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Original Article
A Genome-Wide Association Study Identifies KNG1 as
a Genetic Determinant of Plasma Factor XI Level and
Activated Partial Thromboplastin Time
Maria Sabater-Lleal, Angel Martinez-Perez, Alfonso Buil, Lasse Folkersen, Juan Carlos Souto,
Maria Bruzelius, Montserrat Borrell, Jacob Odeberg, Angela Silveira, Per Eriksson, Laura Almasy,
Anders Hamsten,* José Manuel Soria*
Downloaded from http://atvb.ahajournals.org/ by guest on December 22, 2016
Objective—Elevated plasma levels of coagulation factor XI (FXI) are implicated in the pathogenesis of venous
thromboembolism and ischemic stroke, and polymorphisms in the F11 gene are associated both with risk of venous
thromboembolism and an elevated plasma FXI level.
Methods and Results—Here, we report the first hypothesis-free genome-wide genetic analysis of plasma FXI levels.
Two genome-wide significant loci were detected in the family-based Genetic Analysis of Idiopathic Thrombophilia 1
cohort: one located in the kininogen 1 gene (KNG1) (rs710446; P=7.98×10−10) and one located in the structural F11 gene
(rs4241824; P=1.16×10−8). Both associations were replicated in a second population-based Swedish cohort. A significant
effect on KNG1 mRNA expression was also seen for the 2 most robustly FXI-associated single nucleotide polymorphisms
located in KNG1. Furthermore, both KNG1 single nucleotide polymorphisms were associated with activated partial
thromboplastin time, suggesting that FXI may be the main mechanistic pathway by which KNG1 and F11 influence
activated partial thromboplastin time and risk of thrombosis.
Conclusion—These findings contribute to the emerging molecular basis of venous thromboembolism and, more importantly,
help in understanding the biological regulation of a phenotype that has proved to have promising therapeutic properties in
relation to thrombosis. (Arterioscler Thromb Vasc Biol. 2012;32:00-00.)
Key Words: factor XI ◼ kininogen ◼ genome-wide associations ◼ activated partial thromboplastin time ◼ thrombosis
C
deficiency have a reduced incidence of venous thromboembolism (VTE) and stroke.10,13,14
Recent molecular genetic studies of VTE have identified
polymorphisms in the F11 gene located on chromosome 4,
which are associated both with risk of disease and elevated
plasma FXI level,15–17 findings that have subsequently been
replicated in other VTE studies in whites and blacks.18–20
Of note, the fact that common genetic factors underlie both
traits had been suggested earlier based on a family study of
thrombophilia, indicating a significant genetic correlation
between FXI levels and risk of VTE (0.56; P=0.02).21
Although the evidence of an important role for FXI in the
pathogenesis of VTE is strong, knowledge about the regulation of the plasma concentration of this protein remains sparse.
However, it is notable, in this context, that the additive heritability of plasma FXI has been estimated to be as high as 45%.22
Despite the substantial genetic regulation, a hypothesis-free
genome-wide association study (GWAS) has so far not been
oagulation factor XI (FXI) is a component of the contact
(activation) pathway and circulates in the blood in complex with high-molecular-weight kininogen (HK). FXI has an
important role in propagation and stabilization of the evolving
thrombus in vivo, which is independent of coagulation factor XII (FXII).1 Interestingly, congenital FXI deficiency only
confers a mild bleeding phenotype compared with deficiencies in coagulation factors VIII and IX (hemophilia A and B,
respectively). There is also growing evidence that FXI plays
a key role in the pathogenesis of thrombosis.2–6 Individuals
with elevated plasma levels of FXI (defined as values above
the 90th percentile of the distribution seen in control subjects)
run an ≈2-fold increased risk of venous thrombosis.4,7 Smaller
studies of younger patients also support an association with
ischemic stroke, whereas the FXI level seems to be of minor
importance in myocardial infarction.2,6,8–11 Conversely, FXIdeficient mice are protected from experimentally induced
thrombosis or stroke,12 and human subjects with severe FXI
Received on: February 24, 2012; final version accepted on: May 14, 2012.
From the Department of Medicine, Cardiovascular Genetics and Genomics Group, Atherosclerosis Research Unit, Karolinska Institutet, Karolinska
University Hospital Solna, Stockholm, Sweden (M.S.-L., L.F., M.B., J.O., A.S., P.E., A.H.); Unit of Genomics of Complex Diseases, Research Institute
of Hospital de la Santa Creu i Sant Pau, Barcelona, Spain (A.M.-P., A.B., J.M.S.,); Department of Genetic Medicine and Development, University of
Geneva Medical School, Geneva, Switzerland (A.B.); Department of Hematology, Haemostasis and Thrombosis Unit, Hospital de la Santa Creu i Sant
Pau, Barcelona, Spain (J.C.S., M.B.); and Department of Population Genetics. Southwest Foundation for Biomedical Research. San Antonio, TX (L.A.).
*These authors contributed equally to this work.
Correspondence to José Manuel Soria, Unit of Genomics of Complex Diseases, Research Unit Hospital de la Santa Creu i Sant Pau, C/ Sant Antoni M.
Claret 167, 08025, Barcelona, Spain. E-mail [email protected]
© 2012 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1
DOI: 10.1161/ATVBAHA.112.248492
2 Arterioscler Thromb Vasc Biol August 2012
conducted to reveal additional gene loci, influencing the variation in FXI level. Against this background, we performed a
GWAS of the plasma FXI concentration in extended Spanish
families, half of which were ascertained through thrombophilia, with replication of the key findings in a populationbased sample of healthy Swedish subjects. Expression analysis
in human liver was also undertaken to confirm the functional
significance of the identified candidate gene.
Patients and Methods
Subjects
The discovery GWAS was performed in the Genetic Analysis of
Idiopathic Thrombophilia 1 (GAIT-1) cohort, which is a family-based
cohort consisting of 398 individuals belonging to 21 Spanish families, 12 of whom were selected through a proband with idiopathic
thrombophilia and 9 were randomly selected. Of these, 339 had FXI
and aPPT measurements. A detailed description of the GAIT-1 sample
has been reported elsewhere.21 All subjects gave informed consent to
the use of their data, and the GAIT-1 study protocols were approved
by the Institutional Review Board of the Hospital de la Santa Creu i
Sant Pau.
Replication was performed in population-based, unrelated Swedish
individuals, 340 men and 322 women with a similar age distribution
(33–80 years), who had been recruited at random from the greater
Stockholm area for inclusion as control subjects in the Precocious
Coronary Artery Disease (PROCARDIS) study (cohort acronym
OLIVIA).23 Of these, 658 had FXI measurements. Informed consent
was provided by all participants, and the study was granted approval
by the Ethics Committee of the Karolinska Institutet.
Expression analyses in human liver were conducted in 211 liver
biopsy samples from the Advanced Study of Aortic Pathology
(ASAP) study. ASAP enrolls patients undergoing aortic valve surgery at the Karolinska University Hospital, Stockholm, Sweden.24
Biopsies were obtained during open-heart surgery. Informed consent
was provided by all the participants, and approval was given by the
ethics committee of the Karolinska Institutet.
Phenotype and Genotype Determinations
Venous blood samples were obtained after an overnight fast, and
DNA was extracted from whole blood samples using a standard protocol in both cohorts. Plasma FXI concentration was determined in
GAIT-1 using platelet-poor citrate plasma, which had been prepared
by centrifugation at 2000g for 20 minutes at room temperature.
Assays for activated partial thromboplastin time (aPTT) and FXI
were performed on fresh plasma samples. Briefly, aPTT was measured in an automated coagulometer (ACL 3000; Instrumentation
Laboratory SpA, Milan, Italy) with the use of bovine cephalin and
silica, and FXI was assayed with deficient plasma from Diagnostica
Stago (Asnières, France). Intra-assay and interassay coefficients of
variation were between 2% and 6% for all phenotypes in GAIT1.22 For OLIVIA, FXI antigen level was determined in EDTA
plasma samples by ELISA, using a matched pair antibody set for
assay of human FXI antigen from Affinity Biologicals Inc (Ontario,
Canada), essentially according to the instructions provided by
the manufacturer. The VisuCal Antigen from Affinity Biologicals
Inc was used as calibrator, and results were expressed as U/mL.
Coagulation reference plasma from Technoclone GmbH (Vienna,
Austria) was used as control plasma. The interassay coefficient of
variation was 10.6%.
Genome-wide, high-coverage, single nucleotide polymorphism
(SNP) genotyping in GAIT-1 was performed using the Illumina
Infinium 317K Beadchip (Illumina), as previously described.25 SNPs
with a call rate <0.95, a minor allele frequency <0.025, or deviating
from Hardy-Weinberg equilibrium at P<5×10−7 were excluded. After
quality control (QC) filtering, 283 437 SNPs remained for analysis.
OLIVIA samples were genotyped using the Illumina 1M Beadchip.
QC filters (call rate ≤0.95, minor allele frequency <0.01, and
deviation from Hardy-Weinberg equilibrium [P<10−6]) were applied
before imputation. After QC filtering, 498 717 SNPs remained available for imputation. Dosage imputation was then conducted using
MaCH 1.0.16 and the 1000 Genomes Caucasian (CEU) reference
panel providing a total of 2 543 887 SNPs for analysis. Genotyping
in ASAP was performed on Illumina Human 610W-Quad Bead arrays
(Illumina), and filters identical to the ones applied in OLIVIA were
used for QC.
As GAIT-1 sample is a family-based study and the origin of individuals is homogeneous, we did not perform a stratification analysis.
However, in OLIVIA, multidimensional scaling coordinates, reflecting genetic distances between individuals (population substructure),
were calculated based on the genotype data using PLINK1.05, and
outliers in the nearest neighborhood analysis for multidimensional
scaling 1 versus multidimensional scaling 2 were excluded.
Total RNA from human ASAP liver specimens was isolated using
RNAlater (Ambion, Austin, TX), Trizol (BRL-Life Technologies,
Grand Island, NY), and Rneasy Mini kit (Qiagen, Hilden, Germany),
including treatment with RNase-free DNase set (Qiagen) according to the manufacturer’s instructions. RNA quality was determined
with an Agilent 2100 bioanalyzer (Agilent Technologies Inc, Palo
Alto, CA), and quantity was measured by a NanoDrop (Thermo
Scientific, Waltham, MA). Global gene expression was generated
using Affymetrix ST 1.0 Exon arrays (Affymetrix, Santa Clara, CA)
and the core subset of Affymetrix meta probe sets (Affymetrix). QC
procedures and preparations preceding meta probe set level investigations (ie, the analysis of whole-gene variation with genotype) have
been reported in detail.24
Statistical Analysis
Association analyses in GAIT-1 between SNPs and the natural
logarithm-transformed FXI level were performed using variance
components methods in the SOLAR version 4.0 software package
(Southwest Foundation for Biomedical Research, San Antonio, TX),
with sex and age as covariates. From the genome-wide significant
loci, we selected the 5 most significantly associated SNPs for further
exploration in the OLIVIA replication cohort, comprising 658
Swedish controls. Linear regression analysis according to an additive
model of inheritance was applied in OLIVIA to test for associations
between the selected SNPs and natural logarithm-transformed FXI
antigen level, using STATA version 11 (StataCorp, College Station,
TX), and adjusting for sex and age. Fine mapping of candidate
regions using 1000 Genomes−imputed SNP data was conducted in
OLIVIA by analyzing associations between all imputed SNPs located
within ±50 kb of the 2 lead SNPs showing the strongest association in
GAIT-1 (rs410446 in the KNG1 locus and rs4241824 in the FXI locus)
and logarithm-transformed FXI level, applying an additive model of
inheritance with adjustment for sex and age. The 3 SNPs that reached
Bonferroni-corrected significant P values in the replication analysis
(rs710446, rs5030062, and rs4241824) were subsequently tested for
association with expression levels of F11 and KNG1 in a total of 211
human liver samples obtained from the ASAP study. Genotypes from
the 3 candidate SNPs were tested for association with gene expression
levels using additive linear models in R, with each genotype being
coded as 0, 1, or 2.
Results
Sample Characteristics
Table 1 shows sample characteristics of participants in the
GAIT-1 and OLIVIA studies. Briefly, both were European
samples with similar proportion of men and women and similar body mass index distributions, with mean values going
from 24.7 to 25.9 kg/m2. OLIVIA differed from the GAIT-1
sample, in that all participants were healthy (compared with
10.5% of individuals with VTE in GAIT-1) and it was a
population-based sample compared with the family-based
design of GAIT-1. Also, participants in the OLIVIA study
Sabater-Lleal et al KNG1 is a Novel Genetic Determinant of FXI Level 3
Table 1. Characteristics of Study Participants in the
Discovery and Replication Cohorts
Characteristic
Counts
GAIT-1
OLIVIA
339
658
37.7 (19.83)
60.3 (7.01)
Men, %
48.7
51.16
European ancestry, %
100
100
24.7 (4.9)
25.9 (4.0)
Arterial disease, %
6.5
0
Venous disease, %
10.5
0
3.8
4.47
Age
BMI, kg/m2
T2D, %
FXI, %
FXI quartiles
aPTT
aPTT quartiles
100.7 (21.1)
105.9 (44.9)
86.5; 100; 113.5
76.0; 98.0; 125.2
0.95 (0.10)
ND
0.87; 0.95; 1.02
ND
Values are mean (SD) or number of subjects or percentage of subjects in
group. aPTT levels are expressed as a ratio of the aPTT time (in s) between the
individual value and the aPTT reference value of the laboratory.
GAIT-1 indicates Genetic Analysis of Idiopathic Thrombophilia 1; BMI, body
mass index; T2D, type-2 diabetes mellitus; FXI, coagulation factor XI; aPTT,
activated partial thromboplastin time; ND, not determined.
were significantly older (60.3 years old) than the subjects
enrolled in GAIT-1 (37.7 years old), explaining why levels of FXI were higher in OLIVIA (105.9 ± 44.9%) than in
GAIT-1 (100.7 ± 21.1%). In addition, mean and SD values
for aPTT were 0.95 (0.10) in GAIT-1, and the correlation
between FXI and aPTT was high (R2=−0.60, P=9.11×10−29).
Discovery Analysis
Figure 1 illustrates the results of the discovery association
analyses performed for 283 437 SNPs in relation to FXI level,
organized by chromosomal position in a Manhattan plot.
Two SNPs passed the genome-wide significance level of
P<5×10−8 for their association with the plasma FXI activity
level (Table 2); the most robustly associated SNP (rs710446;
P=7.98×10−10) was located in the kininogen 1 (KNG1) gene.
This gene encodes HK, which acts as a cofactor for the activation of kallikrein and FXII, initiating the contact pathway
of coagulation. The other SNP attaining genome-wide significance (rs4241824; P=1.16×10−8) was located in an intron
within F11, the structural gene coding for FXI. Further
adjustments for disease status resulted in minor alterations
to the strength of the associations (rs710446; P=3.49×10−10,
and rs4241824; P=3.78×10−8). No additional genomesignificant signals appeared when we conducted the analysis
conditioned on the 2 most significant SNPs (rs710446 and
rs4241824).
Replication Analysis and Fine Mapping
From the 2 genome-wide significant loci we selected the 5
most significant SNPs for further exploration in the OLIVIA
replication cohort, comprising 658 Swedish controls. These
5 SNPs were all associated with log-transformed FXI levels
at P<10−7 in the discovery cohort. Linkage disequilibrium
measures between rs710446 and rs5030062 were R2=0.93
and D′=1, indicating that the 2 SNPs are highly linked
according to the 1000 Genomes Pilot 1 release. The pairwise linkage disequilibrium measures between the other
combinations of these 5 SNPs were all <0.8. Nonetheless,
we decided to apply a standard Bonferroni correction for
replication of 5 SNPs (P<0.01). Three of these SNPs passed
the Bonferroni-corrected level for replication, 2 of which
were located in the KNG1 gene (rs710446; P=1.81×10−4, and
rs5030062; P=1.57×10−4), and 1 was located in the FXI gene
(rs4241824; P=5.06×10−3) (Table 2). Only 1 SNP reached
nominal significance (rs4253399; P=3.96×10−2), whereas
1 SNP (rs4686799) did not replicate at all. The ranges of
Figure 1. Manhattan plot representing the genome-wide association results between the 283 437 single nucleotide polymorphisms
(SNPs) in the discovery cohort and natural log-transformed FXI levels adjusted for age and sex. Every dot represents an SNP organized
by chromosomal order and position. The y axis is proportional to statistical significance (expressed as −log10 of the P values).
Table 2. Associations Between Plasma FXI Concentration and the Most Significantly FXI-Associated SNPs in the Discovery and
Replication Cohorts
CHR
SNP
Gene
Position
A1
A2
FreqA1 FreqA1
GAIT-1 OLIVIA
P GAIT-1
β GAIT-1 SE GAIT-1
P OLIVIA
β OLIVIA
SE OLIVIA
Imputation
Quality OLIVIA
3
rs710446
KNG1
187942621
C
T
0.45
0.42
7.98E−10
0.094
0.016
1.81E−04*
0.08
0.021
0.9979
3
rs5030062
KNG1
187936874
C
A
0.43
0.38
1.19E−07
0.083
0.017
1.57E−04*
0.082
0.022
0.9987
3
rs4686799
KNG1
187933930
C
T
0.76
0.77
2.00E−07
0.099
0.019
7.80E−01
0.007
0.024
0.9999
4
rs4241824
F11
187428781
G
A
0.48
0.5
1.16E−08
−0.088
0.016
5.06E−03*
−0.062
0.022
0.9758
rs4253399
F11
187425088
T
G
0.58
0.62
6.22E−08
−0.088
0.017
3.96E−02
−0.047
0.023
0.9628
4
CHR indicates chromosome; SNP, single nucleotide polymorphism; GAIT-1, Genetic Analysis of Idiopathic Thrombophilia 1.
*Significant after Bonferroni correction for multiple testing.
plasma FXI levels associated with the different genotypes are
shown in Table 3.
We then looked at all imputed SNPs in OLIVIA that were
located within ±50 kb of the lead SNP in the KNG1 locus
detected in GAIT-1 (rs710446) (Figure 2A). Among the 90
SNPs located within this region, 9 had P values ≤0.01. The
strongest associations in this locus in OLIVIA were found for
SNPs rs698078 (P=1.55×10−4), rs5030062 (P=1.57×10−4),
rs710446 (P=1.81×10−4), rs5030072 (P=1.88×10−4), and
rs2304456 (P=4.86×10−4). Linkage disequilibrium measures between the strongest associated SNP in the discovery
sample (rs710446) and the strongest associated SNP in the
fine mapping (rs698078) were R2=1 and D′=1, indicating
that the 2 SNPs are in complete allelic association, according to the 1000 Genomes Pilot 1 release. After adjusting for
SNP rs710446, only SNP rs2304456 among the 90 SNPs
analyzed had a P value <0.05 (P=0.003), although being
substantially higher than in the original analysis, indicating
that only 1 SNP is responsible for the association observed
at the KNG1 locus.
When we performed the same analysis for the region
around the F11 locus lead SNP (rs4241824, P=1.16×10−8),
we found that among 110 SNPs, 11 had P<0.01. (Figure
2B). The strongest association in OLIVIA was found for an
SNP located 45.5 kb downstream of rs4241824 (rs6839415;
P=2.35×10−3), which was not linked to the discovery lead
SNP (R2=0.010; D´=0.492). However, conditional analysis
with further adjustment for SNP rs6839415 resulted in a
substantial reduction in the significance level for all associations, including the one for SNP rs4241824 (P value change
from 5.06×10−3 to 2.09×10−2).
Association With Expression Level in Human Liver
Because FXI is primarily synthesized in the liver, we inves
tigated associations between the 2 most robustly associated
SNPs in the KNG1 locus (rs710446 and rs5030062)
and expression levels of KNG1 in a total of 211 human
liver samples. As shown in Figure 3, both SNPs showed
significant associations with KNG1 mRNA expression in the
liver (P values of 0.048 and 0.031, respectively) that were
consistent with the effects observed on plasma levels of FXI
in GAIT-1 and OLIVIA. Furthermore, we found a highly
significant correlation (R2=0.62, P<0.001) between KNG1
mRNA expression and FXI mRNA expression in liver. The
corresponding association between the strongest plasma FXIassociated SNP in the FXI locus (rs4241824) and expression
level of FXI mRNA in the liver was not significant.
Associations With aPTT and Other
Related Phenotypes
To explore the effects of the most robustly FXI-associated
SNPs on thrombosis-related proteins involved in the contact pathway, we also tested the associations between the 5
top SNPs and FXII, histidine-rich glycoprotein (HRG), and
prekallikrein in the GAIT-1 sample. We also tested the effects
on aPTT, as an indicator of effects on coagulation factors
belonging to the intrinsic pathway. Interestingly, all 3 top
SNPs located in the KNG1 locus were also found to be strongly
associated with aPTT (rs710446; P=1.77×10−9, rs5030062;
P=3.35×10−7, and rs4686799; P=1.67×10−7). This association
was substantially attenuated when adjustment was made for
FXI levels (in addition to sex and age), but it did not disappear
Table 3. Ranges of Plasma FXI Levels According to Genotype for the 3 Replicated SNPs
N_GAIT-1
rs710446
rs5030062
rs4241824
Mean_GAIT-1
SD_GAIT-1
N_OLIVIA
Mean_OLIVIA
SD_OLIVIA
CC
80
111.209
22.22307
112
117.82
48.25
CT
192
103.461
20.83135
307
105.62
45.61
TT
115
90.6566
17.62139
228
100.15
41.79
AA
126
91.8909
17.77347
248
99.79
41.22
CA
185
104.323
21.05324
303
106.93
46.36
CC
70
110.807
22.97315
96
117.77
48.45
AA
102
109.522
20.92292
170
112.87
47.67
GA
178
101.343
19.3923
325
103.47
45.07
GG
87
20.63006
152
102.89
41.58
90.9067
FX1 indicates coagulation factor XI; SNP, single nucleotide polymorphism; GAIT-1, Genetic Analysis of Idiopathic Thrombophilia 1.
Figure 2. A, Regional plot showing the fine mapping within the KNG1 locus performed in OLIVIA. Squares represent single nucleotide
polymorphisms (SNPs) plotted by chromosome position. The y axis reflects −log P of the associations between SNPs and coagulation factor XI (FXI) concentration. Color intensity represents the level of linkage disequilibrium between all SNPs and the reference SNP
(rs710446).40 B, Regional plot showing the fine mapping within the F11 locus performed in OLIVIA. Squares represent SNPs plotted by
chromosome position. The y axis reflects −log P of the associations between SNPs and FXI concentration. Color intensity represent the
level of linkage disequilibrium between all SNPs and the reference SNP (rs4241824) (figures created with SNAP software).40
Figure 3. Box-plots (median, 50% and 95% CIs) showing the differences in expression levels of KNG1 and F11 according to genotypes
of the replicated single nucleotide polymorphisms (SNPs). A, Expression level of KNG1 transcript in the liver according to genotype distribution of KNG1 SNP rs710446 (P=0.031). B, Expression level of KNG1 transcript in the liver according to genotype distribution of KNG1
SNP rs5030062 (P=0.048). C, Expression level of F11 transcript in the liver according to genotype distribution of F11 SNP rs4241824
(P=0.145).
completely (rs710446; P=3.33×10−5, rs5030062; P=7.00×10−4,
and rs4686799; P=3.37×10−3). Moreover, the proportion of
variance in aPTT accounted for by these 3 SNPs (after adjustment for age and sex) dropped from nearly 20% to almost
0 (Table 4). However, the 3 top SNPs located in the KNG1
locus were also found to be strongly associated with prekallikrein (rs710446; P=7.15×10−9, rs5030062; P=2.19×10−9,
and rs4686799; P=0.016). In addition, SNP rs4686799 was
also significantly associated with FXII (P=0.032) and HRG
(P=6.09×10−7). The corollary of these findings is that SNPs in
KNG1 affect aPTT mainly by influencing not only the regulation of FXI, but also by regulating other closely related proteins contained in the contact pathway. This effect would be
mediated by an increase in KNG1 levels, as indicated in our
RNA expression analyses.
In comparison, SNPs located in the F11 locus showed
weaker associations with aPTT (rs4253399; P=3.97×10−3, and
rs4241824; P=9.49×10−4) that completely disappeared when
controlling for FXI concentration (Table 4). As for the KNG1
SNPs, the proportion of variance in aPTT accounted for by
FXI SNPs (after adjustment for age and sex) became almost
0 (Table 4) after adjusting for FXI levels. SNPs located in the
F11 locus were not associated with any of the other tested
proteins belonging to the contact pathway.
Discussion
Elevated plasma levels of FXI have been associated with
venous thrombosis4 and ischemic stroke,6,13 which constitute
important, disabling, and potentially fatal diseases with an
incidence of 0.1% to 0.3% per year in whites.26,27 Although
recent studies have identified some common polymorphisms
within the F11 gene showing an association with FXI levels
both in white and black populations,15–17 a hypothesis-free
genome-wide study, which could potentially identify additional genetic loci influencing the plasma FXI level, had yet to
be performed. In this report, we present the first GWAS aimed
at identifying novel genetic factors that contribute to FXI
regulation conducted in European-ancestry Spanish families.
The most robustly associated locus in our discovery analysis is the KNG1 gene. This association was replicated in a second sample of population-based, healthy Swedish individuals,
and genotype-gene expression level association analyses provided evidence that the most robustly FXI-associated SNPs
harbored in KNG1 are related in an allele-specific manner
to KNG1 mRNA expression. Taken together, these findings
suggest that KNG1 is a good candidate gene for the regulation of plasma FXI concentration. Of note, KNG1 encodes
HK, which is known to play an important role in the contact
pathway of coagulation. Almost all FXI and 70% to 90% of
prekallikrein circulate in blood as a complex with HK, HK
enhances FXI activation by FXII,28–30 and severe HK deficiency is usually associated with low FXI levels31 Thus, it is
plausible to conclude that SNPs in KNG1 influence FXI levels by altering levels of HK, which complexes with and stabilizes FXI in plasma. In this regard, the lead SNP in KNG1
(rs710446) causes a nonsynonymous substitution (Ile581Thr)
in HK, which is predicted to be benign according to SIFT
and PolyPhen softwares but which is suggested to be part of
a potential regulatory region (functional-SNP).32 Supporting
this prediction, our analysis of RNA expression in the liver
showed that SNP rs710446 (associated with higher FXI level)
was also associated with higher KNG1 expression.
Interestingly, we also found a significant association between
the lead KNG1 rs710446 SNP and aPTT, an association which
is in agreement with a recent GWAS analysis that identified
this SNP as one of the SNPs that is most strongly associated
with aPTT.32 When we performed the same analysis, except
that we adjusted for FXI levels, the associations between SNPs
located in the KNG1 locus and aPTT, as well as the proportion
of variance explained by these SNPs, were substantially
attenuated. This is in agreement with the interpretation of our
results that SNPs in KNG1 affect aPTT mainly by altering FXI
regulation. However, the remaining association may indicate
that KNG1 also influences other members of the contact
pathway that together alter aPTT. In this regard, we found
highly significant associations of KNG1 SNPs with HRG
and prekallikrein levels in the GAIT-1 cohort. HRG, which
was found to be an important determinant of aPTT,33,34 has
been shown to bind FXIIa with high affinity, a mechanism by
which it may modulate the contact pathway.33 It should also be
emphasized in this context that a highly significant association
has been previously found between aPTT and SNP rs9898 in
the HRG gene,33 an association that could be confirmed in
GAIT-1 (P=6.6×10−4). This SNP was, however, not associated
with FXI level in our sample, indicating that KNG1 SNPs
affect FXI and HRG independently and that these 2 proteins,
probably among others, determine the variation in aPPT in the
population.
1.646
−1.453
0.377
2.200
−2.995
0.176
2.710
−4.810
0.058
4.83E−05
0.001
0.004
0.596
0.13
0.001
0.022
0.004
G
T
rs4253399
4
187425088
rs4241824
4
CHR indicates chromosome; FXI, coagulation factor XI; SNP, single nucleotide polymorphism; aPTT, activated thromboplastin time; FXII, coagulation factor XII; HRG, histidine-rich glycoprotein; VarExpl, variance explained by the
SNP.
1.894
1.675
−1.550
0.318
2.097
−1.077
0.556
2.723
0.327
1.512
rs4686799
187428781
G
A
0.001
0.025
0.001
0.13
0.211
0.009
0.001
≈0
−2.455
4.596
0.016
2.371
12.025
6.09E-07
2.970
6.075
0.032
0.04
0.001
0.023
0.003
0.19
0.001
0.043
1.67E−07
T
C
1.505
3
187933930
9.528
9.083
7.151E−09
2.188E−09
2.118
2.062
−1.994
−3.344
0.116
0.334
2.514
2.595
3.009
3.878
0.104
0.218
0.05
0.07
0.001
0.001
0.024
0.028
3.33E−05
0.001
0.19
0.22
0.001
0.001
0.037
0.042
1.77E−09
3.35E−07
A
T
C
C
187936874
rs5030062
3
187942621
rs710446
3
β
SE
β
CHR
SNP
Position
A1
A2
P
β
SE
VarExpl
P
β
SE
VarExpl
P
β
SE
P
HRG
FXII
aPTT (age, sex, FXI)
aPTT (age, sex)
Table 4. Associations Between the Most Significantly FXI-Associated SNPs in the Discovery Cohort and Other Related Phenotypes of the Contact Pathway
P
Prekallikrein
SE
Accordingly, we hypothesize that KNG1 may play a central
role in the regulation of aPTT (and thereby risk of thrombosis)
by modulating the FXI concentration, as well as the level of
other related proteins of the contact pathway, such as HRG
and prekallikrein. Further mechanistic studies are warranted
to elucidate these mechanisms.
A prolonged aPTT is an indicator of deficiencies in coagulation factors belonging to the intrinsic pathway of coagulation,
and a short aPTT is a stable predictor of risk of thrombosis.35,36 In this regard, it is important to point out that SNP
rs710446 has also recently been found to be associated with
increased risk of venous thrombosis in 2 independent samples
from Marseille (odds ratio=1.20 [1.07–1.34]).37 Our findings,
taken together with other observations in KNG1 knockout
mice,38 suggest that genetic variation in KNG1 may be important in determining both the function of the intrinsic pathway
of coagulation and the risk of thrombosis. Furthermore, the
mechanistic pathway by which KNG1 affects aPTT and risk of
thrombosis may be to a large extent through regulation of FXI
concentration, which has in fact been proposed as a risk factor
for deep vein thrombosis too.
The second genome-wide significant locus in our discovery
analysis was found to be contained in the structural gene for
FXI, with the most robustly associated SNP being rs4241824.
These results were also confirmed in our replication cohort
and are in agreement with previous studies showing that
genetic variants within or in the vicinity of the F11 gene show
suggestive evidence of association with FXI concentration.16
In fact, among the 2 independent SNPs that were found to significantly influence FXI level by Li et al, one is in almost perfect linkage disequilibrium with SNP rs4241824 (R2=0.97).
The second was not genotyped in our discovery analysis but
showed significant association with FXI level in our replication cohort (rs2289252; P=0.025). Interestingly, the second
most robustly associated SNP in the F11 locus in our study
(rs4253399) showed suggestive evidence of association with
aPTT both in our study and in a previous study,33 an effect
that disappeared completely after further adjustment for FXI
concentration. Finally, SNPs located in the F11 locus have
also been associated with VTE both in white and black populations,16,20 providing further evidence that the effect of these
SNPs on the aPTT and VTE phenotypes is mediated through
modulation of FXI concentration.
The importance of FXI in VTE has been supported by work
in animal models (mice, rabbits, and rats), in which targeting
of FXI with inhibitory antibodies and peptidomimetic inhibitors potently reduced thrombus formation. Moreover, inhibition of FXIa in baboons is efficacious in preventing formation
of platelet-rich thrombi in arterial grafts, demonstrating the
role of FXI-dependent thrombin generation, platelet activation, and fibrin formation.5 Given the evidence supporting FXI
as a key player in the pathogenesis of thrombosis in humans,2–6
and the fact that human subjects with severe FXI deficiency
have a reduced incidence of VTE and stroke10,13,14 but nonetheless a mild bleeding phenotype compared with other bleeding
disorders, FXI has recently gained increasing attention as an
anticoagulant drug target that could potentially improve the
ratio of efficacy to bleeding compared with currently available
anticoagulant agents.39,40
One limitation of this study is the restricted sample size of
GAIT-1, which results in a limited power to detect associations with low effect. Thus, it is plausible that other signals
with lower effect sizes have remained undetected; hence, further studies with larger sample sizes are warranted to identify
any additional genetic factors determining FXI concentration.
In summary, we report the first hypothesis-free genomewide genetic analysis of the FXI activity level, which reveals
that KNG1 and F11 are the main genetic regulators of plasma
FXI level. More importantly, we suggest that FXI may be the
main mechanistic pathway by which KNG1 and F11 determine aPTT and risk of venous thrombosis, although other
proteins contained in the contact pathway, such as HRG and
prekallikrein, may also add to the KNG1 effect on aPTT. These
findings may contribute to elucidation of the molecular basis
determining risk of VTE, and more importantly, may help in
understanding the biological regulation of a phenotype that
has proved to have promising therapeutic properties.
Sources of Funding
The GAIT-1 study was supported partially by grants PI-08/0420,
PI-08/0756, PI-11/0184, SAF2008/01859, and RECAVA-RD06/0014.
J.M. Soria was supported by Programa d’Estabilització d’Investigadors
de la Direcció d’Estrategia i Coordinació del Departament de Salut
(Generalitat de Catalunya). The OLIVIA study was supported by
the European Community Sixth Framework Program (LSHMCT- 2007-037273), the Swedish Research Council, the Knut and Alice
Wallenberg Foundation, the Swedish Heart-Lung Foundation, the
Torsten and Ragnar Söderberg Foundation, the Strategic Cardiovascular
Program of Karolinska Institutet and the Stockholm County Council,
the Foundation for Strategic Research, and the Stockholm County
Council. The ASAP study was supported by the Swedish Research
Council (12660), the Swedish Heart-Lung foundation (20090541), the
European Commission (FAD, Health-F2-2008–200647), and a private
donation from Fredrik Lundberg. M. Sabater-Lleal is a recipient of
a Marie Curie Intra European Fellowship within the 7th Framework
Programme of the European Union (PIEF-GA-2009–252361).
None.
Disclosures
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A Genome-Wide Association Study Identifies KNG1 as a Genetic Determinant of Plasma
Factor XI Level and Activated Partial Thromboplastin Time
Maria Sabater-Lleal, Angel Martinez-Perez, Alfonso Buil, Lasse Folkersen, Juan Carlos Souto,
Maria Bruzelius, Montserrat Borrell, Jacob Odeberg, Angela Silveira, Per Eriksson, Laura
Almasy, Anders Hamsten and José Manuel Soria
Arterioscler Thromb Vasc Biol. published online June 14, 2012;
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
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