P.(Asp47Asn) and p.(Thr62Met): non deleterious LDL receptor missense variants functionally characterized in vitro

A Benito-Vicente,H Ostolaza,U Galicia-Garcia,C Martin,K B Uribe,S Jebari,H Siddiqi,A Larrea-Sebal,M Stef,L Palacios

doi:10.1038/s41598-018-34715-x

A Benito-Vicente, H Ostolaza + Show 8 more

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https://doi.org/10.1038/s41598-018-34715-x

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Abstract

Familial Hypercholesterolemia (FH) is a common genetic disorder caused most often by mutations in the Low Density Lipoprotein Receptor gene (LDLr) leading to high blood cholesterol levels, and ultimately to development of premature coronary heart disease. Genetic analysis and subsequent cascade screening in relatives allow diagnosis of FH at early stage, especially relevant to diagnose children. So far, more than 2300 LDLr variants have been described but only a minority of them have been functionally analysed to evaluate their pathogenicity in FH. Thus, identifying pathogenic mutations in LDLr is a long-standing challenge in the field. In this study, we investigated in vitro the activity p.(Asp47Asn) and p.(Thr62Met) LDLr variants, both in the LR1 region. We used CHO-ldlA7 transfected cells with plasmids carrying p.(Asp47Asn) or p.(Thr62Met) LDLr variants to analyse LDLr expression by FACS and immunoblotting, LDL binding and uptake was determined by FACS and analysis of mutation effects was assessed in silico. The in vitro activity assessment of p.(Asp47Asn) and p.(Thr62Met) LDLr variants shows a fully functional LDL binding and uptake activities. Therefore indicating that the three of them are non-pathogenic LDLr variants. These findings also emphasize the importance of in vitro functional LDLr activity studies to optimize the genetic diagnosis of FH avoiding the report of non-pathogenic variants and possible misdiagnose in relatives if cascade screening is carried out.

Highlights

Familial Hypercholesterolemia (FH) is a common genetic disorder caused most often by mutations in the Low Density Lipoprotein Receptor gene (LDLr) leading to high blood cholesterol levels, and to development of premature coronary heart disease
Expression of p.(Asp47Asn) and p.(Thr62Met) LDLr variant was analysed by Western blot in CHO-ldlA7 transfected cells as detailed above
Quantitative densitometry was performed to determine relative levels of LDLr expression (Fig. 1B). These results were further corroborated by assessing LDLr expression by FACS, as shown in Fig. 1C expression of p.(Asp47Asn) and p.(Thr62Met) LDLr variants, determined by IgG-C7 antibody resulted similar to the wt LDLr

Summary

Introduction

Familial Hypercholesterolemia (FH) is a common genetic disorder caused most often by mutations in the Low Density Lipoprotein Receptor gene (LDLr) leading to high blood cholesterol levels, and to development of premature coronary heart disease. Indicating that the three of them are non-pathogenic LDLr variants These findings emphasize the importance of in vitro functional LDLr activity studies to optimize the genetic diagnosis of FH avoiding the report of non-pathogenic variants and possible misdiagnose in relatives if cascade screening is carried out. Familial Hypercholesterolemia (FH) (OMIM 143890) is a common genetic disease caused most often by mutations in the Low Density Lipoprotein Receptor gene (LDLr; MIM# 606945) These heterogeneous LDLr variants impair the function of the receptor pathway, leading to high blood cholesterol levels, xanthomas, cholesterol accumulation in peripheral tissues and development of atherosclerosis at an early age, leading to development of premature coronary heart disease (CHD)[1]. Experimental reproducibility between laboratories all over the world ensures rigorous verification of all functional studies performed for each variant

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