ORIGINAL ARTICLE
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Hormozgan University of Medical
Sciences
The association of Factor XIII 100 G/T polymorphism and the risk of deep venous
thromboembolism: An in silico and experimental study
Nasrin Bargahi 1, Ahmad Poursadegh Zonouzi 1, Hedye Fardmanesh2, Saeid Ghorbian3, Ali Akbar Poursadegh Zonouzi 2,
1
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2
Department of Genetics and Medical Biotechnology, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
3
Department of Molecular Biology, Ahar Branch, Islamic Azad University, Ahar, Iran
Accepted 28 February 2016; Spring-Summer2016
ABSTRACT
Introduction: Several lines of evidence suggest that the Factor XIII (FXIII) 100 G/T
polymorphism may influence susceptibility to deep venous thromboembolism (DVT). To
explore this hypothesis, we investigated whether this polymorphism is associated with the
predisposition to DVT. We also predict the possible impact of residue substitution at
codons 34 (Val34Leu) in A subunits of the FXIII coagulation factor on the structure and
function of it.
Methods: A total of 693 individuals were included in the current study, 193 patients with
DVT clinical symptoms, and 500 healthy subjects without both personal and family
histories of thromboembolic disorders. Genotyping was carried out using the amplification
refractory mutation system-polymerase chain reaction (ARMS-PCR) technique. To predict
the potentially effects of residue changes at codons 34 on the structure and function of the
FXIII, we used Polymorphism Phenotyping (PolyPhen) online software.
Results: No significant difference in the genotypes distribution of the FXIII 100 G/T gene
polymorphism was detected between DVT patients and healthy controls (P>0.05). Our
results showed that the frequency of the mutated FXIII 100T allele was not statistically
significant different between two group. Using PolyPhen online software, residue
substitution at codons 34 of the FXIII was predicted to be benign.
Conclusion: Our in silico and experimental findings indicated that the FXIII 100 G/T gene
polymorphism is not associated with DVT.
Key words: Deep Venous Thromboembolism, FXIII 100 G/T Polymorphism,
Thrombophilia, PolyPhen
*Corresponding author: Tel: +989148149131
E-mail addres: [email protected] (Ali Akbar Poursadegh Zonouzi)
Molecular Medicine Journal 2016, 2(1), 18-23
The Association of Factor XIII 100 G/T Polymorphism
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Introduction:
Deep venous thromboembolism (DVT) is part
of a group of diseases called venous
thromboembolism (VTE) which is accompanied by
thrombosis formation in the deep venous of the legs
[1]. Despite the incidence of DVT, the etiology of it
is not completely understood [2]. DVT is a
multifactorial disorder with many known inherited
and acquired risk factors. Surgery, trauma, age,
hormone
therapy,
pregnancy,
prolonged
immobilization, plaster cast, and some types of
cancers are main acquired risk factors for DVT
[3,4]. It seems that the role of inherited risk factors
in the pathogenesis of DVT far outweighs the
environmental parameters on the basis of family
and twin studies [5].
Inherited susceptibility to DVT may be the
result of variations in various genes that involved in
natural procoagulant, anticoagulant and fibrinolytic
systems [6]. However, not all these genetic variants
are consistently associated with risk of DVT.
Several studies have investigated that the
association of FXIII 100 G/T (rs5985)
polymorphism with susceptibility to the
thromboembolic events [7- 8]. FXIII is a
protransglutaminase enzyme with tetrameric
structure (A2B2) containing two catalytic A
subunits (FXIII-A) and two non-catalytic B
subunits (FXIII-B) that are held together noncovalently [9]. The FXIII activated by thrombin
and Ca2+ via cleavage the Arg37-Gly38 peptide
bond of FXIII-A subunit. Activated FXIII (FXIIIa)
play a fundamental role in the final stage of
coagulation cascade and stabilizes fibrin clots by
forming fibrin γ-chain dimers that lead to increased
rigidity of the thrombus [9]. FXIIIa also increases
resistance of the fibrin clot to plasmin degradation.
A100 G/T polymorphism in A subunits of the
FXIII, which is characterized by a valine to leucine
substitution at codon 34 (Val34Leu), three amino
Molecular Medicine Journal 2016, 2(1), 18-23
Nasrin Bargahi, et al
acids from the thrombin-cleavage site, has been
shown to change the function of FXIII [9].
Recently, various studies have been conducted
to discover the link between FXIII 100 G/T
polymorphism and DVT, but these studies have
shown conflicting results [7-11]. In the present
study, our aim is to address the question of
“whether FXIII 100 G/T polymorphism can be a
risk factor for DVT or not,” by examining this
polymorphism in DVT patients. Moreover, we
used PolyPhen online software (http://genetics.
bwh.harvard.edu/pph2/index.shtml) to disclose the
effect of amino acid substitution at codons 34 of the
FXIII (Val34Leu) on the functional and structural
properties of it.
Material & Methods:
Preparation of samples
A total of 193 individuals (103 male and 90
female) with DVT clinical symptoms were
recruited as case group, and 500 healthy subjects
(275 male and 225 female) without both personal
and family histories of thromboembolic disorders
were selected as control group, during 2012 to
2014. All case subjects were referred by clinician
after confirmation of the existence of thrombus
using Doppler ultrasonography (ALOKA, SSd1700, Japan). Recognized environmental factors
that may increase the risk of DVT, including
surgery, major trauma, hospitalization, prolonged
immobilization and cancer were excluded from
criteria. None of the healthy control subjects had
any evidence of thrombotic events. The mean and
range of age for the case and control groups were
displayed in Table 1. The present study was
approved by the Ethics and Human Rights
Committee of Tabriz University of medical
sciences, Tabriz, Iran and the informed consents
were filled out by all participants.
19
The Association of Factor XIII 100 G/T Polymorphism
Nasrin Bargahi, et al
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Table 1. The mean and range of age in all study groups
Mean age ± SD (years) a
Range (years)
Case group (n=193)
46.18 ± 4.72
38 - 57
Control group (n=500)
46.27 ± 5.82
36 - 59
Men of case group (n=103)
45.38 ± 4.44
38 - 54
Women of case group (n=90)
47.10 ± 4.89
39 - 57
Men of control group (n=275)
45.79 ± 6.23
36 - 59
Women of control group (n=225)
46.85 ± 5.23
37 - 58
a
Data are means ± SD.
DNA extraction and genotype screening
Peripheral blood samples were taken from all
subjects and total genomic DNA was extracted
from leukocytes using a QIAamp DNA Blood Mini
kit (Qiagen, USA), according to the manufacturer's
instruction.We applied Amplification Refractory
Mutation System-Polymerase Chain Reaction
(ARMS-PCR) for identification of FXIII 100 G/T
polymorphism. ARMS-PCR was performed using
three primers for this polymorphism, one forward
primer and two reverse primers specific forthe wild
type and mutant alleles [12]. The PCR
amplifications were carried out on total volume 25
μl solution containing 100 ng genomic DNA, 1×
PCR buffer, 10 pmol of each primers, 10 nmol each
deoxyribonucleotide triphosphates,1.5mmolMg2+
and 1 U Taq polymerase. PCR conditions were
started with an initial denaturation step (96 °C, 2
min) was followed by 10 cycles of denaturation (96
°C, 15 s) andannealing/extension (65 °C, 60 s),
followed by a final 20 cycles of denaturation (96
°C, 10 s), annealing (61 °C, 50 s), andextension (72
°C, 30 s) [12]. The PCR products were separated on
2 % agarose gel and visualized with ethidium
bromide.
Statistical analysis
Statistical analysis was performed using SPSS
software version 16.0 (SPSS Inc., Chicago, IL,
USA). The difference in genotype distributions of
polymorphism and frequencies of heterozygous,
homozygous and mutated alleles among the case
and control groups was evaluated using Pearson’s
chi-square (χ2 test) and Fisher’s exact tests. The
homozygote and heterozygote genotypes of each
20
group were unified as a new group and then odds
ratios and 95% confidence intervals (CI) were
calculated. P values less than 0.05 were statistically
considered as significant.
In silico analysis of Val34Leu polymorphism
in FXIII gene
Bioinformatics procedures are applicative tools
for prediction the possible impact of a residue
substitution on the structure and function of a
protein. We used polymorphism Phenotyping
(PolyPhen) online software to predict the putative
effects of amino acid substitutions at codon 34 on
the structure and function of FXIII coagulation
factor.
Results:
Factor XIII 100G/T genotype analysis
The genotype distribution of the FXIII 100 G/T
polymorphism in the case and control groups is
displayed in Table 2. Our findings showed that the
FXIII 100 G/T polymorphism were prevalent in
DVT patients compared with the controls, but these
differences was not statistically significant (P>0.05)
(see Table 2).
The heterozygosity and homozygosity
frequencies for FXIII 100 G/T polymorphism in
both groups are given in Table 3. The frequencies
of heterozygous and homozygous genotypes for
FXIII 100 G/T polymorphism was higher but not
significantly different in DVT patients compared
with the control group (P>0.05) (see Table 3).
Molecular Medicine Journal 2016, 2(1), 18-23
The Association of Factor XIII 100 G/T Polymorphism
Nasrin Bargahi, et al
Table 2. The genotype distribution of the FXIII 100 G/T polymorphism in case and control groups
Case group (n=193) %
Control group (n=500) %
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FXIII 100 G/T b
P value a
0.517
GG
72.02
74.60
GT
24.87
23.60
TT
3.11
1.80
Abbreviations: F XIII; Factor XIII.
a
P value calculated by chi-square test
b
FXIII, at nucleotide position 100 on the gene a G was exchanged by a T
Table 3. The heterozygote and homozygote genotypes and mutant allele frequencies of the FXIII 100 G/T
polymorphism in case and control groups
Case group
(n=193)
%
Control group
(n=500)
%
GT
24.87
23.60
0.766
0.933
0.634 - 1.373
TT
3.11
1.80
0.381
0.571
0.201 - 1.627
Frequency of T allele
15.54
13.60
0.346
0.855
0.615 - 1.189
Carrier (GT+ TT) c
27.98
25.40
0.500
0.876
0.603 - 1.273
P value a
OR
95 % CI
FXIII 100 G/T b
Abbreviations: F XIII; Factor XIII, OR; Odds Ratio, CI; Confidence Interval
a
b
c
P value calculated by chi-square test
FXIII, at nucleotide position 100 on the gene a G was exchanged by a T
Carriers: individuals who had either heterozygous or homozygous specified mutation
The frequencies of mutated FXIII 100T allele in
case and control subjects was also calculated (see
Table 3). We found that the prevalence of mutated
FXIII 100T allele was more frequent in the case
subjects but, did not show a statistically significant
difference (P>0.05) (see Table 3).
Finally, we unified the heterozygote and
homozygote genotypes of each group into a new
group (carrier group) and then the odds ratios (OR)
and 95% confidence intervals (CI) were calculated.
Our data showed that the FXIII 100 G/T
polymorphism is not associated with the increased
risk of DVT (see Table 3).
Molecular Medicine Journal 2016, 2(1), 18-23
Prediction of Functional Effect of Factor
XIII Val34Leu polymorphism
The substitution of valine by leucine at codon
34 was predicted to be benign by PolyPhen online
software (see Figure 1).
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The Association of Factor XIII 100 G/T Polymorphism
Figure 1. In silico analysis of the Factor XIII
100 G/T polymorphism using PolyPhen. The
substitution of leucine a valine for a valine at
codon 34 in FXIII coagulation factor was
predicted to be benign.
Conclusion:
As indicated earlier, increasing evidences
suggest that the procoagulant, anticoagulant and
fibrinolytic systems deficiencies play a
fundamental role in susceptibility to
thromboembolic disorders [6]. To our
knowledge, here, the possible association
between FXIII 100 G/T polymorphism with
DVT in Northwestern Iran was investigated in the
current study for the first time. Our results
revealed that the prevalence of the FXIII 100 G/T
polymorphism was not statically significant
different between case and control subjects in our
population (P>0.05).
However, a high prevalence of this
polymorphism in the patient group was detected.
We also found that the frequency of mutated
FXIII 100T allele was higher in the DVT patients
than in healthy controls, but this difference was
not statistically significant (P>0.05). Therefore, it
seems that the presence of the FXIII 100T allele
is not associated with DVT, based upon our
experimental results. Our results are in
accordance with the findings of several previous
studies that have been published in this field.
Corral et al, reported that Factor XIII 100T allele
does not has any effect on the risk of venous
thrombosis in the Spanish population [10]. Le Gal
et al, studied 286 patients with idiopathic VTE
and 286 healthy controls for FXIII 100 G/T
polymorphism and reported that FXIII 100T
allele was significantly associated with a lower
risk of VTE [11]. Similar data were obtained in a
22
Nasrin Bargahi, et al
study by Renner et al, who investigated FXIII
100 G/T polymorphism in 154 DVT patients and
308 healthy controls in Austrian population and
reported that presence of the FXIII 100T allele is
associated with a decreased risk for DVT [13].
Moreover, Van Hylckama Vlieg et al,
demonstrated that FXIII 100T allele was
associated with a slightly decreased thrombotic
risk [14]. In light of these studies and metaanalysis studies by Wells et al, and Gohil et al,
provided additional evidence to support the
hypothesis that the FXIII 100 G/T polymorphism
has a significant protective effect against VTE
[15, 16]. It has been proposed that the Leu34
FXIII is activated by thrombin more rapidly than
their Val34 variant [17]. Although, the presence
of FXIII Leu34 allele increases the catalytic
activity of FXIII but it decreases clot stability and
resistance to fibrinolysis through alteration of clot
structure [16]. A study by Ariens et al, suggested
that fibrin clots formed in the presence of Leu34
FXIII allele have finer structures with thinner
fibers, smaller pores, and altered permeation
characteristics when compared with fibrin clots
formed in the presence of the Val34 variant [17,
18]. The findings of these studies have suggested
that thinner and less porous fibrin clots relatively
resistant to plasmin degradation [17,18]. Hence, it
is conceivable that the FXIII 100 G/T
polymorphism is associated with pathogenesis of
VTE. Therefore, the role of FXIII 100 G/T
polymorphism in pathogenesis of DVT remained
controversial.
In conclusion, our in silico and experimental
results revealed that the FXIII 100 G/T
polymorphism is not associated with DVT.
However, in spite of the negative association
reported in the current study, the possible role of
FXIII 100 G/T polymorphism in DVT
pathogenesis could not be completely ruled out,
and further studies will be needed to conclusively
find association between this polymorphism and
DVT.
Acknowlegment:
The authors would like to express our sincerest
appreciation to all the subjects for participating in
this study.
Molecular Medicine Journal 2016, 2(1),18-23
The Association of Factor XIII 100 G/T Polymorphism
Declaration of conflicting interests
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There are no potential conflicts of interest for
each author, concerning the submitted manuscript.
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