Validation of LDLr Activity as a Tool to Improve Genetic Diagnosis of Familial Hypercholesterolemia: A Retrospective on Functional Characterization of LDLr Variants

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by high blood-cholesterol levels mostly caused by mutations in the low-density lipoprotein receptor (LDLr). With a prevalence as high as 1/200 in some populations, genetic screening for pathogenic LDLr mutations is a cost-effective approach in families classified as 'definite' or 'probable' FH and can help to early diagnosis. However, with over 2000 LDLr variants identified, distinguishing pathogenic mutations from benign mutations is a long-standing challenge in the field. In 1998, the World Health Organization (WHO) highlighted the importance of improving the diagnosis and prognosis of FH patients thus, identifying LDLr pathogenic variants is a longstanding challenge to provide an accurate genetic diagnosis and personalized treatments. In recent years, accessible methodologies have been developed to assess LDLr activity in vitro, providing experimental reproducibility between laboratories all over the world that ensures rigorous analysis of all functional studies. In this review we present a broad spectrum of functionally characterized missense LDLr variants identified in patients with FH, which is mandatory for a definite diagnosis of FH.

Keywords: Low Density Lipoprotein receptor (LDLr); familial hypercholesterolemia; functional validation; in vitro; is silico; variants.

Figure 1

Domain organization of LDLr and LDLr pathway and its dysregulation by defective mutations. (A) Schematic representation of LDLr domains; (B) LDLr cycle. LDLr is synthesed at ER, transproted to Golgi where is further processed with glycosilations. Mature LDLr is transported to the plasma membrane, where the ligand-binding domain binds to the apo B100 moiety on LDL particles. The LDLr/LDL complex undergoes endocitosis and within the cell, LDL particle components are targeted for lysosomal degradation, whereas the LDLR is recycled to the cell surface. LDLr mutations affecting different LDLr cycle results in dysregulation of the cycle.

Figure 2

Flowchart of the used methodologies to functionally characterize LDLr variants ex vivo and in vitro. Functional studies of LDLr variants are mainly conducted using two major approaches: 1. ex vivo methods, using cells from Familial Hypercholesterolemia (FH) patients (left-hand panel); 2. in vitro methods using cell lines transfected with the LDLr variant (right-hand panel). LDLr activity determination is based in combination of different methodologies: Western blot to analyse LDLr expression followed by fluorescence-activated cell sorting (FACS) and Confocal Laser Scanning Microscopy (CLSM) that allow assessment of Class type mutation. The ex vivo approach is adequate for Class 1, Class 2a and Class 3 LDLr variants. In vitro characterization allows identification of Class 2b mutations by colocalizing the LDLr variants in the ER with calrgulin; using a solid-phase immunoassay it is possible to determine LDLr-LDL EC₅₀ values for Class 3 mutations which is important to understand mild pathogenic variants; Class 4 variants are classified by complementing CLSM with a colocalization assay with clathrin and, identification of Class 5 mutants is performed by absence of LDLr colocalization with calregulin, LDLr colocalization with a lysosome marker complemented by a FACS analysis of LDL binding to LDLr at different pH (7.4–5.2).