Characterization of selected LDLR substitutions in patients with familial hypercholesterolemia

Familial hypercholesterolemia is a genetic disorder caused by pathogenic or likely pathogenic variants in four key genes: LDLR, APOB, PCSK9, and APOE. It leads to elevated levels of low-density lipoprotein cholesterol in the bloodstream and significantly increases the risk of coronary artery disease. This study aimed to functionally characterize LDLR variants identified in Polish FH patients. Experimental data were used to learn about variants' phenotypes and incorporate them into the ACMG/AMP variant classification framework.

The functional analysis was performed using the HEK293T-ldlrG1 cells with the expression vectors pTetRedLDLR carrying the mutated LDLR gene variants. Receptor expression was evaluated using Western blot and immunofluorescence. The low-density lipoprotein uptake and ligand binding capacity were examined with fluorescent dye-labeled LDL by confocal microscopy. A functional study was performed to analyze the variants under assessment and compare them to known benign and pathogenic control variants.

The experimental study revealed an impaired activity of the c.662A > G p. (Asp221Gly), c.1775G > A p. (Gly592Glu), and c.2483delA p. (Tyr828Phefs∗101) LDLR variants, classifying them as functionally abnormal. In contrast, in vitro activity assessment of the c.91G > A p. (Glu31Lys) LDLR variant showed fully functional low-density lipoprotein binding and uptake activities. These results suggested that c.91G > A p. (Glu31Lys) is unlikely to be a disease-causing variant.

The results provide functional evidence for the activity of selected LDLR variants in a cellular model based on confocal techniques that meets the ACMG/AMP variant classification criteria. These findings highlight the importance of in vitro assays in evaluating the functional impact of LDLR variants and contribute valuable insights for clinical interpretation and genetic counseling.