Determination Of The Functionality Of Common Apoa5 Polymorphisms

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 -1131T--> C, -3A--> G, 751G--> T, and 1891T--> C;
and APOA5*3, distinguished by the c56C--> G (S19W). Molecular modeling of the
apoAV signal peptide (SP) showed an increased angle of insertion (65 degrees ) at
the lipid/water interface of Trp-19 SP compared with Ser-19 SP (40 degrees ),
predicting reduced translocation. This was confirmed by 50% reduction of
Trp-19-encoded SP.secretory alkaline phosphatase (SEAP) fusion protein secreted
into the medium from HepG2 cells compared with the Ser-19.SEAP 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 -3A--> G in the Kozak sequence, in vitro
transcription/translation assays and primer extension inhibition assays showed no
alternate AUG initiation codon usage, demonstrating that -3A--> G did not
influence translation efficiency. Although 1891T--> C in the 3'-untranslated
region disrupts a putative Oct-1 transcription factor binding site, when inserted
3' of the luciferase gene the T--> 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.