Familial hypercholesterolemia (FH) is a disorder of cholesterol metabolism characterized by elevated LDL-cholesterol levels. The most common cause of FH is pathogenic variants in the LDL receptor (LDLR) gene. To shed light on the functional impact of selected LDLR variants, we functionally characterized 16 LDLR genetic variants alongside 10 control variants. We performed in vitro assays based on transient expression of WT and mutant LDLRs in LDLR-deficient Chinese hamster ovary cells. We used flow cytometry to analyze the relative amount of LDLRs expressed on the cell surface and the relative amount of internalized LDL. In addition, we analyzed the expression and maturation of LDLR protein by Western blotting. Of the 16 studied variants, two variants (p.(Asn272Thr) and p.(Arg574Leu)) did not exhibit a defect in LDLR function, one variant (p.(Ala540Thr)) exhibited a defect in LDL binding and/or internalization despite normal LDLR cell surface expression, and the remaining 13 variants had a detrimental effect on both LDLR cell surface expression and LDL internalization. The information presented in this study contributes to the clinical classification of LDLR variants and a more precise diagnosis of FH patients, highlighting the type of defect each variant produces.

Central European Institute of Technology Masaryk University Brno Czech Republic

Centre of Molecular Biology and Genetics University Hospital Brno Brno Czech Republic; Faculty of Medicine Masaryk University Brno Czech Republic

Centre of Molecular Biology and Genetics University Hospital Brno Brno Czech Republic; National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic

Centre of Molecular Biology and Genetics University Hospital Brno Brno Czech Republic; National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic; Faculty of Medicine Masaryk University Brno Czech Republic

Departamento de Promoção da Saúde e Doenças não transmissíveis Instituto Nacional de Saúde Dr Ricardo Jorge Lisboa Portugal; BioISI Biosystems and Integrative Sciences Institute Faculty of Sciences University of Lisboa Lisboa Portugal

Faculty of Medicine Masaryk University Brno Czech Republic

Faculty of Medicine Masaryk University Brno Czech Republic; Centre for Cardiovascular Surgery and Transplantation Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic; Central European Institute of Technology Masaryk University Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic; Centre for Cardiovascular Surgery and Transplantation Brno Czech Republic

Zobrazit více v PubMed

Musunuru K., Hershberger R.E., Day S.M., Klinedinst N.J., Landstrom A.P., Parikh V.N., et al. Genetic testing for inherited cardiovascular diseases: a scientific statement from the American heart association. Circ. Genom. Precis. Med. 2020;13 PubMed

Ference B.A., Ginsberg H.N., Graham I., Ray K.K., Packard C.J., Bruckert E., et al. Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the european atherosclerosis society consensus panel. Eur. Heart J. 2017;38:2459–2472. PubMed PMC

Vuorio A., Docherty K.F., Humphries S.E., Kuoppala J., Kovanen P.T. Statin treatment of children with familial hypercholesterolemia--trying to balance incomplete evidence of long-term safety and clinical accountability: are we approaching a consensus? Atherosclerosis. 2013;226:315–320. PubMed

Futema M., Ramaswami U., Tichy L., Bogsrud M.P., Holven K.B., Roeters van Lennep J., et al. Comparison of the mutation spectrum and association with pre and post treatment lipid measures of children with heterozygous familial hypercholesterolaemia (FH) from eight European countries. Atherosclerosis. 2021;319:108–117. PubMed

Südhof T.C., Goldstein J.L., Brown M.S., Russell D.W. The LDL receptor gene: a mosaic of exons shared with different proteins. Science. 1985;228:815–822. PubMed PMC

Yamamoto T., Davis C.G., Brown M.S., Schneider W.J., Casey M.L., Goldstein J.L., et al. The human LDL receptor: a cysteine-rich protein with multiple alu sequences in its mRNA. Cell. 1984;39:27–38. PubMed

Cummings R.D., Kornfeld S., Schneider W.J., Hobgood K.K., Tolleshaug H., Brown M.S., et al. Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J. Biol. Chem. 1983;258:15261–15273. PubMed

He G., Gupta S., Yi M., Michaely P., Hobbs H.H., Cohen J.C. ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2. J. Biol. Chem. 2002;277:44044–44049. PubMed

Anderson R.G., Goldstein J.L., Brown M.S. A mutation that impairs the ability of lipoprotein receptors to localise in coated pits on the cell surface of human fibroblasts. Nature. 1977;270:695–699. PubMed

Maurer M.E., Cooper J.A. The adaptor protein Dab2 sorts LDL receptors into coated pits independently of AP-2 and ARH. J. Cell Sci. 2006;119:4235–4246. PubMed

Strøm T.B., Laerdahl J.K., Leren T.P. Mutations affecting the transmembrane domain of the LDL receptor: impact of charged residues on the membrane insertion. Hum. Mol. Genet. 2017;26:1634–1642. PubMed

Tolleshaug H., Goldstein J.L., Schneider W.J., Brown M.S. Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia. Cell. 1982;30:715–724. PubMed

Zou P., Ting A.Y. Imaging LDL receptor oligomerization during endocytosis using a co-internalization assay. ACS Chem. Biol. 2011;6:308–313. PubMed PMC

Brown M.S., Anderson R.G., Goldstein J.L. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983;32:663–667. PubMed

Basu S.K., Goldstein J.L., Anderson R.G., Brown M.S. Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts. Cell. 1981;24:493–502. PubMed

Leren T.P. Sorting an LDL receptor with bound PCSK9 to intracellular degradation. Atherosclerosis. 2014;237:76–81. PubMed

Hobbs H.H., Brown M.S., Goldstein J.L. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia. Hum. Mutat. 1992;1:445–466. PubMed

Hobbs H.H., Russell D.W., Brown M.S., Goldstein J.L. The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu. Rev. Genet. 1990;24:133–170. PubMed

Koivisto U.M., Hubbard A.L., Mellman I. A novel cellular phenotype for familial hypercholesterolemia due to a defect in polarized targeting of LDL receptor. Cell. 2001;105:575–585. PubMed

Strøm T.B., Tveten K., Laerdahl J.K., Leren T.P. Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum. FEBS Open Bio. 2014;4:321–327. PubMed PMC

Strøm T.B., Laerdahl J.K., Leren T.P. Mutation p.L799R in the LDLR, which affects the transmembrane domain of the LDLR, prevents membrane insertion and causes secretion of the mutant LDLR. Hum. Mol. Genet. 2015;24:5836–5844. PubMed

Landrum M.J., Lee J.M., Riley G.R., Jang W., Rubinstein W.S., Church D.M., et al. ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014;42:D980–D985. PubMed PMC

Tichý L., Freiberger T., Zapletalová P., Soška V., Ravčuková B., Fajkusová L. The molecular basis of familial hypercholesterolemia in the Czech Republic: spectrum of LDLR mutations and genotype-phenotype correlations. Atherosclerosis. 2012;223:401–408. PubMed

Tichý L., Fajkusová L., Zapletalová P., Schwarzová L., Vrablík M., Freiberger T. Molecular genetic background of an autosomal dominant hypercholesterolemia in the Czech Republic. Physiol. Res. 2017;66(Suppl 1):S47–S54. PubMed

Chora J.R., Iacocca M.A., Tichý L., Wand H., Kurtz C.L., Zimmermann H., et al. The clinical genome resource (ClinGen) familial hypercholesterolemia variant curation expert panel consensus guidelines for LDLR variant classification. Genet. Med. 2022;24:293–306. PubMed PMC

Ranheim T., Kulseth M.A., Berge K.E., Leren T.P. Model system for phenotypic characterization of sequence variations in the LDL receptor gene. Clin. Chem. 2006;52:1469–1479. PubMed

Dušková L., Nohelová L., Loja T., Fialová J., Zapletalová P., Réblová K., et al. Low density lipoprotein receptor variants in the beta-propeller subdomain and their functional impact. Front Genet. 2020;11:691. PubMed PMC

Gudnason V., Patel D., Sun X.M., Humphries S., Soutar A.K., Knight B.L. Effect of the StuI polymorphism in the LDL receptor gene (Ala 370 to Thr) on lipid levels in healthy individuals. Clin. Genet. 1995;47:68–74. PubMed

Alves A.C., Azevedo S., Benito-Vicente A., Graça R., Galicia-Garcia U., Barros P., et al. LDLR variants functional characterization: contribution to variant classification. Atherosclerosis. 2021;329:14–21. PubMed

Benito-Vicente A., Alves A.C., Etxebarria A., Medeiros A.M., Martin C., Bourbon M. The importance of an integrated analysis of clinical, molecular, and functional data for the genetic diagnosis of familial hypercholesterolemia. Genet. Med. 2015;17:980–988. PubMed

Sørensen S., Ranheim T., Bakken K.S., Leren T.P., Kulseth M.A. Retention of mutant low density lipoprotein receptor in endoplasmic reticulum (ER) leads to ER stress. J. Biol. Chem. 2006;281:468–476. PubMed

Etxebarria A., Benito-Vicente A., Palacios L., Stef M., Cenarro A., Civeira F., et al. Functional characterization and classification of frequent low-density lipoprotein receptor variants. Hum. Mutat. 2015;36:129–141. PubMed

Rubinsztein D.C., Jialal I., Leitersdorf E., Coetzee G.A., van der Westhuyzen D.R. Identification of two new LDL-receptor mutations causing homozygous familial hypercholesterolemia in a South African of Indian origin. Biochim. Biophys. Acta. 1993;1182:75–82. PubMed

Chang J.H., Pan J.P., Tai D.Y., Huang A.C., Li P.H., Ho H.L., et al. Identification and characterization of LDL receptor gene mutations in hyperlipidemic Chinese. J. Lipid Res. 2003;44:1850–1858. PubMed

Bertolini S., Cassanelli S., Garuti R., Ghisellini M., Simone M.L., Rolleri M., et al. Analysis of LDL receptor gene mutations in Italian patients with homozygous familial hypercholesterolemia. Arterioscler. Thromb. Vasc. Biol. 1999;19:408–418. PubMed

Romano M., Di Taranto M.D., Mirabelli P., D'Agostino M.N., Iannuzzi A., Marotta G., et al. An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations. J. Lipid Res. 2011;52:2095–2100. PubMed PMC

Sun X.M., Patel D.D., Knight B.L., Soutar A.K. Comparison of the genetic defect with LDL-receptor activity in cultured cells from patients with a clinical diagnosis of heterozygous familial hypercholesterolemia. The Familial Hypercholesterolaemia Regression Study Group. Arterioscler. Thromb. Vasc. Biol. 1997;17:3092–3101. PubMed

Costantini L.M., Baloban M., Markwardt M.L., Rizzo M.A., Guo F., Verkhusha V.V., et al. A palette of fluorescent proteins optimized for diverse cellular environments. Nat. Commun. 2015;6:7670. PubMed PMC

Krieger M., Martin J., Segal M., Kingsley D. Amphotericin B selection of mutant Chinese hamster cells with defects in the receptor-mediated endocytosis of low density lipoprotein and cholesterol biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 1983;80:5607–5611. PubMed PMC

Emmer B.T., Sherman E.J., Lascuna P.J., Graham S.E., Willer C.J., Ginsburg D. Genome-scale CRISPR screening for modifiers of cellular LDL uptake. PLoS Genet. 2021;17 PubMed PMC

Lucero D., Dikilitas O., Mendelson M.M., Aligabi Z., Islam P., Neufeld E.B., et al. Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen. J. Lipid Res. 2022;63 PubMed PMC

Reimund M., Dearborn A.D., Graziano G., Lei H., Ciancone A.M., Kumar A., et al. Structure of apolipoprotein B100 bound to the low-density lipoprotein receptor. Nature. 2024;11 doi: 10.1038/s41586-024-08223-0. PubMed DOI

Wurm F.M. CHO Quasispecies—Implications for manufacturing processes. Processes. 2013;1:296–311.

Li C., Zhang J. Stop-codon read-through arises largely from molecular errors and is generally nonadaptive. PLoS Genet. 2019;15 PubMed PMC

van Driel I.R., Goldstein J.L., Südhof T.C., Brown M.S. First cysteine-rich repeat in ligand-binding domain of low density lipoprotein receptor binds Ca2+ and monoclonal antibodies, but not lipoproteins. J. Biol. Chem. 1987;262:17443–17449. PubMed

Jeon H., Blacklow S.C. Structure and physiologic function of the low-density lipoprotein receptor. Annu. Rev. Biochem. 2005;74:535–562. PubMed

Pisciotta L., Tarugi P., Borrini C., Bellocchio A., Fresa R., Guerra D., et al. Pseudoxanthoma elasticum and familial hypercholesterolemia: a deleterious combination of cardiovascular risk factors. Atherosclerosis. 2010;210:173–176. PubMed

Thormaehlen A.S., Schuberth C., Won H.H., Blattmann P., Joggerst-Thomalla B., Theiss S., et al. Systematic cell-based phenotyping of missense alleles empowers rare variant association studies: a case for LDLR and myocardial infarction. PLoS Genet. 2015;11 PubMed PMC

Junna N., Ruotsalainen S., Ripatti P., FinnGen Ripatti S., Widén E. Novel Finnish-enriched variants causing severe hypercholesterolemia and their clinical impact on coronary artery disease. Atherosclerosis. 2023;386 PubMed

Humphrey W., Dalke A., Schulten K. VMD: visual molecular dynamics. J. Mol. Graph. 1996;14:33–38. PubMed

Huang S., Henry L., Ho Y.K., Pownall H.J., Rudenko G. Mechanism of LDL binding and release probed by structure-based mutagenesis of the LDL receptor. J. Lipid Res. 2010;51:297–308. PubMed PMC

Gent J., Braakman I. Low-density lipoprotein receptor structure and folding. Cell Mol. Life Sci. 2004;61:2461–2470. PubMed PMC

Pavloušková J., Réblová K., Tichý L., Freiberger T., Fajkusová L. Functional analysis of the p.(Leu15Pro) and p.(Gly20Arg) sequence changes in the signal sequence of LDL receptor. Atherosclerosis. 2016;250:9–14. PubMed

Richards S., Aziz N., Bale S., Bick D., Das S., Gastier-Foster J., et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015;17:405–424. PubMed PMC

Feingold K.R. Lipid and lipoprotein metabolism. Endocrinol. Metab. Clin. North Am. 2022;51:437–458. PubMed

Schmidt H.H., Tietge U.J., Buettner J., Barg-Hock H., Offner G., Schweitzer S., et al. Liver transplantation in a subject with familial hypercholesterolemia carrying the homozygous p.W577R LDL-receptor gene mutation. Clin. Transplant. 2008;22:180–184. PubMed

Benito-Vicente A., Uribe K.B., Jebari S., Galicia-Garcia U., Ostolaza H., Martin C. Validation of LDLr activity as a tool to improve genetic diagnosis of familial hypercholesterolemia: a retrospective on functional characterization of LDLr variants. Int. J. Mol. Sci. 2018;19:1676. PubMed PMC

Lin S., Hu T., Wang K., Wang J., Zhu Y., Chen X. In vitro assessment of the pathogenicity of the LDLR c.2160delC variant in familial hypercholesterolemia. Lipids Health Dis. 2023;22:77. PubMed PMC

Jiang L., Wu W.F., Sun L.Y., Chen P.P., Wang W., Benito-Vicente A., et al. The use of targeted exome sequencing in genetic diagnosis of young patients with severe hypercholesterolemia. Sci. Rep. 2016;6 PubMed PMC

Jiang L., Benito-Vicente A., Tang L., Etxebarria A., Cui W., Uribe K.B., et al. Analysis of LDLR variants from homozygous FH patients carrying multiple mutations in the LDLR gene. Atherosclerosis. 2017;263:163–170. PubMed

Wang H., Xu S., Sun L., Pan X., Yang S., Wang L. Functional characterization of two low-density lipoprotein receptor gene mutations in two Chinese patients with familial hypercholesterolemia. PLoS One. 2014;9 PubMed PMC

Rodríguez-Jiménez C., Pernía O., Mostaza J., Rodríguez-Antolín C., de Dios García-Díaz J., Alonso-Cerezo C., et al. Functional analysis of new variants at the low-density lipoprotein receptor associated with familial hypercholesterolemia. Hum. Mutat. 2019;40:1181–1190. PubMed

Rodríguez-Nóvoa S., Rodríguez-Jiménez C., Alonso C., Rodriguez-Laguna L., Gordo G., Martinez-Glez V., et al. Familial hypercholesterolemia: a single-nucleotide variant (SNV) in mosaic at the low density lipoprotein receptor (LDLR) Atherosclerosis. 2020;311:37–43. PubMed

Costantini L.M., Fossati M., Francolini M., Snapp E.L. Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions. Traffic. 2012;13:643–649. PubMed PMC

Snapp E.L., Hegde R.S., Francolini M., Lombardo F., Colombo S., Pedrazzini E., et al. Formation of stacked ER cisternae by low affinity protein interactions. J. Cell Biol. 2003;163:257–269. PubMed PMC