279 - High-throughput microscopy profiling of LDLR variants (ID 955)
Abstract
Background and Aims
Mutations in the Low Density Lipoprotein Receptor (LDLR) gene are the major cause of familial hypercholesterolaemia (FH), with over 2600 variants described. However, less than 15% of the LDLR variants identified in clinical FH patients have functional evidences to prove their pathogenicity. The aim of the present work is to stablish a quantitative high-throughput in vitro microscopy approach to functionally characterize rare LDLR variants.
Methods
Wild-type or mutant LDLR variants were overexpressed 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 characterized, including 20 previously described control variants, for assay validation, and 20 rare missense variants identified in Portuguese patients, with a clinical FH diagnose. The latter classified as 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 the identification of 13 variants affecting LDLR function and 7 functionally normal missense variants among the studied VUS. This approach takes one-third of the time consumed by the reference method for functional characterization (FACS).
Conclusions
To distinguish disruptive rare variants from silent rare ones is the challenge of contemporary genetics. We have established a time and cost-effective high-throughput assay to functionally profile LDLR variants that can be easily scaled-up. This strategy allows to accurately discriminate the biological effects of LDLR variants, contributing to an improved variant classification that leads to a better diagnosis.
