In this analysis of pooled data from 12 studies with 26,118 participants we identified a clear association of Lp-PLA2 activity with risk of CHD, but the effect size was sensitive to the degree of adjustment for co-variables including blood lipids, with which Lp-PLA2 activity is also associated. Several PLA2G7 variants that were associated with Lp-PLA2 activity, did not exhibit any association with a wide range of established and novel cardiovascular risk factors, suggesting that their association with CHD events or angiographic CHD should be less prone to confounding. Despite some of the variants showing association with Lp-PLA2 activity, none showed a clear association with angiographic coronary disease or CHD events. However, this null result needs to be interpreted in the light of the relatively weak influence of these SNPs on Lp-PLA2 activity, the available sample size and therefore the statistical power of the study.
The association of Lp-PLA2 activity with CHD risk was modified by the adjustment for other cardiovascular risk factors in a multivariate analysis with the attenuation in effect size being most marked when blood lipids were entered into the model. This involves a judgement on which variables should be considered as potential mediators of the association and which as confounders. Confounding factors should be balanced among groups of individuals categorised by genotype for common SNPs in PLA2G7
that affect Lp-PLA2 activity, because genotype is determined by randomised allocation at conception. We therefore identified variants in PLA2G7
associated with Lp-PLA2 activity and studied their effect on other cardiovascular risk factors and CHD events. A careful selection of the PLA2G7
SNPs was conducted to minimise the likelihood of a false-negative finding due to inadequate selection of genetic tools. This included the identification of common tag-SNPs for European-descent individuals which were initially evaluated in the NPHS-II study, and whose results were then complemented with published-evidence on the effects of PLA2G7
variants on Lp-PLA2 activity and disease risk21
, enabling a final selection of the seven variants utilised in the genetic component of the analysis. Of the seven SNPs used, four (rs1051931, rs1421378, rs1805017 and rs2216465) exhibited association with Lp-PLA2 activity (), although the magnitude of these associations was small to moderate. Indeed, rs1051931 which showed the strongest association, displayed only a relative difference on Lp-PLA2 activity of 7.2% and 3% for rare allele homozygotes and heterozygotes, respectively (see ); the other variants showed associations of similar or lower magnitude.
Lp-PLA2 activity itself was correlated with a range of blood lipids in the current analysis, and the findings are consistent with those of the Framingham Heart Study.23
However, we found that rs1051931, which displayed the largest effect on Lp-PLA2 activity, showed no association with blood lipids. This observation is consistent with data from the two short-term randomised trials in humans using the Lp-PLA2 inhibitor, darapladib.6,24
Even though Lp-PLA2 activity was reduced by about 60% (at a dose of 160 mg daily), darapladib did not modify the concentrations of the lipid particles (LDL-, HDL-cholesterol or triglycerides). These findings in combination suggest that blood lipids may be confounder rather than mediators for the association between Lp-PLA2 activity and CHD risk.
In the current study, variants that affect Lp-PLA2 activity were not associated with coronary atheroma detected by angiography. The absence of an effect on the degree of coronary stenosis could be consistent with the experimental studies in a pig model of diabetic hyperlipidaemia and a human trial of darapladib.5,6,24
In the pig study, 24 weeks treatment with darapladib led to only a modest reduction in coronary lesion size, but treated animals showed a significant alteration in their plaque composition with a smaller necrotic core populated by fewer inflammatory cells suggesting that darapladib treatment may reduce plaque composition and vulnerability, independent of blood lipid changes. Similarly, in the human studies of darapladib, there were detectable reductions in the area of necrotic core of the athermatous plaques in coronary arteries in patients receiving the drug.24
The hypothesis that this may translate to a reduction in plaque rupture and clinical events is now being tested in the STABILITY trial.
There was no clear signal for an effect of PLA2G7
variants on CHD, despite the inclusion of more than 10,494 cases (, ). However, given the small to modest effect of common alleles at the PLA2G7
locus on Lp-PLA2 activity, and considering the strength of the association of Lp-PLA2 activity and CHD risk (), a per-allele OR as low as 1.05 (which we were insufficiently powered to detect) could still be compatible with a contributory role of Lp-PLA2 to CHD. Other genetic studies examining this question have yielded inconsistent findings.25,26
In a Taiwanese study of MIs before 45yrs, the rare allele of rs1051931 was associated with increased risk of an event (OR= 1.66 [95%CI: 1.14.1, 5.80]), despite being associated with lower Lp-PLA2 activity, while the V279F variant showed no association with enzyme activity or risk of MI.27
In a case-control study in Chinese subjects of the seven SNPs in PLA2G7
studied singly or as haplotypes, only the promoter SNP rs13210554 (not included in our analysis) was associated with increased CHD risk, while the V279F variant and rs1805018 (but not rs1805017 or rs1051931) showed association with lower Lp-PLA2 activity but were not associated with CHD risk.28
However, Sutton et al, reported a significant association of PLA2G7
variants, including rs1805017 and rs1051931 with CHD risk in two multi-ethnic studies.21
Lp-PLA2 activity levels are stable among healthy subjects or patients with stable coronary disease.29
It is important to consider whether other genetic variants might help delineate the mechanisms underlying the association between Lp-PLA2 activity and CHD. Genetic variants other than in PLA2G7
itself, in the genes MEF2A, CXCL12, VEGF, PTGIS and CD44
(presumably acting in trans
) may influence Lp-PLA2 activity23
. We also previously reported that variants in the APOE
cluster explained ~4% of the variance in Lp-PLA2.30
However, variants in the vicinity of PLA2G7
itself (acting in cis
) provide a much more specific genetic tool than variants located outside this gene which could influence other pathways. A number of approaches could be undertaken to overcome the problem that the PLA2G7
SNPs studied so far are weak genetic instruments, which compromises statistical power. These include expanding the dataset of European subjects included in a Mendelian randomisation analysis; resequencing of the PLA2G7
gene in individuals of European ancestry to identify less frequent variants with more extreme effects on Lp-PLA2 activity; and undertaking more studies in subjects of Asian ancestry among whom there is a common loss-of function variant (V279F) associated with a very substantial reduction in enzyme activity with a frequency of heterozygotes of 25% and homozygotes of 1-4%.31,32
The results from the association studies on this V279F variant have so far been inconclusive. Earlier hospital based studies have indicated that subjects carrying the mutant allele are at higher risk of arterial events (MI or stroke), as indicated in the study by Yamada et al. including up to 850 cases and 1684 controls33
. However, more recently a Korean case-control study (532 cases: 670 controls) showed opposite results34
while the Chinese study of 827 cases and 947 controls did not find a significant association between the null variant and CHD.28
In summary, in a tagging SNP approach using common PLA2G7 variants in a large scale collaboration including approximately 26,000 European individuals, four SNPs were identified with small to moderate association with Lp-PLA2 activity, but with no effect on lipid variables or CHD risk. However, the modest effect of these variants on Lp-PLA2 activity means that their expected effect on CHD risk, were Lp-PLA2 to be contributory, would be predicted to be small, and even through the collaboration included over 10,400 cases, it may lack sufficient power to detect a genetic effect consistent with a causal role for Lp-PLA2 in CHD. The STABILITY trial should inform on the efficacy and safety of darapladib in particular and Lp-PLA2 inhibition in general for the prevention of CHD events in a number of ethnic groups.