Functional characterization of two APOB variants from exon 29 found in individuals with clinical diagnosis of Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is an inherited lipid disorder characterized by increased levels of LDL cholesterol. About 5-10% of FH cases occur due to variants in the APOB gene, but these alterations can be a more common cause of FH than expected since most of APOB variants identified is still unknown their effect on the metabolism. The majority of the variants are missense but there are a few nonsense variants and small indels in exon 29 identified in individuals with hypercholesterolemia phenotype that can cause FH. The aim of this project was to functional characterize APOB variants from exon 29 identified in individuals referred to the Portuguese FH Study to assess if these are the genetic cause of disease. LDL from index cases and relatives was isolated through sequential ultracentrifugation. ED-LDLR was purified from HEK293 cells transfected with the pcDNA3.1-EC-LDLR-His plasmid by affinity chromatography. Purified ED-LDLR fragments were coated onto 96-well plates and incubated with the different APOB variants. Antibodies were used for ligand detection, and absorbance was determined at 405 nm. CHO-ldlA7 cells were transfected with wt LDLR plasmid and incubated with FITC-labeled LDL to determine LDL binding and uptake by flow cytometry. p.(Gln4316*) and p.(Glu4387Asnfs*7) alterations from exon 29 showed reduced affinity for the LDL receptor. Uptake and binding assays results were similar, so these variants may affect the binding of apoB to the LDL receptor. The alterations studied were not present in a normolipidemic panel. APOB variants studied in this work produce truncated forms of apoB, but they are unlikely to lead to nonsense-mediated decay processes due to their location near the end of the gene. Functional studies can provide important evidence for variant pathogenicity assessment being these essential to provide an accurate diagnosis. These assays can confirm the clinical diagnosis by highlighting the cause of disease, and contribute to a personalized treatment and stratify patient associated cardiovascular risk.N/

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.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). GL is supported by the Agence Nationale de la Recherche (Paris, France) Program Grant CHOPIN (CHolesterol Personalized Innovation) ANR-16-RHUS-0007 and Project Grant KRINGLE2 ANR-20-CE14-0009 as well as by La Fondation De France (FDF-00096274). U.G-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019-2020. A.L.-S. was supported by a grant PIF (2019–2020), Gobierno Vasco, and partially supported by Fundación Biofísica Bizkaia. KC and AKJ received a scholarship from the European Union (European Regional Development Fund INTERREG V) and the Région Réunion (Saint-Denis, Réunion, France)

Pathophysiology of Atherosclerosis

Atherosclerosis is the main risk factor for cardiovascular disease (CVD), which is the leading cause of mortality worldwide. Atherosclerosis is initiated by endothelium activation and, followed by a cascade of events (accumulation of lipids, fibrous elements, and calcification), triggers the vessel narrowing and activation of inflammatory pathways. The resultant atheroma plaque, along with these processes, results in cardiovascular complications. This review focuses on the different stages of atherosclerosis development, ranging from endothelial dysfunction to plaque rupture. In addition, the post-transcriptional regulation and modulation of atheroma plaque by microRNAs and lncRNAs, the role of microbiota, and the importance of sex as a crucial risk factor in atherosclerosis are covered here in order to provide a global view of the disease.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019/2020; U.G-G. was supported by Margarita Salas Grant; and S.J. and A.L-S were supported by a grant PIF (2017–2018) and PIF (2019–2020) Gobierno Vasco, respectively

Statin Treatment-Induced Development of Type 2 Diabetes: From Clinical Evidence to Mechanistic Insights

Statins are the gold-standard treatment for the prevention of primary and secondary cardiovascular disease, which is the leading cause of mortality worldwide. Despite the safety and relative tolerability of statins, observational studies, clinical trials and meta-analyses indicate an increased risk of developing new-onset type 2 diabetes mellitus (T2DM) after long-term statin treatment. It has been shown that statins can impair insulin sensitivity and secretion by pancreatic β-cells and increase insulin resistance in peripheral tissues. The mechanisms involved in these processes include, among others, impaired Ca2+ signaling in pancreatic β-cells, down-regulation of GLUT-4 in adipocytes and compromised insulin signaling. In addition, it has also been described that statins’ impact on epigenetics may also contribute to statin-induced T2DM via differential expression of microRNAs. This review focuses on the evidence and mechanisms by which statin therapy is associated with the development of T2DM. This review describes the multifactorial combination of effects that most likely contributes to the diabetogenic effects of statins. Clinically, these findings should encourage clinicians to consider diabetes monitoring in patients receiving statin therapy in order to ensure early diagnosis and appropriate management.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J. and A.L.-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia

Pathophysiology of Type 2 Diabetes Mellitus

Type 2 Diabetes Mellitus (T2DM), one of the most common metabolic disorders, is caused by a combination of two primary factors: defective insulin secretion by pancreatic β-cells and the inability of insulin-sensitive tissues to respond appropriately to insulin. Because insulin release and activity are essential processes for glucose homeostasis, the molecular mechanisms involved in the synthesis and release of insulin, as well as in its detection are tightly regulated. Defects in any of the mechanisms involved in these processes can lead to a metabolic imbalance responsible for the development of the disease. This review analyzes the key aspects of T2DM, as well as the molecular mechanisms and pathways implicated in insulin metabolism leading to T2DM and insulin resistance. For that purpose, we summarize the data gathered up until now, focusing especially on insulin synthesis, insulin release, insulin sensing and on the downstream effects on individual insulin-sensitive organs. The review also covers the pathological conditions perpetuating T2DM such as nutritional factors, physical activity, gut dysbiosis and metabolic memory. Additionally, because T2DM is associated with accelerated atherosclerosis development, we review here some of the molecular mechanisms that link T2DM and insulin resistance (IR) as well as cardiovascular risk as one of the most important complications in T2DM.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especializaci.n de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J. and A.L-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia

Cholesterol Efflux Efficiency of Reconstituted HDL Is Affected by Nanoparticle Lipid Composition

Cardiovascular disease (CVD), the leading cause of mortality worldwide is primarily caused by atherosclerosis, which is promoted by the accumulation of low-density lipoproteins into the intima of large arteries. Multiple nanoparticles mimicking natural HDL (rHDL) have been designed to remove cholesterol excess in CVD therapy. The goal of this investigation was to assess the cholesterol efflux efficiency of rHDLs with different lipid compositions, mimicking different maturation stages of high-density lipoproteins (HDLs) occurring in vivo. Methods: the cholesterol efflux activity of soybean PC (Soy-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC:Chol:1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPC) and DPPC:18:2 cholesteryl ester (CE):LysoPC rHDLs was determined in several cell models to investigate the contribution of lipid composition to the effectiveness of cholesterol removal. Results: DPPC rHDLs are the most efficient particles, inducing cholesterol efflux in all cellular models and in all conditions the effect was potentiated when the ABCA1 transporter was upregulated. Conclusions: DPPC rHDLs, which resemble nascent HDL, are the most effective particles in inducing cholesterol efflux due to the higher physical binding affinity of cholesterol to the saturated long-chain-length phospholipids and the favored cholesterol transfer from a highly positively curved bilayer, to an accepting planar bilayer such as DPPC rHDLs. The physicochemical characteristics of rHDLs should be taken into consideration to design more efficient nanoparticles to promote cholesterol efflux.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J.-B. and A.L.-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia

Functional Characterization of p.(Arg160Gln) PCSK9 Variant Accidentally Found in a Hypercholesterolemic Subject

Familial hypercholesterolaemia (FH) is an autosomal dominant dyslipidaemia, characterised by elevated LDL cholesterol (LDL-C) levels in the blood. Three main genes are involved in FH diagnosis: LDL receptor (LDLr), Apolipoprotein B (APOB) and Protein convertase subtilisin/kexin type 9 (PCSK9) with genetic mutations that led to reduced plasma LDL-C clearance. To date, several PCSK9 gain-of-function (GOF) variants causing FH have been described based on their increased ability to degrade LDLr. On the other hand, mutations that reduce the activity of PCSK9 on LDLr degradation have been described as loss-of-function (LOF) variants. It is therefore important to functionally characterise PCSK9 variants in order to support the genetic diagnosis of FH. The aim of this work is to functionally characterise the p.(Arg160Gln) PCSK9 variant found in a subject suspected to have FH. Different techniques have been combined to determine efficiency of the autocatalytic cleavage, protein expression, effect of the variant on LDLr activity and affinity of the PCSK9 variant for the LDLr. Expression and processing of the p.(Arg160Gln) variant had a result similar to that of WT PCSK9. The effect of p.(Arg160Gln) PCSK9 on LDLr activity is lower than WT PCSK9, with higher values of LDL internalisation (13%) and p.(Arg160Gln) PCSK9 affinity for the LDLr is lower than WT, EC50 8.6 ± 0.8 and 25.9 ± 0.7, respectively. The p.(Arg160Gln) PCSK9 variant is a LOF PCSK9 whose loss of activity is caused by a displacement of the PCSK9 P’ helix, which reduces the stability of the LDLr-PCSK9 complex.This research was funded by Grupos Consolidados Gobierno Vasco 2021, grant number IT1720-22. A.L.-S. was supported by a grant PIF (2019–2020), Gobierno Vasco and partially supported by Fundación Biofísica Bizkaia. S.J-B. was supported by a Margarita Salas Grant 2022 from the University of the Basque Country

Replacement of Cysteine at Position 46 in the First Cysteine-Rich Repeat of the LDL Receptor

Background and aims Pathogenic mutations in the Low Density Lipoprotein Receptor gene (LDLR) cause Familial Hypercholesterolemia (FH), one of the most common genetic disorders with a prevalence as high as 1 in 200 in some populations. FH is an autosomal dominant disorder of lipoprotein metabolism characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis. To date, 2500 LDLRvariants have been identified in the LDLR gene; however, only a minority of them have been experimentally characterized and proven to be pathogenic. Here we investigated the role of Cys46 located in the first repeat of the LDL receptor binding domain in recognition of apolipoproteins. Methods Activity of the p.(Cys46Gly) LDLR variant was assessed by immunoblotting and flow cytometry in CHO-/d/A7 expressing the receptor variant. Affinity of p.(Cys46Gly) for LDL and VLDL was determined by solid-phase immunoassays and in silico analysis was used to predict mutation effects. Results and conclusion Functional characterization of p.(Cys46Gly) LDLRvariant showed impaired LDL and VLDL binding and uptake activity. Consistent with this, solid-phase immunoassays showed the p. (Cys46Gly) LDLR variant has decreased binding affinity for apolipoproteins. These results indicate the important role of Cys46 in LDL receptor activity and highlight the role of LR1 in LDLr activity modulation. This study reinforces the significance of in vitro functional characterization of LDL receptor activity in developing an accurate approach to FH genetic diagnosis. This is of particular importance because it enables clinicians to tailor personalized treatments for patients' mutation profile.Progenika Biopharma SA, is an Spanish biotech company founded in 2000 with headquarters in Derio, Bizkaia (SPAIN). Progenika Biopharma SA did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries to M.S. and L.P. This work was supported by Gobernio Vasco, ELKARTEK BIOGUNE 2015 (Codigo KK-2015/0000089) and Basque Government (Grupos Consolidados IT849-13). A. B-V. was supported by a grant (PIF 2014/2015) Eusko Jaurlaritza and S.J. was supported by grant (PIF 2018/2019) Eusko Jaurlaritza. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript

Hiperkolesterolemia eta Hiperlipemia Familiarra eragiten duten aldaera genetikoak karakterizatzeko eta aurresateko in vitro eta in silico tresnen garapena

320 p.El objetivo de mi tesis se centra en el desarrollo de técnicas in silico e in vitro para la predicción y caracterización de variantes causantes de hipercolesterolemia familiar e hiperlipemias. El uso de modelos y software para la predicción de patogenicidad de variantes facilita la identificación de variantes patogénicas. Estos modelos necesitan una base de datos robusta para poder ser desarrollados. Por esa razón, no se pueden aplicar a cualquier proteína. El receptor de lipoproteínas de baja densidad (en inglés, LDLR) ha sido ampliamente estudiado y cuenta con cientos de variantes caracterizadas, lo que nos ha permitido desarrollar dos modelos predictivos basados en distintas metodologías: MLb-LDLR y OptiMo-LDLR.En cambio, otros genes implicados en el desarrollo de hiperlipemias, como pueden ser PCSK9 o APOE, no cuentan con una base de datos tan amplia. En estos casos, la caracterización funcional de las variantes sigue siendo clave para la identificación de variantes patogénicas. Por esa razón, en esta tesis proponemos dos metodologías para la caracterización sistemática de variantes de PCSK9 y APOE.CSIC; Instituto Biofisik

Cardiovascular Disease, Atherosclerosis and Familial Hypercholesterolemia: From Molecular Mechanisms Causing Pathogenicity to New Therapeutic Approaches

This Special Issue, “Cardiovascular Disease, Atherosclerosis and Familial Hypercholesterolemia: From Molecular Mechanisms Causing Pathogenicity to New Therapeutic Approaches”, contributes to advancing our knowledge of the molecular mechanisms that drive cardiovascular disease, atherosclerosis and familial hypercholesterolemia and the development of state-of-the-art research in the field [...