PCSK9 Loss-of-function Research Articles

Detrimental effects of PCSK9 loss-of-function in the pediatric host response to sepsis are mediated through independent influence on Angiopoietin-1

BackgroundSepsis is associated with significant mortality. Yet, there are no efficacious therapies beyond antibiotics. PCSK9 loss-of-function (LOF) and inhibition, through enhanced low-density lipoprotein receptor (LDLR) mediated endotoxin clearance, holds promise as a potential therapeutic approach among adults. In contrast, we have previously demonstrated higher mortality in the juvenile host. Given the potential pleiotropic effects of PCSK9 on the endothelium, beyond canonical effects on serum lipoproteins, both of which may influence sepsis outcomes, we sought to test the influence of PCSK9 LOF genotype on endothelial dysfunction.MethodsSecondary analyses of a prospective observational cohort of pediatric septic shock. Genetic variants of PCSK9 and LDLR genes, serum PCSK9, and lipoprotein concentrations were determined previously. Endothelial dysfunction markers were measured in day 1 serum. We conducted multivariable linear regression to test the influence of PCSK9 LOF genotype on endothelial markers, adjusted for age, complicated course, and low- and high-density lipoproteins (LDL and HDL). Causal mediation analyses to test impact of select endothelial markers on the association between PCSK9 LOF genotype and mortality. Juvenile Pcsk9 null and wildtype mice were subject to cecal slurry sepsis and endothelial markers were quantified.ResultsA total of 474 patients were included. PCSK9 LOF was associated with several markers of endothelial dysfunction, with strengthening of associations after exclusion of those homozygous for the rs688 LDLR variant that renders it insensitive to PCSK9. Serum PCSK9 was not correlated with endothelial dysfunction. PCSK9 LOF influenced concentrations of Angiopoietin-1 (Angpt-1) upon adjusting for potential confounders including lipoprotein concentrations, with false discovery adjusted p value of 0.042 and 0.013 for models that included LDL and HDL, respectively. Causal mediation analysis demonstrated that the effect of PCSK9 LOF on mortality was mediated by Angpt-1 (p = 0.0008). Murine data corroborated these results with lower Angpt-1 and higher soluble thrombomodulin among knockout mice with sepsis relative to the wildtype.ConclusionsWe present genetic and biomarker association data that suggest a potential direct role of the PCSK9-LDLR pathway on Angpt-1 in the developing host with septic shock and warrant external validation. Further, mechanistic studies on the role of PCSK9-LDLR pathway on vascular homeostasis may lead to the development of pediatric-specific sepsis therapies.Graphical abstract

A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants.

Background: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted. Objective: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants. Methods: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay. Results: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants. Conclusions: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand.

PCSK9 loss of function is protective against extra-coronary atherosclerotic cardiovascular disease in a large multi-ethnic cohort.

BackgroundTherapeutic inhibition of PCSK9 protects against coronary artery disease (CAD) and ischemic stroke (IS). The impact on other diseases remains less well characterized.MethodsWe created a genetic risk score (GRS) for PCSK9 using four single nucleotide polymorphisms (SNPs) at or near the PCSK9 locus known to impact lower LDL-Cholesterol (LDL-C): rs11583680, rs11591147, rs2479409, and rs11206510. We then used our GRS to calculate weighted odds ratios reflecting the impact of a genetically determined 10 mg/dL decrease in LDL-C on several pre-specified phenotypes including CAD, IS, peripheral artery disease (PAD), abdominal aortic aneurysm (AAA), type 2 diabetes, dementia, chronic obstructive pulmonary disease, and cancer. Finally, we used our weighted GRS to perform a phenome-wide association study.ResultsGenetic and electronic health record data that passed quality control was available in 312,097 individuals, (227,490 White participants, 58,907 Black participants, and 25,700 Hispanic participants). PCSK9 mediated reduction in LDL-C was associated with a reduced risk of CAD and AAA in trans-ethnic meta-analysis (CAD OR 0.83 [95% CI 0.80–0.87], p = 6.0 x 10−21; AAA OR 0.76 [95% CI 0.68–0.86], p = 2.9 x 10−06). Significant protective effects were noted for PAD in White individuals (OR 0.83 [95% CI 0.71–0.97], p = 2.3 x 10−04) but not in other genetic ancestries. Genetically reduced PCSK9 function associated with a reduced risk of dementia in trans-ethnic meta-analysis (OR 0.86 [95% CI 0.78–0.93], p = 5.0 x 10−04).ConclusionsGenetically reduced PCSK9 function results in a reduction in risk of several important extra-coronary atherosclerotic phenotypes in addition to known effects on CAD and IS, including PAD and AAA. We also highlight a novel reduction in risk of dementia, supporting a well-recognized vascular component to cognitive impairment and an opportunity for therapeutic repositioning.

P5322Oxidized phospholipids on apolipoprotein B-100 among black US adults with and without PCSK9 loss-of-function variants

Abstract Background High oxidized phospholipid-apolipoprotein B-100 (OxPL-apoB) levels are associated with an increased risk for coronary heart disease (CHD). Genetic PCSK9 loss-of-function (LOF) variants result in life-long lower levels of LDL-C and lipoprotein(a) and reduced CHD risk, but the association with OxPL-apoB is unknown. Purpose To estimate the association between PCSK9 LOF variants and OxPL-apoB levels among black adults. Methods Genotyping for LOF variants (Y142X and C679X) was conducted for 10,196 black Reasons for Geographic And Racial Differences in Stroke study participants. OxPL-apoB was measured using antibody E06 for all participants with LOF variants (n=241) and randomly selected participants, matched at a 1:3 ratio, without LOF variants (n=723). Low OxPL-apoB was defined as the bottom quartile of the population distribution (<1.6 nM). Prevalence ratios (PR) and 95% confidence intervals (CI) were calculated for the association between PCSK9 LOF variants and low OxPL-apoB levels adjusting for age, sex, and estimated glomerular filtration rate. Results Adults with versus without PCSK9 LOF variants had lower LDL-C and lipoprotein(a) and were less likely to be taking a statin. (Table) A higher proportion of adults with versus without PCSK9 LOF variants had low OxPL-apoB levels (30.3 vs 23.4, p=0.03). After adjustment for covariates, the PR of low OxPL-apoB was increased for participants with compared to without LOF variants (PR 1.31, 95% CI 1.00, 1.72). Characteristics of REGARDS participants PCSK9 loss-of-function variant p-value Yes (n=241) No (n=723) Age, years, mean (SD) 63.7 (9.2) 63.8 (8.6) 0.81 Female, % 61.4 60.6 0.82 Diabetes, % 34.4 27.4 0.04 LDL-C, mg/dL, mean (SD) 85 (32) 118 (37) <0.001 Lp(a), nmol/L, median (25th, 75th percentile) 63.2 (30.4, 119.6) 80.4 (39.7, 138.4) 0.02 Statin use, % 13.3 30.4 <0.001 OxPL-apoB <1.6 nM, % 30.3 23.4 0.03 Abbreviations: LDL-C, low-density lipoprotein cholesterol; Lp(a), lipoprotein(a); LOF, loss-of-function; nM, nanomolar; OxPL-apoB, oxidized phospholipids on apolipoprotein B-100; PCSK9, proprotein convertase subtilisin/kexin type-9; REGARDS, REasons for Geographic And Racial Differences in Stroke; SD, standard deviation. Conclusion Among black adults, PCSK9 LOF variants were associated with lower OxPL-apoB levels. Acknowledgement/Funding Industry/academic collaboration between Amgen Inc., University of Alabama at Birmingham and the Icahn School of Medicine at Mt. Sinai; and U01NS041588

Functional analysis of natural PCSK9 mutants in modern and archaic humans.

PCSK9 is the last member of the proprotein convertases (PCs) family and its gene is mutated in ~2% to 3% of individuals with familial hypercholesterolemia (FH). This protein enhances the degradation of the low-density lipoprotein receptor (LDLR) and hence increases the levels of circulating LDL-cholesterol (LDLc). Studies of the underlying mechanism(s) regulating the activity of different mutations in the PCSK9 gene are ongoing as they enhance our understanding of the biology and clinical relevance of PCSK9 and its partners. In an attempt to unravel the regulation of PCSK9 transcription and possibly identify mutation 'hot spot' regions with alterations in CpG methylation, we present for the first time the complete methylome profile of the PCSK9 gene in modern and archaic humanoids. Our data showed that the genomes of modern humans and archaic PCSK9 exhibit a similar methylation pattern. Next, we defined the mechanistic consequences of three PCSK9 natural mutations (PCSK9-R96L, -R105W, and -P174S) and one archaic Denisovan mutation (PCSK9-H449L) using various complementary cellular and invitro binding assays. Our results showed that the PCSK9-H449L is a loss-of-function (LOF) mutation, likely due to its lower binding affinity to the LDLR. Similarly, PCSK9-R96L and -R105W are LOF mutations, even though they have been identified in FH patients. The PCSK9-R105W mutation leads to a significantly lower autocatalytic processing of proPCSK9. PCSK9-P174S resulted in a LOF in both extracellular and intracellular pathways. In conclusion, our extensive analyses revealed that all studied mutations result in PCSK9 LOF, via various mechanisms, leading to lower levels of LDLc.

Soluble LDLR Associates with Multiple Lipoprotein Parameters in a Caucasian Population and its Association with Serum PCSK9 is Affected by PCSK9 Loss of Function

Soluble LDLR Associates with Multiple Lipoprotein Parameters in a Caucasian Population and its Association with Serum PCSK9 is Affected by PCSK9 Loss of Function

Loss-of-Function PCSK9 Mutations Are Not Associated With Alzheimer Disease.

Hypercholesterolemia is a major risk factor for the late-onset form of Alzheimer disease (AD). Loss-of-function (LOF) mutations of PCSK9 and PCSK9 inhibitors lower low-density lipoprotein cholesterol (LDL-C) and have been associated with a reduced risk of cardiovascular disease. The aim of this study was to examine the effect of PCSK9 LOF variants on risk and age of onset of AD. A total of 878 participants (410 controls and 468 AD cases) from the Quebec Founder Population were included in the study. Fifty-four (6.2%) participants carried the R46L mutation, whereas 226 (26.2%) participants carried the InsLEU mutation. There was no protective or no deleterious effect of carrying PCSK9 LOF mutations on AD prevalence nor on age of onset, even when stratified by apolipoprotein E epsilon 4 genotype or by gender. Our data indicate that carrying PCSK9 LOF mutations has a neutral effect on neurocognitive health and the prevalence of AD.

The anti-adipogenic effect of peripheral blood mononuclear cells is absent with PCSK9 loss-of-function variants.

To determine the effect of (1) an oral fat load and (2) pro-protein convertase subtilisin/kexin type (PCSK) 9 loss-of-function (LOF) variant status on the ability of peripheral blood mononuclear cells (PBMC) to inhibit human adipogenesis. PBMC from subjects with one or more PCSK9 LOF variants versus non-variant controls were compared in the fasting state and after an oral fat load. Fasting triglyceride (TG) levels were lower in the LOF variant versus non-variant group but rose to the same level after the oral fat load. Conditioned medium from PBMC was obtained in fasting (PBMC-CM-F) and 4-h postprandial (PBMC-CM-PP) states. PBMC-CM-PP from non-variant controls inhibited adipogenesis of human preadipocytes more than did PBMC-CM-F. In contrast, PBMC-CM-F or -PP from PCSK9 LOF variant subjects had no effect on adipogenesis. After the oral fat load, PBMC from PCSK9 LOF variant subjects showed significant increases in mRNA levels of interleukin-1β, tumor necrosis factor-α, sterol regulatory element binding protein-1c, CD36, and monocyte chemoattractant protein-1 (MCP-1), only MCP-1 mRNA levels increased in PBMC from non-variant controls. The absence of anti-adipogenic action of PBMC from PCSK9 LOF variant subjects points to a novel role for PCSK9 in PBMC-adipose cell interactions.

Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain.

PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9.

Abstract 216: PCSK9 Loss-of-Function Mutation Q152H is Associated with Reduced Dietary Fat Clearance and Insulin Resistance in Humans

Introduction: PCSK9 regulates plasma LDL-C concentrations. Loss-of-function (LOF) mutations are associated with cardiovascular protection via increased clearance of LDL particles. Work from our and other labs has shown that increased cellular uptake of LDL is proapoptotic for pancreatic β-cells and impairs adipose tissue function. Moreover, obese women with high concentration of LDL (ie plasma apoB) have lower insulin sensitivity and delayed dietary fat clearance. We recently identified a novel PCSK9 LOF mutation, Q152H, causing a 79% reduction of plasma PCSK9 and 48% reduction of plasma LDL-C. The impact of this mutation on the metabolic profile remains unexplored. Hypothesis: We hypothesize that despite a cardioprotective profile, carriers of Q152H have delayed dietary TG clearance and impaired insulin sensitivity similar to subjects with high apoB. Methods/Results: The heterozygous Q152H proband woman was compared to 2 groups of obese, age, sex and BMI-matched controls with low (n=6) and high (n=7) apoB. Fasting apoB, PCSK9, total cholesterol and LDL-C concentrations were lower in the proband (0.84g/l,126ng/ml, 4.22mM and 2.11mM, respectively) compared to low apoB (0.75 ± 0.05 g/l, 267 ± 18ng/ml, 5.1 ± 0.2mM and 2.8 ± 0.3nM, respectively) and high apoB (1.25 ± 0.09 g/l, 412 ± 79ng/ml, 6.9 ± 0.4mM and 4.3 ± 0.3nM, respectively). Women with high apoB had delayed dietary TG clearance over 6 hours as compared to women with low apoB (incremental TG AUC: 6.75 ± 1.65 vs 4.06 ± 0.77 mM/6h, p=NS). Incremental AUC TG in the proband was 106% greater than that of women with high apoB and 243% greater than that of low apoB. Botnia clamp was also conducted to measure insulin secretion and sensitivity. There was no difference in insulin secretion between the proband and the control groups measured via an intravenous glucose tolerance test. However, insulin sensitivity, as measured by glucose infusion rates (GIR) during a hyperinsulinemia clamp, was 7.5mg/kg/min in the proband, 32% lower than women with high apoB (11.1 ± 1.1 mg/kg/min) and 48% lower than women with low apoB (14.3 ± 0.9 mg/kg/min). Conclusion: PCSK9 deficiency favours a cardioprotective profile; however increased clearance of LDL by peripheral tissue may induce peripheral insulin resistance in obese subjects.