Determination of the Functionality of Common APOA5 Polymorphisms

Abstract

Common variants of APOA5 have consistently shown association with differences in plasma triglyceride (TG) levels. These single nucleotide polymorphisms (SNPs) fall into three common haplotypes: APOA5*1, with common alleles at all sites; APOA5*2, with rare alleles of ؊1131T3 C, ؊3A3 G, 751G3 T, and 1891T3 C; and APOA5*3, distinguished by the c56C3 G (S19W). Molecular modeling of the apoAV signal peptide (SP) showed an increased angle of insertion (65°) at the lipid/water interface of Trp-19 SP compared with Ser-19 SP (40°), predicting reduced translocation. This was confirmed by 50% reduction of Trp-19-encoded SP⅐secretory alkaline phosphatase (SEAP) fusion protein secreted into the medium from HepG2 cells compared with the Ser-19⅐SEAP fusion protein (p < 0.002). Considering APOA5*2 SNPs, there was no significant difference in the relative luciferase expression in Huh7 cells transiently transfected with a ؊1131T construct compared with the ؊1131C (fragments ؊1177 to ؊516 or ؊1177 to ؊3). Similarly, for the ؊3A3 G in the Kozak sequence, in vitro transcription/translation assays and primer extension inhibition assays showed no alternate AUG initiation codon usage, demonstrating that ؊3A3 G did not influence translation efficiency. Although 1891T3 C in the 3-untranslated region disrupts a putative Oct-1 transcription factor binding site, when inserted 3 of the luciferase gene the T3 C change demonstrated no significant difference in luciferase expression. Thus, association of APOA5*2 SNPs with TG levels is not due to the individual effects of any of these SNPs, although cooperativity between the SNPs cannot be excluded. Alternatively, the effect on TG levels may reflect the strong linkage disequilibrium with the functional APOC3 SNPs.There is strong evidence from clinical studies, epidemiological studies, and animal models (reviewed in Ref. 1) that apoAV plays a key role in triglyceride (TG) 1 metabolism. Transgenic and knock-out mouse models identified an inverse relationship between apoAV and plasma TG levels (2). APOA5 variants determine between individual differences in plasma TG in all ethnic groups studied to date (3, 4). The role of apoAV in TG metabolism is further supported by the recently identified rare mutation Q145X, introducing a premature termination of apoAV and resulting in apoAV deficiency and severe hypertriglyceridemia (5). However the exact function(s) of apoAV remain to be clarified. APOA5 expression is up-regulated in liver regeneration after rat hepatectomy (6), suggesting that it acts as a break on very low density lipoprotein assembly (7). ApoAV is found preferentially on high density lipoprotein but is thought to transfer to very low density lipoprotein in the postprandial state (8) and has been shown to activate lipoprotein lipase in vitro and in vivo (9).The APOA5 gene itself is relatively polymorphic. The five most common APOA5 haplotypes can be defined by seven SNPs (10). Two haplotypes, APOA5*2 and APOA5*3, are of particular interest as they show association with raised plasma TG leve...

Plasma apolipoprotein A5 and triglycerides in type 2 diabetes

“…The present study was too small to perform meaningful haplotype analyses, but it has been suggested that the minor allele of the APOA1 promoter polymorphism is on the same haplotype as the minor allele of c56C→G, which could provide an explanation for the observed relationship [30]. The S19W (c56C→G) polymorphism located in the signal peptide is predicted to have an effect on APOA5 gene transcription [33], whereas results from in vitro expression studies suggested that the −1131T→C may not be a functional polymorphism [33]. However, we do find a significant association with plasma APOA5 levels, although in the opposite direction to the c56C→G polymorphism.…”

A single nucleotide polymorphism in APOA5 determines triglyceride levels in Hong Kong and Guangzhou Chinese

“…4 A peroxisome proliferator response element is present in the promoter region of APOA5, 42 but a functional effect of this SNP has not been demonstrated in studies in a luciferase reporter assay. 43 Instead, its effect could be due to strong linkage with functional variants in the nearby APOC3 gene. 43,44 In conclusion, our study shows convincingly in two large independent populations that one SNP only, namely rs662799, is a major determinant of plasma triglyceride levels in Chinese, independent of other known determinants of triglyceride levels.…”

“…43 Instead, its effect could be due to strong linkage with functional variants in the nearby APOC3 gene. 43,44 In conclusion, our study shows convincingly in two large independent populations that one SNP only, namely rs662799, is a major determinant of plasma triglyceride levels in Chinese, independent of other known determinants of triglyceride levels. As hypertriglyceridaemia is recognized as a risk factor for coronary heart disease and stroke, 45 this SNP may influence the susceptibility of the individual to cardiovascular disease.…”

Protease Inhibitor-Associated Dyslipidemia in HIV-Infected Patients Is Strongly Influenced by the APOA5–1131T→C Gene Variation

“…Because APOA5 expression is downregulated by SREBP1c (20 ), we assume that apo A-V plays a determining role in PI-induced hyperlipidemia. Whether the -1131C allele is a functional variant or is acting as a marker of a functional variant elsewhere in the gene is not clear (21 ).…”