Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment

Abstract

Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1∶500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of 125I-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with 125I-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the 125I methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than 125I-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations.

Identification of the (Pro)renin Receptor as a Novel Regulator of Low-Density Lipoprotein Metabolism

“…To test this we made use of LDLA7 cells, which is a CHO-derived cell line that lacks functional LDLR resulting in strongly diminished uptake of LDL. 19,35 Similar to the other cell lines, silencing (P)RR expression reduced SORT1 abundance in LDLA7 cells, and vice versa, implying that a functional (P)RR-SORT1 interaction does not require the presence of the LDLR ( Figure 3C). Overall LDL uptake in LDLA7 cells is low, but nevertheless silencing (P) RR or SORT1 significantly reduced LDL uptake in these cells.…”

The importance of an integrated analysis of clinical, molecular, and functional data for the genetic diagnosis of familial hypercholesterolemia

“…In silico analysis was also not conclusive for the majority of the alterations, having assessed correctly only 7/13 alterations. So, functional study is essential to determine a variant's pathogenicity, and these studies can be performed by any research laboratory with access to a flow cytometer and a confocal microscopy since the protocol has been published, 24 or collaborations can be established. Nevertheless, when the lipid and molecular profiles of affected and nonaffected relatives are known, this information is useful for the pathogenicity assessment of a variant, and it should always be taken into consideration.…”

“…Cell transfection and semiquantitative immunoblotting were performed as described before. 24 ldl isolation and lipoprotein labeling LDL was isolated from 3 ml serum samples from healthy individuals using two-step centrifugation (Supplementary Methods online). LDL was labeled with fluorescein isothiocyanate as previously described.…”

“…25 Quantification of ldlr expression and activity by flow cytometry LDLR cell surface expression and LDL binding and uptake were determined by flow cytometry, specifically fluorescenceactivated cell sorting, as previously described. 24 confocal laser scanning microscopy Confocal laser scanning microscopy was used to analyze LDLR expression and intracellular colocalization of LDLR, as described before 24 (Supplementary Methods online).…”

Low-density lipoprotein receptor mutational analysis in diagnosis of familial hypercholesterolemia

“…The major difference is that radioactive iodine is replaced by fluorescent labeling of the LDL particle to follow the cycle [28].…”