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bioRxiv · 10.64898/2026.03.13.711724

Functional impact of PCSK9 variants on LDL uptake in a knockout hepatic model

Li, H.·Liu, H.·Xu, W.·Zeng, Y.·Huang, P.·Guo, J.·Cai, B.·Chen, Y.·Lin, Y.·Zhang, C.
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Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central regulator of low-density lipoprotein (LDL) cholesterol metabolism, yet the functional consequences of many clinically observed PCSK9 variants remain unknown. To establish a rigorous system for quantitative variant assessment, we generated a PCSK9 knockout (KO) HepG2 cell line through CRISPR/Cas9-mediated deletion of exons 2-8, effectively removing both the prodomain and catalytic regions required for PCSK9 function. This null background enabled systematic functional mapping of wild-type (WT) PCSK9 and multiple clinically relevant variants representing well-characterized, recurrent, and previously understudied alleles. Functional assays revealed pronounced heterogeneity among variant activities. The classical gain-of-function (GOF) variants D374Y and R496W exhibited robust suppression of LDL uptake, whereas A443T--an infrequently reported and previously uncharacterized variant--demonstrated a loss-of-function (LOF)-like phenotype with significantly enhanced LDL uptake. Additional poorly characterized variants, including V4I, R104C/V114A, and R496W/N425S, displayed minimal functional profiles, providing novel mechanistic insights. Surface LDL receptor (LDLR) levels generally correlated with LDL uptake but revealed unique patterns for specific variants. This KO-based rescue system provides a high-resolution framework for mechanistic classification of both established and poorly characterized PCSK9 variants, bridging the gap between genetic discovery and functional interpretation while supporting precision lipid-lowering strategies.

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Li, H., Liu, H., Xu, W., Zeng, Y., Huang, P., Guo, J., Cai, B., Chen, Y., Lin, Y., Zhang, C.. 2026-03-17. Functional impact of PCSK9 variants on LDL uptake in a knockout hepatic model. https://doi.org/10.64898/2026.03.13.711724

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