Functional analysis from ex-vivo characterization of LDLR exon 13-15 duplication associated to familial hypercholesterolemia.

Background: Familial hypercholesterolemia (FH) is an inherited semidominant disorder characterized by high plasma cholesterol levels and increased risk of premature cardiovascular disease. More than 3,000 low-density lipoprotein receptor (LDLR) variants have been identified, most lack functional evidence to determine their pathogenicity. One of them is the exon13_15dup, the most frequent FH-causing variant in Chile. However, its functional impact is poorly understood. Objective: To determine the functional impact of the exon13_15dup variant in the LDLR, in familial hypercholesterolemia patients. Methods: Three heterozygous carriers of an exon 13-15 duplication and five wild type subjects were recruited. The peripheral blood mononuclear cells were isolated and differentiated to macrophages. The LDLR expression levels on the cell membrane were evaluated by flow cytometry, subcellular localization by confocal microscopy and LDL incorporation by LDL-FITC uptake assays. Results: The exon13_15dup variant leads to significantly increased cell-surface LDLR expression and enhanced localization in the endoplasmic reticulum. This results in a reduced capacity for LDL uptake in patient cells, with principal component analysis highlighting distinct differences in LDLR localization compared to wild-type samples. Conclusions: The functional analysis showed that the mutation affects the proper transport and function of LDLR, resulting in a dysfunctional protein that cannot effectively internalize LDL.