Functional profiling of LDLR variants: Important evidence for variant classification: Functional profiling of LDLR variants
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Updated: Sep 23, 2025
Identification and Classification of Position-specific GABAA Receptor Subunit Missense Variants for Their Role In Hippocampal Pyramidal Neurons
Published on: June 6, 2025
Rafael Graça1, Ana Catarina Alves1, Magdalena Zimon2
1Unidade de I&D, Grupo de Investigação Cardiovascular, Departamento de Promoção da Saúde e Prevenção de Doenças Não Transmissíveis, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1600-609 Lisboa, Portugal (Drs Graça, Alves, and Bourbon,); BioISI - Biosystems & Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal (Drs Graça, Alves, and Bourbon).
Insights
Functional studies reclassified 7 variants of unknown significance in Familial Hypercholesterolemia (FH). This improves diagnostic accuracy for lipid metabolism disorders and coronary heart disease risk.
Area of Science:
- Genetics
- Biochemistry
- Cardiology
Background:
- Familial Hypercholesterolemia (FH) is a genetic lipid metabolism disorder.
- It significantly increases the risk of premature atherosclerosis and coronary heart disease.
- Thousands of Low-Density Lipoprotein Receptor (LDLR) variants exist, but most lack functional validation for disease classification.
Purpose of the Study:
- To functionally characterize 13 rare missense LDLR variants found in Familial Hypercholesterolemia patients.
- To determine the impact of these variants on LDLR function.
- To improve the diagnostic classification of genetic variants associated with FH.
Main Methods:
- Utilized LDLR-deficient Chinese Hamster Ovary (CHO-ldlA7) cells.
- Transfected cells with plasmids containing specific LDLR variants created via site-directed mutagenesis.
- Assessed LDLR activity and expression using Fluorescence-Activated Cell Sorting (FACS).
Main Results:
- 11 out of 13 variants demonstrated an impact on LDLR function.
- Two variants yielded inconclusive results.
- Seven variants initially classified as Variants of Unknown Significance (VUS) were reclassified as Likely Pathogenic or Pathogenic based on functional data.
Conclusions:
- Functional characterization is crucial for accurate diagnosis of Familial Hypercholesterolemia.
- This study successfully reclassified several VUS, aiding in precise genetic diagnosis.
- Further functional studies are essential for understanding and managing FH.
Background:
Familial Hypercholesterolemia (FH) is a semidominant disorder of the lipid metabolism associated with premature atherosclerosis and coronary heart disease. So far, about 3,000 unique LDLR variants have been described, most of which lack functional evidence proving their effect on LDLR function, despite the important role that functional studies play in variant classification.
Objective:
In this work, we aimed to functionally characterize 13 rare missense variants, identified worldwide and in Portugal, in clinical FH patients.
Methods:
LDLR-deficient CHO-ldlA7 cells were transfected with plasmids carrying different LDLR variants generated by site-directed mutagenesis. LDLR activity and expression were assessed by FACS.
Results:
11/13 variants affect LDLR function (p.Cys109Phe; p.Cys143Arg; p.Glu267Lys; p.Cys352Ser; p.Ile451Thr; p.His485Gln; p.Asp492Asn; p.Val500Ala; p.Gly529Arg; p.Phe614Ile; p.Glu626Lys) and 2/13 are inconclusive (p.Arg81Cys; p.Gly98Arg;).
Conclusion:
Of the 13 variants studied, 8 were classified as VUS by ACMG criteria, but for 7 of these 8, our functional studies were able to reassign them as Likely pathogenic or Pathogenic. For an accurate diagnosis, an effort must be made to improve functional characterization of putative disease-causing variants.
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