Graça, Rafael2022-10-312022-10-312021-02-26http://hdl.handle.net/10400.18/8283Aim: Mutations in the Low Density Lipoprotein receptor (LDLR) gene are the major cause of familial hypercholesterolaemia (FH), with over 2300 variants described in clinical FH patients. However, less than 15 % of these variants have functional evidence to prove their pathogenicity. The aim of the present work is to establish a quantitative high-throughput in vitro microscopy approach to functional characterise rare LDLR variants. Methods: Wild type or mutant LDLR variants were over expressed in LDLR-deficient CHO-ldlA7 cells. LDLR expression at cell surface and functional activity were quantified by multiparametric analysis of images acquired by high-content automated microscopy. A total of 40 variants were studied, 20 previously characterised (controls) used for assay validation, and 20 rare missense variants identified in Portuguese patients, with a clinical FH diagnosis. The latter were classified as variants of unknown significance (VUS) according to the ACMG/AMP guidelines. Results: Analysis of control variants confirmed the effectiveness of this approach to correctly classify LDLR variants according to their pathogenicity. Moreover, this work allowed to identify 13 functionally abnormal missense variants and 7 functionally normal missense variants that do not affect LDLR activity among studied VUS. Moreover, this procedure is performed in 1/3 of the time needed with the reference method for functional characterisation (flow cytometry). Conclusions: Distinguish disruptive rare variants from silent rare variants is a fundamental challenge of contemporary genetics. We have established a time and cost-effective high-throughput assay to functionally profile LDLR variants, that can be scaled-up to a higher number of variants. This strategy allows to firmly discriminate the biological effects and likely disease relevance of rare LDLR missense variants, contributing to an improved variant classification, and consequently to a better diagnosis and patients’ prognosis.engFamilial HypercholesterolemiaDoenças Cardio e Cérebro-vascularesColesterolPortugallecture