Lipoprotein lipase (LPL) is a key enzyme in the metabolism of triglyceride- rich and high density lipoproteins. Defective functional enzyme is the diagnostic feature of a rare autosomal recessive syndrome, familial LPL deficiency. We have already identified several mutations of the gene in LPL deficient subjects and shown that the heterozygous state for LPL deficiency (LPLn) accounted for familial hypertriglyceridemia (HTG) in a large pedigree. We intend to pursue a systematic genetic and biochemical investigation of LPL in classical deficiency states as well as in a variety of clinical contexts where the pathophysiological significance of HTG is not understood, including coronary heart disease, essential hypertension, diabetes, pregnancy-induced hypertension or diabetes, and acute pancreatitis, recruiting subjects through multiple sources in both Salt Lake City and San Francisco. To achieve these scientific objectives, we propose to: (1) identify mutations of the LPL gene in familial LPL deficiency patients by sequencing LPL exons and intron-exon boundaries; (2) search for mutations of LPL in a large series of HTG subjects in various clinical contexts, screening for known mutations by dot-blot hybridization and for unidentified mutations by direct detection of nucleotide variants (single strand conformation polymorphism or chemical mismatch detection) and DNA sequencing; (3) confirm the functional significance of these mutations by in vitro mutagenesis and expression in mammalian cells; (4) identify the functional domain affected by each mutation by testing catalytic activity with particulate and soluble substrates, affinity for lipid, apolipoprotein C-II and heparin, and self-association to a dimer; (5) perform in vitro expression at residues where substitutions have been identified to investigate structure-function relationships of the domains involved.
