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Tagging SNP haplotype analysis of the secretory
PLA2-V gene, PLA2G5, shows strong association
with LDL and oxLDL levels, suggesting
functional distinction from sPLA2-IIA:
results from the UDACS study
Peter T.E. Wootton
1,{
, Nupur L. Arora
1,{
, Fotios Drenos
1
, Simon R. Thompson
1
,
Jackie A. Cooper
1
, Jeffrey W. Stephens
2
, Steven J. Hurel
3
, Eva Hurt-Camejo
4,5
,
Olov Wiklund
5
, Steve E. Humphries
1
and Philippa J. Talmud
1,
*
1
Division of Cardiovascular Genetics, Department of Medicine, Royal Free and University College Medical School,
5 University Street, London WC1E 6JF, UK,
2
The Medical School, University of Wales Swansea, Singleton Park,
Swansea SA2 8PP, UK,
3
Department of Diabetes and Endocrinology, UCL Hospitals, London W1T 3AA, UK,
4
AstraZeneca, R&D, Molecular Pharmacology, Mo
¨
lndal S-43183, Sweden and
5
Wallenberg Laboratory for
Cardiovascular Research, l, Goteborg SE-413 45, Sweden
Received January 25, 2007; Revised March 19, 2007; Accepted April 6, 2007
Animal and human st udies suggest that both secretory PLA2 (sPLA2)-V and sPLA2-IIA (encoded, respect-
ively, by the neighbouring PLA2G5 and PLA2G2A genes) contribute to atherogenesis. Elevated plasma
sPLA2-IIA predicts coronary heart disease (CHD) risk, but no mass assay for sPLA2-V is available. We pre-
viously reported that tagging single nucleotide polymorphism (tSNP) haplotypes of PLA2G2A are strongly
associated with sPLA2-IIA mass, but not lipid levels. Here, we use tSNPs of the sPLA2-V gene to investigate
the associ ation of PLA2G5 wi th CHD risk markers. Seven PLA2G5 tSNPs genotypes, explaining >92% of the
locus genetic variability, were determined in 519 patients with Type II diabetes (in whom PLA2G2A tSNP data
was available), and defined seven common haplotypes (frequencies >5%). PLA2G5 an d PL A2G2A tS NP s
showed linkage disequilibrium (LD). Compared to the common PLA2G5 haplotype, H1 (frequency 34.9%),
haplotypes H2–7 were associated with overall higher plasma LDL (P < 0.00004) and total cho lesterol
(P < 0.00003) levels yet lower oxLDL/LDL (P 5 0.006) and sPLA2-IIA mass (P 5 0.0 4), probably reflecting LD
with PLA2G2A. Intronic tSN P (rs11573248), unlike ly itself to be func tional, distinguished H1 from
LDL-raising haplotypes and may mark a functional site. In conclusion, PLA2G5 tSNP haplotypes demonstrate
an association with total and LDL ch olesterol and oxLDL/LDL, not seen with PLA2G2A, thus confirming
distinct functional roles for these two sPLA2s.
INTRODUCTION
Secretory phospholipase A2 (PLA2) group V (sPLA2-V)
enzyme is a member of the superfamily of PLA2 enzymes
characterized by their ability to hydrolyse the sn-2 ester
bond of phospholipids and cell membranes, generating
non-esterified free fatty acids (NEFAs) and lyso-phospholipids
(1). The sPLA2-V gene (PLA2G5) is tightly linked and in a
negative orientation to the sPLA2-IIA gene (PLA2G2A)on
chromosome 1p34–36.1 (2), with both enzymes sharing
structural and functi onal similarities (3). While the role of
sPLA2-IIA in atherogenesis has been well studied, the
# The Author 2007. Published by Oxford University Press. All rights reserved.
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{
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
*To whom correspondence should be addressed. Tel: þ44 2076796968; Fax: þ44 2076796212; Email: p.talmud@ucl.ac.uk
Human Molecular Genetics, 2007, Vol. 16, No. 12 1437–1444
doi:10.1093/hmg/ddm094
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involvement of sPLA2-V is less well understood and questions
about functional redundancy have been raised (4).
sPLA2-IIA is an acute phase protein, elevated in cell types in
response to pro-inflammatory stimuli (3). sPLA2-IIA hydrolyses
LDL phospholipids generating oxidation-susceptible, small-
dense LDL (sdLDL) particles, with an altered configuration of
apolipoprotein B (5), leading to LDL-receptor-independent
uptake into the arterial wall (6). Within the arterial wall, these
modified LDL particles bind proteoglycans present in the
intima and are further modified, leading to increased retention
(7). In addition, the release by sPLA2s of potent lipid
mediators, in particular arachidonic acid and lyso-phospholipids,
promotes pro-inflammatory responses in the arterial wall
(reviewed in 8). The involvement of sPLA2-IIA in athero-
genesis is confirmed by its localization in atherosclerotic
plaques (9,10).
Observational studies have also identified sPLA2-IIA as a
marker for coronary heart disease (CHD) risk (11), with elev-
ated levels associated with an increased probability of recur-
rent coronary events (12,13). In prospective analysis, serum
levels of sPLA2-IIA were associated with a higher risk of
future CHD in healthy individuals independent of classical
and inflammatory markers (14). In animal models,
sPLA2-IIA transgenic mice show an increased susceptibility
to atherosclerosis (15,16).
Less information exists regarding the potential
pro-atherogenic actions of the functionally similar sPLA2-V
enzyme. In vitro, sPLA2-V is able to hydrolyse HDL and
LDL phosphatidylcholine (PC) 20 times more efficiently
than sPLA2-IIA (17), thus hydrolysing the most common
phospholipid species present in lipoproteins and cell surface
membranes (18), leading to arachidonic acid and lyso-PC
generation (19). Since several atherogenic susceptible mouse
strains have a naturally occurring mutation in pla2g2a, other
sPLA2s have been implicated in the development of athero-
sclerosis in these strai ns. sPLA2-V is a likely candidate, and
recent immunohistochemical analysis (supported by mRNA
expression studies) has shown sPLA2-V to be associated
with smooth muscle cells and foam cells in the lipid cores
of both human and mouse athe rosclerotic lesions (20). LDL
receptor knock-out mice, either over-expressing or deficient
in Pla2g5, provide the first in vivo evidence that sPLA2-V
contributes to atherosclerosis (21).
Although these studies support an independent role for
sPLA2-V in atherogenesis, currently no commercially avail-
able assay for sPLA2-V mass exists, and therefore human
observational studies to confirm the CHD risk association
cannot be undertaken.
By utilizing a tagging single nucleotide polymorphism
(tSNP) haplotype approach, we have made use of common
genetic variations to test the hypothesis that variation in
PLA2G5 influences both plasma lipid levels and oxidative
stress. These tSNPs were genotyped in a cohort of patients
with Type II diabetes (T2D) mellitu s, a group associated
with a higher CHD risk, where oxidative stress and
lipid measurements were available (22). In addition, we
have sPLA2-IIA mass measures and PLA2G2A tSNP haplo-
type data in the same cohort (22), thus enabling us to
compare the relationship of these two genes with CHD
risk markers.
RESULTS
The baseline characterist ics of the Caucasian men and women
with T2D, with or without CHD, are shown in Table 1. Those
who had CHD were significantly older, and had a longer dur-
ation of diabetes, and their higher usage of statins and angio-
tensin converting enzyme (ACE) inhibitors, most likely
explains their lower diastolic blood pressure (BP), LDL and
total cholesterol levels compared with CHD-free men and
women.
Seven tSNPs (21437C. A, 21251G. A, 2423G. A,
1640C. T, 1742Gin/del, 11069TAin/del, 22507T. G) were
identified in the PLA2G5 gene region. The genotype distri-
bution of all tSNPs was as expected for Hardy –Weinberg
equilibrium as tested by THESIAS (Table 2 gives a summary
of the observed allele frequencies). The locations of the
chosen tSNPs (three in the promoter region, three within
introns and one variant 3
0
of exon 5) are shown schematically
in Figur e 1A. Lewontin
0
sD
0
for each individual tSNP is shown
below the SNP map in Figure 1B, demonstrating strong LD
across the PLA2G5 gene.
Univariate analyses of tSNPs with total, LDL- and
HDL-cholesterol and measures of total antioxidant status,
LDL size and oxidized LDL to LDL ratio are presented in
Supplementary Material, Tables S2–S8. Of the tSNPs,
21437C.A, 1742Gin/del and 11069TAin/del showed signifi-
cant association with cholesterol and LDL levels (P-values
ranged from P , 0.012,0.0001); Supplementary Material,
Tables S2, S6 and S7, respectively). 2423G.A and
1742Gin/del were also strongly associated with differences
in sPLA2-IIA levels (P , 0.01; Supplementary Material,
Tables S4 and S6, respectively).
Table 1. Baseline characteristics of Caucasian patients with Type II diabetes
from UDACS
No CHD, N ¼ 383 CHD, N ¼ 136 P-value
Age (years) 65.5 (11.3) 69.5 (9.7) 0.0003
BMI (kg/m
2
)
a
29.2 (5.5) 29.5 (4.7) 0.67
HbA1c (%)
a
7.7 (1.7) 7.5 (1.5) 0.27
Glucose (mmol/l)
a
10.02 (4.40) 9.58 (4.25) 0.31
Cholesterol (mmol/l) 5.19 (1.07) 4.71 (1.12) ,0.0001
LDL (mmol/l)
b
2.81 (0.93) 2.32 (0.89) ,0.0001
HDL (mmol/l)
a
1.30 (0.38) 1.23 (0.37) 0.06
TG (mmol/l)
a
1.90 (1.06) 1.92 (1.07) 0.84
SBP (mmHg)
a
141.5 (20.6) 140.0 (20.9) 0.47
DBP (mmHg) 81.2 (11.4) 78.4 (10.0) 0.01
Duration of diabetes
(years)
c
8.0 (4–16) 11.0 (6–17) 0.005
Gender (% male) 57.2% (219) 66.2% (90) 0.07
Smoking (% current) 17.0% (64) 12.0% (16) 0.18
TAOS (%)
c
44.9 (36.7–52.5) 42.9 (34.1–50.7) 0.13
Ox-LDL/LDL (U/mmol)
a
16.8 (7.8) 18.6 (10.3) 0.08
sd-LDL (%)
c
71.9 (58.5–81.4) 71.5 (54.9–80) 0.74
CRP (mg/L)
a
1.66 (1.42) 1.77 (1.59) 0.49
sPLA2-IIA (ng/ml)
a
3.08 (2.20) 3.45 (2.62) 0.12
Statin (%) 23.0 60.0 ,0.0001
ACE I (%) 26.5 38.9 0.003
a
Log-transformed.
b
Square-root transformed.
c
Median (IQR).
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Figure 1B shows the D
0
for PLA2 G2A tSNPs used pre-
viously (22), indicating that despite the 90 Kb distance, there
is a certain degree of LD across the region, with 2423G.A
and the 1742Gin/del, PLA2G5 tSNPs that showed significant
association with sPLA2-IIA mass, showing LD with
PLA2G2A tSNPs 2655T.C and the 763C.G, which had
shown strong association with differences in sPLA2-IIA
mass (22).
Since the seven PLA2G5 tSNPs were primarily identified
for tagging purposes, chosen to cover maximum genetic varia-
bility of PLA2G5, haplotype analysis was examined. Complete
haplotype analysis was available on 351 individ uals. Of the
potential 119 haplotypes defined by the tSNPs, 18 inferred
haplotypes were observed in the sample. Of these, seven
occurred at frequencies .5% and accounted for 92% of the
haplotypic variation within the gene (Table 3). Table 4
shows the association of these haplotypes with intermediate
phenotypes, giving the mean value for one copy of each hap-
lotype as determined by THESIAS (23,24). Compared to the
common haplotype, H1, haplotypes H2–4 were associa ted
with significantly higher cholesterol (9.1%, P ¼ 0.008; 13%,
P ¼ 0.001; 14.0%, P , 0.001, respectively) and H2 was
associated with higher LDL (23%; P , 0.001) and lower
sPLA2-IIA mass (49%; P ¼ 0.001). H6 was associated with
47% (P ¼ 0.01) lower oxLDL/LDL than H1. This effect on
cholesterol and LDL could not be accounted for by statin
usage, as there was no difference in statin used either by indi-
vidual genotypes or haplotypes (all .0.4).
Since the effects of H2 –7 showed no significant evidence
for heterogeneity on any trait, their combined effects, com-
pared to H1, were examined. The unadjusted P-values of
these combined haplotypes are presented in Table 4. Com-
pared to H1, H2– 7 carriers had significantly lower
sPLA2-IIA mass (P ¼ 0.04), and higher cholesterol
(P , 0.00003) and LDL (P , 0.00004) and lower oxLDL/
LDL (P ¼ 0.006). These P-values remained statistically sig-
nificant even after adjustment for age, gender, CHD status
and statin use (Table 4).
The mean LDL- cholesterol levels in subjects with 0 (H1/
H1), 1 (H1/H2–7) and 2 (H2– 7/H2–7) copies of the com-
bined H2– 7 haplotypes are presented in Figure 2, showing a
strong co-dominant raising effect.
The UDAC study is cross-sectional in design and did not
have the power to assess the association of these haplotypes
with CHD risk since the number of CHD positive individuals
in each haplotype group was small. Attempts at cladogram
analysis similar to that demonstrated in the haplotype analysis
of the closely related PLA2G2A gene (22) was not achievable
since several different combinational changes of tSNPs were
possible for each haplotype. As such, no individual tSNP
could be identified as being responsible for the associations
described. However, H2 – 7, which showed significant associ-
ation with LDL cholesterol, in contrast to H1, have in
common the 11609TAin allele, while H1 is defined by the
rare 11609TAdel. H5, which also carrie s the 11609TAdel
allel
e, has trait values most similar to H1. The 11609TAin/
del variant (rs11573248) is located 3 kb 5
0
of exon 2, and it
is therefore doubtful that it is of functional importance, but
is likely to be in strong LD with a yet undetermined functional
variant.
DISCUSSION
PLA2G5 tSNP haplotypes are associated with differences
in LDL levels and sPLA2-IIA mass
We have used a genetic approach to distinguish the effects of
the sPLA2 enzymes group V and IIA. tSNPs that capture 92%
of the locus genetic variation were used to investigate the
association of PLA2G5 variants (encoding sPLA2-V) with
plasma lipid levels and with markers of oxidative stress, con-
trasted in the same study to those previously reported for
PLA2G2A (encoding sPLA2-IIA) (22). The common
PLA2G5 tSNP haplotype H1 (with a frequency of 39%),
when compared with the rarer haplotypes H2– 7, showed
strong association with lower plasma levels of LDL
(P , 0.00004) and total cholesterol (P , 0.00003) , yet
higher oxLDL/LDL (P ¼ 0.006). In this same sample, we pre-
viously showed that tSNP haplotypes of PLA2G2A were
strongly associated with sPLA2-IIA mass levels (P ,
0.00001), yet showed no association with lipid levels or
measures of oxidative stress (22). These data implied that in
vivo, sPLA2-IIA is not having a major impact on determining
the hydrolysis of lipids in the circulation. In contrast, PLA2G5
tSNP haplotypes are having a major effect on LDL levels,
reflected also in levels of total cholesterol, and oxLDL levels.
Functional differences in sPLA2-V and sPLA2-IIA
The contrasting effects of PLA2G5 and PLA2G2A on LDL
levels are supported by in vitro data demonstrating that
sPLA2-V is 20 times more active than sPLA2-IIA in its
ability to hydrolyse phospholipids (17). The recent studies
by Rosengren et al. (20,25) strongly support distinct roles
for sPLA2-V and sPLA2-IIA on circulating lipids and in
atherogenic lesions. Recombinant sPLA2-V but not sPLA2–
IIA hydrolysed lipoprotein phospholipids in human sera and
isolated VLDL, HDL and LDL (preferentially in that order,
probably due to differences in the sphingomyelin content),
accompanied by an increase in lyso-phospholipids (20). This
enzymatic difference between the two sPLA2s is suggested
to be due to the tryptophan residues in the interfacial-binding
region of sPLA2-V, absent in sPLA2-IIA, which would enable
enhanced penetration of sPLA2 -V into the phospholipid
monolayer (20).
Table 2. SNP rs number and minor allele frequency of the PLA2G5 tSNPs in
UDACS
tSNP rs number Minor allele frequency
(95% CI)
2 1437C. A rs11573185 0.45 (0.43–0.48)
2 1251G. A rs2148911 0.07 (0.05– 0.08)
2 423G. A rs11573191 0.18 (0.16–0.19)
1640C. T rs640022 0.15 (0.13– 0.16)
1742Gin/del rs11573203 0.28 (0.26–0.30)
11069TAin/del rs11573248 0.36 (0.34–0.39)
22507T. G rs622450 0.14 (0.12–0.15)
Human Molecular Genetics, 2007, Vol. 16, No. 12 1439
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Further differences between sPLA2-V and sPLA2-IIA were
seen by Rosengren et al. (20). In studies of C57BL/6 mice
and ldlr/apoe double knockout mice, which lack pla2g2a due
to deletion of exon 3, sPLA2-V expression was upregulated
in the aorta when mice were fed a Western diet. No effect of
a high fat diet was seen on sPLA2-II A expression in the
PLA2G2A transgenic mice. However, when mice were treated
with inter-peritoneal injection of lipopolysaccharide to
promote inflammation, expression of sPLA2-IIA but not
sPLA2-V was stimulated. Difference in the localization of
these two enzymes in the atherosclerotic plaques of mice and
humans further support a distinct functional divergence (20,25).
Inverse relationship between LDL levels and sPLA2
Surprisingly, compared to PLA2G5 H2–7 carriers, H1 carriers
had significantly lower LDL cholesterol (P , 0.00004), but
significantly higher oxLDL/LDL (P ¼ 0.006) and borderline
higher sPLA2-IIA mass (P ¼ 0.04), so in part the lower
LDL levels could reflect higher LDL hydrolysis by
sPLA2-IIA. This seems unlikely, since as discussed above,
PLA2G2A haplotypes that had a strong association with
sPLA2-IIA levels showed no effect on LDL cholesterol
levels. PLA2G2A and PLA2G5 lie head to tail on chromosome
1p34– 36.1 (2) (in a cluster with other sPLA2 genes), thus it is
Figure 1. (A) Map of the PLA2G5 gene showing the selected tagging SNPs numbered from the start of exon 1. (B) Haploview LD (D
0
) of tSNPs of both PLA2G5
and PLA2G2A. The darker boxes represent the stronger LD. The D
0
for any two SNPs is presented in the box representing their intersection. No number indicates
aD
0
of 1. The head to tail orientation of the two genes and the tSNPs used in this and our previous study (22) are presented.
1440 Human Molecular Gen etics, 2007, Vol. 16, No. 12
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possible that variation within this intergenic region may be
influencing the expression of both genes. PLA2G5 SNPs
2423G.A and 1742 Gin/del are both associated with signifi-
cant differences in sPLA2-IIA levels (see Supplementary
Material, Tables), and from Figure 1B it is clear that both
these SNPs show significant LD with PLA2G2A 763C. G,
which shows strong statistical significant association with
sPLA2-IIA levels (P , 0.0001) (see Supplementary Material,
Tables in 22). Therefore, it is probable that the association of
PLA2G5 haplotypes with sPLA2-IIA mass (explaining only
0.6% of the sPLA2-IIA variance) is simply reflecting the LD
that exists in this gene cluster. The effect on lipid levels there-
fore suggest that the association of PLA2G5 haplotypes with
LDL levels are due to the haplotypic effects associated with
differences in sPLA2-V activity/mass itself.
Thus, we speculate that, in comparison to haplotypes H2 –7,
H1 should be associated with higher sPLA2-V, and since high
sPLA2 activity is reported to increase risk in humans (13) and
mice (5), this would lead to increased CHD risk. On the face
of it this appears counter–intuitive, since the lower LDL chol es-
terol levels in H1 carriers, compared to H2 –7, should be associ-
ated with decreased risk. However, the lower plasma LDL levels
seen in the H1 carriers could be the result of LDL conversion to
sdLDL particles due to increased phospholipid hydrolysis. This
would result in increased ret ention of LDL in the arterial wall,
increased oxidation and thus lead to increased atherosclerosis.
Since sdLDL is more prone to oxida tion, the oxLDL/LDL
ratio should also be higher. We did not see an effect of
PLA2G5 haplotyp e on sdLDL levels but we did see a
significant effect of haplotypes on oxLDL/LDL. Carriers of
haplotype H1 had oxLDL/LDL levels which were 22% higher
(10.19 U/mmol) compared to H2–7 (8.35 U/mmol, P ¼ 0.006).
There is support for these associations and the predicted
outcomes from the study by Mallat et al. (13), who measured
both sPLA2 activity and mass in patients with acute coronary
syndrome. While the sPLA2-IIA mass assay shows no cross
Table 3. Inferred haplotype frequencies in UDACS
Haplotype name Haplotype combination Frequency in UDACS
H1 AGGCGinTAdelT 0.359
H2 CGACGdelTAinT 0.169
H3 CGGCGinTAinG 0.128
H4 AGGCGdelTAinT 0.094
H5 AGGCGinTAinT 0.060
H6 CAGTGinTAinT 0.059
H7 CGGTGinTAinT 0.055
AGGTGinTAinT 0.032
CGGCGinTAinT 0.026
CGGCGdelTAinG 0.003
AGGCGinTAinG 0.003
CAGTGinTAinT 0.002
CGACGdelTAdelT 0.002
AGGTGdelTAinT 0.002
CGGTGinTAinT 0.002
AGGTGinTAdelT 0.001
CGGCGinTAdelG 0.001
CAGCGinTAinT 0.001
tSNP order from left to right is: 21437C. A, 21251G. A, 2423G. A,
1640C. T, 1742G in/del, 11609TA in/del and 22507T. G.
Table 4. Association of mean values (95% CI) of intermediate phenotypes with common haplotypes H1–7 in UDACS
Quantitative traits and
P-values compared to
H1: AGGCGinTAdelT
H1
a
:
AGGCGinTAdelT
H2
a
:
CGACGdelTAinT
H3
a
:
CGGCGinTAinG
H4
a
:
AGGCGdelTAinT
H5
a
:
AGGCGinTAinT
H6
a
:
CAGTGinTAinT
H7
a
:
CGGTGinTAinT
P-value H1 versus
H2– 7 unadjusted
P-value H1 versus
H2– 7 adjusted
sPLA2 IIA levels (ng/ml)
b
2.36 (2.07–2.69) 1.58 (1.28 –1.96) 1.88 (1.47 –2.42) 1.89 (1.41–2.53) 2.23 (1.49 –3.36) 2.06 (1.43– 2.97) 1.97 (1.20 –3.25) 1.46 (0.81–0.90) 1.85 (0.91–1.01)
P-value – 0.001 0.13 0.19 0.81 0.51 0.50 0.04 0.01
Cholesterol (mmol/l) 2.59 (2.50–2.67) 2.85 (2.71 –3.00) 2.93 (2.76 –3.12) 2.97 (2.74–3.20) 2.67 (2.41 –2.94) 2.87 (2.62– 3.14) 2.79 (2.52 –3.07) 2.64 (2.57–2.72) 2.87 (2.79– 2.94)
P-value – 0.008 0.001 0.001 0.59 0.04 0.18 0.00003 0.001
HDL (mmol/l)
b
0.72 (0.69–0.75) 0.69 (0.64 –0.74) 0.75 (0.70 –0.82) 0.76 (0.68–0.85) 0.64 (0.56 –0.73) 0.69 (0.60– 0.79) 0.78 (0.66 –0.92) 0.64 (0.55–0.58) 0.72 (0.59–0.61)
P-value – 0.37 0.36 0.42 0.11 0.53 0.35 0.83 0.90
LDL (mmol/l)
c
1.34 (1.27–1.42) 1.65 (1.50 –1.81) 1.63 (1.46 –1.82) 1.65 (1.41–1.92) 1.48 (1.22 –1.79) 1.62 (1.33– 1.98) 1.48 (1.22 –1.81) 1.61 (0.87–0.92) 1.61 (0.87–0.92)
P-value – ,0.001 0.01 0.02 0.38 0.07 0.37 0.00004 0.00005
oxLDL/LDL (U/mmol)
b
9.40 (8.60–10.10) 7.8 (6.40–9.40) 7.60 (5.82–9.75) 7.20 (5.13– 9.71) 9.68 (7.31 –12.34) 6.36 (4.58–8.45) 7.45 (5.55– 9.76) 8.35 (7.60 –9.14) 7.61 (6.91–8.35)
P-value – 0.1 0.13 0.11 0.87 0.01 0.13 0.006 0.004
a
Mean value for one copy of each haplotype adjusted for age, gender, CHD status and statin use.
b
Log-transformed.
c
Square root transformation.
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reactivity with sPLA2-V, the activity assay measures most
plasma sPLA2s (i.e. including both -IIA and -V) (13). Those
individuals without hyperlipidaemia had significantly higher
sPLA2 activity compared to those with hyperlipidaemia
(2.88 versus 2.33 nmol/min/ml , respectively; P ¼ 0.001), and
the cumulative incidence of death or MI according to tertiles
of sPLA2 in patients with cholesterol 197 mg/dl was 8.4%
compared to 5.2% in those with cholesterol.197 mg/dl (13).
Thus, this stud y also shows an inverse relationship between
plasma lipids and sPLA2 act ivity (13).
Limitations of the study
The tSNPs identified for this study were derived from the rese-
quenced data from the NIEHS database and have an r
2
¼ 0.92.
A comparison cannot be made directly to those tSNPs
obtained from the recent HAPMAP release (21). This high-
lights the issue of tSNP identification in a rapidly changing
field where both the databases are constantly upgraded, as
well as the algorithms for tSNP identification. The use of
additional tSNPs might possibly sub-divide haplotype H1,
allowing a clearer identification of the relevant haplotype for
functional studies. However, the use of a limited set of
tSNPs cannot be a confounder of an association that is ident-
ified. In the absence of a specific sPLA2-V assay, we cannot
exclude the possibility that these haplotypic effects on LDL
cholesterol levels are independent of sPLA2-V activity or
mass, and we cannot determine what the association of these
PLA2G5 haplotypes actually are with sPLA2-V levels. In
addition, because of the relatively small sample size, the
UDAC study was insufficiently powered to detect any associ-
ations between PLA2G5 inferred haplotypes and CHD risk.
The association between haplotype H1 and lower LDL
levels has been reported here in patients with T2D, and the
association may not be the same in non-T2D individuals.
Finally, the strong LD across the gene cluster means that it
is not possible to distinguish PLA2G5 and PLA2G2A effects
entirely, although LD may be lower in non-Caucasians,
which could allow for better discrimination of these effects.
CONCLUSION
The association of PLA2G5 haplotypes with markers of CHD
provides the foundation for further investigations in case-
control and prospective studies, specifically investigating
whether these haplotypes are associated with CHD risk and
with LDL levels in non-diabetic subjects. Despite these limit-
ations, this study represents the first investigation of genetic
variation in PLA2G5 and its association with markers of
CHD and strongly supports the recent studies by Rosengren
et al. (20,25) showing a distinct functional difference
between the two closely related sPLA2 enzymes sPLA2-V
and sPLA2-IIA.
MATERIALS AND METHODS
Study design
The University College London Diabetes And Cardiovascular
Disease Study (UDACS) has been described in detail else-
where (26,27). In brief, the UDACS consists of 1014 consecu-
tive subjects recruited from the diabetes clinic at Universi ty
College London Hospitals NHS Trust (UCLH) 2001 –02
(629 men; 600 Caucasians with T2D). All patients had dia-
betes according to WHO criteria (28). Analysis was restricted
to the Caucasian subjects with T2D to remove possible hetero-
geneity within the sample.
Clinical measurements
CHD event was recorded if any patient had positive coronary
angiography/angioplasty, coronary artery bypass, cardiac thal-
lium scan, exercise tolerance test, myocardial infarction or symp-
tomatic/treated angina (27). Routine plasma traits were measured
including plasma oxidised LDL (oxLDL) by ELISA (Mercodia,
Uppsala, Sweden), expressed as the ratio of oxLDL divide by
total LDL to generate a specific measure of LDL oxidation
(27,29). Plasma total antioxidant status (TAOS), which is inver-
sely related to oxidative stress, was measured by a photometric
assay (30). The percentage sdLDL is derived from the percentage
of LDL sub-classes I and II from the four sub-classes I– IV
obtained by ultra-centrifugation (31). Serum sPLA2-IIA levels
were measured by a commercially available ELISA (Cayman
Chemical Company, Ann Arbor, MI, USA). The intra- and inter-
assay coefficients of variation were 6.0 and 10.3%, respectively.
Full ethical approval was granted by the UCLH NHS trust and all
patients included in the study had given written consent.
DNA extraction, tagging-SNP identification
and genotyping
DNA was extracted using the salting out method (32). Tagging
SNPs were identified using the STRAM algorithm (33) on
the PHASE (34) output from the National Institute of Environ-
mental Health Sciences SNP database website (http://egp.gs.
Figure 2. The mean LDL-cholesterol levels (95% CI) in subjects with 0 (H1/
H1), 1 (H1/H2–4) and 2 (H2–4/H2 –4) copies of the H1 and combined H2 –4
haplotypes.
1442 Human Molecular Gen etics, 2007, Vol. 16, No. 12
by guest on May 29, 2013http://hmg.oxfordjournals.org/Downloaded from
washington.edu/data/PLA2G5/). Seven tSNPs of PLA2G5 were
identified (rs11573185, rs2148911, rs11573191, rs11573203,
rs640022, rs11573248, rs622450). All SNPs except rs640022
were genotyped using TaqMan technology (Applied Bio-
sciences, ABI, Warrington, UK). Oligonucleotides and MGB
probes are detailed in Supplementary Material, Table S1. The
rs640022 tSNP was determined using a polymerase chain reac-
tion (PCR) with sense (5
0
-GGACTGTTGATGGTGGGAGT-3
0
)
and anti-sense (5
0
-CCAGGTATGATGGTGCACAG-3
0
) oligo-
nucleotides flanking the variant of interest. Restriction of the
PCR product with the PvuII enzyme results in fragment sizes
of 166/20 bp in common homozygotes, 186/166/20 bp in hetero-
zygotes and 186 bp in rare homozygotes. Fragments were
resolved using Microtitre Array Diagonal Gel Electrophoresis
(MADGE) (35). Two negative controls were included in each
PCR run.
Statistical methods
Hardy–Wei nberg equilibrium was assessed using THESIAS
(23,24). Linkage disequilibrium (LD) as measured by D
0
was assessed using Haploview (http://www.broad.mit.edu/
mpg/haploview/). All analyses were performed on normally
distributed data after appropriate transformation (log or
square root). Results are presented as mean and sta ndard
deviation (SD). Parametric or non-parametric (Krusk all –
Wallis) analysis of variance was used, when appropriate, to
compare the changes of the continuous variables across the
SNPs categories. Adjusted P-values were obtained from the
analysis of covariance for continuous data, and logistic
regression for categorical data. Haplotypes were inferred
using both THESIAS (23,24) and PHASE (34) excluding indi-
viduals with missing values. Th e haplotypic pair for each
subject was calculated by PHASE and only the haplotypes
with frequencies 5% were used for further analysis.
Because of multiple testing, the significance level was taken
as P , 0.01, instead of an inappropriately conservative
Bonferroni-like adjustment of the P-values (36,37).
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG Online.
ACKNOWLEDGEMENTS
We thank Dr Birit Johansson for helpful discussion and critical
appraisal of the manuscript. We would like to thank contribu-
tors to the University College London Diabetes and Cardio-
vascular Study. P.J.T., P.T.E.W., S.R.T. and S.E.H. are
supported by the British Heart Foundation Grants RG2000/
15 and FS/2002/087/14762 and FS/2004/039. Diabetes UK
supported J.W.S. (BDA: RD01/0001357) and the creation of
UDACS. O.W. is supported by the Swedish Heart and Lung
Foundation Grants 200441654 and 20041243.
Conflict of Interest statement. None declared.
REFERENCES
1. Dennis, E.A. (1994) Diversity of group types, regulation, and function
of phospholipase A2. J. Biol. Chem., 269, 13057– 13060.
2. Tischfield, J.A., Xia, Y.R., Shih, D.M., Klisak, I., Chen, J., Engle, S.J.,
Siakotos, A.N., Winstead, M.V., Seilhamer, J.J., Allamand, V. et al.
(1996) Low-molecular-weight, calcium-dependent phospholipase
A2 genes are linked and map to homologous chromosome regions in
mouse and human. Genomics, 32, 328–333.
3. Jaross, W., Eckey, R. and Menschikowski, M. (2002) Biological effects
of secretory phospholipase A(2) group IIA on lipoproteins and in
atherogenesis. Eur. J. Clin. Invest., 32, 383– 393.
4. Webb, N.R. (2005) Secretory phospholipase A2 enzymes in
atherogenesis. Curr. Opin. Lipidol., 16, 341–344.
5. Sartipy, P., Camejo, G., Svensson, L. and Hurt-Camejo, E. (1999)
Phospholipase A(2) modification of low density lipoproteins forms
small high density particles with increased affinity for proteoglycans
and glycosaminoglycans. J. Biol. Chem., 274, 25913 –25920.
6. Galeano, N.F., Al Haideri, M., Keyserman, F., Rumsey, S.C. and
Deckelbaum, R.J. (1998) Small dense low density lipoprotein has increased
affinity for LDL receptor-independent cell surface binding sites: a potential
mechanism for increased atherogenicity. J. Lipid Res., 39, 1263 –1273.
7. Sartipy, P., Johansen, B., Camejo, G., Rosengren, B., Bondjers, G. and
Hurt-Camejo, E. (1996) Binding of human phospholipase A2 type II to
proteoglycans. Differential effect of glycosaminoglycans on enzyme
activity. J. Biol. Chem., 271, 26307 –26314.
8. Hurt-Camejo, E., Camejo, G., Peilot, H., Oorni, K. and Kovanen, P.
(2001) Phospholipase A(2) in vascular disease. Circ. Res., 89, 298– 304.
9. Elinder, L.S., Dumitrescu, A., Larsson, P., Hedin, U., Frostegard, J. and
Claesson, H.E. (1997) Expression of phospholipase A2 isoforms in human
normal and atherosclerotic arterial wall. Arterioscler. Thromb. Vasc. Biol.,
17, 2257–2263.
10. Hurt-Camejo, E., Andersen, S., Standal, R., Rosengren, B., Sartipy, P.,
Stadberg, E. and 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, 300–309.
11. Kugiyama, K., Ota, Y., Takazoe, K., Moriyama, Y., Kawano, H.,
Miyao, Y., Sakamoto, T., Soejima, H., Ogawa, H., Doi, H. et al. (1999)
Circulating levels of secretory type II phospholipase A(2) predict
coronary events in patients with coronary artery disease. Circulation,
100, 1280–1284.
12. Kugiyama, K., Ota, Y., Sugiyama, S., Kawano, H., Doi, H., Soejima, H.,
Miyamoto, S., Ogawa, H., Takazoe, K. and Yasue, H. (2000) Prognostic
value of plasma levels of secretory type II phospholipase A2 in patients
with unstable angina pectoris. Am. J. Cardiol., 86, 718 –722.
13. 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. et al. (2005)
Circulating secretory phospholipase A2 activity predicts recurrent events in
patients with severe acute coronary syndromes. J. Am. Coll. Cardiol., 46,
1249– 1257.
14. 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., Hack, C.E. et al.
(2005) Serum levels of type II secretory phospholipase A2 and the risk of
future coronary artery disease in apparently healthy men and women: the
EPIC-Norf
olk Prospective Population Study. Arterioscler. Thromb. Vasc.
Biol., 25, 839 –846.
15. Ivandic, B., Castellani, L.W., Wang, X.P., Qiao, J.H., Mehrabian, M.,
Navab, M., Fogelman, A.M., Grass, D.S., Swanson, M.E., de Beer, M.C.
et al. (1999) Role of group II secretory phospholipase A2 in
atherosclerosis: 1. Increased atherogenesis and altered lipoproteins in
transgenic mice expressing group IIa phospholipase A2. Arterioscler.
Thromb. Vasc. Biol., 19, 1284– 1290.
16. Leitinger, N., Watson, A.D., Hama, S.Y., Ivandic, B., Qiao, J.H.,
Huber, J., Faull, K.F., Grass, D.S., Navab, M., Fogelman, A.M. et al.
(1999) Role of group II secretory phospholipase A2 in atherosclerosis:
2. Potential involvement of biologically active oxidized phospholipids.
Arterioscler. Thromb. Vasc. Biol., 19, 1291–1298.
17. Pruzanski, W., Lambeau, L., Lazdunsky, M., Cho, W., Kopilov, J. and
Kuksis, A. (2005) Differential hydrolysis of molecular species of
lipoprotein phosphatidylcholine by groups IIA, V and X secretory
phospholipases A2. Biochim. Biophys. Acta, 1736, 38 –50.
Human Molecular Genetics, 2007, Vol. 16, No. 12 1443
by guest on May 29, 2013http://hmg.oxfordjournals.org/Downloaded from
18. Kim, K.P., Han, S.K., Hong, M. and Cho, W. (2000) The molecular basis
of phosphatidylcholine preference of human group-V phospholipase A2.
Biochem. J., 3, 643– 647.
19. Satake, Y., Diaz, B.L., Balestrieri, B., Lam, B.K., Kanaoka, Y.,
Grusby, M.J. and Arm, J.P. (2004) Role of group V phospholipase A2 in
zymosan-induced eicosanoid generation and vascular permeability
revealed by targeted gene disruption. J. Biol. Chem, 279, 16488– 16494.
20. Rosengren, B., Peilot, H., Umaerus, M., Jonsson-Rylander, A.C.,
Mattsson-Hulten, L., Hallberg, C., Cronet, P., Rodriguez-Lee, M. and
Hurt-Camejo, E. (2006) Secretory Phospholipase A2 Group V. Lesion
Distribution, Activation by Arterial Proteoglycans, and Induction in Aorta
by a Western Diet. Arterioscler. Thromb. Vasc. Biol, 26, 1579–1585.
21. Bostrom, M.A., Boyanovsky, B.B., Jordan, C.T., Wadsworth, M.P.,
Taatjes, D.J., de Beer, F.C. and Webb, N.R. (2007) Group V Secretory
Phospholipase A2 Promotes Atherosclerosis. Evidence From Genetically
Altered Mice. Arterioscler. Thromb. Vasc. Biol.; Epub ahead of print.
22. Wootton, P.T., Drenos, F., Cooper, J.A., Thompson, S.R., Stephens, J.W.,
Hurt-Camejo, E., Wiklund, O., Humphries, S.E. and Talmud, P.J. (2006)
Tagging-SNP haplotype analysis of the secretory PLA2IIa gene
PLA2G2A shows strong association with serum levels of sPLA2IIa:
results from the UDACS study. Hum. Mol. Genet., 15, 355– 361.
23. Tregouet, D.A., Barbaux, S., Escolano, S., Tahri, N., Golmard, J.L.,
Tiret, L. and Cambien, F. (2002) Specific haplotypes of the P-selectin
gene are associated with myocardial infarction. Hum. Mol. Genet., 11,
2015– 2023.
24. Tregouet, D.A., Ricard, S., Nicaud, V., Arnould, I., Soubigou, S.,
Rosier, M., Duverger, N., Poirier, O., Mace, S., Kee, F. et al. (2004)
In-depth haplotype analysis of ABCA1 gene polymorphisms in relation to
plasma ApoA1 levels and myocardial infarction. Arterioscler. Thromb.
Vasc. Biol., 24, 775–781.
25. Rosengren, B., Jonsson-Rylander, A.C., Peilot, H., Camejo, G. and
Hurt-Camejo, E. (2006) Distinctiveness of secretory phospholipase A(2)
group IIA and V suggesting unique roles in atherosclerosis. Biochim.
Biophys. Acta, 1761, 1301– 1308.
26. Dhamrait, S.S., Stephens, J.W., Cooper, J.A., Acharya, J., Mani, A.R.,
Moore, K., Miller, G.J., Humphries, S.E., Hurel, S.J. and Montgomery, H.E.
(2004) Cardiovascular risk in healthy men and markers of oxidative stress in
diabetic men are associated with common variation in the gene for
uncoupling protein 2. Eur. Heart J., 25, 468–475.
27. Stephens, J.W., Hurel, S.J., Acharya, J. and Humphries, S.E. (2004) An
interaction between the interleukin-6-174G.C gene variant and urinary
protein excretion influences plasma oxidative stress in subjects with type 2
diabetes. Cardiovasc. Diabetol., 3,2.
28. Alberti, K.G. and Zimmet, P.Z. (1998) Definition, diagnosis and
classification of diabetes mellitus and its complications. Part 1: diagnosis
and classification of diabetes mellitus provisional report of a WHO
consultation. Diabet. Med., 15, 539–553.
29. Scheffer, P.G., Bos, G., Volwater, H.G., Dekker, J.M., Heine, R.J. and
Teerlink, T. (2003) Associations of LDL size with in vitro oxidizability
and plasma levels of in vivo oxidized LDL in Type 2 diabetic patients.
Diabet. Med., 20, 563– 567.
30. Sampson, M.J., Gopaul, N., Davies, I.R., Hughes, D.A. and Carrier, M.J.
(2002) Plasma F2 isoprostanes: direct evidence of increased free radical
damage during acute hyperglycemia in type 2 diabetes. Diabetes Care,
25, 537–541.
31. Krauss, R.M. and Burke, D.J. (1982) Identification of multiple subclasses
of plasma low density lipoproteins in normal humans.
J. Lipid Res., 23,
97
–104.
32. Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988) A simple salting out
procedure for extracting DNA from human nucleated cells. Nucleic Acids
Res., 16, 1215.
33. Stram, D.O. (2005) Software for tag single nucleotide polymorphism
selection. Hum. Genomics, 2, 144–151.
34. Stephens, M., Smith, N.J. and Donnelly, P. (2001) A new statistical
method for haplotype reconstruction from population data. Am. J. Hum.
Genet., 68, 978 –989.
35. Day, I.N., Humphries, S.E., Richards, S., Norton, D. and Reid, M. (1995)
High-throughput genotyping using horizontal polyacrylamide gels with
wells arranged for microplate array diagonal gel electrophoresis
(MADGE). Biotechniques, 19, 830 –835.
36. Perneger, T.V. (1999) Adjusting for multiple testing in studies is less
important than other concerns. BMJ, 318, 1288.
37. Rothman, K.J. (1990) No adjustments are needed for multiple
comparisons. Epidemiology, 1, 43 –46.
1444 Human Molecular Gen etics, 2007, Vol. 16, No. 12
by guest on May 29, 2013http://hmg.oxfordjournals.org/Downloaded from
