Abstract
BackgroundSecretory phospholipase A2 group IIA (sPLA2-IIA) has been identified as a biomarker of atherosclerosis in observational and animal studies. The protein is encoded by the PLA2G2A gene and the aim of this study was to test the functionality of two PLA2G2A non-coding SNPs, rs11573156 C>G and rs3767221 T>G where the rare alleles have been previously associated with higher and lower sPLA2-IIA levels respectively.Methodology/Principal FindingsLuciferase assays, electrophoretic mobility shift assays (EMSA), and RNA expression by RT-PCR were used to examine allelic differences. For rs3767221 the G allele showed ∼55% lower luciferase activity compared to the T allele (T = 62.1 (95% CI 59.1 to 65.1) G = 27.8 (95% CI 25.0 to 30.6), p = 1.22×10−35, and stronger EMSA binding of a nuclear protein compared to the T-allele. For rs11573156 C >G there were no luciferase or EMSA allelic differences seen. In lymphocyte cell RNA, from individuals of known rs11573156 genotype, there was no allelic RNA expression difference for exons 5 and 6, but G allele carriers (n = 7) showed a trend to lower exon 1–2 expression compared to CC individuals. To take this further, in the ASAP study (n = 223), an rs11573156 proxy (r2 = 0.91) showed ∼25% higher liver expression of PLA2G2A (1.67×10−17) associated with the G allele. However, considering exon specific expression, the association was greatly reduced for exon 2 (4.5×10−5) compared to exons 3–6 (10−10 to 10−20), suggesting rs11573156 G allele-specific exon 2 skipping.ConclusionBoth SNPs are functional and provide useful tools for Mendelian Randomisation to determine whether the relationship between sPLA2-IIA and coronary heart disease is causal.
Highlights
Elevated levels of secretory phospholipases show association with several diseases such as coronary heart disease (CHD), rheumatoid arthritis and asthma [1,2,3]
The genotype effect of rs10732279 on PLA2G2A liver mRNA is presented in Figure 3a; compared to the common A homozygotes, carriers of the rare G allele had roughly 25% higher expression of PLA2G2A (p = 1.67610217)
The expression results were confirmed by the results from the Advanced Study of Aortic Pathology (ASAP) study which showed that the SNP rs10732279, acting as proxy for rs11573156, was associated with significantly lower differential expression of exon 2 compared to the following four exons of the gene, but significantly increased total mRNA expression across all exons
Summary
Elevated levels of secretory phospholipases (sPLA2s) show association with several diseases such as coronary heart disease (CHD), rheumatoid arthritis and asthma [1,2,3]. SPLA2 enzymes further contribute to atherogenesis by hydrolysing the outer phospholipid layer of low density lipoprotein (LDL) particles in the circulation, generating smalldense LDL (sd-LDL) particles which can transverse the endothelial cell layer of the artery wall into the intima, where they are further modified [7]. This modification increases the propensity of the particles to aggregate and bind to proteoglycans, resulting in aggregation of sd-LDL in the intima. The protein is encoded by the PLA2G2A gene and the aim of this study was to test the functionality of two PLA2G2A non-coding SNPs, rs11573156 C.G and rs3767221 T.G where the rare alleles have been previously associated with higher and lower sPLA2-IIA levels respectively
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