Phd in INNOVATIVE BIOMEDICAL TECHNOLOGIES IN CLINICAL MEDICINE

Thesis title: FUNCTIONAL CHARACTERIZATION OF VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) IN PATIENTS WITH HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA (HeFH) USING A FLOW CYTOMETRY ASSAY

ABSTRACT Background: Familial hypercholesterolemia (FH) is an autosomal lipid disorder increasing cardiovascular risk. It is primarily caused by pathogenic variants in the low-density lipoprotein receptor (LDLR) gene. However, some variants are classified as variants of uncertain significance (VUS), which presents a challenge in diagnosing FH. Functional tests are recommended to confirm variant pathogenicity. Aim: To assess the functional impact of VUS in LDLR identified in FH patients by using a flow-cytometry technique. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 33 FH patients carrying VUS LDLR and from three normocholesterolemic individuals. PBMCs isolated from three FH patients carrying well established functional LDLR variants served as positive controls. These cells were stimulated with CD3/CD28 beads in a lipoprotein-deficient serum (LPDS) and the entire LDLR cycle functionality (cell surface expression, LDL binding, and uptake) was assessed by using flow-cytometry. Results were expressed as the ratio of the mean fluorescence intensity (gMFI) of activated CD4 T-lymphocytes to gMFI of wild-type controls. Measurements were repeated three times and presented as median (25th-75th percentiles). The American College of Medical Genetics and Genomics (ACMG) guidelines were followed to classify variant function as normal or abnormal based on thresholds of >95% and <85% activity of wild-type levels. Results: Out of 19 unique VUS tested, 14 (73.7%) showed deleterious effects on at least one of the LDLR functions. The c.1530_1532del, c.*34C>T and c.1007A>G variants disrupted the entire LDLR cycle. The remaining variants exhibited defects in LDLR expression on the cell surface localization (n=5 - 35.7%) or a combination of binding/expression or binding/uptake (n=6 - 42.8%). Thus, 28 out of 33 FH-VUS patients (85%) showed at least one defect in LDLR function. Interestingly, these patients had slightly higher untreated LDL-C levels than those with non-defective variants (220 ± 72.8 vs. 180.1 ± 31.9 mg/dl; P=0.06). Furthermore, VUS with proven abnormal functionality were more prevalent among patients with a Dutch Lipid Clinic Network (DLCN) score ≥6 (81.5%), suggesting that DLCN may serve as a valuable predictor of biological effects. Conclusions: This study illustrates the significance of functional testing for LDLR variants to clarify biological effects and include VUS in FH diagnosis.