Flow cytometric assessment of LDL ligand function for detection of heterozygous familial defective apolipoprotein B-100.

BACKGROUND Familial defective apolipoprotein (apo) B-100 (FDB) is caused by a mutation in the apoB gene and characterized by decreased binding of LDL to LDL receptors because of reduced function of the apoB-100 ligand. FDB may be associated with severe hypercholesterolemia and cannot always be distinguished from familial hypercholesterolemia phenotypically. METHODS We used a fluorescence flow cytometry assay with Epstein-Barr virus-transformed lymphocytes to detect reduced LDL ligand function by competitive binding with fluorescently conjugated LDL (DiI-LDL). The assay was tested and validated using LDL from patients heterozygous for the Arg(3500)-Gln mutation and their first-degree relatives. Knowing the actual apoB genotype of patients and relatives allowed us to assess the ability of the assay to predict the results of DNA analysis. The results were compared to measurements of LDL ligand function in unrelated healthy control subjects to characterize functionally the Arg(3500)-Gln mutation. RESULTS Fluorescence was significantly increased in cells incubated with DiI-LDL in competition with unlabeled LDL from FDB(R3500Q) heterozygotes compared with cells incubated with DiI-LDL in competition with unlabeled LDL from relatives or unrelated healthy control subjects. Thus, patients heterozygous for the Arg(3500)-Gln mutation had significantly reduced LDL ligand function. The binding affinity of LDL from FDB(R3500Q) heterozygotes was 32% of that in non-FDB relatives and healthy controls. The assay had a diagnostic sensitivity of 0.95 and diagnostic specificity of 0.89. CONCLUSIONS The diagnostic accuracy of the assay was too low to allow reliable diagnosis of individual cases of heterozygous FDB(R3500Q). However, fluorescence flow cytometry may supplement genetic identification of FDB and functionally characterize gene mutations associated with major reductions in LDL ligand function.