Percorrer por autor "Pfisterer, Simon"
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- From Uncertainty To Diagnosis: Functional Reclassification Of LDLR Variants In Familial HypercholesterolemiaPublication . Chora, Joana Rita; Islam, Mohammad Majharul; Alves, Ana Catarina; Pfisterer, Simon; Bourbon, MafaldaObjectives: Familial hypercholesterolaemia (FH) is a common disorder of lipid metabolism marked by an increased risk of premature atherosclerotic cardiovascular disease. While genetic testing confirms diagnosis, many LDLR variants are classified as variants of uncertain significance (VUS) due to lack of evidence, limiting clinical actionability. Functional assays can support reclassification, but conventional approaches are time-consuming. This study aims to use a high-throughput functional pipeline to evaluate LDLR VUS and improve diagnostic precision in FH. Methods: We analysed 133 LDLR VUS lacking functional evidence using an automated analysis platform based on multiplexed high-content imaging to quantify LDL uptake. Variants were classified based on uptake relative to wild-type: <70% for PS3_Supporting (pathogenic) and >90% for BS3_Supporting (benign), according to LDLR-specific ACMG/AMP guidelines. A subset of 46 “hot VUS” required only one supporting criterion to reach a likely benign or likely pathogenic classification. Results: Of the 133 variants, 53 showed <70% uptake and were assigned PS3_Supporting, while 41 showed >90% uptake and received BS3_Supporting. The remaining 39 variants exhibited intermediate activity (70–90%). Among the 46 hot VUS, 12 were reclassified as likely benign and 11 as likely pathogenic. An additional 43 variants are now one supporting criterion away from potential reclassification. Conclusions: High-throughput functional testing provides robust and rapid evidence for LDLR variant interpretation. By adding evidence about LDLR activity these assays contribute to resolve diagnostic uncertainty and enables earlier and more accurate diagnosis, optimizes cascade screening, and enables better risk stratification to inform treatment options. These findings support a shift toward personalised genomic medicine in lipid disorders, with direct implications for patient care and cardiovascular risk reduction.
- Re-classification of LDLR Variants through High-Throughput Functional Characterisation: Advancing Diagnosis in Familial HypercholesterolaemiaPublication . Chora, Joana Rita; Islam, Mohammad Majharul; Alves, Ana Catarina; Pfisterer, Simon; Bourbon, MafaldaBackground: 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.