Characterization of Two Variants at Met 1 of the Human LDLR Gene Encoding the Same Amino Acid but Causing Different Functional Phenotypes.

Rafael Graça,Rafael Fernandes,Luísa Romão,Juliane Menezes,Mafalda Bourbon,Ana Catarina Alves

doi:10.3390/biomedicines9091219

Abstract

Familial hypercholesterolemia (FH) is the most common genetic disorder of lipid metabolism, characterized by increased levels of total and LDL plasma cholesterol, which leads to premature atherosclerosis and coronary heart disease. FH phenotype has considerable genetic heterogeneity and phenotypic variability, depending on LDL receptor activity and lifestyle. To improve diagnosis and patient management, here, we characterized two single nucleotide missense substitutions at Methionine 1 of the human LDLR gene (c.1A>T/p.(Met1Leu) and c.1A>C/p.(Met1Leu)). We used a combination of Western blot, flow cytometry, and luciferase assays to determine the effects of both variants on the expression, activity, and synthesis of LDLR. Our data show that both variants can mediate translation initiation, although the expression of variant c.1A>T is very low. Both variants are in the translation initiation codon and codify for the same amino acid p.(Met1Leu), yet they lead to different levels of impairment on LDLR expression and activity, corroborating different efficiencies of the translation initiation at these non-canonical initiation codons. The functional data of these variants allowed for an improved American College of Medical Genetics (ACMG) classification for both variants, which can allow a more personalized choice of the lipid-lowering treatment and dyslipidemia management, ultimately improving patients’ prognosis.

Highlights

Familial hypercholesterolemia (FH) (OMIM 143890) is an autosomal disorder of lipid metabolism characterized by increased plasma cholesterol levels and lipid accumulation in arteries and tendons, promoting premature atherosclerosis and coronary heart disease (CHD) [1]
To quantify the expression and activity of both LDLR variants, CHO-ldlA7 cells were transiently transfected with WT LDLR, c.1A>T, p.(Met1Leu), or c.1A>C, p.(Met1Leu) constructs to determine the ability of the receptor to bind and internalize labeled LDL
To quantify the expression and activity of both LDLR variants, CHO-ldlA7 cells were transiently transfected with WT LDLR, c.1A>T, p.(Met1Leu), or c.1A>C, p.(Met1Leu) con6 of 11 structs to determine the ability of the receptor to bind and internalize labeled LDL

Summary

Introduction

Familial hypercholesterolemia (FH) (OMIM 143890) is an autosomal disorder of lipid metabolism characterized by increased plasma cholesterol levels and lipid accumulation in arteries and tendons, promoting premature atherosclerosis and coronary heart disease (CHD) [1]. FH is one of the most common genetic disorders with a frequency of around. The underlying genetics of the FH phenotype is complex, more than 90% of FH cases result from LDLR defects associated with an autosomal dominant inheritance pattern [3,4]. There are more than 2300 LDLR variants exclusively identified in FH patients [7]. Another key feature of FH genetics is the phenotypic variability [3,8]; Biomedicines 2021, 9, 1219.

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