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

We previously carried out genetic and metabolic studies in a partially inbr ed herd of pig carrying cholesterol-elevating mutations. Quantitative pedig ree analysis indicated that apolipoprotein (apo)B and a second major gene w ere responsible for the hypercholesterolemia in these animals. In this stud y, we assessed LDL receptor function by three different methods: ligand blo ts of liver membranes using beta-very low density lipoprotein (VLDL) as a l igand; low density lipoprotein (LDL)-dependent proliferation of T-lymphocyt es; and direct binding of I-125-labeled LDL to cultured skin fibroblasts, A ll three methods demonstrated that LDL receptor ligands bound with decrease d affinity to the LDL receptor in these animals. In skin fibroblasts from t he hypercholesterolemic pig, the K-d of binding was about 4-fold higher tha n in cells from normal pig, The cDNA of the pig LDL receptor from normal an d hypercholesterolemic pigs was isolated and sequenced, We identified a mis sense mutation that results in an Arg-->Cys substitution at the position co rresponding to Arg(94) Of the human LDL receptor. The mutation is in the th ird repeat of the ligand binding domain of the receptor. By single-stranded conformational polymorphism (SSCP) analysis, we studied the relationship b etween LDL receptor genotype and plasma cholesterol phenotype, In contrast to humans, the hyper-cholesterolemia associated with the LDL receptor mutat ion in pig was expressed as a recessive trait. The LDL receptor mutation ma de a far more significant contribution to hypercholesterolemia than did the apoB mutation, consistent with observations made in human subjects with ap oB mutations. Within each genotypic group (mutated apoB or mutated receptor ), there was a wide range in plasma cholesterol, As the animals were on a w ell-controlled low-fat diet, this suggests that there are additional geneti c factors that influence the penetrance of cholesterol-elevating mutations. -Grunwald, K. A. A., K, Schueler; P. J. Uelmen, B. A Lipton, M. Kaiser, K. Buhman, and A. D. Attie. Identification of a novel Arg->Cys mutation in th e LDL receptor that contributes to spontaneous hypercholesterolemia in pig.