Functional characterisation of ABCA1 variation

Abstract

Low levels of high-density lipoprotein cholesterol (HDL-C) are an independent risk factor for the development of cardiovascular disease (CVD). The ATP-binding cassette transporter A1 (ABCA1) is a major genetic determinant of circulating HDL-C level. ABCA1 is a membrane bound transporter that is crucial to the formation of mature HDL particles though its role in facilitating efflux of cellular cholesterol and phospholipids onto lipid-poor apoA-I protein. Mutations in ABCA1 are associated with low HDL-C levels, Tangier Disease and familial hypoalphalipoprotienemia (FHA). The major aim of this study was to determine the impact of ABCA1 variation on expression and function. Six ABCA1 mutations, identified in local low HDL-C subjects; A594T, I659V, Y1767D, R2004K, A2028V and Q2239N, were characterised in vitro using HEK293 cells transfected with ABCA1-GFP cDNA expression vectors. Functional testing, using cholesterol efflux assays, showed the mutants to have varying degrees of perturbed efflux function and confocal microscopy indicated many of the mutant proteins to be mislocalised. A further three ABCA1 mutations; C978fsX988, T1512M and N1800H, were identified and characterised within the context of a FHA pedigree. Cholesterol efflux assays, ex vivo using primary fibroblasts and in vitro using transfected HEK293 cells, showed all three mutations to impair efflux function and to be associated with disrupted sub-cellular localisation. The level of C978fsX988 mRNA was shown to be negligible in the fibroblast cultures, which precluded a dominant negative effect of the truncated allele on full-length ABCA1 alleles. As mislocalisation correlated with impaired function for many of the mutants, the effect of the chemical chaperone, sodium 4-phenylbutyrate (4-PBA), was investigated. Treatment with 4- PBA improved localisation and efflux function for the majority of mutants in transfected HEK293 cells. This was associated with an increase in the level of ABCA1-GFP protein, likely due to stimulation of the ABCA1 expression vector CMV promoter by 4-PBA. Functional testing using primary skin fibroblasts from the available mutants however, showed an improved efflux function without any increase in ABCA1 protein expression. The prevalence of two ABCA1 promoter polymorphisms, C-14T and G-278C was determined in a local population, where the -14T variant and the -14T, -278C haplotype were found to be associated with increased HDL-C level. Functional analysis of the two polymorphisms in isolation and in combination showed cell type specific effects on transcriptional activity, with a heightened response of the -14T genotype to agonist stimulation in HepG2 cells compared to RAW264.7 cells.

Finally, comparison of the lipid profile for subjects with low and normal HDL-C levels revealed low HDL-C to be associated with triglyceride-enrichment of apoB-containing lipoproteins, particularly very low-density lipoprotein (VLDL) and in extreme cases of FHA, low-density lipoprotein (LDL) also. The results of this study highlight that ABCA1 variation manifests in alternate ways, dependent on the specific nature of the alteration and the resulting influence on ABCA1 regulation. Mutations in ABCA1 were found to have variable effects on expression, sub- cellular localisation and cholesterol efflux function. It was shown that correction of mislocalisation by a chemical chaperone was sufficient to rescue function for many ABCA1 mutants and may be relevant in the development of therapies to increase HDL-C level and reduce CVD risk. The impact of promoter polymorphisms on ABCA1 was discrete between cell types and the disproportionate response of the -14T promoter variant in liver cells supported the observed association with increased HDL-C level. It can be concluded that ABCA1 is controlled by a complex regulatory system that is influenced by rare coding mutations, common promoter polymorphisms and pharmacological agents. Further, the observed relationship of HDL-C level with an altered composition of apoB-containing lipoproteins likely contributes to the CVD risk that is associated with reduced HDL-C levels.