Abstract 1: Molecular Basis of Hypertriglyceridemia Associated with the APOA5 c553.G>T SNP

Abstract

Objectives Apolipoprotein (apo) A-V is a low abundance protein with profound effects on plasma triglyceride (TG) levels. Several APOA5 SNPs correlate with hypertriglyceridemia (HTG). The c.553 G>T SNP, substituting a Cys for Gly at position 162 of mature apoA-V, is prevalent in Asian populations and correlates with HTG. To investigate the mechanism underlying this association, gene transfer studies were performed in apoa5-/- mice. Methods Adeno-associated virus (AAV2/8) harboring the coding sequence for wild type apoA-V (AAV2/8-apoA-V), G162C apoA-V (AAV2/8-G162C) and LacZ (AAV2/8-LacZ) were injected (1x10e12 virus genome) into the tail vein of 8 apoa5-/- mice per group. Blood samples were collected weekly for 4 weeks and TG and apoA-V levels measured. FPLC was performed on plasma obtained from AAV2/8-apoA-V and AAV2/8-G162C mice. VLDL, LDL, HDL and the lipoprotein-free region were characterized. Results Compared to AAV2/8-LacZ mice, AAV2/8-apoA-V mice had significantly lower plasma TG levels (50% ±5). Unlike AAV2/8-apoA-V, mice injected with AAV2/8-G162C displayed little or no reduction in TG despite similar amounts of plasma apoA-V protein. Immunoblot analysis of FPLC fractionated plasma revealed that, whereas wild-type apoA-V was lipoprotein associated (VLDL and HDL), G162C apoA-V was largely recovered in the lipoprotein-free fraction. Immunoblot analysis following SDS-PAGE under non-reducing conditions revealed that lipoprotein-associated wild type apoA-V and G162C apoA-V are monomeric; by contrast, the electrophoretic mobility of G162C apoA-V recovered in the lipoprotein-free fraction was retarded. Conclusions Gene transfer of wild type apoA-V induces a significant reduction in plasma TG levels of apoa5-/- mice. By contrast, G162C apoA-V failed to induce a corresponding decrease in plasma TG, recapitulating effects observed in human populations harboring this SNP. The propensity of G162C apoA-V to form a disulfide bond with one or more plasma proteins interferes with its lipoprotein binding ability, resulting in loss of function. The results provide a molecular explanation for HTG associated with a common APOA5 SNP. The gene transfer strategy employed provides a platform for studies of other common apoA-V SNPs.