A unique strain of pigs, exhibiting inherited plasma hypercholesterolemia, associated with the development of atherosclerosis, which resembles the human disease, will be used in this project. The goals of this proposal are to; identify genetic variants in apolipoproteins, carry out biochemical and physiological studies and molecular analysis of these variants and determine their contribution to plasma cholesterol variations and atherosclerosis when fed a standard or elevated fat diet.
The aims are: 1) Produce alloantibodies recognizing ApoE, CII and AI variants associated with hypercholesterolemia; search for DNA markers at apoB, S, AI, E, CII loci, and test the cosegregation of DNA markers with the allotypes and lipid phenotypes; 2) Using pigs of defined allotypes, DNA genotypes and hypercholesterolemia phenotypes, carry out multigeneration studies by outcross and intercross matings to obtain informative segregation data for examining the contribution of candidate loci variants to hypercholesterolemia and atherosclerosis; 3) Test gene-diet effect on plasma cholesterol variations in respective homozygous pigs using a diet with 17% of common food fats; 4) Use immunosorbers with available apolipoprotein epitope specific alloantibodies to separate monoallelic LDL populations from plasma of pigs heterozygous for the normolipidemic and hypercholesterolemic phenotypes, characterize their lipid and lipoprotein composition and concentrations to obtain an estimate of the heritable differential contribution of each variant to the plasma hyperlipidemia; 5) Characterize Apo-E, CII and AI, including amino acid analysis and protein sequencing of fragments containing the polymorphic sites; map apoE, CII and AI loci; and perform histological and immunohistochemical analysis of aorta and coronary arteries from pigs with different lipid phenotypes, and DNA and apoprotein genotypes; 6) continue propagation and preservation of pig-lines with identified apolipoprotein variants; Thirty litters of pigs, obtained from planned matings will be produced yearly, and multiple plasma samples per animal collected. Physiochemical and immunochemical methods, alloantibodies for Apo-B epitopes and isospecific antibodies for Apo-B, E and AI will be used to phenotype and genotype apolipoproteins of the experimental pigs. Southern blot analyses will be used to detect DNA markers. Analysis of variance will be used to calculate the significance of differences in lipid and apolipoprotein levels between the genotypes.
