Elevated levels of low density lipoprotein cholesterol are recognized risk factors for premature heart disease. Although genetic factors are involved, the cause of the majority of cases of hypercholesterolemia is unknown. Apolipoprotein B100 is the ligand of low density lipoproteins responsible for receptor mediated clearance of these lipoproteins which transport more than 60% of plasma cholesterol. It is well-established that aberrant forms of the low density lipoprotein receptor derived from mutant alleles of the receptor can be responsible for impaired low density lipoprotein binding. The overall aim of this proposal is to test the proposition that defective apo-B100 resulting from mutations in the apo-B100 can also disturb this ligand-receptor binding and lead to defects in cholesterol metabolism. The proposal outlines approaches to detecting and defining human and mouse apo-B100 gene mutations. The genomes of patients with defective low density lipo-protein will be screened for mutations leading to changes in the receptor binding domain of apo-B100 or to abnormal levels of plasma LDL. These mutation will be localized, sequenced by rapid recently developed methods, and analyzed by comparison to the recently completed human apo-B100 gene sequence. In parallel, many inbred mouse strains, including those known to be sensitive or resistant to diet induced atherosclerosis, will be screened for naturally occurring mutations that affect low density lipoprotein metabolism. In addition, transgenic animals will be produced by infecting various plasmid constructs into early mouse embryos; these constructs are designed to elevate or depress low density lipoprotein levels by adding extra copies of the apo-B100 gene or by inhibiting transcription of endogenous genes by means of anti-sense RNA. These experiments are designed to produce mouse models of human diseases of lipid metabolism. Because these considerations raise the issue as to the mechanism of the control of apo-B100 gene expression, a detailed characterization of the promoter region of this gene is planned. Sites for binding of control proteins will be mapped by nuclease digestion and direct binding methods and correlated with mutations that affect activity in vivo and in cells transfected with chimeric gene constructs.
Showing the most recent 10 out of 11 publications
