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Rare variants in a PCSK9 distal-enhancer as drivers of LDL-cholesterol levels

ABSTRACT: BACKGROUND. Discoveries of genetic causes of dyslipidemia have significantly advanced our understanding of lipoprotein metabolism and its implications in cardiometabolic diseases. However, a significant number of patients with dyslipidemia still have unidentified genetic causes. Leveraging insights from genome-wide association studies, where most common associated variants are located in non-coding regions, we propose that rare non-coding variants located in genomic regulatory regions may be responsible for dyslipidemia. METHODS. Using whole-genome sequencing data from the UK Biobank (n > 200,000), we performed region-based rare-variant association analyses, focusing on liver specific genomic regulatory regions (assessed by transposase-accessible chromatin assay coupled with sequencing (ATAC-seq)), to assess whether the cumulative burden of rare genetic variants (allele frequency < 0.01) in these genomic regulatory regions was associated with LDL-cholesterol levels. To validate our findings, we used an advanced cellular model of human induced pluripotent stem cells (hiPSCs) stably expressing a CRISPR-interference system. This system enables, when combined with guide-RNAs, to target regions of interest and silence regulated genes. RESULTS. We found that the cumulative burden of rare genetic variants in 18 among 53,039 ATAC-seq peaks in human liver, is significantly associated with LDL-cholesterol. We focus our proof-of-concept study on a peak located ~17 kb upstream PCSK9, strongly associated with LDL-cholesterol (p=5.85e-52), apolipoprotein-B (p=3.82e-49) and PCSK9 plasma levels (p=1.15e-67). We showed that this regulatory region is also active in hiPSCs and hiPSC-derived liver organoids. When transfected with guide-RNAs targeting this region, we showed that CRISPRi-hiPSCs present a hypermethylation of the targeted region which in terms leads to a substantial decrease in PCSK9 gene expression (-48%), intracellular protein (-65%) and secretion (-36%) (p<0.001). CONCLUSIONS. This study identifies a non-coding genetic region upstream PCSK9 where rare variants lead to decreased PCSK9 expression and lowered LDL-cholesterol. Such findings advocate for the genetic screening of non-coding regulatory regions in patients with unidentified genetic causes of extreme dyslipidemia and open novel perspectives for genetic regulatory therapeutic intervention.

ORGANISM(S): Homo sapiens

PROVIDER: GSE339521 | GEO | 2026/07/23

REPOSITORIES: GEO

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Project description:Background: The low-density lipoprotein receptor (LDLR) in the liver plays a crucial role in clearing low-density lipoprotein cholesterol (LDL-C) from the bloodstream. This process takes place mainly in the liver. Under atherogenic conditions, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), secreted by the liver, binds to LDLR on hepatocytes, preventing its recycling and enhancing its lysosomal degradation. This process reduces LDL-C clearance, promoting hypercholesterolemia. Epsins, a family of ubiquitin-binding endocytic adaptors, are key regulators of atherogenesis in lesional cells, including endothelial cells and macrophages. Given epsins' canonical role in regulating endocytosis of cell-surface receptors, we aimed to determine whether and how liver epsins contribute to PCSK9-mediated LDLR endocytosis and degradation, thereby impairing LDL-C clearance and accelerating atherosclerosis. Methods: Liver-specific epsin knockout (Liver-DKO) atherosclerotic models were generated in ApoE-/- and PCSK9-AAV8-induced atheroprone mice fed on a Western diet. We utilized single-cell RNA sequencing, along with molecular, cellular, and biochemical analyses, to investigate the physiological role of liver epsins in PCSK9-mediated LDLR degradation. Additionally, we explored the therapeutic potential of nanoparticle-encapsulated siRNAs targeting epsins 1 and 2 in ApoE-/- mice with established atherosclerosis. Results: Western diet (WD)-induced atherosclerosis was significantly attenuated in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, as well as in PCSK9-AAV8-induced Liver-DKO mice compared with PCSK9-AAV8-induced wild-type (WT) mice, accompanied by reductions in blood cholesterol and triglyceride levels. Mechanistically, single-cell RNA sequencing of hepatocytes and aortas isolated from atherosclerotic ApoE-/- and ApoE-/-/Liver-DKO mice revealed epsin-deficient Ldlrhi hepatocytes with diminished lipogenic potential. Notably, pathway analysis of hepatocytes revealed increased LDL particle clearance and enhanced LDLR-cholesterol interactions under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, which correlated with decreased plasma LDL-C levels. Furthermore, pathway analysis of the aortas showed attenuated inflammation and endothelial activation, coupled with reduced lipid uptake, and enhanced cholesterol efflux under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls. Moreover, the absence of liver epsins led to an upregulation of LDLR protein expression in hepatocytes. We further demonstrated that epsins bind LDLR via the ubiquitin-interacting motif (UIM), enabling PCSK9-mediated LDLR degradation. Depleting epsins abolished this degradation, thereby preventing atheroma progression. Lastly, targeting liver epsins with nanoparticle-encapsulated epsins siRNAs effectively ameliorates dyslipidemia and inhibits atherosclerosis progression. These results are consistent with findings showing increased expression of epsin1 and epsin2 in patients with atherosclerotic cardiovascular disease. Conclusions: Liver epsins drive atherogenesis by promoting PCSK9-mediated LDLR degradation, thereby elevating circulating LDL-C levels and heightening lesional inflammation. As such, targeting epsins in the liver represents a promising therapeutic strategy to mitigate atherosclerosis by preserving LDLR and enhancing LDL-C clearance.

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Rare variants in a PCSK9 distal-enhancer as drivers of LDL-cholesterol levels

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