Matthieu Pichelin

Characterization of Autosomal Dominant Hypercholesterolemia Caused by PCSK9 Gain of Function Mutations and its Specific Treatment with Alirocumab, a PCSK9 Monoclonal Antibody

Background—Patients with PCSK9 gene gain of function (GOF) mutations have a rare form of autosomal dominant hypercholesterolemia. However, data examining their clinical characteristics and geographic distribution are lacking. Furthermore, no randomized treatment study in this population has been reported. Methods and Results—We compiled clinical characteristics of PCSK9 GOF mutation carriers in a multinational retrospective, cross-sectional, observational study. We then performed a randomized placebo-phase, double-blind study of alirocumab 150 mg administered subcutaneously every 2 weeks to 13 patients representing 4 different PCSK9 GOF mutations with low-density lipoprotein cholesterol (LDL-C) ≥70 mg/dL on their current lipid-lowering therapies at baseline. Observational study: among 164 patients, 16 different PCSK9 GOF mutations distributed throughout the gene were associated with varying severity of untreated LDL-C levels. Coronary artery disease was common (33%; average age of onset, 49.4 years), and untreated LDL-C concentrations were higher compared with matched carriers of mutations in the LDLR (n=2126) or apolipoprotein B (n=470) genes. Intervention study: in PCSK9 GOF mutation patients randomly assigned to receive alirocumab, mean percent reduction in LDL-C at 2 weeks was 62.5% (P<0.0001) from baseline, 53.7% compared with placebo-treated PCSK9 GOF mutation patients (P=0.0009; primary end point). After all subjects received 8 weeks of alirocumab treatment, LDL-C was reduced by 73% from baseline (P<0.0001). Conclusions—PCSK9 GOF mutation carriers have elevated LDL-C levels and are at high risk of premature cardiovascular disease. Alirocumab, a PCSK9 antibody, markedly lowers LDL-C levels and seems to be well tolerated in these patients. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique Identifier: NCT01604824.

Urine-sample-derived human induced pluripotent stem cells as a model to study PCSK9-mediated autosomal dominant hypercholesterolemia.

ABSTRACT Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (−38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (−89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19±0.77-fold in HLC-S127R compared to 1.38±0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function. Summary: The authors used urine-sample-derived patient-specific human induced pluripotent stem cells to generate hepatocytes carrying gain- or loss-of-function mutations of PCSK9, and mimicking the pathophysiology in vitro.

Homozygous Familial Hypercholesterolemia Patients With Identical Mutations Variably Express the LDLR (Low-Density Lipoprotein Receptor)Highlights: Implications for the Efficacy of Evolocumab

Objective— Evolocumab, a PCSK9 (proprotein convertase subtilisin kexin type 9)–neutralizing antibody, lowers low-density lipoprotein cholesterol (LDL-C) in homozygous familial hypercholesterolemic (HoFH) patients with reduced LDLR (low-density lipoprotein receptor) function. However, their individual responses are highly variable, even among carriers of identical LDLR genetic defects. We aimed to elucidate why HoFH patients variably respond to PCSK9 inhibition. Approach and Results— Lymphocytes were isolated from 22 HoFH patients enrolled in the TAUSSIG trial (Trial Assessing Long Term Use of PCSK9 Inhibition in Subjects With Genetic LDL Disorders). Ten patients were true homozygotes (FH1/FH1) and 5 identical compound heterozygotes (FH1/FH2). Lymphocytes were plated with or without mevastatin, recombinant PCSK9 (rPCSK9), or a PCSK9-neutralizing antibody. Cell surface LDLR expression was analyzed by flow cytometry. All HoFH lymphocytes had reduced cell surface LDLR expression compared with non-FH lymphocytes, for each treatment modality. Lymphocytes from FH1/FH2 patients (LDLR defective/negative) displayed the lowest LDLR expression levels followed by lymphocytes from FH1/FH1 patients (defective/defective). Mevastatin increased, whereas rPCSK9 reduced LDLR expression. The PCSK9-neutralizing antibody restored LDLR expression. Lymphocytes displaying higher LDLR expression levels were those isolated from patients presenting with lowest levels of LDL-C and apolipoprotein B, before and after 24 weeks of evolocumab treatment. These negative correlations remained significant in FH1/FH1 patients and appeared more pronounced when patients with apolipoprotein E3/E3 genotypes were analyzed separately. Significant positive correlations were found between the levels of LDLR expression and the percentage reduction in LDL-C on evolocumab treatment. Conclusions— Residual LDLR expression in HoFH is a major determinant of LDL-C levels and seems to drive their individual response to evolocumab.

Glycaemic control influences the relationship between plasma PCSK9 and LDL cholesterol in type 1 diabetes.

Pro‐protein convertase subtilisin/kexin type 9 (PCSK9) is a critical regulator of LDL cholesterol metabolism. Little is known, however, about the regulation of PCSK9 in patients with type 1 diabetes (T1D). In the present study, we aimed to determine the relationship between circulating PCSK9 and metabolic variables in T1D. Plasma PCSK9 levels were measured in 195 people with T1D (mean age 38.8 years, mean diabetes duration 17.2 years, mean glycated haemoglobin [HbA1c] 8.3%), who were free of any lipid‐lowering agent. Plasma PCSK9 was positively correlated with LDL cholesterol (P = .0007), triglycerides (P = .004), apolipoprotein B (P = .005), HbA1c (P = .003), systolic (P = .003) and diastolic (P = .001) blood pressure and body mass index (0.02). In multivariate analysis, PCSK9 concentration was independently associated with HbA1c (P = .02) and LDL cholesterol (P = .03). After classifying patients according to HbA1c tertile, the correlation between PCSK9 and LDL cholesterol was only observed in the highest tertile (P = .0006; Rho = 0.43), whereas no correlation was found in the lowest and intermediate tertiles. This study suggests that good glycaemic control abolishes the positive relationship between PCSK9 and LDL cholesterol in patients with T1D; however, the underlying molecular mechanisms remain to be established.

One-year metreleptin therapy decreases PCSK9 serum levels in diabetic patients with monogenic lipodystrophy syndromes

Diabetes & Metabolism - In Press.Proof corrected by the author Available online since mercredi 28 septembre 2016

Circulating PCSK9 levels in acute coronary syndrome: Results from the PC-SCA-9 prospective study

BACKGROUND Serum proprotein convertase subtilisin/kexin type 9 (PCSK9) concentrations have been shown to be positively associated with LDL cholesterol (LDL-C), but the relationship between PCSK9 and coronary atherosclerosis lesions remains unclear. OBJECTIVE This study aims to investigate the correlation between serum PCSK9 levels and coronary damage severity in patients hospitalized for acute coronary syndrome (ACS). METHODS In this prospective proof-of-concept study, coronary lesions were assessed using SYNTAX scores. Serum PCSK9 concentrations were measured on admission (Day 0) for ACS by Elisa, and on every day of hospitalization. Spearmans correlations were used to determine the association between PCSK9 levels, SYNTAX score and metabolic parameters. RESULTS A total of 174 patients (mean age: 59±14 years, 79% male) with ACS (on Day 0, 119 patients were not taking statins, but 55 were) were included. After initiation of high-intensity statin therapy, serum PCSK9 concentrations increased significantly, reaching maximum levels on Day 2 (+31% vs. Day 0), and remained stable up to Day 4 (P<0.001, by mixed model). Serum PCSK9 on Day 0 was associated with LDL-C (rho=0.226, P=0.017) and apolipoprotein B (rho=0.282, P=0.005) in the statin-naïve group only, and with triglycerides and non-HDL-C in all groups. More important, PCSK9 levels on Day 0 were positively associated with SYNTAX scores in the statin-naïve group (rho=0.239, P=0.009), but not in the statin-treated group (P=NS). This association was maintained after adjusting for LDL-C (P=0.014) and major CV risk factors (P=0.008). CONCLUSION Serum PCSK9 levels are positively associated with severity of coronary artery lesions independently of LDL-C concentrations in patients hospitalized for ACS. This reinforces the potential importance of PCSK9 inhibition in the management of ACS.

Identification of novel APOB mutations by targeted next-generation sequencing for the molecular diagnosis of familial hypobetalipoproteinemia

BACKGROUND AND AIMS Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by decreased plasma levels of LDL-cholesterol and apolipoprotein B (ApoB). Currently, genetic diagnosis in FHBL relies largely on Sanger sequencing to identify APOB and PCSK9 gene mutations and on western blotting to detect truncated ApoB species. METHODS Here, we applied targeted enrichment and next-generation sequencing (NGS) on a panel of three FHBL genes and two abetalipoproteinemia genes (APOB, PCSK9, ANGPTL3, MTTP and SAR1B). RESULTS In this study, we identified five likely pathogenic heterozygous rare variants. These include four novel nonsense mutations in APOB (p.Gln845*, p.Gln2571*, p.Cys2933* and p.Ser3718*) and a rare variant in PCSK9 (Minor Allele Frequency <0.1%). The affected family members tested were shown to be carriers, suggesting co-segregation with low LDL-C. CONCLUSIONS Our study further demonstrates that NGS is a reliable and practical approach for the molecular screening of FHBL-causative genes that may provide a mean for deciphering the genetic basis in FHBL.

P212: Effet de l’eau minéralisée sur le métabolisme lipidique chez le sujet adulte modérément hypercholestérolémique après une ingestion chronique et aiguë

Introduction Les maladies cardiovasculaires sont fortement modulees par les habitudes alimentaires. L’objectif de cette etude etait d’analyser l’effet de l’eau mineralisee sur les lipides plasmatiques apres ingestion chronique a l’etat de jeune et postprandial immediat (aigue). Materiels et methodes Une etude clinique monocentrique, controlee, en cross-over, randomisee en double aveugle comparant une eau mineralisee a une eau peu mineralisee a ete realisee chez 12 sujets mâles moderement hypercholesterolemiques apres huit semaines d’ingestion d’eau mineralisee (Saint Yorre) ou faiblement mineralisee (Ogeu) separees par une periode d’une semaine de wash-out. Resultats La comparaison intragroupe a montre une baisse significative des TG (triglycerides) plasmatiques a l’etat basal et apres ingestion chronique (p Cependant l’analyse intergroupes n’a pas montre de difference pour tous ces parametres mesures a jeun ou a l’etat postprandial. L’efficacite des efflux de cholesterol cellulaire mesure dans le but de tester l’effet de l’augmentation des HDL sur ce parametre n’a montre aucune difference significative. Conclusion En conclusion, les resultats ont montre une diminution des triglycerides a jeun et en aigu ainsi que l’augmentation du cholesterol des HDL en aigu et suggerent un fort impact positif de l’eau mineralisee sur le metabolisme lipidique.