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 b y 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 m ay be associated with severe hypercholesterolemia and cannot always be dist inguished from familial hypercholesterolemia phenotypically. Methods: We used a fluorescence flow cytometry assay with Epstein-Barr viru s-transformed lymphocytes to detect reduced LDL ligand function by competit ive binding with fluorescently conjugated LDL (DiI-LDL). The assay was test ed and validated using LDL from patients heterozygous for the Arg(3500)-Gln mutation and their first-degree relatives. Knowing the actual apoB genotyp e of patients and relatives allowed us to assess the ability of the assay t o predict the results of DNA analysis. The results were compared to measure ments of LDL ligand function in unrelated healthy control subjects to chara cterize functionally the Arg(3500)-Gln mutation. Results: Fluorescence was significantly increased in cells incubated with D iI-LDL in competition with unlabeled LDL from FDBR3500Q heterozygotes compa red with cells incubated with DiI-LDL in competition with unlabeled LDL fro m relatives or unrelated healthy control subjects. Thus, patients heterozyg ous for the Arg(3500)-Gln mutation had significantly reduced LDL ligand fun ction. The binding affinity of LDL from FDBR3500Q 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 reli able diagnosis of individual cases of heterozygous FDBR3500Q. However, fluo rescence flow cytometry may supplement genetic identification of FDB and fu nctionally characterize gene mutations associated with major reductions in LDL ligand function. (C) 2000 American Association for Clinical Chemistry.