Wootton, PTE; (2006) Genetic and functional studies of lipoprotein-associated phospholipase A2 and secretory phospholipase A2 variants: Their potential role in atherosclerosis. Doctoral thesis , UCL (University College London).

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Abstract

The aim of this thesis was to investigate the role of Lipoprotein-associated PLA2 (Lp-PLA2, PLA2G7) and two secretory PLA2 enzymes (IIA and V, PLA2G2A and PLA2G5) in the progression of atherosclerosis. All three enzymes may be implicated in the oxidative modification of lipoproteins, and the release/removal of powerful inflammatory mediators. The first objective of this thesis was to try and resolve the contribution of Lp-PLA2 to CHD. The relationship between statins and PLA2G7 expression was also investigated. Secondly, the association of haplotypes in the PLA2G2A and PLA2G5 genes with lipid markers and SPLA2-IIA mass levels was determined for the first time. PLA2G7 Using SSCP analysis, the PLA2G7 promoter was screened for novel polymorphisms. A G to A change was identified 1230 base pairs 5' of the transcription start site (rare allele frequency of 0.22), and between potential RORalpha and OCT1 transcription factor binding sites. The G-1230A and activity-altering A379V polymorphisms, were then investigated in several studies with regards to measures of Lp-PLA2 activity, LDL particle oxidation, CHD risk, body composition, and other intermediate phenotypes related to the enzyme's potential role in atherosclerosis. In the prospective NPHS II study, quartiles of Lp-PLA2 activity showed no association with CHD risk (p=0.37 after adjustment for age and sex). However, the 379V allele was associated with higher Lp- PLA2 activity (p=0.05) and specific activity (p=0.001). In the EPIC-Norfolk case-control study, higher Lp-PLA2 activity was associated with higher CHD risk (p<0.0001), although adjustment for LDL removed this association (p=0.45). In agreement with the NPHS II study, the 379V allele was associated with a higher Lp-PLA2 activity (p=0.03). In the UDAC study of Caucasian diabetic men and women, individuals with the metabolic syndrome (MS) were associated with higher Lp-PLA2 activity compared to those without (p=0.02). There was also an inverse relationship of Lp-PLA2 activity and oxLDL/LDL (p=0.03). There was no significant association of the A379V genotype with either Lp-PLA2 enzyme activity (p=0.34) or oxLDL/LDL levels (p=0.32). The A379V allele was not found to be associated with CHD risk in any of the investigated studies to<0.06). Finally, the association of the A379V variant with body composition changes was investigated in a longitudinal study of 123 male Caucasians over 10 weeks of physical training (BH2 study). After exercise training, the 379V allele was associated with a decrease in percentage adipose tissue mass (p=0.01) and increase in percentage lean mass (p=0.01) compared to the other genotype groups. The results presented in this thesis raise further questions about the causal relationship of the Lp- PLA2 enzyme with CHD and its suitability as a clinical marker of disease. In parallel to these investigations, in vitro work was performed to determine whether the novel G-1230A variant influenced gene transcription. Human liver Huh-7 cells were transiently transfected with different G-1230A reporter constructs driven by the PLA2G7 promoter, and assayed for differences in luciferase activity. No difference in activity was observed between the -1230 alleles over six experiments (p=0.94). In addition, the novel G-1230A SNP was not associated with enzyme activity (p=0.77) and oxLDL/LDL levels (p=0.07) in the UDAC study as well as % adipose tissue and % lean mass (p=0.91 and p=0.92 respectively) in the BH2 study. The G-1230A variant was not associated with CHD risk (p>0.22 in all models) in the case-control HIFMECH study. Taken together, these in vitro and epidemiological analyses suggest that the novel G-1230A variant does not represent a putatively functional variant. The previously observed association of lower Lp-PLA2 activity with statin use in UDACS (p=0.04) was further investigated in vitro. Human macrophages of known A379V genotype were exposed to Simvastatin for 48hrs. Overall there was a 17.6% and 27.2% reduction in gene expression (RT-PCR) when treated with 10//M and 25//M of Simvastatin, respectively (p<0.01). There was no heterogeneity of effect between A379 or 379V homozygotes (p=0.96). This reduction in expression suggests that the pleiotropic effects of statins may independently influence PLA2G7 expression PLA2G2A and PLA2G5 Using publicly available re-sequencing data, tagging SNPs (tSNP) and common haplotypes were identified in the UDAC study for the PLA2G2A and PLA2G5 genes. Overall, haplotypic variation in the PLA2G2A gene was associated with significant effects on sPLA2 HA mass levels (p<0.0001). This study represents the first investigation of genetic variation in the PLA2G2A and PLA2G5 genes with regards to atherosclerotic risk traits and SPLA2-IIA levels. (Abstract shortened by UMI.).

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