Optimized flow cytometry assay for functional characterization of variants of uncertain significance in familial hypercholesterolemia

Background: The definitive diagnosis of Familial Hypercholesterolemia (FH) relies on the identification of deleterious variants in FH-related genes, but many detected variants are classified as of uncertain significance (VUS). This study aims to evaluate the functional impact of LDLR VUS using an optimized flow cytometry assay in autologous activated CD4+ T lymphocytes. Methods: Twenty unique LDLR variants were tested. Six known pathogenic variants served as positive controls, while T lymphocytes from normocholesterolemic individuals were used as negative controls. Peripheral blood mononuclear cells were stimulated with CD3/CD28 beads in lipoprotein-deficient serum. LDLR expression, LDL binding, and uptake were quantified by flow cytometry using Bodipy-labeled LDL and expressed as the geometric mean fluorescence intensity ratio between patient and wild-type CD4+ T cells. Following ClinGen FH Expert Panel Specifications guidelines variants were classified as defective if activity was <85% of wild-type in at least one parameter. For a VUS subset, LDLR activity was further tested by site-directed mutagenesis and optical microscopy. Results: At flow cytometry assay, 15 variants (75%) showed deleterious effects in at least one functional LDLR parameter; the others affected expression (n = 5; 35.7%) or binding/expression or binding/uptake (n = 6; 42.8%); only 4 (21.5%) impaired the entire LDLR cycle. Site-directed mutagenesis and/or microscopy of selected variants [c.(-97)G > A, c.367T > C, c.929T > A, c.1007A > G, c.1530_1532del, c.1945C > T, c.2282C > T, c.2479G > A, c.∗34C > T], confirmed impaired LDLR activity, supporting the flow cytometry findings. Conclusions: This optimized flow cytometry assay provides robust functional assessment of LDLR VUS, improving definitive FH diagnosis and supporting its inclusion in FH diagnostic workflows.