Identification of a novel Arg-->Cys mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia...

We previously carried out genetic and metabolic studies in a partially inbred herd of pigs carrying cholesterol-elevating mutations. Quantitative pedigree analysis indicated that apolipoprotein (apo)B and a second major gene were responsible for the hypercholesterolemia in these animals. In this study, we assessed LDL receptor function by three different methods: ligand blots of liver membranes using beta-very low density lipoprotein (VLDL) as a ligand; low density lipoprotein (LDL)-dependent proliferation of T-lymphocytes; and direct binding of 125I-labeled LDL to cultured skin fibroblasts. All three methods demonstrated that LDL receptor ligands bound with decreased affinity to the LDL receptor in these animals. In skin fibroblasts from the hypercholesterolemic pigs, the Kd of binding was about 4-fold higher than in cells from normal pigs. The cDNA of the pig LDL receptor from normal and hypercholesterolemic pigs was isolated and sequenced. We identified a missense mutation that results in an Arg'Cys substitution at the position corresponding to Arg94 of the human LDL receptor. The mutation is in the third repeat of the ligand binding domain of the receptor. By single-stranded conformational polymorphism (SSCP) analysis, we studied the relationship between LDL receptor genotype and plasma cholesterol phenotype. In contrast to humans, the hypercholesterolemia associated with the LDL receptor mutation in pigs was expressed as a recessive trait. The LDL receptor mutation made a far more significant contribution to hypercholesterolemia than did the apoB mutation, consistent with observations made in human subjects with apoB mutations. Within each genotypic group (mutated apoB or mutated receptor), there was a wide range in plasma cholesterol. As the animals were on a well-controlled low-fat diet, this suggests that there are additional genetic factors that influence the penetrance of cholesterol-elevating mutations.