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Flow cytometric assessment of LDL ligand function for detection of heterozygous familial defective apolipoprotein
B Raungaard1, F Heath, P S Hansen
1Department of Internal Medicine and Cardiology, Aarhus Amtssygehus University Hospital, Tage Hansens Gade 2, DK-8000 Aarhus C, Denmark. rau@dadlnet.dk
Insights
Familial defective apolipoprotein B-100 (FDB) diagnosis can be aided by a new fluorescence flow cytometry assay. This method detects reduced LDL ligand function, but its diagnostic accuracy is currently too low for individual FDB diagnoses.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Familial defective apolipoprotein B-100 (FDB) is a genetic disorder caused by apoB gene mutations.
- It leads to reduced low-density lipoprotein (LDL) binding to LDL receptors, potentially causing severe hypercholesterolemia.
- FDB can be phenotypically indistinguishable from familial hypercholesterolemia.
Purpose of the Study:
- To develop and validate a fluorescence flow cytometry assay for detecting reduced LDL ligand function.
- To functionally characterize the Arg(3500)-Gln mutation in FDB.
- To assess the diagnostic accuracy of the assay for FDB.
Main Methods:
- Utilized a fluorescence flow cytometry assay with Epstein-Barr virus-transformed lymphocytes.
- Employed competitive binding with fluorescently conjugated LDL (DiI-LDL) to measure LDL ligand function.
- Validated the assay using LDL from patients with the Arg(3500)-Gln mutation and their relatives, comparing results to healthy controls.
Main Results:
- The assay detected significantly reduced LDL ligand function in patients heterozygous for the Arg(3500)-Gln mutation.
- LDL binding affinity in FDB(R3500Q) heterozygotes was 32% of that in controls.
- The assay demonstrated a diagnostic sensitivity of 0.95 and specificity of 0.89.
Conclusions:
- The fluorescence flow cytometry assay's diagnostic accuracy is insufficient for reliable individual FDB diagnosis.
- However, the assay can supplement genetic identification of FDB.
- It offers a method to functionally characterize gene mutations impacting LDL ligand function.
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.
