Abstract 1179: Identification of a Novel Lipid Efflux Defect Causing Low HDL Cholesterol that is not due to Mutations in the ABCA1 Gene but to Regulation of ABCA1 Protein
HDL deficiency is the most frequent abnormality in atherosclerotic cardiovascular disease. Mutations in several genes have been identified as causing HDL deficiency, the most common are mutations in ABCA1, apoA 1 and LCAT. In a cohort of French-Canadian patients with severe HDL-c deficiency (< 5th percentile of the population) we have identified 42 subjects in whom mutations in ABCA1, ApoA 1 and LCAT have been excluded. We carried out cellular lipid efflux assays in skin fibroblasts from the patients to identify individuals in whom the low HDL phenotype is due to impaired cellular cholesterol metabolism. We loaded the fibroblasts with free cholesterol (24 h) and subsequently incubated them with lipid-free apoA 1 (12 h) to stimulate cellular lipid efflux. The assay results indicated two patients in which phospholipid efflux is defective in response to cholesterol stimulation (20ug/mL), while cholesterol efflux is normal. The extent of the phospholipids efflux defect is similar to the defect observed in ABCA1 mutation heterozygote subjects. Stimulation with the physiological oxysterols - 22-, 25-, and 27-hydroxycholesterol, and 24,25 epoxycholesterol (2.5 ug/mL for all) normalized the defective phospholipids efflux response in patient cells. To investigate the cause of the efflux defect in the patients, we utilized real time RT-PCR and Western blot approaches. We investigated the expression of ABCA1 mRNA in response to both cholesterol stimulation and oxysterol stimulation. We observed no difference in ABCA1 mRNA expression between normal control and patient fibroblasts in response to either cholesterol or oxysterol stimulation. Interestingly, Western blot results showed that ABCA1 protein expression is upregulated in response to both cholesterol and oxysterol stimulation in normal control cells but in patient cells only oxysterol stimulation resulted in increased ABCA protein expression, whereas cholesterol stimulation did not. This identifies a new lipid efflux defect in humans that is characterized by defective phospholipid efflux but normal cholesterol efflux. The defect is not due to mutations in the ABCA1 gene but rather to impaired ABCA1 protein regulation.