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Human Molecular Genetics, 2004, Vol. 13, No. 13 1341-1351
DOI: 10.1093/hmg/ddh145
Human Molecular Genetics, Vol. 13, No. 13 © Oxford University Press 2004; all rights reserved

Platelet-activating factor-acetylhydrolase and PAF-receptor gene haplotypes in relation to future cardiovascular event in patients with coronary artery disease

Ewa Ninio1,*, David Tregouet1, Jean-Luc Carrier1, Dominique Stengel1, Christoph Bickel2, Claire Perret1, Hans J. Rupprecht2, François Cambien1, Stefan Blankenberg1,2 and Laurence Tiret1 for the AtheroGene Investigators

1INSERM U525/IFR14 Coeur Muscle Vaisseaux and Université PM Curie/Faculté de Médecine Pitié-Salpêtrière, Paris, France and 2Department of Medicine II, Johannes Gutenberg-University Mainz, Mainz, Germany

Received February 26, 2004; Revised April 14, 2004; Accepted April 21, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 APPENDIX
 REFERENCES
 
Oxidation of low density lipoproteins is an initial step of atherogenesis that generates pro-inflammatory phospholipids, including platelet-activating factor (PAF) and its analogs. PAF is degraded by PAF-acetylhydrolase (PAF-AH), a circulating enzyme having both pro- and anti-inflammatory activities. PAF-AH activity has been postulated to be a risk factor for coronary artery disease (CAD); however, whether PAF-AH has a causal role or is simply a marker of risk is unclear. The aim of this study was to relate the variability of the genes encoding PAF-AH (PLA2G7) and the PAF-receptor (PTAFR) to the risk of CAD and its complications. All polymorphisms located in putatively functional regions were investigated in a prospective cohort of CAD patients (n=1314) and a group of healthy controls (n=485). The whole gene variability was investigated in relation to case–control status, prospective cardiovascular outcome and plasma PAF-AH levels by means of haplotype analyses. All analyses indicated an effect of the PLA2G7/A379V polymorphism independent of the other polymorphisms. The V379 allele was less frequent in CAD patients than in controls and was associated with a lower risk of future cardiovascular events, suggesting that this allele might be protective against the development of CAD. The V379 allele was also associated with a weak increase of plasma PAF-AH activity that was unlikely to explain the protective effect of the allele on risk. A more likely interpretation is that the A379V polymorphism might modify the enzyme function towards a more anti-atherogenic form. Polymorphisms of the PTAFR gene were not related to any phenotype.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 APPENDIX
 REFERENCES
 
The recognition that atherosclerosis has a strong inflammatory component (1) has stimulated a great deal of research on the role of inflammatory mediators in the atherosclerotic disease process. Oxidation of low density lipoproteins (LDL) is an initial step of atherogenesis that generates a myriad of pro-inflammatory phospholipids, including platelet-activating factor (PAF) (2) and its analogs (3), which are implicated in signaling and activation of pro-inflammatory cells such as platelets, leukocytes and macrophages (4). PAF exerts its various effects via the G-protein-coupled PAF-receptor that binds PAF with high affinity (5). PAF and its biologically active analogs are degraded by PAF-acetylhydrolase (PAF-AH), a circulating enzyme bound mainly to LDL, and to a lower extent to high density lipoprotein (HDL) (6,7), which is also known as lipoprotein-associated phospholipase A2 (Lp-PLA2) or LDL-PLA2. Besides having an anti-inflammatory activity by degrading PAF, PAF-AH may also exert a pro-inflammatory activity by massively hydrolyzing phospholipids to generate lysophosphatidylcholine (lyso-PC) and free oxidized fatty acids, both pro-inflammatory mediators largely responsible for the pro-atherogenic activity of oxidized LDL (8).

During recent years, attention has focused on the role of PAF-AH in atherosclerosis. Experimental data support both the pro- and the anti-atherogenic roles of this enzyme. PAF-AH has been detected in human and rabbit atherosclerotic lesions, hence potentially contributing to the release of lyso-PC and free oxidized fatty acids, although it might also prevent biological activities of PAF-like substances in situ (9). Supporting the pro-atherogenic role of PAF-AH is the fact that its inhibition leads to the reduction of atherosclerotic lesion formation in hypercholesterolemic rabbits (10). In favor of the anti-atherogenic role is the fact that recombinant PAF-AH shows anti-inflammatory properties in animal models (11). In the same line of evidence, the adenoviral overexpression of PAF-AH in atherosclerosis prone apoE–/– mice has been shown: (i) to diminish substantially the macrophage homing to aortic roots (12); (ii) to decrease the neointima formation upon endothelial damage and to inhibit spontaneous atherosclerosis in aortic roots (13) and (iii) to protect plasma lipoproteins from oxidation and to inhibit foam cell formation by facilitating cholesterol efflux from macrophages (14).

Epidemiological data also support the dual roles of PAF-AH. Genetic deficiency of PAF-AH caused by a missense mutation (Val279Phe) in exon 9 of the PAH-AH gene is present in 4% of the Japanese population and leads to a complete loss of catalytic activity (15). This loss-of-function mutation has been reported to be associated with an increased risk of coronary artery disease (CAD) (16,17) and stroke (18), supporting the anti-atherogenic role of PAF-AH. Studies in European populations, including ours, have consistently shown that plasma PAF-AH activity was elevated in CAD patients, even though it is unclear whether plasma PAF-AH has a causal role in atherosclerosis or is simply a marker of risk (19–22).

One way to demonstrate that a factor is causative is to study its role at a genetic level. The gene coding for PAF-AH (PLA2G7) contains 12 exons, the first exon being untranslated (15). The coding sequence of the PLA2G7 gene has been screened to detect variants, and besides the Val279Phe mutation found in Japan and further reported in other Eastern populations (23), three missense mutations have been described in European populations: Arg92His in exon 4, Iso198Thr in exon 7 and Val379Ala in exon 11 (24,25). The I198T and A379V variants were reported to have a functional role and to be associated with susceptibility to atopy and asthma (25). Recently, homozygosity for the V379 allele was found to be associated with a lower risk of myocardial infarction (MI) in the HIFMECH study, a European multicenter case–control study (26).

Another important player in the PAF signaling pathway is the PAF-receptor, encoded by the PTAFR gene. The PTAFR gene has two non-coding exons that are alternatively spliced to a common splice acceptor site on exon 3 according to the type of cell (27). A functional missense mutation Ala224Asp of this gene has been described in the Japanese population (28), but no polymorphism has been described in European populations so far.

The aim of the present study was to investigate the variability of the PLA2G7 and PTAFR genes in relation to the risk of CAD in the AtheroGene study. We first performed a molecular screening of the yet unexplored functional regions of the two genes in order to identify frequent polymorphisms. All relevant polymorphisms were then genotyped in a cohort of CAD patients prospectively followed up and in a group of healthy control subjects (29,30). The overall genetic variability of the PLA2G7 and PTAFR genes was then related to the case–control status, the prospective outcome and the plasma PAF-AH activity, by means of haplotype-based analyses (31).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 APPENDIX
 REFERENCES
 
Screening of the PLA2G7 and PTAFR genes
PLA2G7 gene.
The organization of the PLA2G7 gene is shown in Figure 1. The 5' region (1239 bp) and the untranslated first exon (127 bp) were screened for detection of polymorphisms. Two polymorphisms were identified in the 5' region (T–403C and C–209G, numbered from the start of transcription): one rare mutation in exon 1 which was not further found in the SIPLAC study (see Materials and Methods) and one T/C substitution affecting the 107th nucleotide of intron 1 which was in almost complete association with the T–403C polymorphism. The T–403C and C–209G polymorphisms, as well as the three non-synonymous polymorphisms already described (R92H, I198T and A379V), were further genotyped in the AtheroGene study.



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  		Figure 1. Organization of the PLA2G7 gene and polymorphisms identified by molecular screening as well as non-synonymous polymorphisms previously described (24,25). The lengths of the screened sequences are given below the 5' region and the untranslated first exon. Polymorphisms in 5' are numbered from the start of transcription. The G+108/ex1ntT polymorphism was not genotyped because of its low frequency and the T+107/in1C polymorphism was in almost complete association with the T–403C polymorphism.

 
PTAFR gene.
The organization of the PTAFR gene is shown in Figure 2. We screened the complete exonic sequence (1931 bp) as well as two regions located upstream from exon 1 and exon 2 (3326 bp in total) and a region located downstream from exon 3 (249 bp). Two C/T substitutions were detected 5' of exon 2 at positions –838 and –414, respectively, from the start of transcription of exon 2. The C–414T polymorphism was not further found in the SIPLAC study (see Materials and Methods). One A/T substitution was found 5' of exon 1 at position –1209 from the start of transcription of exon 1, but this mutation was carried by only four subjects (<1%) in the SIPLAC study. One A/G substitution was found in position +503 after the stop codon (A+503G). Finally, only the C–838/ex2ntT and the A+503G polymorphisms were genotyped in the AtheroGene study.



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  		Figure 2. Organization of the PTAFR gene and polymorphisms identified by molecular screening. The gene has two 5' non-coding exons with distinct transcriptional initiation sites (TIS), which are alternatively spliced to a common splice acceptor site on exon 3. Exons are numbered according to (27), although exon 2 precedes exon 1 in the genomic sequence. The lengths of the screened sequences are given below the corresponding regions. Polymorphisms in 5' of each untranslated exon are numbered from the respective TIS. The polymorphism in 3' is numbered from the stop codon. Polymorphisms C–414/ex2ntT and A–1209/ex1ntT were not genotyped because of their low frequency.

 
Allele frequencies and pairwise linkage disequilibrium (LD) coefficients between polymorphisms estimated in control subjects are shown in Table 1. None of the genotype distributions significantly deviated from Hardy–Weinberg expectations, either in cases or in controls.


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  		Table 1. Allele frequency and pairwise LD between polymorphisms of the PLA2G7 and PTAFR genes in control subjects (n=485)
 
Baseline characteristics of cases and controls
Baseline characteristics of CAD patients (n=1318) and control subjects (n=485) are presented in Table 2, separately in males (74%) and females (26%). Male cases were similar in age to male controls whereas female cases were older than their respective controls. As expected, CAD patients of both sexes were more often smokers, had a higher prevalence of diabetes and hypertension and had a more unfavorable lipid profile compared with controls. The paradoxically lower levels of total and LDL-cholesterol levels in patients might be explained by the large fraction of them taking statin medication (35%). All inflammatory markers were markedly increased in patients of both sexes. As previously reported (22), PAF-AH activity was increased in female cases compared with controls but no difference was observed in males.


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  		Table 2. Baseline characteristics of cases and controls according to sex
 
Association of PLA2G7 and PTAFR gene polymorphisms with case–control status
By univariate analysis, the R92H (P=0.015) and the A379V (P<0.001) polymorphisms of the PLA2G7 gene were significantly associated with the case–control status, the H92 allele being more frequent and the V379 allele being less frequent in cases than in controls (Table 3). These differences were not significantly heterogeneous between males and females. The genotype distribution of the two PTFAR gene polymorphisms did not significantly differ between the two groups (Table 3).


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  		Table 3. Genotype distribution of PLA2G7 and PTAFR gene polymorphisms in CAD patients and control subjects
 
We next compared haplotype frequencies of the PLA2G7 gene between cases and controls. The five polymorphisms generated six common haplotypes accounting for >95% of the chromosomes observed. The global haplotype distribution significantly differed between cases and controls (P=0.004) (Fig. 3). The difference was mainly due to a lower frequency in cases than in controls (0.18 versus 0.23) of the only haplotype carrying the V379 allele (H2). In a logistic regression analysis, the odds ratio (OR) for CAD associated with this haplotype was estimated as 0.74 (95% CI: 0.60–0.91) (P=0.004) by comparing with haplotype H1 carrying all major alleles (Fig. 3). Comparison of pairs of haplotypes differing by a single substitution (H3 versus H4, H4 versus H5, H3 versus H6) provided a test for the effect of each polymorphism given a fixed haplotypic background. None of these comparisons reached statistical significance. In univariate analyses because only the R92H and the A379V polymorphisms were associated with CAD risk, we examined the association of haplotypes combining these two sites with risk. Owing to complete negative LD, these two polymorphisms generated only three haplotypes, R92A379, H92A379 and R92V379. Taking the first one as the reference, haplotype H92A379 was associated with an increase of risk (OR=1.22, 95% CI: 1.02–1.47, P=0.03) whereas haplotype R92V379 was associated with a decrease of risk (OR=0.80, 95% CI: 0.67–0.97, P=0.02).



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  		Figure 3. ORs for CAD associated with haplotypes of the PLA2G7 gene, by reference to the most common haplotype (H1). Polymorphisms are ordered according to their position in the genomic sequence: T–403C, C–209G, R92H, I198T and A379V. The allele underlined corresponds to the minor allele of each polymorphism. Haplotype frequencies in cases and controls are given in brackets. The {chi}2 test is a test of global association (5 df).

 
Owing to their strong negative LD, the two polymorphisms of the PTAFR gene generated only three common haplotypes accounting for >99% of observed chromosomes. The haplotype distribution did not differ between cases and controls (P=0.41).

Association of PLA2G7 and PTAFR gene polymorphisms with plasma PAF-AH activity
Plasma levels of the PAF-AH activity were measured in all control subjects and in a subsample of the CAD patients (n=945). The effect of each polymorphism on PAF-AH activity was tested individually by analysis of variance adjusted for sex and case–control status (Table 4). Only the PLA2G7/A379V polymorphism was significantly associated with PAH-AH activity, the V379 allele being associated with increased activity in a codominant fashion (P=0.02). A borderline association was also observed with the R92H polymorphism (P=0.055). No significant heterogeneity for association was observed between cases and controls or between males and females for any of the PLA2G7 polymorphisms (Table 4).


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  		Table 4. Mean plasma levels of PAF-AH activity adjusted for sex and case–control status according to PLAG2G7 and PTAFR gene polymorphisms
 
A haplotype-based regression analysis of the PLA2G7 polymorphisms revealed that haplotype 2, carrying the V379 allele, was the only one significantly associated with increased PAF-AH activity (P=0.008) (Fig. 4). In haplotype analysis combining only the R92H and the A379V polymorphisms, haplotype H92A379 had a non-significant effect (P=0.23) when comparing with haplotype R92A379, whereas haplotype R92V379 was associated with an increase of PAF-AH activity (P=0.004), suggesting that only the A379V polymorphism significantly influenced the intermediate phenotype. Mean levels of PAF-AH (log-transformed) according to the A379V polymorphism are shown in Figure 5, separately in cases and controls (P for homogeneity=0.76) and in women and men (P for homogeneity=0.53). Further adjustment on smoking, age and body mass index did not alter the association of the A379V polymorphism with PAF-AH activity (P=0.02 after adjustment for these covariates). Taken together, age, sex, body mass index, smoking and case–control status explained 4.2% of the inter-individual variability of PAF-AH (sex alone contributing to 3.7%), and the A379V subsequently added a 0.8% contribution to this variability.



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  		Figure 4. Mean effects of PLA2G7 gene haplotypes on sex- and status-adjusted levels of PAF-AH activity (log-transformed), by reference to the most common haplotype (H1). Polymorphisms are ordered according to their position on the genomic sequence: T–403C, C–209G, R92H, I198T and V379A. The nucleotide underlined corresponds to the minor allele of each polymorphism. The {chi}2 test is a test of global association (5 df).

 


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  		Figure 5. Mean levels of PAF-AH activity (log-transformed) according to the PLA2G7/A379V polymorphism, separately in cases and controls and in men and women.

 
None of the two PTAFR polymorphisms was associated with PAF-AH activity (Table 4). However, there was a significant interaction between the PTAFR/C–838/ex2ntT and sex (P=0.02). Actually, female carriers of the T allele had higher PAF-AH levels than CC homozygotes (3.64±0.05 versus 3.56±0.02 on log-transformed variable) whereas an opposite difference was seen in males (3.64±0.04 versus 3.72±0.01). Haplotypes of the PTAFR gene were not associated with PAF-AH activity (P=0.27).

Association of PLA2G7 and PTAFR gene polymorphisms with cardiovascular events during the follow-up period
The incidence of cardiovascular events during the follow-up period was lower in CAD patients heterozygous or homozygous for the PLA2G7/V379 allele than in patients homozygous for the A379 allele (P=0.033, Table 5). None of the other PLA2G7 polymorphisms was significantly associated with survival outcome by univariate analysis. A haplotype-based survival analysis revealed that the haplotype carrying the V379 allele (H2) had a borderline protective effect on cardiovascular outcome [hazard risk ratio (HRR)=0.67, 95% CI: 0.44–1.01, P=0.055] (Fig. 6). The haplotype analysis combining the R92H and the A379V polymorphisms indicated that the R92V379 haplotype was associated with a significant protective effect when comparing with the R92A379 haplotype (HRR=0.63, 95% CI: 0.43–0.91, P=0.014) whereas the haplotype H92A379 was not associated with outcome.


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  		Table 5. Frequency of cardiovascular events during follow-up in the cohort of CAD patients according to PLA2G7 and PTAFR gene polymorphisms
 


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  		Figure 6. HRRs of cardiovascular event associated with haplotypes of the PLA2G7 gene, by reference to the most common haplotype (H1). Polymorphisms are ordered according to their position on the genomic sequence: T–403C, C–209G, R92H, I198T and V379A. The nucleotide underlined corresponds to the minor allele of each polymorphism. The {chi}2 test is a test of global association (5 df).

 
The haplotype-based survival analysis of the PTAFR gene indicated a borderline significant association of the haplotype T–838A+503 in comparison with the most common haplotype C–838A+503 (HRR=1.50, 95% CI: 0.98–2.30, P=0.061).

Association of the PLA2G7/A379V polymorphism with cardiovascular risk factors
Given the prominent role of the PLA2G7/A379V polymorphism identified in all analyses, we further investigated whether this polymorphism might affect conventional cardiovascular risk factors. The polymorphism did not significantly influence lipid levels or inflammatory markers (C-reactive protein, fibrinogen, interleukin-6). In cases, it was not associated with diabetes, hypertension, ejection fraction or medication by statin or ACE-inhibitor. Genotype and allele frequencies did not differ between patients with stable and unstable angina. Moreover, none of these factors modified the association between PAF-AH activity and the PLA2G7/A379V polymorphism. In particular, the strong positive correlation between PAF-AH activity and LDL-cholesterol reported in our previous paper (22) did not appear to be modulated by genotype.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 APPENDIX
 REFERENCES
 
The present study reports a comprehensive analysis of the genetic variability of the PLA2G7 and PTAFR genes in relation to three different cardiovascular phenotypes: plasma levels of PAF-AH activity, CAD risk assessed in a case–control setting and risk of future cardiovascular event evaluated in a prospective cohort of CAD patients. The strengths of this study lie in the large sample size of the AtheroGene cohort, its prospective follow-up, the fact that all polymorphisms of functionally important regions of the genes were investigated, and that haplotype-based models were used to relate the different phenotypes to the overall gene variability. Moreover, this study constitutes the first application of haplotype analysis to survival data using a Cox formulation.

Whatever the phenotype considered, all analyses pointed towards a role of the PLA2G7/A379V polymorphism in cardiovascular pathophysiology. Haplotype association analyses were used to distinguish the own effect of each polymorphism from the association due to LD with other polymorphisms of the gene, and all analyses indicated an independent effect of the A379V polymorphism. The V379 allele appeared less frequent in CAD cases than in controls and was associated with a lower risk of future cardiovascular event during the follow-up period. These consistent findings suggest that the V379 allele might confer a protective effect against the development of CAD and its complications. This is in accordance with the result from the HIFMECH study reporting a lower frequency of homozygotes for the V379 allele in MI cases than in controls from different European centers (26). In the HIFMECH study, only VV homozygotes were at lower risk of MI, whereas AV heterozygotes were at similar risk as AA homozygotes, a finding at variance with the present study showing a decreased risk in heterozygotes as well. This discrepancy might be explained by the difference in the phenotypes studied (i.e. MI versus coronary angiography defined atherosclerosis). It might be speculated that the protective effect of the V379 allele against acute complications of atherosclerosis, such as MI, depends on a threshold effect in PAF-AH activity.

In vitro functional experiments have shown that the V379 allele resulted in decreased affinity of PAF-AH to its substrate (PAF), and therefore this variant was postulated to prolong the pro-inflammatory activities of PAF in plasma (25). In accordance with this pro-inflammatory effect, the latter study reported an association of the V379 allele with increased susceptibility to atopy and asthma (25). However, given the dual role played by PAF-AH in inflammatory mechanisms, it is also conceivable that the V379 allele might have a protective effect against atherosclerosis by attenuating the release of products of LDL oxidation into the arterial wall, this effect being more prominent than the reduced degradation of PAF.

In the present study, the V379 allele was also associated with an increased plasma PAF-AH activity, consistently observed in men and women and in cases and controls. The findings that the V379 allele was associated both with a decreased CAD risk and with an increased PAF-AH activity would suggest a protective effect of the enzyme. This might appear, however, in contradiction to the fact that plasma levels of PAF-AH are generally increased in CAD patients (16–18), although it is still unknown whether this increase has a causal role by itself or is simply a marker of risk. The elevation of PAF-AH might alternatively reflect an adaptive mechanism to disease. Anyway, the A379V polymorphism explained <1% of the variability of plasma PAF-AH activity after adjustment for main confounders, hence this weak effect was unlikely to explain the effect on risk. It might be rather speculated that the protective effect, instead of being mediated by quantitative variations of plasma enzyme activity, is explained by a qualitative modification of the functionality of the protein, compatible with the fact that A379V polymorphism modifies the protein sequence.

After controlling for LD with the A379V polymorphism, the R92H polymorphism was associated with CAD risk but not with survival or PAF-AH activity. The H92 allele has been shown in an experimental assay to have an increased substrate inhibition compared with the R92 enzyme form, but substrate concentrations used in the assay were far above physiological concentrations of PAF measured in plasma (25). In the latter study, the R92H polymorphism was not associated with asthma or atopy (25). Further studies are needed to confirm if the R92H polymorphism has an effect independent of that of the A379V polymorphism. Another possibility might be that the haplotype H92A379 confers a different functionality to the enzyme.

None of the other polymorphisms of the PLA2G7 gene influenced CAD risk or PAF-AH activity. Variability of the PTAFR gene also did not appear to have an important effect. Only the PTAFR/C–838/ex2ntT polymorphism was found associated with a moderate effect on PAF-AH activity, but in opposite ways in men and women. None of the polymorphisms was associated with lipids or inflammatory markers.

Some aspects of the present study deserve discussion. First, PAF-AH activity was measured only in a subsample of the cohort of cases, preventing us from studying its association with the prospective outcome. Second, other enzymes such as paraoxonase have similar substrate specificities to PAF-AH and might contribute to PAF-AH activity in plasma. However, an argument against this possibility stems from the observation that the serum from a donor carrying the V279F inactivating mutation did not hydrolyze oxidized phospholipids or PAF, yet displaying full paraoxonase activity (32). Third, haplotype-based models assumed an additive effect of haplotypes on phenotype. We cannot preclude the possibility that some haplotypic effect remained undetected because of this assumption. However, the fact that the A379V polymorphism was detected by both single-locus and haplotype analyses reinforces the confidence in the effect of this polymorphism.

In conclusion, this study combining a case–control and a prospective approach showed a protective effect of the V379 allele of the PLA2G7 gene on the risk of CAD and its complications. This allele was also associated with a weak increase of plasma PAF-AH activity that was unlikely to explain by itself the protective effect on risk. It is more likely that the A379V polymorphism has a qualitative effect by modifying the enzyme function towards a more anti-atherogenic form. Polymorphisms of the PTAFR gene were not related to any phenotype.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
APPENDIX
 REFERENCES
 
Study population
Detailed description of the study has been provided elsewhere (29,30). Between November 1996 and July 2000, 1318 CAD patients were recruited at the Department of Medicine II of the Johannes Gutenberg-University Mainz and the Bundeswehrzentralkrankenhaus Koblenz at the occasion of a diagnostic coronary angiography. Inclusion criterion was the presence of a diameter stenosis >30% in at least one major coronary artery. Exclusion criteria were evidence of significant concomitant diseases, in particular, hemodynamic valvular heart disease, known cardiomyopathy and malignant diseases, as well as febrile condition. Patients were followed up during a median of 3.9 (maximum 5.2) years. Follow-up information was obtained about death from cardiovascular causes (n=89), death from non-cardiovascular causes (n=26) and non-fatal MI (n=54). Information about the cause of death or clinical events was obtained by the hospital or the general practitioner.

Healthy control subjects (n=485) were recruited either from general practitioners' offices in the course of a routine check-up visit or by newspaper announcement. The newspaper announcement described briefly the study design and invited healthy German individuals aged ≥40 years to participate in the AtheroGene study as control subjects. Of the individuals who presented, those without any clinical or anamnestic evidence of a history of atherosclerosis and without evidence of any pathological ECG pattern were selected. All individuals who presented received results of classical and treatable risk factors for personal use later.

Study participants had German nationality and were inhabitants of the Rhein-Mainz area. The study was approved by the ethics committee of the University of Mainz. Participation was voluntary and each study subject gave written informed consent.

Laboratory methods
PAF-AH activity was measured from plasma stored at –80°C by the trichloroacetic acid precipitation procedure in 96-well plates as previously described (22,33). Samples were measured in duplicate. A pool of 10 control plasma samples served as an internal standard for all measurements. The within-assay variability was <5%.

Serum lipid levels (total cholesterol, Roche Diagnostics, Germany; HDL-cholesterol, Rolf Greiner Biochemica, Flacht, Germany; LDL-cholesterol, calculated according to the Friedewald formula; triglycerides, Roche Diagnostics) were determined immediately. C-reactive protein was determined by a highly sensitive, latex particle enhanced immunoassay (Roche Diagnostics), fibrinogen by derived method, and interleukin-6 by ELISA technique (EASIATM, Biosource Europe, Fleurus, Belgium) according to the manufacturer instructions.

Screening of genes and genotyping of polymorphisms
Screening of PLA2G7 and PTAFR genes for detection of polymorphisms was performed by comparing 190 chromosomes from 95 unrelated MI patients from the ECTIM study (34). The method of detection was PCR/SSCP followed by direct sequencing. Polymorphisms identified were then genotyped in the SIPLAC study by allele-specific oligonucleotides in order to estimate allele frequencies and pairwise LD coefficients. SIPLAC is a subsample of the ECTIM study (n=600) used as a first step to select the subset of polymorphisms that will be further genotyped in larger studies after exclusion of rare polymorphisms and polymorphisms in complete association (see our web site GeneCanvas http://www.genecanvas.org for a description). Polymorphisms selected at this step were genotyped in the AtheroGene study using the TaqMan technology [Applied Biosystem (ABI), Warrington, UK]. Briefly, PCR primers and TaqMan MGB probes were designed using Primer Express version 2.0. Reactions were performed in 96-well microplates with GeneAmp 9700 thermal cyclers. Fluorescence was measured using an ABI Prism 7000 sequence detection system and analyzed with the ABI Prism 7000 SDS software version 1.0. Primer and probe sequences as well as amplification conditions for genotyping can be found at our web site GeneCanvas http://www.genecanvas.org.

Statistical analysis
Allele frequencies were estimated by gene counting. Pairwise LD coefficients were estimated by log-linear analysis (35). Departure from Hardy–Weinberg equilibrium was tested by a {chi}2 test with 1 df. Single-locus association between polymorphisms and case–control status was tested by a {chi}2 test. Association of polymorphisms with plasma levels of PAF-AH activity was tested by ANOVA adjusted for sex and case–control status after having tested the homogeneity of effects between cases and controls and between men and women. PAF-AH activity was log-transformed to remove positive skewness and geometric means (95% CI) were reported. Association of polymorphisms with prospective outcome was tested by Cox regression analysis. The combined endpoint considered was death from cardiovascular cause and non-fatal MI.

We performed haplotype-based association analyses of the PLA2G7 and PTAFR genes with the different phenotypes (case–control status, PAF-AH activity and survival outcome). Haplotype analyses for binary and continuous phenotypes were performed by use of our previously described model (36) implemented in the SEM algorithm (31) and extended to survival analysis using a Cox formulation for the present analysis. This method allows one to estimate haplotype frequencies as well as haplotype effects by comparison with a reference haplotype, assuming additive effects of haplotypes on phenotype. Haplotype effects (95% CI) are expressed as ORs for a binary trait, mean effects for a continuous trait and HRRs for a survival outcome. The reference haplotype was chosen as the most frequent one. A global test of association between haplotypes and the phenotype considered was performed by a likelihood ratio test ({chi}2 with m–1 df in the case of m haplotypes). It is also possible, by setting appropriate constraints on parameters, to compare the effects between any pair of haplotypes, in particular those differing only by one nucleotide substitution ({chi}2 with 1 df). Effects associated with rare haplotypes (frequency<0.02) were not estimated and fixed to 0.

In all analyses, P<0.05 was considered to be significant. Except for haplotype analyses, all analyses were carried out with the SAS software version 8.01 (SAS Institute Inc., Cary, NC, USA).


   ACKNOWLEDGEMENTS
 
This work was supported by a grant of the ‘Stiftung Rheinland-Pfalz für Innovation,’ Ministry for Science and Education (AZ 15202-386261/545), Mainz, by the MAIFOR grant 2001 of the Johannes Gutenberg-University Mainz, Germany, and by grants from the Fondation de France (no. 2002005211 and 2002004994).


   APPENDIX
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 APPENDIX
REFERENCES
 
The AtheroGene Group: Hans-Jürgen Rupprecht, Stefan Blankenberg, Christoph Bickel, Christine Espinola-Klein, Jürgen Meyer (Department of Medicine II), Karl J. Lackner, Gerd Hafner (Institute of Laboratory Medicine and Clinical Chemistry): Johannes Gutenberg-University, Mainz, Germany. Laurence Tiret, Odette Poirier, Tiphaine Godefroy, Claire Perret, Viviane Nicaud, Jean-Louis Georges, David-Alexandre Tregouet, François Cambien: INSERM U525, Faculté de Médecine Pitié-Salpêtrière, Paris, France. AtheroGene recruitment centers: Department of Medicine II, Johannes Gutenberg-University Mainz; Bundeswehrzentralkrankenhaus, Koblenz, Germany.


   FOOTNOTES
 
* To whom correspondence should be addressed at: INSERM U525, Faculté de Médecine Pitié-Salpêtrière, 91 bd de l'Hôpital, 75634 Paris cedex 13, France. Tel: +33 140779725; Fax: +33 140779768; Email: ninio{at}chups.jussieu.fr


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 APPENDIX
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