ABCA1 TRANSPORTER IN MONOGENIC DISORDERS OF CELL CHOLESTEROL METABOLISM: TANGIER DISEASE AND FAMILIAL HYPERCHOLESTEROLEMIA.

Background: ABCA1 is an ubiquitous plasma membrane transporter that promotes the efflux of cholesterol and phospholipids from the cell membrane to an extracellular acceptor, known as apolipoprotein A-I (apoAI), the main protein constituent of plasma high density lipoprotein (HDL). ABCA1 mediated cholesterol/phospholipids efflux leads to the extracellular formation of HDL. Loss of function mutations of both ABCA1 alleles is the cause of Tangier Disease (TD), a recessive disorder characterized by extremely low levels of plasma HDL, increased risk of cardiovascular disease (CAD) and other clinical manifestations. Heterozygous mutations of ABCA1 gene cause Familial HDL Deficiency (FHD), characterized by half normal level of HDL-C and increased CAD risk. Familial Hypercholesterolemia (FH) is a monogenic disorder due to mutations of LDLR gene. In addition to the striking elevation of plasma LDL, FH patients (especially homozygotes) have greatly reduced plasma HDL levels, suggesting that they may have a defect in ABCA1-mediated cholesterol efflux pathway. Aim: Specific aims of this work were: i) the identification and functional characterization of several new ABCA1 mutations found in TD and FHD; ii) the study of ABCA1 regulation in fibroblasts derived from FH patients. Methods: ABCA1 gene was analyzed in 27 subjects with familial low HDL-C levels, by DHPLC and direct sequencing. Among the novel 22 mutations identified, 3 splice site mutations, 6 missense mutations and 1 frameshift mutation were investigated. The effect of splice site mutations was investigated ex-vivo, using patients’ fibroblasts or blood mononuclear cells, or in vitro by looking at transcripts generated by the expression of a mutant ABCA1 minigene in a heterologous cell system. ABCA1 protein expression was studied in eight FH derived fibroblast cell lines under different conditions: basal culture conditions, stimulation with free cholesterol (FC), 22-hydroxycholesterol/9cis-Retinoic Acid (22OH/9cRA), or the LXR agonist T0901317. Cholesterol efflux to ApoA-I was also measured in these fibroblasts. Main results: 22 novel ABCA1 mutations were reported. The effect of 3 splice site mutations (c.814-14InsA, c.2961-2A>C, c.4773+1G>A) was investigated with an ex-vivo or with a minigene approach. The c.814-14InsA resulted to be non-pathogenetic, the c.2961-2A>C resulted in the generation of three abnormally spliced mRNA species, the c.4773+1G>A caused the formation of an ABCA1 protein containing an in-frame deletion, which was poorly synthesized by cells. Five additional fibroblast cell lines derived from TD or FHD patients were studied in terms of ABCA1 protein expression and function. Fibroblasts carrier of R587W, A1046D, D1099Y, H1600R/M586Fs.629X, R130K/N1800H showed a null ABCA1-mediated cholesterol efflux. ABCA1 protein expression was studied in eight FH fibroblasts under different stimuli. Under basal culture conditions ABCA1 expression resulted lower in FH cells with respect to control cells. This reduction was observed also after stimulation of cells with a load of FC or with 22OH/9cRA, known to stimulate ABCA1 expression via the transcription factor LXR. Treatment of cells with a non-steroidal LXR agonist (T0901317) was able to restore an ABCA1 expression similar to that of control fibroblasts. Also ABCA1-mediated cholesterol efflux in FH fibroblasts was lower than in control fibroblasts. Conclusions: Among 27 individuals with low HDL-C, 22 novel mutations in ABCA1 gene were identified. Three splice site, 6 missense and 1 frameshift mutation were functionally characterized in TD/FHD derived cells, allowing us to gain insight on genotype-phenotype correlations. Furthermore we demonstrated a defect in ABCA1 protein expression and function in FH, which may be partly responsible for the low plasma HDL-C levels exhibited by FH patients.