Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia
Dennis Norman, Xi-Ming Sun, Mafalda Bourbon, Brian L. Knight, Rossitza P. Naoumova, Anne K. Soutar
Dennis Norman, Xi-Ming Sun, Mafalda Bourbon, Brian L. Knight, Rossitza P. Naoumova, Anne K. Soutar
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Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia

Abstract

Familial hypercholesterolemia (FH) is characterized by a raised concentration of LDL in plasma that results in a significantly increased risk of premature atherosclerosis. In FH, impaired removal of LDL from the circulation results from inherited mutations in the LDL receptor gene or, more rarely, in the gene for apo B, the ligand for the LDL receptor. We have identified two unrelated clinically homozygous FH patients whose cells exhibit no measurable degradation of LDL in culture. Extensive analysis of DNA and mRNA revealed no defect in the LDL receptor, and alleles of the LDL receptor or apo B genes do not cosegregate with hypercholesterolemia in these families. FACS® analysis of binding and uptake of fluorescent LDL or anti–LDL receptor antibodies showed that LDL receptors are on the cell surface and bind LDL normally, but fail to be internalized, suggesting that some component of endocytosis through clathrin-coated pits is defective. Internalization of the transferrin receptor occurs normally, suggesting that the defective gene product may interact specifically with the LDL receptor internalization signal. Identification of the defective gene will aid genetic diagnosis of other hypercholesterolemic patients and elucidate the mechanism by which LDL receptors are internalized.

Authors

Dennis Norman, Xi-Ming Sun, Mafalda Bourbon, Brian L. Knight, Rossitza P. Naoumova, Anne K. Soutar

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Time course of internalization of transferrin by cultured lymphoblasts. ...
Time course of internalization of transferrin by cultured lymphoblasts. Cultured lymphoblasts (0.5 × 107 cells/mL) were incubated at 4°C for 60 minutes in RPMI-1640 medium containing 125I-labeled transferrin (0.2 μg/mL, specific activity = 1,198 dpm/ng of protein). Excess unlabeled transferrin was then added (10 μg/mL), and the cells were incubated for the indicated times at 37°C. At each time point, the medium was removed, and the cells were washed with 0.2 M acetic acid, 0.5 M NaCl (pH 2.4) to remove surface-bound transferrin and then were solubilized in 1 M NaOH (15). Radioactivity in the medium (transferrin released into the medium), acid wash (surface-bound transferrin), and solubilized cells (internalized transferrin) was determined and expressed as a percentage of the total radioactivity at zero time. Values shown are means of triplicate incubations. At zero time, the total amount of labeled transferrin associated with the cells (ng of transferrin per mg of cell protein) was as follows: FH-1, 41.2; FH-2, 18.6; FH hmz-E387K, 32.3; normal control, 19.3.