Abstract 1347: Cellular Physiology of Cholesterol Efflux Defects in Low HDL-c Individuals with a Normal ABCA1 Gene
Currently, HDL deficiency due to genetic causes is attributed to mutations in three genes- LCAT, Apo A1 and ABCA1. These genes, however, do not account for the majority of cases of low HDL. In the search for new genes in the HDL metabolic pathway, we have identified 42 French-Canadian subjects with severe HDL-c deficiency (<5th percentile of the population) in whom mutations in LCAT, Apo A1 and ABCA1 have been excluded (via candidate gene sequencing, haplotyping and linkage analysis approaches). To further identify individuals in whom the low HDL phenotype is due to defective HDL biogenesis, cellular lipid efflux assays were performed in skin fibroblasts. Fibroblasts were loaded with free cholesterol and incubated with lipid-free apoA 1 (24 h) to stimulate cellular lipid efflux. The assay results indicated three probands in whom both cholesterol and phospholipid efflux are defective (75% and 70% of normal controls). To investigate the genes causing the efflux defect in the probands, we applied RNA from these cells to Affymatrix gene chips. RNA was isolated from individual fibroblasnts subjected to the following conditions: (1) basal with no apo A1; (2) FC+apoA 1; (3) 22 hydroxycholesterol (22OH)+ 9 cis retinoic acid (RA)+ apoA 1. Results from the microarray analyses demonstrated that the major cholesterol regulatory pathways - the SREBP and LXR pathways - were regulated normally in the probands. Several intracellular cholesterol transport genes, however, including the recently characterized oxysterol binding protein (OSBP) and the steroidogenic acute regulatory proteins (STARD-5 and STARD-12), were differentially regulated versus normal controls. We are presently conducting further cellular efflux assays under various conditions (LDL-c loading, FC loading, 22OH/RA stimulation), isolating the mRNA and protein of various intracellular cholesterol transport genes, and using custom-made gene chips and RT-PCR approaches to isolate the particular gene defect(s). Identification of new genes involved in HDL metabolism may lead to the discovery of novel control points in the intracellular HDL cholesterol regulatory pathways and to the development of more effective HDL-raising therapies .