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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
An association of candidate gene haplotypes and bleeding severity
in von Willebrand disease (VWD) type 1 pedigrees
Thomas J. Kunicki, Augusto B. Federici, Daniel R. Salomon, James A. Koziol, Steven R. Head, Tony S. Mondala,
Jeffrey D. Chismar, Luciano Baronciani, Maria Teresa Canciani, and Ian R. Peake
von Willebrand disease (VWD) type 1 is
difficult to diagnose because of bleeding
variability and low heritability of von Willebrand factor (VWF) levels. We compared a bleeding severity score and bleeding times to candidate gene haplotypes
within pedigrees of 14 index cases, using
a covariance components model for multivariate traits (Mendel: QTL Association).
These pedigrees included 13 affected and
40 unaffected relatives, as defined by
plasma ristocetin cofactor (VWF:RCo) levels. The bleeding severity score was de-
rived from a detailed history. Donors were
genotyped using a primer extension
method, and 9 candidate genes were selected for analysis. VWF:RCo levels had
the strongest influence on bleeding severity score and bleeding time. ITGA2 haplotype 2 (807C) and ITGA2B haplotype 1
(Ile843) were each associated with increased bleeding severity scores (P < .01
and P < .01, respectively). GP6 haplotype
b (Pro219) was also associated with increased scores (P ⴝ .03) after adjustment
for donor age. No association was ob-
served with 6 other candidate genes,
GP1BA, ITGB3, VWF, FGB, IL6, or TXA2R.
Increased plasma VWF:Ag levels were
associated with VWF haplotype 1
(ⴚ1793G; P ⴝ .02). These results establish that genetic differences in the adhesion receptor subunits ␣2, ␣IIb, and GPVI
can influence the phenotype of VWD type
1. (Blood. 2004;104:2359-2367)
© 2004 by The American Society of Hematology
Introduction
In arterial flow, the initial transient arrest of platelets on collagen
requires von Willebrand factor (VWF) acting as a molecular bridge
between collagen and the glycoprotein Ib (GPIb) complex.1 Next,
the direct binding of platelet ␣2␤1 and platelet GPVI to collagen
anchors the platelet more firmly to the matrix and facilitates signal
transduction that leads to an activated, procoagulant platelet
monolayer.2 Genetic variation in any of the responsible receptors
might have an impact on platelet function in vivo, particularly in
mild forms of von Willebrand disease (VWD) type 1, which
accounts for at least 60% of all cases of VWD.3
Three criteria must be satisfied to make the correct diagnosis.
First, the bleeding history must be mainly mucocutaneous; second,
the inheritance is usually autosomal dominant; and third, laboratory
findings must demonstrate reduced levels of otherwise functionally
normal VWF, evident by concomitant reduction of VWF ristocetin
cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag) with a
normal VWF multimeric pattern.3 A positive bleeding history since
childhood with symptoms observed in at least 2 different sites is
considered the most important criterion. The true incidence is
difficult to determine with accuracy because the diagnosis is not
always straightforward, especially in patients with very mild
defects and no apparent family history. Genetic follow-up will
often identify mutations that inhibit the synthesis/secretion or
enhance the clearance of VWF.4 VWF multimer assays will
typically demonstrate decreased levels of all multimer sizes. Quite
often, individuals with very similar or identical VWF levels will
exhibit only modest, if any, bleeding tendency. These exceptions to
the paradigm have confounded both the diagnosis and prognostic
predictability in VWD type 1.
We selected haplotypes of 5 platelet glycoprotein genes that
have already been implicated in risk for thrombosis or bleeding or
both. These are (Table 1): GPIb␣ (GP1BA)5,6, GPVI (GP6),7 integrin ␣2
(ITGA2),8 integrin ␣IIb (ITGA2B),9 and integrin ␤3 (ITGB3).10
Quantitative differences in platelet ␣2␤1 have been correlated
with inheritance of 3 major ITGA2 haplotypes. Haplotype 1
(807T/1648G) is associated with highest levels of ␣2␤1; haplotype
3 (807C/1648A), with intermediate to high levels; and haplotype 2
(807C/1648G), with lowest levels.8 A correlation has been found
between haplotype 1 (807T; high receptor density) and risk for
arterial thrombosis in younger men with a history of myocardial
infarction,11,12 women who are heavy smokers,13 patients with
diabetic retinopathy,14 and younger patients with stroke.15 These 3
haplotypes can be defined by 2 single nucleotide polymorphisms
(SNPs) within intron 7 depicted in Table 1 (Bgl II and Ase I sites).
Two major GP6 haplotypes have been defined. Haplotype a
(T13254) encodes a serine at residue 219, whereas haplotype b
From the Roon Research Center for Arteriosclerosis and Thrombosis, Division
of Experimental Hemostasis and Thrombosis, Division of Biostatistics of the
Department of Molecular and Experimental Medicine, and the DNA Array Core
Facility of The Scripps Research Institute, La Jolla, CA; Department of Internal
Medicine and Dermatology, IRCCS Maggiore Hospital and University of Milan,
Italy; and Division of Genomic Medicine, Royal Hallamshire Hospital, Sheffield,
United Kingdom.
work in Milan (L.B., M.T.C., A.B.F.) was supported by a grant from the European
Community QLRT 1999-30387 for the project entitled “Molecular and Clinical
Markers for Diagnosis and Management of type 1 von Willebrand Disease”
(A.B.F.). I.R.P. is the Scientific Coordinator of this European project.
Submitted February 4, 2004; accepted June 2, 2004. Prepublished online as
Blood First Edition Paper, June 29, 2004; DOI 10.1182/blood-2004-01-0349.
Supported by grant R01 HL46979 from the National Heart, Lung and Blood
Institute (T.J.K.). The General Clinical Research Center (GCRC) of Scripps
Clinic is supported by National Institutes of Health grant M01 RR00833. The
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
Reprints: Thomas J. Kunicki, Department of Molecular and Experimental
Medicine, The Scripps Research Institute, 10550 N Torrey Pines Rd,
MEM-150, La Jolla, CA 92037; e-mail: [email protected].
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.
© 2004 by The American Society of Hematology
2359
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2360
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
KUNICKI et al
Table 1. Candidate glycoprotein genes
Gene (GenBank
accession no.) and
haplotype
Other designation
SNP
Haplotype differences in each of these 9 genes can potentially
influence the efficiency of hemostasis, and our objective is to analyze the
association of these haplotypes with risk for bleeding in VWD type 1,
through an analysis of index cases and their family members.
ITGA2 (NT_023081)
1
807T; Bgl II ( ⫹ ); Ase I (neg)
2915969A; 2916690G
2
807C; Bgl II (neg); Ase I ( ⫹ )
2915969G; 2916690A
3
807C; Bgl II (neg); Ase I (neg)
2915969G; 2916690G
⫺ 92C
None
2850525C
⫺ 92G
None
2850525G
⫺ 52C
None
2850565C
⫺ 52T
None
2850565T
a
Ser219
13039T
b
Pro219
13039C
1
HPA-2a; Thr145
757C
2
HPA-2b; Met145
757T
⫺ 5T
None
271T
⫺ 5C
None
271C
1
HPA-1a; P1A1; Leu33
29519T
2
HPA-1b; P1A2; Pro33
29519C
1
HPA-3a; Baka; IIe843
10893T
2
HPA-3b; Bakb; Ser843
10893G
GP6 (NT_011225)
GPIBA (NT_010823)
ITGB3 (NT_010833)
ITGA2B (M33320)
VWF (NT_009731)
1
⫺ 1793G
⫺ 1793G
2
⫺ 1793C
⫺ 1793C
1
⫺ 174G
1510G
2
⫺ 174C
1510C
IL6 (AF372214)
FGB (NM_005141)
1
⫺ 455G
⫺ 455G
2
⫺ 455A
⫺ 455A
TXA2R (NM_001060)
1
924C
1915C
2
924T
1915T
(C13254) encodes a proline.7 A recent report by Joutsi-Korhonen et
al16 presents biologic evidence that platelets homozygous for
haplotype b exhibit a decreased tendency toward thrombosis on a
collagen surface in flowing whole blood or in the Platelet Function
Analyzer-100 (PFA-100; Dade-Behring International, Düdingen,
Switzerland). On the other hand, homozygosity for haplotype b was
associated with risk for myocardial infarction, particularly among older
women (ⱖ 60 years old) who were smokers and carried the fibrinogen
B␤ ⫺455A haplotype (higher fibrinogen level).7
Polymorphism of the ␣IIb gene is responsible for the expression
of the Bak (HPA-3) alloantigen system. The allele Ile843 confers the
Baka (HPA-3a) alloantigen determinant (frequency 0.616), whereas
Ser843 represents the Bakb (HPA-3b) epitope. An initial indication
that HPA-3 is related to mortality after stroke17 was followed by
larger studies that did not find a risk association in either stroke18 or
coronary artery disease.19 However, Reiner et al9 found that
homozygosity for the Ser843 allele was associated with an approximately 5-fold increased risk of ischemic stroke among subgroups
of women who carried a diagnosis of hypertension or diabetes
(OR ⫽ 4.51; 95% CI, 1.01-20.13) or had elevated plasma homocysteine levels (OR ⫽ 5.94; 95% CI, 1.53-23.05).
We included haplotypes of 4 other genes that directly or indirectly
influence thrombotic potential. These include 2 major haplotypes of the
promoter regions of the VWF gene itself (VWF),20 the fibrinogen-B
gene (FBG),21 and the interleukin 6 gene (IL6),22 and 2 major haplotypes
of the thromboxane A2 receptor (TXA2R).23
Patients, materials, and methods
Criteria for enrollment of VWD patients and healthy individuals
This study was organized in 14 different families from the Milan area, part
of the 154 European families already enrolled in the study entitled
“Molecular and Clinical Markers for Diagnosis and Management of Type 1
von Willebrand Disease (MCMDM-1VWD)” under the Fifth Framework
Program of the European Community (Coordinators: Prof I. R. Peake and F.
Rodeghiero). The criteria used in this study were the same as those applied
to the entire population of European VWD. Patients with VWD type 1 were
classified according to the previous definitions of the Scientific Standardization Committee (SSC) on VWF of the International Society on Thrombosis
and Haemostasis (ISTH).24 By these criteria, all index cases previously
diagnosed as VWD type 1 in 12 different European centers were eligible for
enrollment if their family showed at least 2 affected individuals and
additional nonaffected members.
This portion of the project was approved by the local Institutional
Review Board of the University Hospital of Milan. After individuals signed
an informed consent, 14 index cases of VWD type 1 and 18 affected and 38
unaffected relatives were enrolled between April and October 2002 on the
basis of a previous diagnosis of VWD type 1 according to the records of the
Angelo Bianchi Bonomi Hemophilia Thrombosis Center of Milan.
According to the enrollment criteria of the European project, all DNA
and plasma samples derived from VWD type 1 families and controls were
indicated by progressive letter-numerical codes as follows: P03 (partner 3,
Milan), F00 (family number from 1-14), I-IV (generation levels in Roman
numbers, from I-IV), and 00 (individuals in numbers, from 1-10). These
codes are reported in the pedigree charts (Figure 1).
In this blinded study, the subjects were recruited, interviewed, assigned
a bleeding severity score, and tested for all laboratory and clinical
parameters in Milan. DNA samples were then coded without knowledge of
these data or the identification of the family members and their relationships, and the genotyping and statistical analysis were performed at The
Scripps Research Institute (TSRI). Normal unrelated donor pools consisted
of age- and gender-matched individuals from either the Milan area (Milan
controls, n ⫽ 52) or white, non-Hispanic donors recruited through the
General Clinical Research Center (GCRC) of Scripps Clinic and Green
Hospital, La Jolla (white non-Hispanic controls, n ⫽ 145).
Standardized criteria to evaluate bleeding history
A bleeding history was derived from detailed questionnaires, and a score
was compiled, as described.4 This questionnaire was administered to all the
affected and nonaffected members (including index cases) of the different
families from the same well-trained hematologist who was attending the
outpatient care unit at the Angelo Bianchi Bonomi Hemophilia Thrombosis
Center. To avoid bias during the interview, the hematologist was not aware
at that moment which patient would be considered the index case. The
severity of bleeding episodes was ranked from 0 to 3, as shown in Table 2,
in each of 11 bleeding manifestation categories: epistaxis, cutaneous
symptomatic bleeding, bleeding from minor wounds, oral cavity bleeding,
gastrointestinal bleeding, bleeding associated with tooth extraction, surgery,
postpartum hematoma, muscle hematoma, hemarthrosis, and menorrhagia.
The total bleeding score is the numerical sum of the scores for each
category and was calculated in the laboratory area independently by another
hematologist.
Laboratory assays
Blood samples were drawn and processed as previously described.25
Venous blood for hemostasis tests was drawn in 0.125 mM citrate. For
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
CANDIDATE HAPLOTYPES IN VWD TYPE 1
2361
Figure 1. Pedigrees for 14 families affected by VWD. Black symbols indicate affected individuals (VWF: RCo ⱕ 45 U/dL, if blood group non-O; ⱕ 35 U/dL, if blood group O);
gray symbols, individuals with bleeding severity scores ⱖ 3; and white symbols, otherwise healthy individuals. Arrows indicate index cases within each pedigree.
VWF multimeric analysis, blood was drawn in 6 mM EDTA (ethylenediaminetetraacetic acid). Blood samples were centrifuged at 3000g for 20
minutes to obtain platelet-poor plasma. For multimeric analysis, plasma
was transferred to another tube and centrifuged at 40 000g for 20 minutes to
remove residual platelets. All plasma samples were frozen in ethanol-dry
ice and stored at ⫺70°C until tested.
The bleeding time (BT) was measured by the Simplate II device
(General Diagnostics, Morris Plane, NJ). The PFA-100 was not available in
all participating European centers and was therefore not considered a
substitutive assay for primary hemostasis in VWD. VWF antigen (VWF:
Ag) was measured by enzyme-linked immunosorbent assay (ELISA) and
ristocetin cofactor activity (VWF:RCo) was measured by aggregometry of
formalin-fixed platelets, as described.25 After centrifugation at 3500g at 4°C
for 10 minutes, supernatants were frozen at ⫺80°C for VWF:Ag and
VWF:RCo assays. Multimeric analysis was performed on low-resolution
agarose gels. All FVIII/VWF measurements were expressed in international
units (IU), with reference to a plasma pool standardized against the
International Reference Preparation for FVIII/VWF-related activities.
Pedigrees of the families
The 14 family pedigrees are shown in Figure 1, with individuals indicated
by a coded number (see “Criteria for enrollment of VWD patients and
healthy individuals”). Index cases are noted by a bold horizontal arrow, and
below each symbol, the values for (top to bottom) bleeding severity score,
VWF antigen (VWF:Ag), and ristocetin cofactor activity (VWF:RCo) are
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2362
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
KUNICKI et al
Table 2. The bleeding severity scale in patients with VWD
Severity, by symptoms and therapy
Bleeding category
Epistaxis
Cutaneous bleeding
Bleeding from minor wounds
Oral cavity bleeding
Gastrointestinal bleeding
Bleeding after tooth extraction
Bleeding after surgery
Postpartum hemorrhage
Muscle hematomas
Hemarthrosis
Menorrhagia
0
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
1
2
⬍ 10 episodes/y
10 episodes/y
⬍ 10 min/episode
⬍ 10 min/episode
No therapy
No therapy or local therapy
⬍ 10 episodes/y
⬎ 10 episodes/y
No therapy
No therapy or local therapy
⬍ 10 episodes/y
⬎ 10 episodes/y
⬍ 5 min/episode
⬎ 5 min/episode
No therapy
No therapy or local therapy
After minimal trauma only
Spontaneous
No therapy
No therapy or local therapy
1 bleeding episode
⬎ 1 bleeding episode
No therapy
No therapy or local therapy
Sometimes
Always
No therapy or local therapy
Local therapy
Minor bleeding
Major bleeding
No therapy or local therapy
Local therapy
Minor bleeding
Major bleeding
No therapy or local therapy
Local therapy
After major trauma
After minor trauma
No therapy or local therapy
No therapy or local therapy
After major trauma
After minor trauma
No therapy
Local therapy
No therapy or local therapy
Birth control pills
3
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Desmopressin or FVIII/VWF conc
Conc indicates concentrate.
indicated. Affected individuals are designated by black symbols and defined
as those with a VWF:RCo level equal to or less than 45 U/dL, if blood group
non-O, or equal to or less than 35 U/dL, if blood group O. Among these
affected individuals, bleeding severity scores ranged from 3 to 23. Family
members with bleeding severity scores greater than or equal to 3 but
VWF:RCo levels above the cutoff level are indicated by gray symbols.
Individuals with bleeding severity scores less than or equal to 2 (ie, below
the affected donor range) and VWF:RCo levels above the cutoff level are
considered normal and indicated by white symbols.
PCR amplification of the target polymorphic DNA fragment
DNA was isolated from citrate-anticoagulated whole blood using the
DNeasy tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s
instructions. Fifty nanograms gDNA was sufficient for each polymerase
chain reaction (PCR). PCR primer pairs were designed to amplify specific
fragments of DNA encompassing each of SNP, as previously described.26
After polymerase extension of minisequencing primers, antidigoxygenin antibody conjugated to cyanine 5 (Cy5; Jackson ImmunoResearch
Laboratories, West Grove, PA) and streptavidin-phycoerythrin conjugate
(Molecular Probes, Eugene, OR) were added. After incubation for 15
minutes, slides were rinsed and scanned using a ScanArray 5000 (Packard
Bioscience, Downers Grove, IL) confocal scanner equipped with 543-nm
HeNe and 632-nm HeNe lasers to detect the signals of the labeled
dideoxyribonucleotides (ddNTPs). Fluorescent signal data were quantified
using Imagene image analysis software (Biodiscovery, Marina Del Ray,
CA) and exported into Excel (Microsoft, Seattle, WA) for further analysis.
Data collected from samples were compiled on an X-Y scatter plot along
with control samples of known genotype. Genotypes were called based on
data clustering.
Statistical analysis: covariance components model
for multivariate traits
In the QTL Association model, genotypes at the candidate gene locus are
treated as predictors modifying the quantitative trait.27
In our analyses, the QTL Association model was implemented using the
software package MENDEL 5.0 (Dr Kenneth Lange, Dept of Human
Genetics, UCLA).28 For each candidate gene, MENDEL assesses associa-
tion by conducting a likelihood ratio test to determine whether the
haplotype regression coefficients are significantly different from zero.
Continuous variables, such as age, platelet count, plasma VWF:RCo levels,
and plasma VWF:Ag levels are handled as covariates. The haplotypes
associated with minimum or maximum values for each of the quantitative
traits, bleeding time, plasma VWF:Ag levels, bleeding severity score, and
adjusted bleeding severity score (score/age), were computed, and the
significance of the association indicated by P. When necessary to achieve a
normal distribution, quantitative traits were subjected to appropriate
transformations, such as square root, and reciprocal or standardization by
gender and checked for normality by the Kolmogorov-Smirnov test
(SigmaStat 3.0; SYSTAT Software, Richmond, CA).
Results
VWD type 1 pedigrees
All the families were considered VWD type 1 at the time of
enrollment because the diagnosis was based on previous laboratory
analyses reported in the local database. When a diagnosis was
performed again during this study, several affected individuals
showed an apparent reduction of VWF:RCo versus VWF:Ag (F01,
F03, F04, F05, F13, F14), whereas others (F06 and F07) were
characterized by very low VWF measurements. All of these
patients were selected among a large cohort of 150 VWD families
according to the enrollment criteria of the European project (see
“Criteria for enrollment of VWD patients and healthy individuals”) and they have been treated in the past with desmopressin
(DDAVP), a drug than induces VWF release from endothelial
cells (data not shown).
The gene haplotype frequencies among the 14 pedigree members,
when considered as a single population, are not significantly different
from those of a normal control group from Milan (Table 3).
However, when compared to the white non-Hispanic control
group, the frequencies of the GP1BA haplotypes 1 and 2 are
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
CANDIDATE HAPLOTYPES IN VWD TYPE 1
Table 3. Haplotype frequencies
Gene and
haplotype
Pedigree
frequency
M control
frequency
1
0.41
0.35
2
0.50
0.50
P*
WNH control
frequency
P†
.30
0.39
.89
ITGA2
3
0.09
0.15
⫺ 92C
0.82
0.85
⫺ 92G
0.18
0.15
⫺ 52C
0.63
0.59
⫺ 52T
0.37
0.41
a
0.82
0.90
b
0.18
0.10
1
0.86
0.91
2
0.14
0.09
⫺ 5T
0.88
0.93
⫺ 5C
0.12
0.07
0.52
0.09
.66
0.88
.08
0.12
.63
0.65
.77
0.35
GP6
.14
0.86
.33
2363
quantitative trait in question, whereas the maximum haplotype is
that associated with higher values.
In the case of bleeding severity score, not adjusted for age
(Table 5), ITGA2 haplotype 2 is most often associated with higher
bleeding severity scores. Conversely, ITGA2 haplotype 3 is most
often associated with lower bleeding scores. This particular association is statistically significant (P ⫽ .01) and as such is indicated in
bold. These results can be interpreted to mean that haplotype 2 is
associated with bleeding risk, whereas haplotype 3 is protective. A
similar relationship was seen with ITGA2B haplotypes, where
haplotype 1 (Ile843; Baka) was associated with risk for bleeding,
whereas haplotype 2 (Ser843; Bakb) was protective (P ⫽ .03). None
0.14
Table 4. Descriptive statistical analysis
GPIBA
.35
0.92
.02
0.08
.32
0.87
Score
Gene
BT, min
No.
Mean
SD
Mean
SD
.85
ITGA2
0.13
ITGB3
11
10
7.9
8.4
11.4
10.0
1
0.86
0.79
12
18
6.5
4.8
9.4
8.4
2
0.14
0.21
13
5
0.8
1.3
6.9
3.3
22
14
6.3
5.0
7.1
2.9
23
19
4.0
5.4
6.9
4.8
33
1
0
—
6.5
—
.22
0.83
.59
0.17
ITGA2B
1
2
0.57
0.43
0.55
.87
0.45
0.59
.72
0.41
ITGA2-92
VWF
1
0.61
0.59
2
0.39
0.41
.81
0.62
.98
0.38
IL6
1
0.67
0.64
2
0.33
0.36
.69
0.68
.92
cc
42
6.1
6.5
8.6
7.3
cg
23
4.0
3.8
7.9
5.4
gg
2
7.5
4.9
6.8
1.8
5.1
ITGA2-52
cc
25
5.7
4.3
7.1
FGB
ct
29
4.3
5.3
8.4
5.5
1
0.74
0.73
tt
13
7.3
8.3
10.3
10.4
2
0.26
0.27
5.8
0.66
0.79
0.32
.94
0.78
.37
GP6
0.22
TXA2R
1
2
0.34
0.21
.06
0.75
.08
0.25
aa
56
4.7
4.9
7.6
ab
10
8.0
6.8
9.7
bb
1
23
—
30
7.5
—
IL6
Haplotype frequencies within the 14 pedigrees, including affected and unaffected
individuals (n ⫽ 67).
M control indicates Milan controls (n ⫽ 52); WNH control, white non-Hispanic
controls (n ⫽ 145).
*P versus Milan controls by ␹2 (Sigma Stat 3.0).
†P versus white non-Hispanic controls by ␹2 (Sigma Stat 3.0).
significantly different (P ⫽ .02, ␹2 analysis). The frequency of
GP1BA haplotype 1 among the VWD pedigrees (0.86) is significantly lower than that seen in the control white non-Hispanic
population (0.92; P ⫽ .02) and notably, albeit not significantly,
lower than that of the Milan controls (0.91; P ⫽ .35). At the same
time, the fact that haplotype frequencies can vary even among
populations with comparable ethnic backgrounds reinforces the
potential for type 1 errors that can be introduced in case-control
studies. Such errors can be avoided in pedigree analyses.
Furthermore, despite the decrease in GP1BA haplotype 1 among
the VWD pedigrees, the statistical analysis described will show
that GP1BA haplotypes are not associated with a more severe
bleeding phenotype.
For purposes of comparison, the descriptive statistical analyses
of the bleeding severity scores and BTs of pedigree members as a
function of each of the candidate gene haplotypes are summarized
in Table 4.
Genetic correlations
The results are depicted in Table 5. For each gene, the minimum
haplotype is defined as that associated with lower values of the
11
33
5.5
5.4
7.8
6.7
12
20
4.1
4.0
6.8
2.0
22
14
7.6
7.9
11.7
9.4
11
53
5.3
5.8
8.1
6.5
12
13
4.8
5.4
8.6
22
1
GP1BA
10
—
10
7.8
—
GPIBA-5
tt
53
5.4
6.0
8.1
6.9
tc
14
5.5
4.5
8.9
5.3
11
48
6.1
6.0
8.8
6.8
12
16
3.6
4.5
7.3
6.4
22
3
5.0
5.2
5.5
3.0
11
25
7.8
6.6
10.3
9.6
12
31
4.5
4.8
7.4
4.2
22
11
2.4
4.1
6.2
2.0
11
25
6.3
6.5
9.5
7.9
12
27
5.5
5.7
7.9
6.7
22
15
3.9
4.0
6.9
2.9
11
35
5.9
6.0
8.1
6.4
12
24
5.5
5.9
8.0
6.5
22
8
3.3
3.3
8.7
5.1
11
33
5.7
6.2
7.2
6.1
12
26
4.8
5.4
9.4
6.8
22
8
6.3
4.6
7.7
3.8
ITGB3
ITGA2B
VWF
FGB
TXA2R
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2364
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
KUNICKI et al
Table 5. Candidate gene haplotype association: maximum likelihood estimates
Bleeding scores, by gene
Minimum haplotype
Estimate
Maximum haplotype
Estimate
P
ITGA2
3
ⴚ 0.5020
2
0.3718
.01
⫺ 92
G
⫺ 0.0174
C
0.0174
.84
⫺ 52
A
⫺ 0.0769
G
0.0769
.25
GP6
a
⫺ 0.0855
b
0.0855
.48
GPIBA
2
⫺ 0.0718
1
0.0718
.51
⫺5
T
⫺ 0.0417
C
0.0417
.73
ITGB3
2
⫺ 0.1482
1
0.1482
.11
ITGA2B
2
ⴚ 0.1698
1
0.1698
.03
VWF
1
⫺ 0.0584
2
0.0584
.41
IL6
2
⫺ 0.0035
1
0.0035
.96
FGB
2
⫺ 0.0958
1
0.0958
.23
TBXA2R
2
⫺ 0.0088
1
0.0088
.90
ITGA2
3
ⴚ 0.4708
2
0.3374
.01
⫺ 92
G
⫺ 0.0099
C
0.0099
.93
⫺ 52
A
⫺ 0.0961
G
0.0961
.28
GP6
a
ⴚ 0.3312
b
0.3312
.03
GPIBA
1
⫺ 0.1263
2
0.1263
.37
⫺5
C
⫺ 0.0564
T
0.0564
.73
ITGB3
2
⫺ 0.0984
1
0.0984
.40
ITGA2B
2
ⴚ 0.2544
1
0.2544
.01
VWF
2
⫺ 0.1120
1
0.1120
.20
IL6
1
⫺ 0.0170
2
0.0170
.85
FGB
2
⫺ 0.0075
1
0.0075
.94
TBXA2R
1
⫺ 0.0509
2
0.0509
.58
ITGA2
1
⫺ 0.1512
2
0.1364
.37
⫺ 92
C
⫺ 0.1030
G
0.1030
.37
⫺ 52
G
⫺ 0.0802
A
0.0802
.39
GP6
b
⫺ 0.2522
a
0.2522
.09
GPIBA
2
⫺ 0.1868
1
0.1868
.26
⫺5
C
⫺ 0.0307
T
0.0307
.84
ITGB3
1
⫺ 0.0772
2
0.0772
.52
ITGA2B
1
⫺ 0.1572
2
0.1572
.15
VWF
2
ⴚ 0.1939
1
0.1939
.02
IL6
2
⫺ 0.0885
1
0.0885
.38
FGB
2
⫺ 0.0197
1
0.0197
.84
TBXA2R
1
⫺ 0.0480
2
0.0480
.61
Bleeding severity score
Adjusted bleeding severity score, score/age
Plasma VWF:Ag level
Bleeding time
ITGA2
3
0.2144
1
⫺ 0.1321
.35
⫺ 92
G
0.0750
C
⫺ 0.0750
.48
⫺ 52
G
0.0066
A
⫺ 0.0066
.94
GP6
a
0.2329
b
⫺ 0.2329
.11
GPIBA
2
0.0661
1
⫺ 0.0661
.61
⫺5
T
0.2418
C
⫺ 0.2418
.10
ITGB3
2
0.1910
1
⫺ 0.1910
.07
ITGA2B
2
0.0673
1
⫺ 0.0673
.44
.56
VWF
2
0.0482
1
⫺ 0.0482
IL6
1
0.1852
2
⫺ 0.1852
.12
FGB
1
0.0737
2
⫺ 0.0737
.40
TBXA2R
1
0.1132
2
⫺ 0.1132
.18
Boldface indicates P ⬍ .05.
of the other 7 candidate gene haplotypes exhibited a statistically
significant association with bleeding severity score.
When the bleeding severity score was adjusted for age (score/
age; Table 5), the associations of ITGA2 and ITGA2B haplotypes
remained statistically significant (P ⫽ .01 and .01, respectively). In
addition, a statistically significant association was now apparent
with GP6 haplotypes: Haplotype b (Pro219) was associated with
risk for bleeding, whereas haplotype a (Ser219) was protective
(P ⫽ .03). None of the other 6 candidate gene haplotypes exhibited
a statistically significant association with score/age.
The VWF promoter haplotypes did not show an association
with either measure of bleeding severity. On the other hand, with
regard to plasma VWF:Ag levels, VWF promoter haplotype 1
(⫺1793G) was associated with higher levels (P ⫽ .02; Table 5).
With respect to BT (Table 5), there were no statistically
significant association with any of the haplotypes of the 9 candidate genes.
The covariance components model used in this analysis also
permits a simultaneous assessment of the impact of other parameters (covariates). In this manner, the influence of each parameter
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
CANDIDATE HAPLOTYPES IN VWD TYPE 1
(VWF:RCo level, VWF:Ag level, platelet count, gender, and age)
on bleeding severity score and BT, relative to candidate gene
haplotypes, can be determined. A maximum likelihood estimate
and standard error (SE) of the estimate are computed. Under the
null hypothesis, the ratio of the absolute value of the estimate to the
corresponding SE is approximately standard normal. A ratio of 2.0
or greater is arbitrarily accepted to indicate a significant effect, and
a comparison of the ratios provides an indication of the relative
impact of each parameter. In each comparison of parameter
estimates, VWF:RCo and VWF:Ag gave essentially identical
results, so these parameters will be referred to collectively as
VWF level.
With regard to bleeding severity score (Table 6), parameter
estimates clearly show that VWF level has the strongest impact on
the score. This was an inverse effect, as reflected in the negative
estimate, such that a decrease in score was associated with an
increase in VWF level. ITGA2 haplotype 3 greater than ITGAB
haplotype 2 each exerted an inverse effect on the score. Conversely,
those parameters that were associated with an increase in score, in
order of greatest impact, were ITGA2 haplotype 2 and ITGA2B
haplotype 1. It is noteworthy that gender, platelet count, and GP6
haplotype had no significant impact on the score.
These relationships held true when the bleeding severity score
was adjusted for age of the individual (score/age; Table 7), except
that the impact of GP6 haplotypes was now significant. The relative
impact of the 3 protective candidate gene haplotypes of concern
was ITGA2 haplotype 3 more than ITGA2B haplotype 2 more than
GP6 haplotype a.
As noted, none of the candidate gene haplotypes exerted a
significant influence on bleeding time. On the other hand, VWF
level was again the single parameter most responsible for variation
in BT (Table 8).
Discussion
We have tried to correlate integrin haplotypes with bleeding
tendency in a well-characterized group of families previously
diagnosed as VWD type 1. These families were initially recruited
according to predefined enrollment criteria applied to the 154
families enrolled by 12 hemophilia centers from 9 European
countries. The aims of this European project were to determine the
best clinical and molecular markers for diagnosis and management
of previously diagnosed VWD type 1. Because several factors are
known to interfere with VWF levels within the same VWD families
Table 6. Parameter estimates for bleeding severity score
Parameter
Estimate
SE
Ratio*
6.67
VWF:RCo
ⴚ 1.2521
0.1878
VWF:Ag
ⴚ 1.1248
0.1693
6.64
ITGA2 haplotype 3
ⴚ 0.5020
0.1122
4.47
ITGA2B haplotype 2
ⴚ 0.1698
0.0726
2.34
Platelet count
⫺ 0.0017
0.0019
0.89
GP6 haplotype a
⫺ 0.0855
0.1201
0.71
Male
2365
Table 7. Parameter estimates for adjusted bleeding severity score
(score/age)
Estimate
SE
Ratio*
VWF:Ag
Parameter
ⴚ 1.0234
0.1703
6.01
VWF:RCo
ⴚ 1.1309
0.1913
5.91
ITGA2 haplotype 3
ⴚ 0.4708
0.1499
3.14
ITGA2B haplotype 2
ⴚ 0.2544
0.0871
2.92
GP6 haplotype a
ⴚ 0.3312
0.1545
2.14
Platelet count
⫺ 0.0028
0.0019
1.47
Male
⫺ 0.0203
0.0954
0.21
ITGA2 haplotype 2
0.3374
0.1246
2.71
ITGA2B haplotype 1
0.2544
0.0871
2.92
GP6 haplotype b
0.3312
0.1545
2.14
ITGA2 haplotype 1
0.1334
0.1167
1.14
Female
0.0203
0.0954
0.21
*Absolute value (estimate)/SE.
and to influence their clinical behavior in terms of bleeding
tendency, this pilot study on the role of adhesion receptor gene
haplotypes on bleeding tendency in 14 families collected by one
partner (P03, Milan) was approved by all of the other partners of
this European project.
All 14 families with previously diagnosed VWD type 1 satisfied
the criteria of enrollment, but several affected individuals showed
an apparent reduction of VWF:RCo versus VWF:Ag (F01, F03,
F04, F05, F13, F14), whereas others (F06 and F07) were characterized by very low VWF measurements. This is not surprising
because the diagnosis of VWD has been improved in the last 10
years. Updated criteria for the classification of VWD type 1 have
not yet been approved by the SSC of the ISTH. A panel of experts
of this committee is now working on a proposal for an updated
classification of VWD type 1 versus 2.
To better understand the influence of secondary gene polymorphisms on bleeding risk in VWD type 1, we elected to study
pedigrees of index cases. Case-control studies can provide useful
indirect evidence for the contribution of a genetic effect, and we
have previously demonstrated in the initial case-control study of
this kind that the ITGA2 SNP 807C is more common among
individuals with VWD type 1.29 However, case-control studies
suffer from a number of weaknesses that preclude the identification
of many genetic associations.30 These include the introduction of
type 1 errors resulting from hidden population stratification, the
lack of a direct evaluation of familial transmission, and poor ability
to discover novel genes involved in intrapopulation variation in
disease risk. Contributing to these weaknesses are small but
statistically significant differences in haplotype frequency that
might be peculiar to the specific populations under study. One such
frequency difference was seen for GP1BA haplotypes in the
pedigrees that were the subject of this study (Table 3).
Family-based approaches, particularly those that involve large
pedigrees, provide a more powerful approach to the study of
genetic risk associations. The benefit of genetic linkage studies in
Table 8. Parameter estimates for bleeding time
⫺ 0.0311
0.0929
0.33
Estimate
SE
Ratio*
ITGA2 haplotype 2
0.3718
0.0935
3.98
VWF:RCo
Parameter
ⴚ 0.7875
0.2279
3.46
ITGA2B haplotype 1
0.1698
0.0726
2.34
VWF:Ag
ⴚ 0.6093
0.2125
2.87
Age
0.0098
0.0051
1.92
Female
⫺ 0.0231
0.1171
0.20
ITGA2 haplotype 1
0.1302
0.0833
1.56
Platelet count
0.0019
0.0023
0.83
GP6 haplotype b
0.0855
0.1201
0.71
Male
0.0231
0.1171
0.20
Female
0.0311
0.0929
0.33
Age
0.0000
0.0066
0.00
*Absolute value (estimate)/SE.
*Absolute value (estimate)/SE.
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2366
KUNICKI et al
pedigrees derives from the presence of case-parent triads and
multiplex sibships. For studies of genetic risk, designs based on the
genotyping of index cases and their parents are highly informative
and minimize spurious conclusions that might result from hidden
genetic structure, such as stratification or admixture that can afflict
a case-control study.31 The underlying benefit of case-parent triad
lies in the fact that transmission of alleles from parents to offspring
follows mendelian probabilities and for each parental mating type,
the mendelian genotype proportions persist among offspring until
such time as the probands are studied. In addition, multiplex
sibships will be enriched in frequency for a disease susceptibility
allele and thereby create a stronger contrast to parents and
unaffected sibs.32
The paradox of VWD type 1 is that bleeding risk does not
correlate with plasma VWF levels.3 Among healthy donors, plasma
VWF levels vary over a wide range, and 95% of all values fall
within 50% and 200% of the mean. ABO blood type has a major
influence on VWF levels, such that the average level for persons
who are blood type O is 25% to 35% lower than that of persons
who are not type O.33 This accounts for about 30% of the genetic
variance of VWF.34 The SNP G⫺1793C in the VWF promoter also
correlates with small changes in mean VWF level. Among blood
group O donors, mean VWF levels are 77% for genotype CC, 86%
for genotype GC, and 93% for genotype GG.20 Because of the high
allele frequency of ⫺1793G (0.65), it can contribute modestly to
the variation in VWF levels. In our study, we confirm a positive
influence of ⫺1793G on plasma VWF:Ag and VWF:RCo. However, it appears that the impact of the VWF promoter haplotypes are
not sufficiently important that they can be directly associated to
variation in bleeding phenotype.
In an earlier case-control study,29 we showed that the 807C
dimorphism of the integrin gene ITGA2 was associated with
diminished platelet responsiveness to collagen in a high shear
environment, reflected by a prolongation of the PFA-100 collagen/
epinephrine-closure time. However, that study preceded the finding
that there are 2 major haplotypes that share the 807C allele:
haplotype 3 (807C/1648A) is associated with higher levels of the
integrin, whereas haplotype 2 (807C/1648G) is associated with
markedly reduced levels of this receptor.8 The current study refines
our initial association study, demonstrating that haplotype 2
contributes to increased bleeding severity score and increased risk,
whereas haplotype 3 is protective. The protective effect offered by
the presence of haplotype 3 may not be explained exclusively by
increased levels of the integrin and suggests that haplotype 3 may
be associated with increased function of the integrin in addition to
any effect on expression. The unique difference of haplotype 3 is its
expression of the alloantigen HPA-5b (Bra) resulting from a lysine
to glutamate substitution at residue 505. It remains to be determined whether this amino acid substitution has a significant
influence on platelet function tests ex vivo. Aside from this, our
results reinforce the need to distinguish the major haplotypes 2 and
3 of ITGA2, which share the 807C SNP, because each has a
quantitatively and perhaps qualitatively opposite influence on the
function of this integrin.
With regard to GP6 haplotypes, our results show a statistically
significant, albeit weaker, association between haplotype b (Pro219)
and risk for bleeding, which was only apparent when the bleeding
severity score was adjusted for age. Conversely, haplotype a
(Ser219) was protective. These findings are not consistent with the
observation of Croft et al7 that homozygosity for haplotype b would
be associated with risk for acute myocardial infarction, but are
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
consistent with the observations of Joutsi-Korhonen et al16 that
haplotype a confers increased reactivity to the receptor.
The involvement of ITGA2 and GP6 haplotypes in risk for
bleeding in mild VWD is certainly not a coincidence. Although
some have argued that one or the other of GPVI or integrin ␣2␤1 is
the more important for platelet adhesion and signaling on collagens, it is more likely that the concerted activity of both is essential
for optimum platelet function. Receptor cooperation is supported
by several observations. Mice genetically deficient in GPVI exhibit
a modest abnormality in hemostatic function35 whereby platelet
adhesion and thrombus formation persist, albeit with some loss of
thrombus stability. At the same time, similar results occur with
mice that are deficient in ␣2␤1,36 where adhesion and thrombus
formation are moderately impaired but not completely eliminated.
Consistent with this theme, 2 receptor models of platelet adhesion
to collagen and resultant platelet activation emphasize the integrated cross-talk between these 2 receptors. Lastly, mouse strain
differences in platelet aggregation by collagen have been recently
correlated with a difference in expression of integrin ␣2␤1, but not other
prominent receptors, such as GPVI, GPIb␣ or integrin ␣IIb␤3.37
Polymorphism of ITGA2B is responsible for the expression of
the Bak (HPA-3) alloantigen system.38 This polymorphism lies
adjacent to the binding region of the murine monoclonal PMI-1
antibody, which inhibits platelet adhesion and spreading on certain
substrata.39 Adenosine diphosphate (ADP) stimulation of platelets
results in a fibrinogen-dependent increase in binding of the PMI-1
antibody, and peptides containing arginine-glycine-aspartate (RGD)
also reversibly increase the binding of this antibody to cells and to
purified glycoprotein ␣IIb␤3.40 Thus, the ␣IIb region encompassing
HPA-3 and PMI.1 epitopes may participate in adhesive functions
through postreceptor occupancy events. The study of Reiner et al9
is consistent with this hypothesis, wherein homozygosity for the
ITGA2B haplotype 2 (HPA-3b allele) was associated with an
approximately 5-fold increased risk of ischemic stroke among
subgroups of women who carried a diagnosis of hypertension or
diabetes (OR ⫽ 4.51; 95% CI, 1.01-20.13) or had elevated plasma
homocysteine levels (OR ⫽ 5.94; 95% CI, 1.53-23.05). Our findings in a different clinical setting complement the observation of
Reiner et al,9 showing that the complementary haplotype 1 would
decrease the tendency to thrombosis and thus increase risk for
bleeding in VWD type 1.
The results of our study document that, despite its variability
and poor heritability, the level of VWF, measured as antigen or
ristocetin cofactor, remains the single most important parameter
associated with bleeding severity and bleeding times in families of
VWD type 1 index cases. Our results also provide unique
confirmation of the importance of ITGA2, GP6, and ITGA2B
haplotypes in disease outcome, demonstrating that genetically
controlled attenuation of certain adhesion receptors (whether
through expression or activity) can influence risk for morbidity in
clinical settings where hemostasis is compromised. In contrast, a
number of other glycoprotein haplotypes were not predictive,
namely, those represented by the GP1BA, integrin ITGB3, VWF,
FGB, IL6, and TXA2R genes.
Acknowledgments
This is manuscript 16315-MEM from TSRI. We thank Drs Kenneth
Lange and Eric Sobel (Department of Human Genetics, UCLA
School of Medicine) for their excellent genetic and statistical
counseling throughout this study. We also thank Drs Claudia
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
CANDIDATE HAPLOTYPES IN VWD TYPE 1
Mistretta and Francesca Giannello for assistance in interviewing
patients and calculating bleeding scores. We acknowledge the
comments received from all of the other partners of the European
project entitled “Molecular and Clinical Markers for Diagnosis and
Management of Type 1 von Willebrand Disease (MCMDM1VWD): Ann Gooedeve (P01, Sheffield, United Kingdom), Drs
Francesco Rodeghiero and Giancarlo Castaman (P02, Vicenza,
Italy), Dr Javier Battle (P04, La Coruna, Spain), Drs Dominique
2367
Meyer, Mazurier, Goodemand (P05, Paris and Lille, France), Dr
Jeroen Eikenboom (P06, Leiden, The Netherlands), Drs Reinhard
Schneppenheim and Ulrich Budde (P07, Hamburg, Germany), Dr
Jorgen Ingerslev (P08, Aarhus, Denmark), Drs Zdena Vorlova and
David Habart (P09, Prague, Czech Republic), Dr Lars Holmberg
and Stefan Lethagen (P10, Malmo, Sweden), Dr John Pasi (P11,
Leicester, United Kingdom), and Dr Frank Hill (P12, Birmingham,
United Kingdom).
References
1. Savage B, Almus-Jacobs F, Ruggeri ZM. Specific
synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow.
Cell. 1998;94:657-666.
2. Kunicki TJ. The influence of platelet collagen receptor polymorphisms in hemostasis and thrombotic disease. Arterioscler Thromb Vasc Biol.
2002;22:14-20.
3. Sadler JE. Von Willebrand disease type 1: a diagnosis in search of a disease. Blood. 2003;101:
2089-2093.
4. Castaman G, Federici AB, Rodeghiero F, Mannucci PM. von Willebrand’s disease in the year
2003: towards the complete identification of gene
defects for correct diagnosis and treatment.
Haematologica. 2003;88:94-108.
5. Murata M, Furihata K, Ishida F, et al. Genetic and
structural characterization of an amino acid dimorphism in glycoprotein Iba involved in platelet
transfusion refractoriness. Blood. 1992;79:30863090.
6. Afshar-Kharghan V, Li CQ, Khoshnevis-Asl M,
Lopez JA. Kozak sequence polymorphism of the
glycoprotein (GP) Ibalpha gene is a major determinant of the plasma membrane levels of the
platelet GP Ib- IX-V complex. Blood. 1999;94:
186-191.
7. Croft SA, Samani NJ, Teare MD, et al. Novel
platelet membrane glycoprotein VI dimorphism is
a risk factor for myocardial infarction. Circulation.
2001;104:1459-1463.
8. Kritzik M, Savage B, Nugent DJ, et al. Nucleotide
polymorphisms in the alpha 2 gene define multiple alleles which are associated with differences
in platelet alpha 2 beta 1. Blood. 1998;92:23822388.
9. Reiner AP, Kumar PN, Schwartz SM, et al. Genetic variants of platelet glycoprotein receptors
and risk of stroke in young women. Stroke. 2000;
31:1628-1633.
10. Newman PJ, Derbes RS, Aster RH. The human
platelet alloantigens, PLA1 and PLA2, are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are
distinguishable by DNA typing. J Clin Invest.
1989;83:1778-1781.
11. Moshfegh K, Wuillemin WA, Redondo M, et al.
Association of two silent polymorphisms of platelet glycoprotein Ia/IIa receptor with risk of myocardial infarction: a case-control study. Lancet.
1999;353:351-354.
12. Santoso S, Kunicki TJ, Kroll H, Haberbosch W,
Gardemann A. Association of the platelet glycoprotein Ia C807T gene polymorphism with myocardial infarction in younger patients. Blood.
1999;93:2449-2453.
13. Roest M, Banga JD, Grobbee DE, et al. Homozygosity for 807 T polymorphism in alpha(2) subunit
of platelet alpha(2)beta(1) is associated with increased risk of cardiovascular mortality in highrisk women. Circulation. 2000;102:1645-1650.
14. Matsubara Y, Murata M, Maruyama T, et al. Asso-
ciation between diabetic retinopathy and genetic
variations in alpha-2 beta-1 integrin, a platelet
receptor for collagen. Blood. 2000;95:1560-1564.
15. Carlsson LE, Santoso S, Spitzer C, Kessler C,
Greinacher A. The alpha 2 gene coding sequences T807/A873 of the platelet collagen receptor integrin alpha2 beta1 might be a genetic
risk factor for the development of stroke in
younger patients. Blood. 1999;93:3583-3586.
16. Joutsi-Korhonen L, Smethurst PA, Rankin A, et al.
The low-frequency allele of the platelet collagen
signaling receptor glycoprotein VI is associated
with reduced functional responses and expression. Blood. 2003;101:4372-4379.
17. Carter AM, Catto AJ, Bamford JM, Grant PJ. Association of the platelet glycoprotein IIb HPA-3
polymorphism with survival after acute ischemic
stroke. Stroke. 1999;30:2606-2611.
18. Carlsson LE, Greinacher A, Spitzer C, Walther R,
Kessler C. Polymorphisms of the human platelet
antigens HPA-1, HPA-2, HPA-3, and HPA-5 on
the platelet receptors for fibrinogen (GPIIb/IIIa),
von Willebrand factor (GPIb/IX), and collagen
(GPIa/IIa) are not correlated with an increased
risk for stroke. Stroke. 1997;28:1392-1395.
19. Bottiger C, Kastrati A, Koch W, et al. HPA-1 and
HPA-3 polymorphisms of the platelet fibrinogen
receptor and coronary artery disease and myocardial infarction. Thromb Haemost. 2000;
83:559-562.
20. Keightley AM, Lam YM, Brady JN, Cameron CL,
Lillicrap D. Variation at the von Willebrand Factor
(vWF) gene locus is associated with plasma
vWF:Ag levels: identification of three novel single
nucleotide polymorphisms in the vWF gene promoter. Blood. 1999;93:4277-4283.
21. Brull DJ, Dhamrait S, Moulding R, et al. The effect
of fibrinogen genotype on fibrinogen levels after
strenuous physical exercise. Thromb Haemost.
2002;87:37-41.
22. Humphries SE, Luong LA, Ogg MS, Hawe E,
Miller GJ. The interleukin-6 ⫺174 G/C promoter
polymorphism is associated with risk of coronary
heart disease and systolic blood pressure in
healthy men. Eur Heart J. 2001;22:2243-2252.
23. Unoki M, Furuta S, Onouchi Y, et al. Association
studies of 33 single nucleotide polymorphisms
(SNPs) in 29 candidate genes for bronchial
asthma: positive association a T924C polymorphism in the thromboxane A2 receptor gene.
Hum Genet. 2000;106:440-446.
24. Sadler JE. A revised classification of von Willebrand disease. For the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the International Society on
Thrombosis and Haemostasis. Thromb Haemost.
1994;71:520-525.
25. Federici AB, Stabile F, Castaman G, Canciani MT,
Mannucci PM. Treatment of acquired von Willebrand syndrome in patients with monoclonal
gammopathy of uncertain significance: comparison of three different therapeutic approaches.
Blood. 1998;92:2707-2711.
26. Kunicki TJ, Head SR, Salomon DR. Platelet receptor structures and polymorphisms. In: Gibbins
J.M., Mahaut-Smith MP, eds. Platelets and
Megakaryocytes. Methods and Protocols. Totowa, NJ: Humana Press; 2003:455-478.
27. Lange K, Boehnke M. Extensions to pedigree
analysis, IV: covariance components models for
multivariate traits. Am J Med Genet. 1983;14:513524.
28. Lange K, Cantor R, Horvath S, et al. Mendel version 4.0: a complete package for the exact genetic analysis of discrete traits in pedigree and
population data sets [abstract]. Am J Hum Genet.
2001;69:A1886.
29. Di Paola J, Federici AB, Sacchi E, et al. Low
platelet alpha 2 beta 1 levels in type I von Willebrand disease correlate with impaired platelet
function in a high shear stress system. Blood.
1999;93:3578-3582.
30. Blangero J, Williams JT, Almasy L. Novel familybased approaches to genetic risk in thrombosis. J
Thromb Haemost. 2003;1:1391-1397.
31. Umbach DM, Weinberg CR. The use of case-parent triads to study joint effects of genotype and
exposure. Am J Hum Genet. 2000;66:251-261.
32. Risch N. Implications of multilocus inheritance for
gene-disease association studies. Theor Popul
Biol. 2001;60:215-220.
33. Gill JC, Endres-Brooks J, Bauer PJ, Marks WJ Jr,
Montgomery RR. The effect of ABO blood group
on the diagnosis of von Willebrand disease.
Blood. 1987;69:1691-1695.
34. Orstavik KH, Kornstad L, Reisner H, Berg K. Possible effect of secretor locus on plasma concentration of factor VIII and von Willebrand factor.
Blood. 1989;73:990-993.
35. Kato K, Kanaji T, Russell S, et al. The contribution
of glycoprotein VI to stable platelet adhesion and
thrombus formation illustrated by targeted gene
deletion. Blood. 2003;102:1701-1707.
36. Chen J, Diacovo TG, Grenache DG, Santoro SA,
Zutter MM. The alpha(2) integrin subunit-deficient
mouse: a multifaceted phenotype including defects of branching morphogenesis and hemostasis. Am J Pathol. 2002;161:337-344.
37. Li TT, Larrucea S, Souza S, et al. Genetic variation responsible for mouse strain differences in
integrin (alpha)2 expression is associated with
altered platelet responses to collagen. Blood.
2004;103:3396-3402.
38. Lyman S, Aster RH, Visentin GP, Newman PJ.
Polymorphism of human platelet membrane glycoprotein IIb associated with the Baka/Bakb alloantigen system. Blood. 1990;75:2343-2348.
39. Shadle PJ, Ginsberg MH, Plow EF, Barondes SH.
Platelet-collagen adhesion: inhibition by a monoclonal antibody that binds glycoprotein IIb. J Cell
Biol. 1984;99:2056-2060.
40. Frelinger AL III, Lam SC-T, Plow EF. et al. Occupancy of an adhesive glycoprotein receptor
modulates expression of an antigenic site involved in cell adhesion. J Biol Chem. 1988;263:
12397-12402.
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
2004 104: 2359-2367
doi:10.1182/blood-2004-01-0349 originally published online
June 29, 2004
An association of candidate gene haplotypes and bleeding severity in
von Willebrand disease (VWD) type 1 pedigrees
Thomas J. Kunicki, Augusto B. Federici, Daniel R. Salomon, James A. Koziol, Steven R. Head, Tony
S. Mondala, Jeffrey D. Chismar, Luciano Baronciani, Maria Teresa Canciani and Ian R. Peake
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