The use of gene expression microarray analysis has become an effective means to identify the cause of monogenic disorders. Combined with genetic mapping data, ABC1 was seen to be the most likely candidate among the set of underrepresented RNA species from 2 patients with TD. Had the study been performed with material from a message-null patient, it should have been more straightforward to single out this gene. Such analysis also includes potentially useful data concerning the set of genes that are differentially regulated between normal and disease cells. This may help understand the pathophysiology of the disease and identify other genes that act in the same or in compensatory pathways with the primary defect.
Identification of the gene defect in TD should contribute to the elucidation of the pathway of cellular cholesterol efflux that is involved in the accumulation of sterols in phagocytic cells and that contributes to the pathology of atherosclerosis, the major cause of death in industrialized nations. Two distinguishable mechanisms operate in cellular cholesterol efflux whose relative importance depends on cell type and metabolic state (reviewed in refs. 3
). Aqueous diffusion allows bidirectional exchange of cholesterol between cell membranes and HDL particles. This exchange may occur primarily at surface microdomains known as caveolae (31
). Net efflux can be driven by conversion of cholesterol in the extracellular compartment to cholesteryl ester by the action of LCAT. In contrast, the process of apolipoprotein-mediated lipid efflux dominates in macrophages and other scavenger cells when they are cholesterol-loaded and/or growth-arrested. In most cell types, cholesterol content is tightly controlled by feedback regulation of LDL receptors and biosynthetic enzymes (32
). However, macrophages and other cells that contain scavenger receptors accumulate cholesterol in an unregulated manner (33
). Vast stores of internal cholesterol result in conversion to a foam cell phenotype, which is believed to be a major contributor to the development of vascular lesions in atherosclerosis. Although the 2 efflux pathways may operate in concert in certain conditions, the massive accumulation of intracellular cholesterol in the reticuloendothelial system in patients with TD emphasizes the importance of the apolipoprotein-mediated pathway of cholesterol efflux in such cells.
Members of the ABC transporter family are generally composed of 4 domains: 2 hydrophobic domains, each containing 6 transmembrane segments; and 2 hydrophilic nucleotide binding domains containing highly conserved Walker A and B sequence motifs typical of many ATPases (34
). To date, more than 30 members of this family have been identified in the human genome. Most human chromosomes contain at least 1 ABC family member, with no high degree of clustering yet detected (35
). ABC1 now joins other members of this gene family that are associated with diseases including cystic fibrosis, intrahepatic cholestasis, adrenoleukodystrophy, Zellweger’s peroxisomal syndrome, and Stargardt’s ocular disease (15
The accumulated evidence establishes that ABC1 plays a pivotal role in the cellular apolipoprotein-mediated lipid removal pathway. Two pharmacological agents reported to inhibit the activity of ABC1 also inhibited apolipoprotein-mediated sterol efflux from cultured cells. Antisense oligonucleotides had a similar effect, causing a marked reduction in cholesterol efflux. Conversely, transfection of murine macrophages with an ABC1
cDNA expression plasmid led to a severalfold increase in apo A-I–mediated sterol efflux. In addition, expression of ABC1 was induced by incubation conditions previously shown to enhance apolipoprotein-mediated lipid efflux, including serum deprivation, cholesterol loading, and cAMP treatment (3
). Inducible ABC1 was expressed on the cell surface, consistent with a direct role in transport of cellular lipids across the plasma membrane to apolipoproteins. It is possible that ABC1 also resides in intracellular membranes such as the Golgi, where it may play a role in shuttling cholesterol and/or phospholipids between compartments and the cell surface. Taken together, these results suggest that ABC1 is the rate-controlling protein in the pathway of apolipoprotein-mediated lipid transport.
Our studies also provide evidence that mutations in ABC1 are responsible for the impaired lipid efflux in TD. We sequenced DNA from 3 unrelated patients with TD. Two contained charge-altering codon substitutions, at positions conserved in the highly similar mouse ABC1 sequence. These 2 examples occur within 10 residues of one another, in the predicted NH2-terminal hydrophilic domain of the protein. Further studies will seek to identify the possible substrate or protein interactions with this domain and to determine whether this domain of ABC1 is located on the extracellular or intracellular side of the cell membrane. One can only speculate why the expression of ABC1 mRNA was reduced in TD1 and TD2 cells. One allele might not be expressed at all. Alternatively, as has been noted in other genetic diseases, point mutations may result in reduced expression of RNA, either by a mechanism that couples translation to message stability or by the accumulation of mutations/polymorphisms in the control regions of genes that have become nonfunctional. In the case of TD3, insertions in each allele encode proteins that are truncated before their second nucleotide binding domain.
As the likely rate-controlling agent in the pathway of apolipoprotein-mediated lipid removal, ABC1 holds the possibility of increasing cellular cholesterol efflux. A concomitant effect on plasma HDL and the overall flux of cholesterol from peripheral sites to the liver for excretion is a subject for testing in animal models. This study of ABC1 provides a point of departure for characterizing the apolipoprotein-mediated lipid removal pathway as a possible target for the pharmacological treatment of atherosclerosis.
Note added in proof.
Since completion of this work, mutations in the ABC1
gene of patients with TD have been independently reported by other laboratories (36
). The patient we refer to as TD1 is the same individual referred to as TD-2 in ref. 36