Abstract 555: Functional Characterization Uncovered Novel Loss-of-function Mutations in ABCA1

Objectives: ABCA1 encodes the membrane protein ATP-binding cassette transporter A1 (ABCA1), a pivotal player in nascent HDL formation via its ability to facilitate cholesterol and phospholipid efflux to apolipoprotein A-I (ApoA-I). ABCA1 variants are frequently found in subjects with primary hypoalphalipoproteinemia, however, their pathogenicity and causal link with the clinical phenotype are not always known. Methods: In silico analysis (Mutation Assessor, PANTHER, PolyPhen-2, PROVEAN, SIFT, and VEST) were performed to predict the functional consequences of ABCA1 missense variants found in our cohort of hypoalphalipoproteinemia. A subset of novel ABCA1 variants were generated in vitro through site-directed mutagenesis and their abilities in mediating lipid efflux to apoA-I were determined using standard methods. Results: A total of 32 mutations in ABCA1 were identified, among which 15 were classified as missense, 9 as nonsense or frameshift, 7 as intronic, and 1 as ”no-protein”. We selected 5 variants that were labeled as pathogenic or possibly pathogenic by in silico analysis to conduct functional studies. Two newly identified mutations in ABCA1, a nonsense mutation (p.E1005X) and a missense mutation (p.S2046R), resulted in complete loss of the canonical lipid efflux function of ABCA1 (2.5% and 1.8% of wild type cholesterol efflux level respectively). These results were concordant with the phenotypic characteristics of the carriers. Three additional mutations (p.G750W and p.R1341T and p.I1085F) resulted in only a partial loss of function (66-75% of wild type cholesterol efflux level). These results were somewhat discordant with the phenotype of the heterozygote carriers (HDL-C levels of 16, 14 and 38 mg/dl respectively), suggesting the presence of additional causal factors. Conclusions: These results support E1005X and S2046R as ABCA1 loss-of-function mutations and highlight the need to conduct functional studies on unknown variants to determine their pathogenicity.