An APOA5 3′ UTR Variant Associated with Plasma Triglycerides Triggers APOA5 Downregulation by Creating a Functional miR-485-5p Binding Site

APOA5 c.∗158C>T (rs2266788), located in the 3′ UTR, belongs to APOA5 haplotype 2 (APOA5∗2), which is strongly associated with plasma triglyceride levels and modulates the occurrence of both moderate and severe hypertriglyceridemia. Individuals with APOA5∗2 display reduced APOA5 expression at the posttranscriptional level. However, the functionality of this haplotype remains unclear. We hypothesized that the hypertriglyceridemic effects of APOA5∗2 could involve miRNA regulation in the APOA5 3′ UTR. Bioinformatic studies have identified the creation of a potential miRNA binding site for liver-expressed miR-485-5p (MIRN485-5p) in the mutant APOA5 3′ UTR with the c.∗158C allele. In human embryonic kidney 293T (HEK293T) cells cotransfected with an APOA5 3′ UTR luciferase reporter vector and a miR485-5p precursor, c.∗158C allele expression was significantly decreased. Moreover, in HuH-7 cells endogenously expressing miR-485-5p, we observed that luciferase activity was significantly lower in the presence of the c.∗158C allele than in the presence of the c.∗158T allele, which was completely reversed by a miR-485-5p inhibitor. We demonstrated that the rare c.∗158C APOA5 allele creates a functional target site for liver-expressed miR-485-5p. Therefore, we propose that the well-documented hypertriglyceridemic effect of APOA5∗2 involves an APOA5 posttranscriptional downregulation mediated by miR-485-5p

Review of the nutritional benefits and risks related to intense sweeteners.

BACKGROUND: The intense sweeteners currently authorised in Europe comprise ten compounds of various chemical natures. Their overall use has sharply risen in the last 20 years. These compounds are mainly used to formulate reduced-calorie products while maintaining sweetness. METHODS: This extensive analysis of the literature reviews the data currently available on the potential nutritional benefits and risks related to the consumption of products containing intense sweeteners. RESULTS AND CONCLUSIONS: Regarding nutritional benefits, the available studies, while numerous, do not provide proof that the consumption of artificial sweeteners as sugar substitutes is beneficial in terms of weight management, blood glucose regulation in diabetic subjects or the incidence of type 2 diabetes. Regarding nutritional risks (incidence of type 2 diabetes, habituation to sweetness in adults, cancers, etc.), it is not possible based on the available data to establish a link between the occurrence of these risks and the consumption of artificial sweeteners. However, some studies underline the need to improve knowledge of the links between intense sweeteners consumption and certain risks

Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects.

Determination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. Consequently by using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles.Post-heparin plasma was added to the VLDL substrate prepared by ultracentrifugation of heat inactivated normolipidemic human serums, diluted in buffer, pH 8.15. Following incubation at 37°c, the NEFA (non esterified fatty acids) produced were assayed hourly for 4 hours. LPL activity was expressed as µmol/l/min after subtraction of hepatic lipase (HL) activity, obtained following LPL inhibition with NaCl 1.5 mmol/l. Molecular analysis of LPL, GPIHBP1, APOA5, APOC2, APOE genes was available for 62 patients.Our method was reproducible (coefficient of variation (CV): intra-assay 5.6%, inter-assay 7.1%), and tightly correlated with the conventional radiolabelled triolein emulsion method (n = 26, r = 0.88). Normal values were established at 34.8 ± 12.8 µmol/l/min (mean ± SD) from 20 control subjects. LPL activities obtained from 71 patients with documented history of major hypertriglyceridemia showed a trimodal distribution. Among the 11 patients with a very low LPL activity (< 10 µmol/l/min), 5 were homozygous or compound heterozygous for LPL or GPIHBP1 deleterious mutations, 3 were compound heterozygous for APOA5 deleterious mutations and the p.S19W APOA5 susceptibility variant, and 2 were free of any mutations in the usual candidate genes. No homozygous gene alteration in LPL, GPIHBP1 and APOC2 genes was found in any of the patients with LPL activity > 10 µmol/l/min.This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects

Evaluation des bénéfices et des risques nutritionnels des édulcorants intenses.

En 2011, l’Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et dutravail (Anses) a émis un avis examinant deux nouvelles études portant sur d'éventuelseffets sanitaires liés à l'aspartame et l'acésulfame de potassium (K), deux édulcorantsintenses. Suite à cet avis, l’Anses a estimé qu’il était nécessaire de poursuivre l’évaluationdans deux directions. Elle a donc d’une part sollicité l’Efsa (European food safety agency,Autorité européenne de sécurité des aliments) pour que celle-ci anticipe la révision de laDose Journalière Admissible (DJA) de l’aspartame initialement prévue en 2020 par leRèglement européen 257/20101. Les résultats de cette révision ont été publiés en décembre2013 (Efsa, 2013). Parallèlement, constatant qu’en dehors de ces interrogations d’ordretoxicologique, subsistaient des questions récurrentes relatives aux bénéfices et aux risquessanitaires d’ordre nutritionnel des édulcorants intenses, l’Anses a mis en place un groupe detravail (GT) chargé d’étudier ces questions

Evaluation des bénéfices et des risques nutritionnels des édulcorants intenses.

En 2011, l’Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et dutravail (Anses) a émis un avis examinant deux nouvelles études portant sur d'éventuelseffets sanitaires liés à l'aspartame et l'acésulfame de potassium (K), deux édulcorantsintenses. Suite à cet avis, l’Anses a estimé qu’il était nécessaire de poursuivre l’évaluationdans deux directions. Elle a donc d’une part sollicité l’Efsa (European food safety agency,Autorité européenne de sécurité des aliments) pour que celle-ci anticipe la révision de laDose Journalière Admissible (DJA) de l’aspartame initialement prévue en 2020 par leRèglement européen 257/20101. Les résultats de cette révision ont été publiés en décembre2013 (Efsa, 2013). Parallèlement, constatant qu’en dehors de ces interrogations d’ordretoxicologique, subsistaient des questions récurrentes relatives aux bénéfices et aux risquessanitaires d’ordre nutritionnel des édulcorants intenses, l’Anses a mis en place un groupe detravail (GT) chargé d’étudier ces questions

Evaluation des bénéfices et des risques nutritionnels des édulcorants intenses. Avis de l'Anses

Evaluation des bénéfices et des risques nutritionnels des édulcorants intenses. Avis de l'Anse

PCSK9 post-transcriptional regulation: Role of a 3′UTR microRNA-binding site variant in linkage disequilibrium with c.1420G

International audienceBackground and aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol homeostasis. A common variant, the G allele in position c.1420 (c.1420G), has been associated with a decrease of both plasma PCSK9 and LDL-cholesterol concentrations. However, the functional effect of this variant is currently not well understood. We hypothesized that it could be explained by functional variants in linkage disequilibrium (LD), more specifically, by variants located in the PCSK9 3' UTR as targets for miR regulation of PCSK9 expression.Methods: Variations in LD with c.1420G were studied in 1029 patients followed for dyslipidaemia. In silico studies identified potential miRNA binding sites induced by PCSK9 3'UTR variants in LD with c.1420G. Their functionality was studied with a luciferase reporter assay in HuH-7 cells and confirmed by cotransfection of anti-miRNAs.Results: The c.*571C and c.*234T variants located in the PCSK9 3'UTR were found in tight LD with c.1420G (D' = 0.962; LOD = 163.06). The haplotype carrying c.*571C showed a 6.7% decrease in luciferase activity (p = 0.003). Inhibition of hsa-miR-1228-3p and hsa-miR-143-5p counteracted their effect on the haplotype carrying c.*571C allele, suggesting that PCSK9 expression was decreased by the endogenous binding of hsa-miR-1228-3p and hsa-miR-143-5p on its 3'UTR.Conclusions: This post-transcriptional regulation might contribute towards the association between plasma PCSK9 levels and c.1420G. Such regulation of PCSK9 expression may open new perspectives for the treatment of hypercholesterolemia and atherosclerosis cardiovascular diseases

Additive Effect of APOE Rare Variants on the Phenotype of Familial Hypercholesterolemia

International audienceBackground: Autosomal dominant hypercholesterolemia (ADH) is due to deleterious variants in LDLR, APOB, or PCSK9 genes. Double heterozygote for these genes induces a more severe phenotype. More recently, a new causative variant of heterozygous ADH was identified in APOE. Here we study the phenotype of 21 adult patients, double heterozygotes for rare LDLR and rare APOE variants (LDLR+APOE) in a national wide French cohort. Methods: LDLR, APOB, PCSK9, and APOE genes were sequenced in 5743 probands addressed for ADH genotyping. The lipid profile and occurrence of premature atherosclerotic cardiovascular diseases were compared between the LDLR+APOE carriers (n=21) and the carriers of the same LDLR causative variants alone (n=22). Results: The prevalence of LDLR+APOE carriers in this French ADH cohort is 0.4%. Overall, LDL (low-density lipoprotein)-cholesterol concentrations were 23% higher in LDLR+APOE patients than in LDLR patients (9.14±2.51 versus 7.43±1.59 mmol/L, P=0.0221). When only deleterious or probably deleterious variants were considered, the LDL-cholesterol concentrations were 46% higher in LDLR+APOE carriers than in LDLR carriers (10.83±3.45 versus 7.43±1.59 mmol/L, P=0.0270). Two patients exhibited a homozygous familial hypercholesterolemia phenotype (LDL-cholesterol >13 mmol/L). Premature atherosclerotic cardiovascular disease was more common in LDLR+APOE patients than in LDLR carriers (70% versus 30%, P=0.026). Conclusions: Although an incomplete penetrance should be taken into account for APOE variant classification, these results suggest an additive effect of deleterious APOE variants on ADH phenotype highlighting the relevance of APOE sequencing

APOE Molecular Spectrum in a French Cohort with Primary Dyslipidemia

International audiencePrimary hypercholesterolemia is characterized by elevated LDL-cholesterol (LDL-C) levels isolated in autosomal dominant hypercholesterolemia (ADH) or associated with elevated triglyceride levels in familial combined hyperlipidemia (FCHL). Rare APOE variants are known in ADH and FCHL. We explored the APOE molecular spectrum in a French ADH/FCHL cohort of 5743 unrelated probands. The sequencing of LDLR, PCSK9, APOB, and APOE revealed 76 carriers of a rare APOE variant, with no mutation in LDLR, PCSK9, or APOB. Among the 31 APOE variants identified here, 15 are described in ADH, 10 in FCHL, and 6 in both probands. Five were previously reported with dyslipidemia and 26 are novel, including 12 missense, 5 synonymous, 2 intronic, and 7 variants in regulatory regions. Sixteen variants were predicted as pathogenic or likely pathogenic, and their carriers had significantly lower polygenic risk scores (wPRS) than carriers of predicted benign variants. We observed no correlation between LDL-C levels and wPRS, suggesting a major effect of APOE variants. Carriers of p.Leu167del were associated with a severe phenotype. The analysis of 11 probands suggests that carriers of an APOE variant respond better to statins than carriers of a LDLR mutation. Altogether, we show that the APOE variants account for a significant contribution to ADH and FCHL

A new 165-SNP low-density lipoprotein cholesterol polygenic risk score based on next generation sequencing outperforms previously published scores in routine diagnostics of familial hypercholesterolemia

International audienc