Gene 564 (2015) 2934

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Gene

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The association of PLA2G2A single nucleotide polymorphisms with type

IIa secretory phospholipase A2 level but not its activity in patients
with stable coronary heart disease
Yulia A. Shuvalova a,, Zukhra B. Khasanova b, Violetta I. Kaminnaya a, Elena V. Samoilova c,
Alexandra A. Korotaeva c, Alexander V. Rubanovich d, Alexander I. Kaminnyi a
a
Russian Cardiology Research and Production Complex, Department of Atherosclerosis Problems, 3rd Cherepkovskaya str, 15a, Moscow 121552, Russia
b
Russian Cardiology Research and Production Complex, Laboratory of Medical Genetics, 3rd Cherepkovskaya str, 15a, Moscow 121552, Russia
c
Russian Cardiology Research and Production Complex, Laboratory of Biochemical Engineering, 3rd Cherepkovskaya str, 15a, Moscow 121552, Russia
d
Institute of General Genetics named Vavilov N. I., Department of Genetic Safety, Laboratory of Ecological Genetics, Gubkina str, 3, Moscow 117971, Russia

a r t i c l e i n f o a b s t r a c t

Article history: Background: Single nucleotide polymorphisms (SNPs) of the secretory phospholipase A2 type IIa (sPLA-IIa) gene
Received 27 October 2014 (PLA2G2A) affect sPLA2-IIa level and activity in patients with diabetes mellitus, acute coronary syndrome or re-
Received in revised form 5 March 2015 cent cardiovascular surgical interventions. Our study examined the effects of PLA2G2A SNPs on sPLA2-IIa levels
Accepted 16 March 2015 and activity in patients with stable CHD.
Available online 17 March 2015
Methods and results: The study included a total of 396 patients (30% women). Six SNPs of PLA2G2A: rs1774131,
rs11573156, rs3753827, rs2236771, rs876018, and rs3767221, sPLA2-IIa level and activity were determined
Keywords:
sPLA2-IIA level
for all patients. Four SNPs (rs1774131, rs11573156, rs3753827, rs3767221) correlated with sPLA2-IIa level but
sPLA2-IIA activity not activity with the strongest correlation observed for rs11573156 (r = 0.49, p = 3.7 1013). All partial
Polymorphisms of PLA2G2A correlations controlling for rs11573156 became insignicant, whereas, the partial correlation of rs11573156
Stable CHD with sPLA2-IIa level controlling for other SNPs remained signicant. Only rs11573156 showed association with
sPLA2-IIa level in multiple regression analysis. Haplotype CGGGTT was associated with a signicantly higher
sPLA2-IIa level but not activity compared with all other haplotypes after adjustment for gender, age, diabetes
mellitus and statin use (p = 0.0023).
Conclusions: According to our results the examined SNPs affect the sPLA2-IIa level to a greater extent than its
activity in patients with stable CHD. It seems that, the impact of these SNPs on sPLA2-IIa level is caused by
their linkage to rs11573156 whose minor alleles were associated with higher sPLA2-IIa level. At the same time
haplotype CGGGTT, which includes the minor allele of rs11573156, was the dominant haplotype and was asso-
ciated with the highest sPLA2-IIa level.
2015 Elsevier B.V. All rights reserved.

1. Introduction in the atherosclerotic plaques suggests its participation in atherogenesis

Secretory phospholipase A2 type IIa (sPLA2-IIa) is an enzyme capa-
ble of hydrolyzing bonds of phospholipid cell membranes, generating
lysophospholipids and free fatty acids. sPLA2-IIa takes part in the forma-
tion of eicosanoids, thus it has a signicant role in the process of inam-
mation and thrombosis (Dennis, 1994). Also, the presence of sPLA2-IIa
Abbreviations: A, adenine; ACS, acute coronary syndrome; C, cytosine; CHD, coronary
heart disease; DM, diabetes mellitus; DNA, deoxyribonucleic acid; EDTA, ethylenediamine-
tetraacetic acid; G, guanine; PLA2G2A, secretory phospholipase A2 type IIa gene; RT-PCR,
Real-Time polymerase chain reaction; SNP, Single nucleotide polymorphism; sPLA-IIa, se-
cretory phospholipase A2 type IIa; T, thymine.
Corresponding author at: Department of Atherosclerosis Problems, Russian
Cardiology Research and Production Complex, 3rd Cherepkovskaya Street, 15a, Moscow
121552, Russia.
E-mail address: shuvalovaj@mail.ru (Y.A. Shuvalova).
(Elinder et al., 1997; Hurt-Camejo et al., 1997).
In large clinical trials sPLA2-IIa was identied as a marker of coro-
nary heart disease (CHD) (Lind et al., 2012), the increase in its level
and activity was associated with an increase in the risk of CHD in
healthy individuals (Boekholdt et al., 2005; Mallat et al., 2007) and an
increase in the risk of major adverse cardiovascular events in patients
with unstable angina pectoris (Kugiyama et al., 2000), severe acute cor-
onary syndrome (ACS) (Mallat et al., 2005; Simon et al., 2008), undergo-
ing percutaneous coronary intervention (Liu et al., 2003) and coronary
artery bypass grafting (Koenig et al., 2009).
In other clinical trials, it was shown that single nucleotide polymor-
phisms (SNPs) of secretory phospholipase A2 type IIa gene (PLA2G2A)
signicantly affect sPLA2-IIa level and activity (Mallat et al., 2010).
However, it is worth noting that these studies included subjects with
the following factors that may inuence the level and activity of sPLA2-

http://dx.doi.org/10.1016/j.gene.2015.03.030
0378-1119/ 2015 Elsevier B.V. All rights reserved.
30 Y.A. Shuvalova et al. / Gene 564 (2015) 2934

Table 1
The frequency of the studied PLA2G2A gene SNP genotypes and alleles.

rs1774131 rs11573156 rs3753827 rs2236771 rs876018 rs3767221

Homozygotes for the major allele 0.37 0.53 0.39 0.77 0.73 0.44
Heterozygotes 0.46 0.41 0.46 0.17 0.24 0.42
Homozygotes for the minor allele 0.18 0.06 0.16 0.07 0.04 0.14
Major allele 0.59 0.73 0.61 0.85 0.85 0.65
Minor allele 0.41 0.27 0.39 0.15 0.15 0.35
Deviation from HW equilibrium (pHW) 0.46 0.58 0.65 0.0001 0.27 0.34

IIa: type 2 diabetes mellitus (DM) (Wootton et al., 2006), ACS or recent
cardiovascular surgical interventions (Breitling et al., 2011; Simon et al.,
2009). Thus, in our study we tried to avoid these limitations and studied
the inuence of the same previously identied SNPs affecting the level
and activity of sPLA2-IIa in patients with stable CHD.
2. Materials and methods
2.1. Population
The study included male and female patients with stable CHD docu-
mented by the results of exercise tests. Patients who had suffered ACS
or undergone surgical intervention less than 6 months before the begin-
ning of the study, patients with inammatory diseases (in severe chronic
or acute state at the time of inclusion), patients with oncological diseases
and patients with familial hypercholesterolemia were excluded from the
study. All patients signed written informed consent for genetic research.
2.5 U; 10-fold buffer of Taq-polymerase 2.5 ml; DNA solution
(10 ng/l) 5 l and 18.2 mQ water to 25 l. The temperature prole
of the reaction included denaturation (95 C 20 s), 50 cycles of amplica-
tion (95 C 15 s, then 55 C 30 s), primer annealing, and elongation (30 s;
59 C for rs1774131, rs3753827, rs3767221; 57 C for rs11573156,
rs876018; 55 C for rs2236771). In the case of major allele presence
only (homozygous genotype for the major allele), growth curves of
amplication were observed only in the VIC channel. In the case of
minor allele presence only (homozygous genotype for the minor al-
lele), growth curves of amplication were observed only in the ROX
channel. Visualization of the amplication curves in both channels
VIC/ROX indicated the existence of both major and minor alleles
and their corresponding heterozygous genotype. In order to ensure
quality control, 10% of the samples were blind duplicates by RT-PCR
method for every SNP. Cohen's kappa coefcients of agreement were
computed to evaluate the inter-methodology variability in genotyping.
Kappa coefcient for different SNPs ranged from 0.93 to 1.

2.2. Genetic methods

Venous blood was collected with EDTA as an anticoagulant and
stored at 70 C until analysis. Genomic DNA was isolated from
whole blood using the DNA-EXTRAN-1 reagent kit (Synthol, Russia).
DNA concentration was measured using a nanophotometer IMPLEN
(GmbH, Germany), and adjusted to a concentration of 10 ng/l. Determi-
nation of PLA2G2A SNPs was performed by Real-Time polymerase chain
reaction (RT-PCR) using a 7500 Fast Real-Time PCR System amplier (Ap-
plied Biosystems, USA). SNPs of PLA2G2A (dbSNP-polymorphism reposi-
tory: http://www.ncbi.nlm.nih.gov/SNP/): rs1774131, rs11573156,
rs3753827, rs2236771, rs876018 and rs3767221 were determined.
Primers and probes were synthesized by a company Synthol, Russia.
The primer and probe sequences used for amplication are shown in on-
line Supplemental materials.
In addition to primers (10 pmol) and probes (5 pmol) the reac-
tion mixture contained 200 M of each dNTP; Taq-polymerase
2.3. Biochemical methods
Plasma level of sPLA2-IIa was determined with the use of monoclo-
nal antibodies sPLA2-IIa human synovial enzyme immunoassay kit
(Cayman Chemical Company, Ann Arbor, MI, USA). This antibody is
specic for sPLA2-IIa and does not cross react with types I, IV, V, VI
and X sPLA2. The limit of detection was 15.6 pg/ml. The results obtained
are presented in ng/ml.
sPLA2-IIa activity was determined with uorescence-labeled phos-
phatidyl choline in a VICTOR X3 Multilabel PlateReader (Perkin Elmer,
Finland). The reader settings corresponded to those for uorescence
(excitation 485 nm, emission 535 nm). Measurements were carried
out in a 96-well plate. The nal volume in a well was 200 l. The reaction
mixture contained 20 l serum, 0.9 M uorescence-labeled substrate,

Fig. 1. Linkage disequilibrium within the PLA2G2A. D the deviation between the expect-
ed haplotype frequency (under the assumption of no association) and the observed fre- Fig. 2. SNP genotypes of PLA2G2A and sPLA2-IIa level adjusted for gender, age, type 2 DM
quency; r2 Pearson's correlation coefcient between alleles. and statin use. Multiple linear regression. Data are presented as Mean SD.
 Y.A. Shuvalova et al. / Gene 564 (2015) 2934 31

Table 2
Correlation analysis between SNPs of PLA2G2A and sPLA2-IIa level.

rs11573156 rs1774131 rs3753827 rs2236771 rs876018 rs3767221

Correlation (p-value) 0.488 0.333 0.195 0.014 0.059 0.245

(3.7 1013) (1.7 106) (0.006) (0.841) (0.408) (0.001)
Partial correlation controlling for 763C/G (p-value) 0.082 0.087 0.010 0.035 0.088
(0.255) (0.231) (0.888) (0.632) (0.225)

Table 3
Multiple linear regression analysis of sPLA2-IIa level dependence on SNPs of PLA2G2A.

Constant rs1774131 rs11573156 rs3753827 rs2236771 rs876018 rs3767221

B coefcient 1.19 2.09 0.47 0.39 2.47 0.52 0.10 0.43 0.95 0.47 0.11 0.55 0.6 0.45
Betaa 0.092 0.434 0.019 0.144 0.016 0.116
p-Value 0.569 0.234 3.4 106 0.815 0.046 0.834 0.185
a
Standardized coefcient of linear regression.

100 mM TrisHCl pH 8.0, and 2 mM CaCl2. sPLA2-IIa isolated from human
heart myxoma (specic activity 36 mol/min/m) was used as a standard.
sPLA2-IIa activity in blood samples is presented in nmol/ml/min.
2.4. Statistical analysis
Statistical data analysis was carried out using the STATISTICA 6.0
software and SNPStats (http://bioinfo.iconcologia.net/SNPstats). Results
were presented as mean and standard deviation (Mean SD). Cor-
relation analysis was performed by paired and partial correlations. The
SNPStats is statistical web-based tool designed from genetic epidemiol-
ogy point of view for the analysis of SNP association studies (Sole et al.,
2006). We use this software to calculate the following parameters: fre-
quencies of alleles and genotypes, test for HardyWeinberg equilibrium
(2 criterion), associations between SNPs and the response adjusted
for gender, age, type 2 DM and statin use (multiple linear regression),
inheritance models (likelihood ratio test), linkage disequilibrium
(D statistic, Pearson's r2 and associated p-values), haplotype frequency
and associations of haplotypes with the response adjusted for gender,
age, type 2 DM and statin use (multiple linear regression). We have
used False Discovery Rate (FDR) control to correct -level (Benjamini
and Hochberg, 1995) and the differences were considered to be signi-
cant at p b 0.01.
3. Results
The study included 396 patients: 276 men (70%) and 120 women
(30%). The average age of patients was 61.7 9.6 years, 62 patients
(15.7%) had 2 type diabetes mellitus, and 362 patients (93.9%) were
on statin therapy. sPLA2-IIa level and activity and six SNPs of
PLA2G2A: rs1774131, rs11573156, rs3753827, rs2236771, rs876018
and rs3767221, which constitute 92% of the genetic variability
(Wootton et al., 2006), were determined for all patients. Except for
rs2236771, all PLA2G2A SNPs showed proles in accordance with
HardyWeinberg equilibrium. The frequency of the studied SNP ge-
notypes and alleles is presented in Table 1. All SNPs had a high de-
gree of linkage disequilibrium within the PLA2G2A (Fig. 1).
Analyzing the association of these SNPs with sPLA2-IIa levels and
activity with adjustment for factors that traditionally affect sPLA2-IIa
level (gender (Breitling et al., 2011), age (Boekholdt et al., 2005), type
2 DM (Rosenson et al., 2011) and statin use since statints have anti-
inammatory effect), we found that homozygotes for the minor allele
of rs1774131 had a signicantly higher sPLA2-IIa level compared with
heterozygotes and homozygotes for the major allele of the same SNP
(8.55 4.25; 6.59 3.35 and 5.06 3.20 ng/ml, p b 0.0001) (Fig. 2). It
was also noted, that homozygotes for the minor allele of rs11573156
had a signicantly higher serum sPLA2-IIa level compared with heterozy-
gotes and homozygotes for the major allele of that SNP (10.23 4.80;
7.89 3.11 and 4.77 3.09 ng/ml, p b 0.0001). At the same time, our re-
sults showed that homozygotes for the minor allele of rs3767221 had
lower sPLA2-IIa levels compared with other genotype variants of the
same SNP (4.6 3.10; 5.95 3.54 and 7.17 3.55 ng/ml, p = 0.001).
There were no signicant differences in sPLA2-IIa levels in patients with
different genotypes of rs3753827 (5.17 3.07, 6.12 3.45 and 7.18
4.0, p = 0.027), rs2236771 (7.6 5.34, 6.07 3.07 and 6.77 3.64,

Table 4
Inheritance models for SNPs of PLA2G2A.

SNPs Dominant Recessive Additive

p value AIC p value AIC p value AIC

rs1774131 b0.0001 1061.2 b0.0001 1061 b0.0001 1053.4

rs11573156 b0.0001 1028.3 b0.0001 1062 b0.0001 1023.2
rs3753827 0.015 1070.6 0.045 1072.6 0.006 1069
rs2236771 0.79 682.7 0.45 682.2 0.88 682.8
rs876018 0.51 1076.2 0.43 1076 0.41 1075.9
rs3767221 0.003 1041.3 0.008 1043.1 b0.0001 1038.1

AIC Akaike Information Criterion is used to choose the inheritance model that best ts
Fig. 3. SNP genotypes of PLA2G2A and sPLA2-IIa activity adjusted for gender, age, type 2 the data. The model with the less AIC corresponds to minimize the expected entropy,
DM and statin use. Multiple linear regression. Data are presented as Mean SD. and the best inheritance model.
32 Y.A. Shuvalova et al. / Gene 564 (2015) 2934

Table 5
Multiple linear regression. SNP haplotypes of PLA2G2A and their association with sPLA2-IIa level.

rs1774131 rs11573156 rs3753827 rs2236771 rs876018 rs3767221 Frequency sPLA2-IIa level (ng/ml) p-Value p-Value adja
Difference in means (95% CI)

1 C G G G T T 0.2054 8.36
2 T C G G T G 0.127 3.68 (4.551.97) b0.0001 b0.0001
3 T C T G A T 0.1218 2.5 (3.961.58) b0.0001 b0.0001
4 T C T G T T 0.1152 3.12 (4.512.19) b0.0001 b0.0001
5 T C T G T G 0.0509 2.34 (4.131.33) 0.0087 0.0017
a a a a a a
Rare 0.3797 2.09 (3.521.33) b0.0001 b0.0001

Common p-value for the association of haplotypes with sPLA2-IIa level: 0.0016.
Common p-value for the association of haplotypes with sPLA2-IIa level after adjustment: 0.0023.
a
Adjusted for gender, age, type 2 DM and statin use.

p = 0.64) and rs876018 (5.29 4.4, 6.22 2.58 and 6.49 3.94,
p = 0.65).
Four SNPs of PLA2G2A (rs1774131, rs11573156, rs3753827,
rs3767221) signicantly correlated with sPLA2-IIa level with the
strongest correlation observed for rs11573156 (Table 2). All partial
correlations controlling for rs11573156 were statistically insignicant,
whereas, the partial correlation rs11573156 with sPLA2-IIa level control-
ling for remaining SNPs was 0.331 (p = 3.4 106). In multiple regres-
sion analysis only rs11573156 showed signicant association with
sPLA2-IIa level (Table 3).
Mean sPLA2-IIa activity in relation to different genotype of each SNP
is represented in Fig. 3. No signicant differences in the results of these
variables were observed.
We examined different inheritance models for studied SNPs of
PLA2G2A, and the best inheritance model for all SNPs, except rs2236771,
was found to be the additive inheritance model (Table 4).
We have determined and analyzed 5 haplotypes, which occurred
with a frequency of more than 5% during our sampling of patients. Hap-
lotype CGGGTT was the most common haplotype identied in our
group of patients with an incidence of 20.5%, and it was associated
with a signicantly higher sPLA2-IIa level compared with all other haplo-
types (8.36 ng/ml) (p = 0.0016). After adjustment for gender, age, type 2
DM and statin use, the association of haplotype CGGGTT with sPLA2-IIa
level remained statistically signicant (p = 0.0023) (Table 5). There
was no signicant association between sPLA2-IIa activity and any of the
identied haplotypes (Table 6).
4. Discussion
We analyzed the association between six SNPs of PLA2G2A, as well as
their haplotypes, and sPLA2-IIa level and activity in patients with stable
CHD. All SNPs of PLA2G2A showed distribution proles in accordance
with HardyWeinberg equilibrium except for SNP rs2236771 which
showed a sharp deviation caused by an excess in homozygotes for the
minor allele (Table 1). Similar non-signicant tendencies were observed
for the other studied SNPs. World-wide HapMap (http://hapmap.ncbi.
nlm.nih.gov/) for European populations (CEU) offers the following geno-
type frequencies of rs2236771: 0.783, 0.183, and 0.033 at pHW = 0.251. In
our study, the frequency of homozygotes for the minor allele was signi-
cantly higher (0.07). Perhaps this indicates that minor allele carriers of
PLA2G2A SNPs have an increased predisposition for CHD.
In our study it was shown that carriage of the minor allele of differ-
ent SNPs might be associated with either an increase or a decrease in
sPLA2-IIa level (Fig. 2). For example, we have found that minor allele
homozygotes of rs1774131 were associated with signicantly higher
sPLA2-IIa levels compared to heterozygotes and major allele homozy-
gotes of the same SNP. These results are consistent with those obtained
in the UDACS study (Wootton et al., 2006), in which it was shown that
minor allele homozygotes of rs1774131 were associated with sig-
nicantly higher levels sPLA2-IIa levels compared with major allele
homozygotes (p b 0.0001). In our study minor allele homozygotes of
rs11573156 was associated with signicantly higher sPLA2-IIa levels
compared with heterozygous and major allele homozygous of the
same SNP, as it was reported in the UDACS and the KAROLA studies
(Wootton et al., 2006; Breitling et al., 2011). Holmes M.V. et al. have
showed an allele dose-dependent association between rs11573156
and sPLA2-IIa level as in our work (Rosenson et al., 2011). Moreover,
in the KAROLA trial and in our study the rs11573156 was the most
strongly associated with the sPLA2-IIa level. Our study shows that
minor allele homozygotes of rs3767221 were associated with a lower
sPLA2-IIa level compared with other genotype variants of these SNPs.
This is similar to reports of other studies in which minor allele homozy-
gotes of rs3767221 (Wootton et al., 2006; Breitling et al., 2011) were as-
sociated with signicantly lower sPLA2-IIa levels compared with major
allele homozygotes. As it was reported in the UDACS study, patients
with different genotypes of rs2236771 and rs876018 showed no sig-
nicant difference in sPLA2-IIa levels. In our study various genotype
carriers of rs3753827 had no signicant difference in sPLA2-IIa
levels (p = 0.027) as we determined the differences to be signicant
at p b 0.01.

Table 6
Multiple linear regression. SNPs haplotypes of PLA2G2A and their association with sPLA2-IIa activity.

rs1774131 rs11573156 rs3753827 rs2236771 rs876018 rs3767221 Frequency sPLA2-IIa activity (nmol/ml/min) p-Value p-Value adja
Difference in means (95% CI)

1 C G G G T T 0.2054 1.48
2 T C G G T G 0.127 0.3 (0.040.56) 0.092 0.16
3 T C T G A T 0.1218 0.07 (0.20.46) 0.69 0.99
4 T C T G T T 0.1152 0.06 (0.170.51) 0.75 0.93
5 T C T G T G 0.0509 0.13 (0.580.15) 0.59 0.67
a a a a a a
Rare 0.3797 0.14 (0.030.89) 0.29 0.38

Common p-value for the association of haplotypes with sPLA2-IIa level: 0.29.
Common p-value for the association of haplotypes with sPLA2-IIa level after adjustment: 0.38.
a
Adjusted for gender, age, type 2 DM and statin use.
 Y.A. Shuvalova et al. / Gene 564 (2015) 2934
A correlation between four SNPs and sPLA2-IIa level has been
observed, with the strongest being with rs11573156, where the correla-
tion coefcient was r = 0.49 when p = 3.7 1013 and this means that
24% of the variance in sPLA2-IIa level was controlled by the genetic
variation in rs11573156. Apparently, the correlation of other SNPs
with sPLA2-IIa level can be caused by their linkage to the rs11573156.
It is evidenced by the fact that all the partial correlations controlling
for rs11573156 became statistically insignicant (Table 2). On the
contrary, partial correlation of rs11573156 with sPLA2-IIa level con-
trolling for remaining SNPs was 0.331 (p = 3.4 10 6), which is
slightly less than that obtained in paired correlation. A similar con-
clusion may be made by analyzing the results of the multiple regres-
sion model sPLA2-IIa level dependence from SNPs of PLA2G2A,
showing only the rs11573156 to be associated with sPLA2-IIa level
(Table 3).
In our study, as well as in the KAROLA (Breitling et al., 2011) and
FAST-MI trials (Simon et al., 2009), we evaluated the association be-
tween genotypes of these SNPs and sPLA2-IIa activity. In our study
there were no signicant differences in this variable in carriers of differ-
ent genotypes of each SNP (Fig. 3). Our results do not agree with the re-
sults of previous studies, which showing the carrying of the minor allele
of rs11573156 was associated with an increase in sPLA2-IIa activity,
whereas, carrying of the minor alleles of rs3753827 and rs3767221
was associated with decrease in its activity. We believe that this is prob-
ably due to the facts that in the FAST-MI study patients with ACS were
included, and patients included in the KAROLA study could hardly
be considered as being stable, because sPLA2-IIa level and activity
were measured in an average of 43 days after myocardial infarction or
revascularization, whereas in our study only patients with stable CHD
were included. This assumption is conrmed by the results of Holmes'
Mendelian study (Holmes et al., 2013): the effect of rs11573156 allele
on sPLA2-IIa activity was smaller in studies of general population than
in studies of ACS patients.
Upon analyzing various models of inheritance of the studied SNPs,
we found that the best model of inheritance for all SNPs of PLA2G2A, ex-
cept for rs2236771, was the additive model (Table 4). Additive model of
inheritance means that each minor allele modies risk in an additive
form, that is, when compared with major allele homozygotes, minor allele
homozygotes have a two times higher risk than heterozygotes.
We identied ve haplotypes of studied SNPs that occurred at a fre-
quency more than 5%, whereas, all others were included into the group
of rare haplotypes. The haplotype CGGGTT, that only one includes the
minor allele of rs11573156, was found to be the most common in our
patient population with an incidence of 20.5% and it was signicantly
associated with the highest sPLA2-IIa level compared to all other haplo-
types. After adjustment for factors that traditionally affect sPLA2-IIa
level (gender, age, type 2 DM, statin use) the association of haplotype
CGGGTT with sPLA2-IIa level remained statistically signicant (Table 5).
This nding shows that rs11573156 has impact on the sPLA2-IIa level,
while the effect of the other SNPs is due to their linkage with
rs11573156. Analysis of the haplotypes was also carried out in three pre-
vious studies (Wootton et al., 2006; Breitling et al., 2011; Simon et al.,
2009), and other haplotypes were the most common in these studies.
We believe that this is due to differences in patient populations.
5. Conclusion
According to our results, apparently, the studied SNPs had a great
impact on sPLA2-IIa level and lesser extent on its activity in patients
with stable CHD. It seems that, the impact of the studied SNPs on
sPLA2-IIa level was related to their linkage to rs11573156 whose
minor allele was associated with higher level of sPLA2-IIa. At the
same time haplotype CGGGTT, which includes the minor allele of
rs11573156, was the dominant haplotype and was associated with
the highest sPLA2-IIa level.
33
Conict of interest
None.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.gene.2015.03.030.
References
Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and
powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289300 (DOI: http://
www.jstor.org/stable/2346101).
Boekholdt, S.M., Keller, T.T., Wareham, N.J., Luben, R., Bingham, S.A., Day, N.E., Sandhu,
M.S., Jukema, J.W., Kastelein, J.J.P., Hack, C.E., Khaw, K.-T., 2005. Serum levels of type
II secretory phospholipase A2 and the risk of future coronary artery disease in appar-
ently healthy men and women: the EPIC-Norfolk Prospective Population Study.
Arterioscler. Thromb. Vasc. Biol. 25, 839846. http://dx.doi.org/10.1161/01.ATV.
0000157933.19424.
Breitling, L.P., Koenig, W., Fischer, M., Hengstenberg, C., Rothenbacher, D., Brenner, H.,
2011. Type II secretory phospholipase A2 and prognosis in patients with stable coro-
nary heart disease: Mendelian randomization study. PLoS One 6 (7), e22318. http://
dx.doi.org/10.1371/journal.pone.0022318.
Dennis, E.A., 1994. Diversity of group types, regulation, and function of phospholipase A2.
J. Biol. Chem. 269, 1305713060.
Elinder, L.S., Dumitrescu, A., Larsson, P., Hedin, U., Frostegard, J., Claesson, H.E., 1997.
Expression of phospholipase A2 isoforms in human normal and atherosclerotic arte-
rial wall. Arterioscler. Thromb. Vasc. Biol. 17, 22572263. http://dx.doi.org/10.1161/
01.ATV.17.10.2257.
Holmes, M.V., Simon, T., Exeter, H.J., Folkersen, L., Asselbergs, F.W., et al., 2013. Secre-
tory phospholipase A(2)-IIA and cardiovascular disease: a mendelian randomi-
zation study. J. Am. Coll. Cardiol. 62, 19661976. http://dx.doi.org/10.1016/j.
jacc.2013.06.044.
Hurt-Camejo, E., Andersen, S., Standal, R., Rosengren, B., Sartipy, P., Stadberg, E., Johansen,
B., 1997. Localization of nonpancreatic secretory phospholipase A2 in normal and
atherosclerotic arteries. Activity of the isolated enzyme on low-density lipoproteins.
Arterioscler. Thromb. Vasc. Biol. 17, 300309. http://dx.doi.org/10.1161/01.ATV.17.
2.300.
Koenig, W., Vossen, C.Y., Mallat, Z., Brenner, H., Benessiano, J., Rothenbacher, D.,
2009. Association between type II secretory phospholipase A2 plasma con-
centrations and activity and cardiovascular events in patients with coronary
heart disease. Eur. Heart J. 30, 27422748. http://dx.doi.org/10.1093/eurheartj/
ehp302.
Kugiyama, K., Ota, Y., Sugiyama, S., Kawano, H., Doi, H., Soejima, H., Miyamoto, S.,
Ogawa, H., Takazoe, K., 2000. Prognostic value of plasma levels of secretory
type II phospholipase A2 in patients with unstable angina pectoris. Am.
J. Cardiol. 86, 718722. http://dx.doi.org/10.1016/S0002-9149(00)01069-9.
Lind, L., Simon, T., Johansson, L., Kotti, S., Hansen, T., Machecourt, J., Ninio, E., Tedgui, A.,
Danchin, N., Ahlstrm, H., Mallat, Z., 2012. Circulating levels of secretory- and
lipoprotein-associated phospholipase A2 activities: relation to atherosclerotic
plaques and future all-cause mortality. Eur. Heart J. 33, 29462954. http://dx.doi.
org/10.1093/eurheartj/ehs132.
Liu, P.Y., Li, Y.H., Tsai, W.C., Chao, T.H., Tsai, L.M., Wu, H.L., Chen, J.H., 2003. Prognostic
value and the changes of plasma levels of secretory type II phospholipase A2
in patients with coronary artery disease undergoing percutaneous coronary
intervention. Eur. Heart J. 24, 18241832. http://dx.doi.org/10.1016/j.ehj.2003.
07.003.
Mallat, Z., Steg, P.G., Benessiano, J., Tanguy, M.L., Fox, K.A., Collet, J.P., Dabbous, O.H.,
Henry, P., Carruthers, K.F., Dauphin, A., Arguelles, C.S., Masliah, J., Hugel, B.,
Montalescot, G., Freyssinet, J.M., Asselain, B., Tedgui, A., 2005. Circulating secretory
phospholipase A2 activity predicts recurrent events in patients with severe acute cor-
onary syndromes. J. Am. Coll. Cardiol. 46, 12491257. http://dx.doi.org/10.1016/j.jacc.
2005.06.056.
Mallat, Z., Benessiano, J., Simon, T., Ederhy, S., Sebella-Arguelles, C., Cohen, A., Huart, V.,
Wareham, N.J., Luben, R., Khaw, K.-T., Tedgui, A., Boekholdt, S.M., 2007. Circulating se-
cretory phospholipase A2 activity and risk of incident coronary events in healthy men
and women: the EPIC-NORFOLK study. Arterioscler. Thromb. Vasc. Biol. 27, 11771183.
http://dx.doi.org/10.1161/ATVBAHA.107.139352.
Mallat, Z., Lambeau, G., Tedgui, A., 2010. Lipoprotein-associated and secreted
phospholipases A2 in cardiovascular disease: roles as biological effectors and
biomarkers. Circulation 122, 21832200. http://dx.doi.org/10.1161/CIRCULATIONAHA.
110.936393.
Rosenson, R.S., Fraser, H., Goulder, M.A., Hislop, C., 2011. Anti-inammatory effects of
varespladib methyl in diabetic patients with acute coronary syndrome. Cardiovasc.
Drugs Ther. 25, 539544. http://dx.doi.org/10.1007/s10557-011-6344-2.
Simon, T., Benessiano, J., Mary-Krause, M., Ninio, E., Steg, G., Gueret, P., Drouet, E., Tedgui,
A., Danchin, N., Mallat, Z., on behalf of fast-MI investigators, 2008. Impact of circulat-
ing secretory phospholipase A2 (sPLA2) and lipoprotein-associated phospholipase A2
(Lp-PLA2) activities on 6 months-survival, recurrent AMI and incident stroke in pa-
tients with AMI, Abstract. Eur. Heart J. 29 (Suppl. 1), 195. http://dx.doi.org/10.1093/
eurheartj/ehn374.
34 Y.A. Shuvalova et al. / Gene 564 (2015) 2934

Simon, T., Mallat, Z., Kotti-Tounsi, S., Benassiano, J., Lambeau, G., Steg, G., Allard-Latour, G., Wootton, P.T.E., Drenos, F., Cooper, J.A., Thompson, S.R., Stephens, J.W., Hurt-Camejo, E.,
Normand, J.-P., Bourlard, P., Tedgui, A., Ferrires, J., Drouet, E., Mulak, G., Danchin, N., Wiklund, O., Humphries, S.E., Talmud, P.J., 2006. Tagging-SNP haplotype analysis of
2009. Impact of secretory PLA2-IIa gene polymorphism on sPLA2 activity and cardio- the secretory PLA2IIa gene PLA2G2A shows strong association with serum levels of
vascular events following an AMI: results from the French Registry of Acute ST sPLA2IIa: results from the UDACS study. Hum. Mol. Genet. 15, 355361. http://dx.
Elevation or Non-ST-elevation Myocardial Infarction (FAST-MI) Registry, Abstract. doi.org/10.1093/hmg/ddi453.
Circulation 120, S1174.
Sole, X., Guino, E., Valls, J., Iniesta, R., Moreno, V., 2006. SNPStats: a web tool for the anal-
ysis of association studies. Bioinformatics 22, 19281929. http://dx.doi.org/10.1093/
bioinformatics/btl268.