Abstract
Background and aimsPathogenic mutations in the Low Density Lipoprotein Receptor gene (LDLR) cause Familial Hypercholesterolemia (FH), one of the most common genetic disorders with a prevalence as high as 1 in 200 in some populations. FH is an autosomal dominant disorder of lipoprotein metabolism characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis. To date, 2500 LDLR variants have been identified in the LDLR gene; however, only a minority of them have been experimentally characterized and proven to be pathogenic. Here we investigated the role of Cys46 located in the first repeat of the LDL receptor binding domain in recognition of apolipoproteins.MethodsActivity of the p.(Cys46Gly) LDLR variant was assessed by immunoblotting and flow cytometry in CHO-ldlA7 expressing the receptor variant. Affinity of p.(Cys46Gly) for LDL and VLDL was determined by solid-phase immunoassays and in silico analysis was used to predict mutation effects.Results and conclusionFunctional characterization of p.(Cys46Gly) LDLR variant showed impaired LDL and VLDL binding and uptake activity. Consistent with this, solid-phase immunoassays showed the p.(Cys46Gly) LDLR variant has decreased binding affinity for apolipoproteins. These results indicate the important role of Cys46 in LDL receptor activity and highlight the role of LR1 in LDLr activity modulation. This study reinforces the significance of in vitro functional characterization of LDL receptor activity in developing an accurate approach to FH genetic diagnosis. This is of particular importance because it enables clinicians to tailor personalized treatments for patients’ mutation profile.
Highlights
Familial Hypercholesterolemia is an autosomal dominant disorder causing premature coronary heart disease (CHD) [1] characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis [2]
Consistent with this, solid-phase immunoassays showed the p. (Cys46Gly) Low Density Lipoprotein Receptor gene (LDLR) variant has decreased binding affinity for apolipoproteins. These results indicate the important role of Cys46 in Low-Density Lipoproteins (LDL) receptor activity and highlight the role of LR1 in LDLr activity modulation
Ex3_4del LDLr variant that produces a binding-defective LDLr was used as internal controls [10]. These results were corroborated by determining LDLr expression by flow cytometry. Using both a IgG-C7 antibody recognizing the N-terminal ligand binding repeat of the LDLr and a polyclonal anti-LDLr antibody, we found that LDLr expression of p.(Cys45Gly) was similar to wt (Fig 1C and 1D, respectively)
Summary
Familial Hypercholesterolemia is an autosomal dominant disorder causing premature coronary heart disease (CHD) [1] characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis [2]. As of August 31, 2018, a total of 2500 LDLR variants have been identified in the ClinVar Database (https://clinvarminer.genetics.utah.edu). Pathogenic mutations in the Low Density Lipoprotein Receptor gene (LDLR) cause Familial Hypercholesterolemia (FH), one of the most common genetic disorders with a prevalence as high as 1 in 200 in some populations. FH is an autosomal dominant disorder of lipoprotein metabolism characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis. We investigated the role of Cys located in the first repeat of the LDL receptor binding domain in recognition of apolipoproteins
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