.-l. wang*
*division of thrombosis and hemostasis, shanghai institute of hematology, ruijin hospital, shanghai second medical上海市第五人民医院血液内科刘立根
university, shanghai; blood centre of zhejiang province, hangzhou, zhejiang; and department of hematology,
shanghai fifth people?s hospital affiliated to fudan university, shanghai, china
summary. to investigate the molecular defects in
two chinese pedigrees with inherited factor v (fv)
deficiency. a 37-year-old male (proband 1) and an
18-month-old boy (proband 2) were diagnosed as
inherited coagulation fv deficiency by severely
reduced plasma levels of fv activity and antigen.
all 25 exons and their flanking sequence of f5 gene
were amplified by polymerase chain reaction (pcr)
for both probands and the pcr products were
directly sequenced. total rna was extracted from
the peripheral lymphocytes of proband 1 for detecting
the changes at mrna level.the homozygous
deletion ivs8 )2a>g was identified in the f5 gene of
proband 1 and complementary dna (cdna) analysis
revealed the abolishment of the canonical splicing
site by the mutation and the activation of the cryptic
acceptor site 24 bp upstream instead. the insertion
introduced eight additional amino acids (aa) into
the fv protein. two heterozygous mutations of f5
gene were discovered in proband 2. the 2238-9del
ag in exon 13 introduced a premature termination
code at 689 aa and the substitution of g6410 by
t in exon 23 lead to the missense mutation
gly2079val.three f5 gene mutations, ivs8
)2a>g, 2238-9del ag and g6410t, have been
identified in two chinese pedigree with congenital
fv deficiency, respectively.
keywords: coagulation factor v, deficiency, gene
mutation, splice site
introduction
human coagulation factor v (fv), a single chain
glycoprotein with molecular weight ratio (mwr)
about 330 kda, is an essential pro-cofactor in blood
coagulation cascade. fv protein is comprised of
2224 aa residues including 28 aa signal peptides
and six domains that is orientated as a1-a2-b-a3-
c1-c2. fv is activated to its active form (fva) by
thrombin or activated fxa which removed the b
domain, generating a heavy chain (1.709 aa
residues, a1-a2 domain) and a light chain (1546.
2196 aa residues, a3-c1-c2 domain) that are
linked together in the presence of calcium ions.
fva binds to fxa and serves as its cofactor in the
prothrombinase complex that convert prothrombin
to thrombin [1.3].
the f5 gene is mapped to human chromosome
1q21.25 [2], and spans more than 80 kb consisting
of 25 exons and 24 introns. there is a 35.40%
homology existing in a and c domains of fv and
fviii. conversely, extensive divergence exists in the
b domains of the two genes [4].
congenital fv deficiency is a rare bleeding disorder,
inherited as an autosomal recessive trait, with
an incidence of about 1 in 1 million [5,6]. common
symptoms are occasional nose bleeding, easy bruising,
bleeding following surgery and excessive menstrual
bleeding in female. haemarthroses and
haematomas occur in only one-quarter of the
patients, whereas life-threatening bleeding episodes
1qi-hua fu and rong-fu zhou contribute equally to this work
and should be considered as co-first author.
correspondence: prof. hong-li wang, vice director, shanghai
institute of hematology, ruijin hospital, shanghai second medical
university, no. 197 ruijin ii road, shanghai 200025, china.
tel.: +86-21-64370045 (extn 610602); fax: +86-21-64743206;
e-mail: wanghongli602@163.com
accepted after revision 19 february 2004
haemophilia (2004), 10, 264.270 doi: 10.1111/j.1365-2516.2004.00896.x
264 2004 blackwell publishing ltd
in the gastrointestinal tract and in the central nervous
system are rare [7]. up to now, over 200 cases of fv
deficiency have been reported, but the molecular
basis for fv deficiency has been established in only a
few cases [4,7.9,11.27].
in this study, we reported three f5 gene mutations
in two unrelated chinese families with fv deficiency.
in proband 1, a homozygous ivs8 )2a>g transition
in genomic dna and an in-frame insertion of 24 bp
in cdna were identified in f5 gene. these results
suggested that the splice mutation ivs8 )2a>g
abolished the acceptor splice site of intron 8 thus
causing the activation of a cryptic acceptor splice site
24 bp upstream of the physiological one within the
intron. in proband 2, novel compound heterozygous
mutations, 2238-9del ag in exon 13 and g6410t
missense mutation in exon 23 were found.
materials and methods
case history
proband 1, a 37-year-old male, was admitted to
hospital because of severe headache and vomiting for
2 days. during childhood, he mainly suffered from
gingival bleeding. at the age of 16 years, he was
treated with fresh frozen plasma infusion for a
persistent haemorrhage after having had appendectomy.
computerized tomography (ct) scan of the
brain revealed that there was a haematoma of
50 ? 29 ? 60 mm in size in frontal lobe of the
cerebrum. laboratory tests suggested that he had a
normal platelet count, liver function and was negative
for antibodies to hepatitis b and c. he was treated
with transfusion of fresh frozen plasma and medication
for decreasing intracranial pressure. three
months later, a repeated ct scan of the brain showed
that the haematoma was completely absorbed. his
parents were known to be consanguineous. his
grandmother died of severe gastrointestinal bleeding.
proband 2 is an 18-month-old boy. at the age of
6 months, he was treated with transfusion of fresh
frozen plasma for gastrointestinal bleeding. subsequently,
he presented with recurrent epitaxis, easy
bruising and gum bleeding. no history of bleeding is
reported in the other family members and his parents
are not consanguineous.
the pedigrees of these two families are illustrated
in fig. 1a and b.
sample collection for coagulation tests
following informed consent, peripheral venous
blood from the probands and family members was
collected gently into 0.109 mol l)1 sodium citrate
(9:1 v/v). platelet poor plasma was obtained by
centrifugation at 2000 g for 10 min and aliquots
were stored at )80 c until use.
coagulation factor activities and fv antigen assays
the plasma coagulation factors activities were
measured following the protocol of one-stage
clotting assay using acl 3000 plus automated
coagulation apparatus (instrumentation laboratory,
milan, italy); plasma fv antigen levels were measured
by using paired antibodies for fv (cedarlane
laboratories limited, ontario, canada) in an
enzyme-linked immunosorbent assay (elisa). fv
antigen level was expressed as percentage of control
plasma pooled from 30 normal individuals, set as
100%. the linear range of the functional and
immunological tests was 1.200% and 0.78.200%,
respectively.
dna and rna isolation
genomic dna was extracted from whole blood
according to a standard phenol-chloroform protocol.
total rna from peripheral lymphocytes of proband
1 was prepared for further studies [8].
genomic dna amplification and sequencing
the primers for polymerase chain reaction (pcr,
primers sequences are available on request) were
designed according to the published f5 gene
sequence (genbank accession number z99275).
the amplification was performed in final volume of
100 ll, including 10 ll 10x pcr buffer with
(a)
(b)
fig. 1. two pedigrees of factor v (fv) deficiency. (a) pedigree
with ivs8 )2a>g mutation. (b) pedigree with compound
heterozygous mutation g6410t and 2238-9del ag. the probands
are indicated by arrows.
three f5 gene mutations 265
2004 blackwell publishing ltd haemophilia (2004), 10, 264.270
mgcl2 (containing 100 mmol l)1 tris-hcl (ph
8.3), 500 mmol l)1 kcl and 15 mmol l)1 mgcl2),
8 ll 2.5 mmol l)1 dntp, 5 ll of each primer
(10 lmol l)1), and 2.5 u taq dna polymerase
[takara biotechnology (dalian) co., ltd, dalian,
china], 500 ng genomic dna. after denatured at
95 c for 5 min, 30 cycles of 95 c for 30 s,
55.60 c for 30 s, 72 c for 30 s were performed,
then extended at 72 c for another 10 min. the pcr
products were purified from agarose gel using
qiaquick gel extraction kit (qiagen gmbh,
hilde, germany) and subsequently sequenced by
using the abi 377 sequencer (applied biosystems,
foster city, ca, usa). all the 25 exons and their
intron.exon boundaries of f5 gene from the probands
genomic dna were amplified and sequenced,
whereas only corresponding sequences were amplified
and sequenced for other family members of the
two pedigrees.
cdna synthesis and sequencing
the first strand of cdna was synthesized by reverse
transcriptionpcrfrom totalrnaof proband 1, using
random hexamers as primers and mmlv-rt (gibco
brl, life technologies, rockville, md, usa). the
reverse transcription product was then amplified by
pcr with the primers from exon 8 (5?-aca
ggtctagcatttggat-3?) and exon 12 (5?-tc
ctcatgcctctttccata-3?) and sequenced
thereafter.
restriction fragment length polymorphism analysis
restriction fragment length polymorphism (rflp)
analysis was performed to validate the g6410t
mutation. the pcr products of exon 23 and its
flanking regions of proband 2, his parents and 100
random normal individuals unrelated to the patient
were digested with restriction enzyme rsai (mbi,
fermentas, vilnius, lithuania) per the conditions
suggested by the manufacturer. the digested products
were examined by agarose electrophoresis.
results
phenotype identification
the patients had a significantly prolonged activated
partial thromboplastin time (aptt) and prothrombin
time (pt). fv activity and antigen level of proband 1
were 1.6% and 7.2% of the normal control, respectively;
whereas for proband 2, they were <1% and
1.5%, respectively. the activities of other coagulation
factors, including fii, fvii, fviii, fix, fx, fxi
and fxii were in normal ranges for both patients.
other heterozygous family members also had
decreased fv activities and antigens (see table 1).
molecular defects of these two pedigrees
proband 1 direct genomic dna sequencing of f5
gene 25 exons and their boundaries sequence of
proband 1 revealed three homozygous variations,
including one silent mutation in exon 2 (a327g),
one polymorphism in exon 13 (c3930a), which
introduced leu to ile substitution at amino acid (aa)
position 1257, and a previously reported homozygous
putative causal splicing mutation ivs8 )2a>g
[9] (fig. 2a). further analysis of dna samples from
the family showed that all of the members but the
probands wife were heterozygous of this splicing
mutation, in agreement with the autosomal recessive
pattern of inheritance.
table 1. some laboratory test results
of pedigrees.
activated partial
thromboplastin
time (aptt, s)
prothrombin
time (pt, s) fv:c (%) fv:ag (%)
pedigree 1
i1 37.2 12.1 66.3 47.6
i2 35.9 12.4 68 49.6
ii1 33.4 11.0 128.6 89.7
ii2 (proband 1) 123 43.4 1.6 7.2
ii3 36.6 11.3 68 47.4
iii1 37.0 11.5 55.1 37.9
pedigree 2
i1 38.5 12.6 54.3 49.5
i2 37.8 13.1 54.3 56.1
ii1 (proband 2) 249.2 46.6 <1 1.5
control 33.44
(n . 30)
11.2.13.6
(n . 30)
50.0.150.0
(n . 30)
100
266 q.-h. fu et al.
haemophilia (2004), 10, 264.270 2004 blackwell publishing ltd
to clarify where the cryptic acceptor splice site
caused by this splicing mutation was, further analysis
of fv cdna sequence of proband 1 was performed
and a 24 bp insertion between exon 8 and exon 9
was identified (fig. 2b). the insertion sequences
could be traced back to the 3?-flanking sequence of
intron 8, which indicated that the ivs8 )2a>g
mutation abolished the acceptor splice site of intron
8 thus causing the activation of a cryptic acceptor
splice site 24 bp upstream of the physiological one
(fig. 2a). the sequence of the amplified cdna did
not reveal the presence of normal mrna of f5 gene.
proband 2 thednaanalysis disclosed four variations
in the proband?s f5 gene, including one silent mutations
in exon 4 (g642t), one polymorphism in exon
10 (g1628a), which introduced arg to lys substitution
at aa position 485. this polymorphism has been
reported previously and the frequency of lys485 in
chinese population was 0.15 [10]. two heterozygous
putative mutations, 2 bp deletion (2238-9del ag) in
exon 13 and g6410t in exon 23 were identified
(fig. 3a and b), the 2238-9del ag mutation introducing
a frameshift and a premature stop at codon 689,
while the g6410t missense mutation causing the
substitution of gly by val at codon 2079. the
probands father was heterozygous for the g6410t
mutation, whereas the probands mother was heterozygous
for the 2238-9del ag mutation.
restriction enzyme analysis
the nucleotide substitution g6410t creates a new
recognition site for restriction enzyme rsai, which
will cleave the 438 bp pcr product of exon 23 and
its flanking regions with the mutation into two
fragments at the length of 194 and 244 bp (fig. 4).
the digested products from proband 2 and his father
had all three fragments 194, 244 and 438 bp,
indicating both of them were heterozygous for the
gene variation, while that from his mother and 100
normal controls had only one 438 bp fragment,
precluding the possibility of the gene variation as a
common polymorphism.
discussion
since murray et al. [27] reported the molecular
mechanism of fv deficiency for the first time in
1995, 29 f5 gene mutations associated with fv
deficiency have been reported [4,8,9,11.27]. most
of mutations (18 of 29) lead to the production
of truncated fv protein, and three of them are
splice site mutations [9,17,18]. in this study, we
(a) intron8 start exon9
(b) exon8 insertion sequence exon9
fig. 2. dna and cdna analysis of proband 1 with mutation ivs8 )2a>g. (a) the genomic dna analysis:
o
indicates the a>g transition.
the cryptic accept splice site in intron 8 is underlined. (b) the cdna analysis. the sequence between two arrows is inserted into the
presumed junction of exon 8 and 9. the insertion will introduce eight additional amino acids, listed below, into the factor v (fv) peptides.
aat aaa ttt gat tta act ttg tgg
asn lys phe asp leu thr leu trp
three f5 gene mutations 267
2004 blackwell publishing ltd haemophilia (2004), 10, 264.270
investigated two patients with fv deficiency from
unrelated chinese families, and identified three f5
gene mutations associated with fv deficiency.
the ivs8 )2a>g transition identified in proband
1 has been detected in another non-related chinese
patient [9]. in mammalian, 98.71% splice site
sequences have canonical gt-ag pairs [28]. all
f5 exons and introns are canonical gt-ag junctions
except for intron 6 [1], so the mutation might
interfere with the process of pre-mrna modification.
with the analysis of mrna extracted from the
peripheral blood cells, we found that the mutation
abolishes the normal acceptor site of intron 8 and
the cryptic acceptor site 24 bp upstream is conscripted
instead, causing an eight aa (asn-lys-
phe-asp-leu-thr-leu-trp) in-frame insertion
between 404 aa (encoded by exon 8) and 405
aa (encoded by exon 9) of fv protein (fig. 2b).
both exon 8 and exon 9 code part of a2 domain of
fv protein. normally, a2 domain has 316 aa and
is the only region where fv reacts to fiia [29]. the
insertion of 8 aa into the a2 domain would affect
the stereotype of fv protein and might thus impair
the stability and secretion of the protein. it is very
interesting to note that the plasma fv antigen level
of the proband 1 was about 7% of normal while
procoagulation activity only 1.6%. so there might
be some fv mutant exists in the circulation,
however, the eight aa insertion might impair the
procoagulant functions of fv by disturbing its
interaction with fiia. it is of course possible that
some normally spliced mrna and hence some
normal fv protein is produced, but sequence of the
pcr products from the amplified cdna of proband
1 did not reveal the presence of any normal mrna
(fig. 2b).
the exon 13 codes the b domain of fv and eight of
29 mutations occur in this exon. we also discovered
a 2-nucleotide deletion (2238-9del ag) in exon 13 in
1 2 3 4 5
2000 bp
1000 bp
750 bp
500 bp
250 bp
100 bp
438 bp ?
244 bp ?
194 bp ?
fig. 4. restriction fragment length polymorphism (rflp) analysis
of missense mutation gly2079val. the polymerase chain reaction
(pcr) products of exon 23 were digested by restriction enzyme
rsai. lanes 1.4 stands for the normal control, family member i2,
i1 and ii1 of pedigree 2, respectively. lane 5 is the dna marker
(dl-2000, takara).
(a)
(b)
fig. 3. dna sequencing for proband 2 (a) and (b) was part of the sequence of exon 13 and exon 23, respectively. arrows indicated are the
mutation sites.
268 q.-h. fu et al.
haemophilia (2004), 10, 264.270 2004 blackwell publishing ltd
proband 2. the deletion shifts the reading frame and
leads to the termination of translation at codon 689.
this would predict the synthesis of a truncated fv
molecular, lacking 98% part of the b domain and the
complete light chain. the putative mutation might
lead to severe fv deficiency by promoting the
selective degradation of the corresponding mrna
because of the nonsense-mediated mrna decay
pathway and/or by the quality control system of
secretive proteins to retain and intracellular degrade
the truncated fv protein [12,16,30].
the fv c2 domain is encoded by exon 23.25 and
composed of eight antiparallel strands arranged in a
jelly-roll structure, whose lower surface exhibits
three adjacent loops (spikes 1, 2 and 3) [3,31]. the
three spikes are linked to one another and to three
shorter loops by an intricate hydrogen-bonding
network, which extends to residues at the bottom
of the b-barrel. they have been proposed to mediate
binding of activated fv to phospholipid membranes
[31]. another putative causal mutation, g6410t,
also identified in proband 2, lead to the substitution
of gly by val at codon 2079, which is strictly
conserved in species (human, bovine, mouse) and in
fviii c2 domain. the gly2079 is involved in the
formation of the second spike and the second specific
phospholipid-binding site. the gly2079val mutation
might not only affect the formation of phospholipid-
binding site but also change the overall
tertiary structure of the domain, causing the mutant
protein destability. among 10 missense mutations
reported so far with fv deficiency, three mutations
were characterized by expression studies [4,19], and
all resulted in a secretion defect with a rapid
intracellular degradation. the probands father was
heterozygous with this mutation and had levels of fv
of approximately 50% (table 1). similarly, putative
gly2079val mutation is likely to cause the secretion
defect with a rapid intracellular degradation of the
mutant fv protein.
in conclusion, three mutations have been identified
in two unrelated chinese pedigree with fv deficiency.
the splicing mutation (ivs8 )2a>g) abolishes
the acceptor splice site of intron 8 thus causing the
activation of a cryptic acceptor splice site 24 bp
upstream of the physiological one. the compound
heterozygous 2238-9del ag and g6410t missence
mutations are identified for the first time.
acknowledgements
authors thank all family members for their participation
in this study, and dr jining lu (med
pulmonary center, boston university) for critical
reading of the manuscript. the financial support
from natural science foundation of shanghai (grant
no. 02zb14043) and part of clyde wu foundation of
sih is gratefully acknowledged.
references
1 cripe ld, moore kd, kane wh. structure of the gene
for human coagulation factor v. biochemistry 1992;
31: 3777.85.
2 koeleman bpc, reitsma ph, bakker e, bertina rm.
location on the human genetic linkage map of 26
genes involved in blood coagulation. thromb haemost
1997; 77: 873.8.
3 jenny rj, pittman dd, toole jj et al. complete cdna
and derived amino acid sequence of human factor v.
proc natl acad sci usa 1987; 84: 4846.50.
4 duga s, montefusco mc, asselta r et al. r2074
cmissense mutation in the c2-domain of factor v
causing moderately severe factor v deficiency:
molecular characterization by expression of the
recombinant protein. blood 2003; 101: 173.7.
5 peyvandi e, mannucci pm. rare coagulation disorders.
thromb haemost 1999; 82: 1207.14.
6 kane wh. factor v. in: colman rw, hirsh j, marder
vj, clowes aw, george jn, eds. hemostasis and
thrombosis: basic principles and clinical practice, 4th
edn. philadelphia, pa: lippincott williams and wilkins,
2001: 157.69.
7 lak m, sharifian r, peyvandi f, mannucci pm.
symptoms of inherited factor v deficiency in 35 iranian
patients. br j haematol 1998; 103: 1067.9.
8 van wijk r, nieuwenhuis k, van de berg m et al.
five novel mutations in the gene for human blood
coagulation factor v associated with type i factor v
deficiency. blood 2001; 98: 358.67.
9 fu wj, hou j, wang dx, yu rq. a novel molecular
mechanism of congenital fv deficiency: mutation in the
intron acceptor splice site of human blood coagulation
fv gene. zhonghua yi xue za zhi 2003; 83: 24.6.
10 le w, yu j, lu l et al. arg485lys polymorphism of
factor v increases the risk of coronary artery disease in
a chinese population. chin med j (engl) 2000; 113:
963.6.
11 guasch jf, cannegieter s, reitsma ph, van?t
veer-korthof et, bertina rm. severe coagulation
factor v deficiency caused by a 4 bp deletion in the
factor v gene. br j haematol 1998; 101: 32.9.
12 van wijk r, montefusco mc, duga s et al. coexistence
of a novel homozygous nonsense mutation in exon
13 of the factor v gene with the homozygous leiden
mutation in two unrelated patients with severe factor v
deficiency. br j haematol 2001; 114: 871.4.
13 castold e, simioni p, kalafatis m et al. combinations
of 4 mutations (fv r506q, fv h1299r,fv
y1702c,pt 20210g/a) affecting the prothrombinase
complex in a thrombophilic family. blood 2000; 96:
1443.8.
three f5 gene mutations 269
2004 blackwell publishing ltd haemophilia (2004), 10, 264.270
14 bossone a, d?angelo f, santacroce r, de lucia d,
margaglione m. factor v arg2074cys: a novel missense
mutation in the c2 domain of factor v. thromb
haemost 2002; 87: 923.4.
15 ajzner ee, balogh i, szabo t, marosi a, haramura g,
muszbek l. severe coagulation factor v deficiency
caused by 2 novel frameshift mutations: 2952delt in
exon 13 and 5493insg in exon 16 of factor 5 gene.
blood 2002; 99: 702.5.
16 montefusco mc, duga s, asselta r et al. a novel two
base pair deletion in the factor v gene associated with
severe factor v deficiency. br j haematol 2000; 111:
1240.6.
17 schrijver i, koerper ma. jones cd, zehnder jl.
homozygous factor v splice site mutation associated
with severe factorvdeficiency. blood 2002; 99: 3063.5.
18 asselta r, montefusco mc, duga s et al. severe factor
v deficiency: exon skipping in the factor v gene
causing a partial deletion of the c1 domain.
j thromb haemost 2003; 1: 1237.44.
19 montefusco mc, duga s, asselta r et al. clinical and
molecular characterization of 6 patients affected by
severe deficiency of coagulation factor v: broadening
of the mutational spectrum of factor v gene and
in vitro analysis of the newly identified missense
mutations. blood 2003; 102: 3210.6.
20 fu q, wu w, ding q et al. type i coagulation factor v
deficiency caused by compound heterozygous mutation
of f5 gene. haemophilia 2003; 9: 646.9.
21 castoldi e, lunghi b, mingozzi f, muleo g et al. a
missense mutation (y1702c) in the coagulation factor
v gene is a frequent cause of factor v deficiency in the
italian population. haematologica 2001; 86: 629.33.
22 xie f, cheng f, zhu x. studies on hereditary deficiency
of coagulation factor v. zhonghua xue ye xue
za zhi 2001; 22: 453.6.
23 schrijver i, houissa-kastally r, jones cd, garcia kc,
zehnder jl. novel factor v c2-domain mutation
(r2074h) in two families with factor v deficiency and
bleeding. thromb haemost 2002; 87: 294.9.
24 asselta r, tenchini ml, holme r, brosstad f, stormorken
h. the discovery of mary?s mutation.
j thromb haemost 2003; 1: 397.8.
25 fu wj, hou j, wang dx, yu rq. identification of a
novel mutation of human blood coagulation fv gene
associated with congenital fv deficiency. zhonghua
xue ye xue za zhi 2003; 24: 119.21.
26 hou lh, xie f, liu xe et al. a novel mutation causes
congenital factor v deficiency. zhonghua xue ye xue
za zhi 2003; 24: 455.9.
27 murray jm, rand md, egan jo, murphy s, kim hc,
mann kg. factor vnew brunswick: ala221-to-val
substitution results in reduced cofactor activity. blood
1995; 86: 1820.7.
28 burset m, seledtsov ia, solovyev vv. splice db:
database of canonical and non-canonical mammalian
splice sites. nucleic acids res 2001; 29: 255.9.
29 villoutreix bo, dahlback b. structural investigation of
the a domains of human blood coagulation factor v by
molecular modeling. protein sci 1998; 7: 1317.25.
30 frischmeyer pa, dietz hc. nonsense-mediated mrna
decay in health and disease. hum mol genet 1999; 8:
1893.900.
31 macedo-ribeiro s, bode w, huber r et al. crystal
structures of the membrane-binding c2 domain of
human coagulation factor v. nature 1999; 402: 434.9.
270 q.-h. fu et al.
haemophilia (2004), 10, 264.270 2004 blackwell publishing ltd
with inherited coagulation factor v deficiency in two chinese
pedigrees
q.-h. fu,*1 r.-f. zhou,*1 l.-g. liu, w.-b. wang,* w.-m. wu,* q.-l. ding,* y.-q. hu,*
x.-f. wang,* z.-y. wang* and h.-l. wang*
*division of thrombosis and hemostasis, shanghai institute of hematology, ruijin hospital, shanghai second medical上海市第五人民医院血液内科刘立根
university, shanghai; blood centre of zhejiang province, hangzhou, zhejiang; and department of hematology,
shanghai fifth people?s hospital affiliated to fudan university, shanghai, china
summary. to investigate the molecular defects in
two chinese pedigrees with inherited factor v (fv)
deficiency. a 37-year-old male (proband 1) and an
18-month-old boy (proband 2) were diagnosed as
inherited coagulation fv deficiency by severely
reduced plasma levels of fv activity and antigen.
all 25 exons and their flanking sequence of f5 gene
were amplified by polymerase chain reaction (pcr)
for both probands and the pcr products were
directly sequenced. total rna was extracted from
the peripheral lymphocytes of proband 1 for detecting
the changes at mrna level.the homozygous
deletion ivs8 )2a>g was identified in the f5 gene of
proband 1 and complementary dna (cdna) analysis
revealed the abolishment of the canonical splicing
site by the mutation and the activation of the cryptic
acceptor site 24 bp upstream instead. the insertion
introduced eight additional amino acids (aa) into
the fv protein. two heterozygous mutations of f5
gene were discovered in proband 2. the 2238-9del
ag in exon 13 introduced a premature termination
code at 689 aa and the substitution of g6410 by
t in exon 23 lead to the missense mutation
gly2079val.three f5 gene mutations, ivs8
)2a>g, 2238-9del ag and g6410t, have been
identified in two chinese pedigree with congenital
fv deficiency, respectively.
keywords: coagulation factor v, deficiency, gene
mutation, splice site
introduction
human coagulation factor v (fv), a single chain
glycoprotein with molecular weight ratio (mwr)
about 330 kda, is an essential pro-cofactor in blood
coagulation cascade. fv protein is comprised of
2224 aa residues including 28 aa signal peptides
and six domains that is orientated as a1-a2-b-a3-
c1-c2. fv is activated to its active form (fva) by
thrombin or activated fxa which removed the b
domain, generating a heavy chain (1.709 aa
residues, a1-a2 domain) and a light chain (1546.
2196 aa residues, a3-c1-c2 domain) that are
linked together in the presence of calcium ions.
fva binds to fxa and serves as its cofactor in the
prothrombinase complex that convert prothrombin
to thrombin [1.3].
the f5 gene is mapped to human chromosome
1q21.25 [2], and spans more than 80 kb consisting
of 25 exons and 24 introns. there is a 35.40%
homology existing in a and c domains of fv and
fviii. conversely, extensive divergence exists in the
b domains of the two genes [4].
congenital fv deficiency is a rare bleeding disorder,
inherited as an autosomal recessive trait, with
an incidence of about 1 in 1 million [5,6]. common
symptoms are occasional nose bleeding, easy bruising,
bleeding following surgery and excessive menstrual
bleeding in female. haemarthroses and
haematomas occur in only one-quarter of the
patients, whereas life-threatening bleeding episodes
1qi-hua fu and rong-fu zhou contribute equally to this work
and should be considered as co-first author.
correspondence: prof. hong-li wang, vice director, shanghai
institute of hematology, ruijin hospital, shanghai second medical
university, no. 197 ruijin ii road, shanghai 200025, china.
tel.: +86-21-64370045 (extn 610602); fax: +86-21-64743206;
e-mail: wanghongli602@163.com
accepted after revision 19 february 2004
haemophilia (2004), 10, 264.270 doi: 10.1111/j.1365-2516.2004.00896.x
264 2004 blackwell publishing ltd
in the gastrointestinal tract and in the central nervous
system are rare [7]. up to now, over 200 cases of fv
deficiency have been reported, but the molecular
basis for fv deficiency has been established in only a
few cases [4,7.9,11.27].
in this study, we reported three f5 gene mutations
in two unrelated chinese families with fv deficiency.
in proband 1, a homozygous ivs8 )2a>g transition
in genomic dna and an in-frame insertion of 24 bp
in cdna were identified in f5 gene. these results
suggested that the splice mutation ivs8 )2a>g
abolished the acceptor splice site of intron 8 thus
causing the activation of a cryptic acceptor splice site
24 bp upstream of the physiological one within the
intron. in proband 2, novel compound heterozygous
mutations, 2238-9del ag in exon 13 and g6410t
missense mutation in exon 23 were found.
materials and methods
case history
proband 1, a 37-year-old male, was admitted to
hospital because of severe headache and vomiting for
2 days. during childhood, he mainly suffered from
gingival bleeding. at the age of 16 years, he was
treated with fresh frozen plasma infusion for a
persistent haemorrhage after having had appendectomy.
computerized tomography (ct) scan of the
brain revealed that there was a haematoma of
50 ? 29 ? 60 mm in size in frontal lobe of the
cerebrum. laboratory tests suggested that he had a
normal platelet count, liver function and was negative
for antibodies to hepatitis b and c. he was treated
with transfusion of fresh frozen plasma and medication
for decreasing intracranial pressure. three
months later, a repeated ct scan of the brain showed
that the haematoma was completely absorbed. his
parents were known to be consanguineous. his
grandmother died of severe gastrointestinal bleeding.
proband 2 is an 18-month-old boy. at the age of
6 months, he was treated with transfusion of fresh
frozen plasma for gastrointestinal bleeding. subsequently,
he presented with recurrent epitaxis, easy
bruising and gum bleeding. no history of bleeding is
reported in the other family members and his parents
are not consanguineous.
the pedigrees of these two families are illustrated
in fig. 1a and b.
sample collection for coagulation tests
following informed consent, peripheral venous
blood from the probands and family members was
collected gently into 0.109 mol l)1 sodium citrate
(9:1 v/v). platelet poor plasma was obtained by
centrifugation at 2000 g for 10 min and aliquots
were stored at )80 c until use.
coagulation factor activities and fv antigen assays
the plasma coagulation factors activities were
measured following the protocol of one-stage
clotting assay using acl 3000 plus automated
coagulation apparatus (instrumentation laboratory,
milan, italy); plasma fv antigen levels were measured
by using paired antibodies for fv (cedarlane
laboratories limited, ontario, canada) in an
enzyme-linked immunosorbent assay (elisa). fv
antigen level was expressed as percentage of control
plasma pooled from 30 normal individuals, set as
100%. the linear range of the functional and
immunological tests was 1.200% and 0.78.200%,
respectively.
dna and rna isolation
genomic dna was extracted from whole blood
according to a standard phenol-chloroform protocol.
total rna from peripheral lymphocytes of proband
1 was prepared for further studies [8].
genomic dna amplification and sequencing
the primers for polymerase chain reaction (pcr,
primers sequences are available on request) were
designed according to the published f5 gene
sequence (genbank accession number z99275).
the amplification was performed in final volume of
100 ll, including 10 ll 10x pcr buffer with
(a)
(b)
fig. 1. two pedigrees of factor v (fv) deficiency. (a) pedigree
with ivs8 )2a>g mutation. (b) pedigree with compound
heterozygous mutation g6410t and 2238-9del ag. the probands
are indicated by arrows.
three f5 gene mutations 265
2004 blackwell publishing ltd haemophilia (2004), 10, 264.270
mgcl2 (containing 100 mmol l)1 tris-hcl (ph
8.3), 500 mmol l)1 kcl and 15 mmol l)1 mgcl2),
8 ll 2.5 mmol l)1 dntp, 5 ll of each primer
(10 lmol l)1), and 2.5 u taq dna polymerase
[takara biotechnology (dalian) co., ltd, dalian,
china], 500 ng genomic dna. after denatured at
95 c for 5 min, 30 cycles of 95 c for 30 s,
55.60 c for 30 s, 72 c for 30 s were performed,
then extended at 72 c for another 10 min. the pcr
products were purified from agarose gel using
qiaquick gel extraction kit (qiagen gmbh,
hilde, germany) and subsequently sequenced by
using the abi 377 sequencer (applied biosystems,
foster city, ca, usa). all the 25 exons and their
intron.exon boundaries of f5 gene from the probands
genomic dna were amplified and sequenced,
whereas only corresponding sequences were amplified
and sequenced for other family members of the
two pedigrees.
cdna synthesis and sequencing
the first strand of cdna was synthesized by reverse
transcriptionpcrfrom totalrnaof proband 1, using
random hexamers as primers and mmlv-rt (gibco
brl, life technologies, rockville, md, usa). the
reverse transcription product was then amplified by
pcr with the primers from exon 8 (5?-aca
ggtctagcatttggat-3?) and exon 12 (5?-tc
ctcatgcctctttccata-3?) and sequenced
thereafter.
restriction fragment length polymorphism analysis
restriction fragment length polymorphism (rflp)
analysis was performed to validate the g6410t
mutation. the pcr products of exon 23 and its
flanking regions of proband 2, his parents and 100
random normal individuals unrelated to the patient
were digested with restriction enzyme rsai (mbi,
fermentas, vilnius, lithuania) per the conditions
suggested by the manufacturer. the digested products
were examined by agarose electrophoresis.
results
phenotype identification
the patients had a significantly prolonged activated
partial thromboplastin time (aptt) and prothrombin
time (pt). fv activity and antigen level of proband 1
were 1.6% and 7.2% of the normal control, respectively;
whereas for proband 2, they were <1% and
1.5%, respectively. the activities of other coagulation
factors, including fii, fvii, fviii, fix, fx, fxi
and fxii were in normal ranges for both patients.
other heterozygous family members also had
decreased fv activities and antigens (see table 1).
molecular defects of these two pedigrees
proband 1 direct genomic dna sequencing of f5
gene 25 exons and their boundaries sequence of
proband 1 revealed three homozygous variations,
including one silent mutation in exon 2 (a327g),
one polymorphism in exon 13 (c3930a), which
introduced leu to ile substitution at amino acid (aa)
position 1257, and a previously reported homozygous
putative causal splicing mutation ivs8 )2a>g
[9] (fig. 2a). further analysis of dna samples from
the family showed that all of the members but the
probands wife were heterozygous of this splicing
mutation, in agreement with the autosomal recessive
pattern of inheritance.
table 1. some laboratory test results
of pedigrees.
activated partial
thromboplastin
time (aptt, s)
prothrombin
time (pt, s) fv:c (%) fv:ag (%)
pedigree 1
i1 37.2 12.1 66.3 47.6
i2 35.9 12.4 68 49.6
ii1 33.4 11.0 128.6 89.7
ii2 (proband 1) 123 43.4 1.6 7.2
ii3 36.6 11.3 68 47.4
iii1 37.0 11.5 55.1 37.9
pedigree 2
i1 38.5 12.6 54.3 49.5
i2 37.8 13.1 54.3 56.1
ii1 (proband 2) 249.2 46.6 <1 1.5
control 33.44
(n . 30)
11.2.13.6
(n . 30)
50.0.150.0
(n . 30)
100
266 q.-h. fu et al.
haemophilia (2004), 10, 264.270 2004 blackwell publishing ltd
to clarify where the cryptic acceptor splice site
caused by this splicing mutation was, further analysis
of fv cdna sequence of proband 1 was performed
and a 24 bp insertion between exon 8 and exon 9
was identified (fig. 2b). the insertion sequences
could be traced back to the 3?-flanking sequence of
intron 8, which indicated that the ivs8 )2a>g
mutation abolished the acceptor splice site of intron
8 thus causing the activation of a cryptic acceptor
splice site 24 bp upstream of the physiological one
(fig. 2a). the sequence of the amplified cdna did
not reveal the presence of normal mrna of f5 gene.
proband 2 thednaanalysis disclosed four variations
in the proband?s f5 gene, including one silent mutations
in exon 4 (g642t), one polymorphism in exon
10 (g1628a), which introduced arg to lys substitution
at aa position 485. this polymorphism has been
reported previously and the frequency of lys485 in
chinese population was 0.15 [10]. two heterozygous
putative mutations, 2 bp deletion (2238-9del ag) in
exon 13 and g6410t in exon 23 were identified
(fig. 3a and b), the 2238-9del ag mutation introducing
a frameshift and a premature stop at codon 689,
while the g6410t missense mutation causing the
substitution of gly by val at codon 2079. the
probands father was heterozygous for the g6410t
mutation, whereas the probands mother was heterozygous
for the 2238-9del ag mutation.
restriction enzyme analysis
the nucleotide substitution g6410t creates a new
recognition site for restriction enzyme rsai, which
will cleave the 438 bp pcr product of exon 23 and
its flanking regions with the mutation into two
fragments at the length of 194 and 244 bp (fig. 4).
the digested products from proband 2 and his father
had all three fragments 194, 244 and 438 bp,
indicating both of them were heterozygous for the
gene variation, while that from his mother and 100
normal controls had only one 438 bp fragment,
precluding the possibility of the gene variation as a
common polymorphism.
discussion
since murray et al. [27] reported the molecular
mechanism of fv deficiency for the first time in
1995, 29 f5 gene mutations associated with fv
deficiency have been reported [4,8,9,11.27]. most
of mutations (18 of 29) lead to the production
of truncated fv protein, and three of them are
splice site mutations [9,17,18]. in this study, we
(a) intron8 start exon9
(b) exon8 insertion sequence exon9
fig. 2. dna and cdna analysis of proband 1 with mutation ivs8 )2a>g. (a) the genomic dna analysis:
o
indicates the a>g transition.
the cryptic accept splice site in intron 8 is underlined. (b) the cdna analysis. the sequence between two arrows is inserted into the
presumed junction of exon 8 and 9. the insertion will introduce eight additional amino acids, listed below, into the factor v (fv) peptides.
aat aaa ttt gat tta act ttg tgg
asn lys phe asp leu thr leu trp
three f5 gene mutations 267
2004 blackwell publishing ltd haemophilia (2004), 10, 264.270
investigated two patients with fv deficiency from
unrelated chinese families, and identified three f5
gene mutations associated with fv deficiency.
the ivs8 )2a>g transition identified in proband
1 has been detected in another non-related chinese
patient [9]. in mammalian, 98.71% splice site
sequences have canonical gt-ag pairs [28]. all
f5 exons and introns are canonical gt-ag junctions
except for intron 6 [1], so the mutation might
interfere with the process of pre-mrna modification.
with the analysis of mrna extracted from the
peripheral blood cells, we found that the mutation
abolishes the normal acceptor site of intron 8 and
the cryptic acceptor site 24 bp upstream is conscripted
instead, causing an eight aa (asn-lys-
phe-asp-leu-thr-leu-trp) in-frame insertion
between 404 aa (encoded by exon 8) and 405
aa (encoded by exon 9) of fv protein (fig. 2b).
both exon 8 and exon 9 code part of a2 domain of
fv protein. normally, a2 domain has 316 aa and
is the only region where fv reacts to fiia [29]. the
insertion of 8 aa into the a2 domain would affect
the stereotype of fv protein and might thus impair
the stability and secretion of the protein. it is very
interesting to note that the plasma fv antigen level
of the proband 1 was about 7% of normal while
procoagulation activity only 1.6%. so there might
be some fv mutant exists in the circulation,
however, the eight aa insertion might impair the
procoagulant functions of fv by disturbing its
interaction with fiia. it is of course possible that
some normally spliced mrna and hence some
normal fv protein is produced, but sequence of the
pcr products from the amplified cdna of proband
1 did not reveal the presence of any normal mrna
(fig. 2b).
the exon 13 codes the b domain of fv and eight of
29 mutations occur in this exon. we also discovered
a 2-nucleotide deletion (2238-9del ag) in exon 13 in
1 2 3 4 5
2000 bp
1000 bp
750 bp
500 bp
250 bp
100 bp
438 bp ?
244 bp ?
194 bp ?
fig. 4. restriction fragment length polymorphism (rflp) analysis
of missense mutation gly2079val. the polymerase chain reaction
(pcr) products of exon 23 were digested by restriction enzyme
rsai. lanes 1.4 stands for the normal control, family member i2,
i1 and ii1 of pedigree 2, respectively. lane 5 is the dna marker
(dl-2000, takara).
(a)
(b)
fig. 3. dna sequencing for proband 2 (a) and (b) was part of the sequence of exon 13 and exon 23, respectively. arrows indicated are the
mutation sites.
268 q.-h. fu et al.
haemophilia (2004), 10, 264.270 2004 blackwell publishing ltd
proband 2. the deletion shifts the reading frame and
leads to the termination of translation at codon 689.
this would predict the synthesis of a truncated fv
molecular, lacking 98% part of the b domain and the
complete light chain. the putative mutation might
lead to severe fv deficiency by promoting the
selective degradation of the corresponding mrna
because of the nonsense-mediated mrna decay
pathway and/or by the quality control system of
secretive proteins to retain and intracellular degrade
the truncated fv protein [12,16,30].
the fv c2 domain is encoded by exon 23.25 and
composed of eight antiparallel strands arranged in a
jelly-roll structure, whose lower surface exhibits
three adjacent loops (spikes 1, 2 and 3) [3,31]. the
three spikes are linked to one another and to three
shorter loops by an intricate hydrogen-bonding
network, which extends to residues at the bottom
of the b-barrel. they have been proposed to mediate
binding of activated fv to phospholipid membranes
[31]. another putative causal mutation, g6410t,
also identified in proband 2, lead to the substitution
of gly by val at codon 2079, which is strictly
conserved in species (human, bovine, mouse) and in
fviii c2 domain. the gly2079 is involved in the
formation of the second spike and the second specific
phospholipid-binding site. the gly2079val mutation
might not only affect the formation of phospholipid-
binding site but also change the overall
tertiary structure of the domain, causing the mutant
protein destability. among 10 missense mutations
reported so far with fv deficiency, three mutations
were characterized by expression studies [4,19], and
all resulted in a secretion defect with a rapid
intracellular degradation. the probands father was
heterozygous with this mutation and had levels of fv
of approximately 50% (table 1). similarly, putative
gly2079val mutation is likely to cause the secretion
defect with a rapid intracellular degradation of the
mutant fv protein.
in conclusion, three mutations have been identified
in two unrelated chinese pedigree with fv deficiency.
the splicing mutation (ivs8 )2a>g) abolishes
the acceptor splice site of intron 8 thus causing the
activation of a cryptic acceptor splice site 24 bp
upstream of the physiological one. the compound
heterozygous 2238-9del ag and g6410t missence
mutations are identified for the first time.
acknowledgements
authors thank all family members for their participation
in this study, and dr jining lu (med
pulmonary center, boston university) for critical
reading of the manuscript. the financial support
from natural science foundation of shanghai (grant
no. 02zb14043) and part of clyde wu foundation of
sih is gratefully acknowledged.
references
1 cripe ld, moore kd, kane wh. structure of the gene
for human coagulation factor v. biochemistry 1992;
31: 3777.85.
2 koeleman bpc, reitsma ph, bakker e, bertina rm.
location on the human genetic linkage map of 26
genes involved in blood coagulation. thromb haemost
1997; 77: 873.8.
3 jenny rj, pittman dd, toole jj et al. complete cdna
and derived amino acid sequence of human factor v.
proc natl acad sci usa 1987; 84: 4846.50.
4 duga s, montefusco mc, asselta r et al. r2074
cmissense mutation in the c2-domain of factor v
causing moderately severe factor v deficiency:
molecular characterization by expression of the
recombinant protein. blood 2003; 101: 173.7.
5 peyvandi e, mannucci pm. rare coagulation disorders.
thromb haemost 1999; 82: 1207.14.
6 kane wh. factor v. in: colman rw, hirsh j, marder
vj, clowes aw, george jn, eds. hemostasis and
thrombosis: basic principles and clinical practice, 4th
edn. philadelphia, pa: lippincott williams and wilkins,
2001: 157.69.
7 lak m, sharifian r, peyvandi f, mannucci pm.
symptoms of inherited factor v deficiency in 35 iranian
patients. br j haematol 1998; 103: 1067.9.
8 van wijk r, nieuwenhuis k, van de berg m et al.
five novel mutations in the gene for human blood
coagulation factor v associated with type i factor v
deficiency. blood 2001; 98: 358.67.
9 fu wj, hou j, wang dx, yu rq. a novel molecular
mechanism of congenital fv deficiency: mutation in the
intron acceptor splice site of human blood coagulation
fv gene. zhonghua yi xue za zhi 2003; 83: 24.6.
10 le w, yu j, lu l et al. arg485lys polymorphism of
factor v increases the risk of coronary artery disease in
a chinese population. chin med j (engl) 2000; 113:
963.6.
11 guasch jf, cannegieter s, reitsma ph, van?t
veer-korthof et, bertina rm. severe coagulation
factor v deficiency caused by a 4 bp deletion in the
factor v gene. br j haematol 1998; 101: 32.9.
12 van wijk r, montefusco mc, duga s et al. coexistence
of a novel homozygous nonsense mutation in exon
13 of the factor v gene with the homozygous leiden
mutation in two unrelated patients with severe factor v
deficiency. br j haematol 2001; 114: 871.4.
13 castold e, simioni p, kalafatis m et al. combinations
of 4 mutations (fv r506q, fv h1299r,fv
y1702c,pt 20210g/a) affecting the prothrombinase
complex in a thrombophilic family. blood 2000; 96:
1443.8.
three f5 gene mutations 269
2004 blackwell publishing ltd haemophilia (2004), 10, 264.270
14 bossone a, d?angelo f, santacroce r, de lucia d,
margaglione m. factor v arg2074cys: a novel missense
mutation in the c2 domain of factor v. thromb
haemost 2002; 87: 923.4.
15 ajzner ee, balogh i, szabo t, marosi a, haramura g,
muszbek l. severe coagulation factor v deficiency
caused by 2 novel frameshift mutations: 2952delt in
exon 13 and 5493insg in exon 16 of factor 5 gene.
blood 2002; 99: 702.5.
16 montefusco mc, duga s, asselta r et al. a novel two
base pair deletion in the factor v gene associated with
severe factor v deficiency. br j haematol 2000; 111:
1240.6.
17 schrijver i, koerper ma. jones cd, zehnder jl.
homozygous factor v splice site mutation associated
with severe factorvdeficiency. blood 2002; 99: 3063.5.
18 asselta r, montefusco mc, duga s et al. severe factor
v deficiency: exon skipping in the factor v gene
causing a partial deletion of the c1 domain.
j thromb haemost 2003; 1: 1237.44.
19 montefusco mc, duga s, asselta r et al. clinical and
molecular characterization of 6 patients affected by
severe deficiency of coagulation factor v: broadening
of the mutational spectrum of factor v gene and
in vitro analysis of the newly identified missense
mutations. blood 2003; 102: 3210.6.
20 fu q, wu w, ding q et al. type i coagulation factor v
deficiency caused by compound heterozygous mutation
of f5 gene. haemophilia 2003; 9: 646.9.
21 castoldi e, lunghi b, mingozzi f, muleo g et al. a
missense mutation (y1702c) in the coagulation factor
v gene is a frequent cause of factor v deficiency in the
italian population. haematologica 2001; 86: 629.33.
22 xie f, cheng f, zhu x. studies on hereditary deficiency
of coagulation factor v. zhonghua xue ye xue
za zhi 2001; 22: 453.6.
23 schrijver i, houissa-kastally r, jones cd, garcia kc,
zehnder jl. novel factor v c2-domain mutation
(r2074h) in two families with factor v deficiency and
bleeding. thromb haemost 2002; 87: 294.9.
24 asselta r, tenchini ml, holme r, brosstad f, stormorken
h. the discovery of mary?s mutation.
j thromb haemost 2003; 1: 397.8.
25 fu wj, hou j, wang dx, yu rq. identification of a
novel mutation of human blood coagulation fv gene
associated with congenital fv deficiency. zhonghua
xue ye xue za zhi 2003; 24: 119.21.
26 hou lh, xie f, liu xe et al. a novel mutation causes
congenital factor v deficiency. zhonghua xue ye xue
za zhi 2003; 24: 455.9.
27 murray jm, rand md, egan jo, murphy s, kim hc,
mann kg. factor vnew brunswick: ala221-to-val
substitution results in reduced cofactor activity. blood
1995; 86: 1820.7.
28 burset m, seledtsov ia, solovyev vv. splice db:
database of canonical and non-canonical mammalian
splice sites. nucleic acids res 2001; 29: 255.9.
29 villoutreix bo, dahlback b. structural investigation of
the a domains of human blood coagulation factor v by
molecular modeling. protein sci 1998; 7: 1317.25.
30 frischmeyer pa, dietz hc. nonsense-mediated mrna
decay in health and disease. hum mol genet 1999; 8:
1893.900.
31 macedo-ribeiro s, bode w, huber r et al. crystal
structures of the membrane-binding c2 domain of
human coagulation factor v. nature 1999; 402: 434.9.
270 q.-h. fu et al.
haemophilia (2004), 10, 264.270 2004 blackwell publishing ltd