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Re-classification of LDLR Variants through High-Throughput Functional Characterisation: Advancing Diagnosis in Familial Hypercholesterolaemia
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Background: Familial hypercholesterolaemia (FH) is the most common autosomal disorder of lipid metabolism, affecting approximately 1 in 300 individuals. FH is characterised by markedly elevated plasma cholesterol levels from birth, predisposing to premature atherosclerotic cardiovascular disease. The identification of a pathogenic variant in a causative gene provides a definitive diagnosis and enables cascade screening of family members. However, a substantial proportion of FH variants remain classified as variants of uncertain significance (VUS), creating a critical gap in genetic diagnosis and patient management.
Purpose: This study aims to implement a high-throughput pipeline for the functional classification of LDLR variants, thereby enabling their clinical re-interpretation and integration into diagnostic practice.
Methods: Selected LDLR variants were divided into two groups: a validation set and a test set. The validation group included 7 variants previously classified as benign/likely benign and 50 variants previously classified as pathogenic/likely pathogenic. The test group comprised 131 VUS with no prior functional assessment. Functional activity was evaluated using an automated analysis platform based on multiplexed high-content imaging to quantify LDL uptake, as described previously (Islam, Tamlander, Hlushchenko, Ripatti, & Pfisterer, 2024). Variant classification followed FH VCEP LDLR-specific guidelines (Chora et al., 2022).
Results: In total, 187 LDLR variants were analysed. In the validation group, 44 variants demonstrated completely concordant results with previous classifications, 8 fell within an intermediate "grey zone" (70–90% LDL uptake), and only 5 pathogenic variants were misclassified, yielding a sensitivity of 88.4%, specificity and precision of 100%, and overall accuracy of 78.9%.
Among the test group, 51 variants exhibited <70% of wild-type LDL uptake, 41 showed >90% of wild-type uptake, and 39 fell into the intermediate range, requiring additional functional studies. Integrating these findings with other ACMG/AMP evidence codes, 47 variants were considered “hot VUS”, amenable to reclassification. Of these, 9 were reclassified as likely benign and 11 as likely pathogenic.
Conclusions: This large-scale functional study of LDLR variants demonstrates the feasibility and clinical utility of integrating high-throughput functional evidence with high accuracy into variant interpretation. The ability to reclassify previously unresolved VUS represents a major step forward in reducing diagnostic uncertainty in FH. The PerMedFH project paves the way for a personalised medicine approach in FH, improving diagnostic precision, and ultimately enhancing patient care and cardiovascular outcomes.
Descrição
Palavras-chave
Familial Hypercholesterolaemia Genetic Diagnosis Variant Classification Functional Studies Doenças Cardio e Cérebro-vasculares