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Background Lipoprotein lipase (LPL) is a key enzyme in triglyceride (TG) metabolism. LPL gene single nucleotide polymorphisms (SNPs) are associated with TG concentrations however the functionality of many of these SNPs remains poorly understood. MicroRNAs (miR) exert post-transcriptional down-regulation and their target sequence on the 3′UTR may be altered by SNPs. We therefore investigated whether LPL 3′UTR SNPs could modulate plasma TG concentration through the alteration of miR binding-sites. Methods and results We performed genetic association studies of LPL 3′UTR SNPs with TG concentrations in 271 type 2 diabetic patients and in general population samples (2997 individuals). A specific LPL haplotype (Hap4) was associated with lower plasma TG concentration (TG-0.18, IC95% [−0.30, −0.07] mmol/L or logTG-0.13, IC95% [−0.18, −0.08], p = 4.77·10 −8 ) in the meta-analysis. Hap4 comprises seven 3′UTR SNP minor alleles and p.Ser474Ter (rs328) a well-documented nonsense mutation associated with low TG concentration although by an unknown mechanism so far. Bio-informatic studies identified several putative miRNA binding-sites on the wild-type Hap1 haplotype, lost on Hap4. Functional validation performed in HEK-293T cells using luciferase expression constructs with various LPL 3′UTR allele combinations demonstrated a binding of miR-29, miR-1277 and miR-410 on Hap1, lost on Hap4. This loss of specific miR binding-site in presence of Hap4 was independent of the allelic variation of p.Ser474Ter (rs328). Conclusions We report the regulation of LPL by the miR-29, miR-1277 and miR-410 that is lost in presence of Hap4, a specific LPL TG-lowering haplotype. Consequently p.Ser474Ter association with TG concentration could be at least partially explained by its strong linkage disequilibrium with these functional 3′UTR SNPs.

International audience; Aim: Identification and functional study of Lipoprotein Lipase (LPL) 3'UTR polymorphisms associated with triglyceridemia and predicted to modulate miRNA binding.Methods: We sequenced LPL 3'UTR in 271 type 2 diabetic patients with documented lipid phenotypes. Haplotype analysis was performed using PHASE software. These data were validated by meta-analysis across four French general population samples. In silico analysis identified putative miRNA binding-sites created by LPL polymorphisms. Both LPL 3'UTR wild-type (WT) and variant (VAR) sequences were cloned in luciferase reporter gene plasmid. Transfection experiments in HEK293T and co-transfection with candidate miRNA-precursor was performed to study the regulation of the LPL constructs by specific miRNA.Results: A specific haplotype combining 8 LPL polymorphisms including the rs328 and 3'UTR minor alleles was found significantly associated with lower triglycerides concentration both in type 2 diabetes and in general populations. This haplotype was predicted to disrupt several miR-29 binding-sites. In vitro, the LPL-WT construct co-transfected with miR-29 showed a significant luciferase activity decrease (-25±6%, p< 0.001), whereas no difference was observed in the presence of the LPL-VAR construct. Moreover in HEK293T cells expressing endogenously miR-29, LPL-WT haplotype expression was lower compared to LPL-VAR haplotype (-32±7%, p

Sporadic hyperchylomicronemia (type V hyperlipoproteinemia) results from complex interactions between genetic and environmental factors that often remain unknown.Upon investigation of a patient suffering from recurrent hypertriglyceridemic pancreatitis without family history or conventional secondary cause of dyslipidemia, we identified a previously unreported nonsense heterozygous lipoprotein lipase (LPL) gene mutation S172fsX179 associated with an antihuman LPL IgG.This autoantibody partially inhibited wild-type LPL activity in vitro. Furthermore, the patient's plasma triglyceride concentrations were efficiently decreased under immunosuppressive treatment, and this was confirmed by sequential withdrawal/reintroduction tests.We consider that this unique combination of a genetic defect and an autoimmune disease results in chronic major hypertriglyceridemia. Because immunosuppressive treatment can improve this dyslipidemia, assessment of anti-LPL autoantibody is worthwhile in unmanageable chronic major hypertriglyceridemia, even in the presence of a heterozygous LPL deficiency.

International audience; BACKGROUND: 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. METHODS: 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 degrees c, the NEFA (non esterified fatty acids) produced were assayed hourly for 4 hours. LPL activity was expressed as micromol/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. RESULTS: 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 micromol/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 (\textless10 micromol/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 \textgreater10 micromol/l/min. CONCLUSION: This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.

International audience; APOA5 c.*158C\textgreaterT (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.

Context: GPIHBP1 is a new endothelial binding site for lipoprotein lipase (LPL), the key enzyme for intravascular lipolysis of triglyceride-rich lipoproteins (TGRL). We have identified two new missense mutations of the GPIHBP1 gene, C89F and G175R, by systematic sequencing in a cohort of 376 hyperchylomicronemic patients without mutations on the LPL, APOC2, or APOA5 gene. Objective: Phenotypic expression and functional consequences of these two mutations were studied. Design: We performed clinical and genotypic studies of probands and their families. GPIHBP1 functional alterations were studied in CHO pgsA-745 transfected cells. Results: Probands are an adult with a homozygous G175R mutation and a child with a hemizygous C89F neomutation and a deletion of the second allele. C89F mutation was associated with a C14F signal peptide polymorphism on the same haplotype. Both patients had resistant hyperchylomicronemia, low LPL activity, and history of acute pancreatitis. In CHO pgsA-745 cells, both G175R and C14F variants reduce the expression of GPIHBP1 at the cell surface. C89F mutation is responsible for a drastic LPL-binding defect to GPIHBP1. C14F may further potentiate C89F effect. Conclusions: The emergence of hyperchylomicronemia in the generation after a neomutation further establishes a critical role for GPIHBP1 in TGRL physiopathology in humans. Our results highlight the crucial role of C65-C89 disulfide bond in LPL binding by GPIHBP1 Ly6 domain. Furthermore, we first report a mutation of the hydrophobic C-terminal domain that impairs GPIHBP1 membrane targeting.

Objective: To provide phenotypic and functional data in new patients with APOA5 mutations and to identify genetic and metabolic factors influencing their phenotypic expression. Methods and results: By sequencing APOA5 gene in a cohort of 286 hyperchylomicronemic subjects, free of LPL or APOC2 mutations, we identified 4 unrelated carriers of the Q97X mutation (3 heterozygotes and 1 homozygote) and one heterozygote with a new L242P mutation. Postheparin LPL activity level was reduced by about 50% in Q97X heterozygotes and more than 90% in the Q97X homozygote, but was normal in the L242P patient after resolution of hyperchylomicronemia. Plasma apoAV was undetectable in the Q97X homozygote and in the normal range in the L242P and Q97X heterozygous carriers. In Western blot studies, the association of apoAV with plasma lipoproteins was altered in Q97X heterozygous carriers but not in the L242P carrier. Hyperchylomicronemic heterozygotes for both mutations carried an additional APOA5 variant haplotype and/or APOE variant (E2 or E4). Type 2 diabetes or metabolic syndrome were not a major phenotypic determinant. Conclusions: The L242P mutation was present in a hyperchylomicronemic proband but its causal involvement remains to be established. The Q97X mutation was clearly involved in hyperchylomicronemia with evidence of concomitant altered intravascular lipolysis, and a complete apoAV deficiency in the homozygote. The phenotypic expression variability of APOA5 mutations was mostly influenced by compound heterozygosity with APOA5 variant haplotypes plus additional genetic factors, and in a lesser extent by the metabolic environment.

Objectif Etudier les polymorphismes en 3′UTR du gene de la LPL associes a la triglyceridemie et determiner leurs implications dans la regulation de l’expression du gene LPL par la modification de sites de liaison de miRNA. Materiels Genotypage de patients diabetiques de type 2, avec phenotype lipidique documente, pour identifier les polymorphismes associes a la triglyceridemie. Estimation des haplotypes par le logiciel PHASE. Etude bioinformatique des modifications d’interaction miRNA-LPL induites par ces polymorphismes. Validation des interactions LPL-miRNA par co-transfection dans la lignee cellulaire HEK293 T de la sequence 3′UTR de la LPL couplee a un gene rapporteur luciferase avec le precurseur des miR-candidats ou un miR-controle. L’expression de la LPL a ete evaluee en mesurant l’activite luciferase. Resultats Le genotypage de 278 sujets a identifie un haplotype specifique compose de huit polymorphismes significativement associes a une diminution des triglycerides. Cet haplotype est present exclusivement chez les sujets normotriglyceridemiques (frequence allelique 7,9 %). In silico, cinq polymorphismes sont susceptibles de supprimer des sites de liaison de miR-29 . In vitro, on observe une diminution significative (–25 ± 6 %, p miR-29 comparee au miR -controle, non observee avec l’haplotype mute. Discussion Nous avons identifie huit polymorphismes appartenant a un haplotype de la LPL significativement associe a une diminution de la triglyceridemie. Cet haplotype supprime le site de liaison de miR-29 in vitro. La levee d’inhibition ainsi induite, pourrait augmenter l’expression de la LPL et expliquer la diminution de la triglyceridemie chez les porteurs de l’haplotype.