👤 Cyrielle Caussy

The LMF1 (lipase maturation factor 1) gene encodes a protein involved in lipoprotein lipase and hepatic lipase maturation. Homozygous mutations in LMF1 leading to severe hypertriglyceridemia (SHTG) are rare in the literature. A few additional rare LMF1 variants have been described with poor functional studies. The aim of this study was to assess the frequency of LMF1 variants in a cohort of 385 patients with SHTG, without homozygous or compound heterozygous deleterious mutations identified in lipoprotein lipase (LPL), apolipoprotein A5 (APOA5), apolipoprotein C2 (APOC2), glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) genes, and to determine their functionality. LMF1 coding variants were screened using denaturing high-performance liquid chromatography followed by direct sequencing. In silico studies of LMF1 variants were performed, followed by in vitro functional studies using human embryonic kidney 293T (HEK-293T) cells cotransfected with vectors encoding human LPL and LMF1 cDNA. LPL activity was measured in cell culture medium after heparin addition using human VLDL-TG as substrate. Nineteen nonsynonymous coding LMF1 variants were identified in 65 patients; 10 variants were newly described in SHTG. In vitro, p.Gly172Arg, p.Arg354Trp, p.Arg364Gln, and p.Arg537Trp LMF1 variants decreased LPL activity, and the p.Trp464Ter variant completely abolished LPL activity. We identified a young girl heterozygote for the p.Trp464Ter variant and a homozygote carrier of the p.Gly172Arg variant with a near 50% decreased LPL activity in vitro and in vivo. The study confirms the rarity of LMF1 variants in a large cohort of patients with SHTG. LMF1 variants are likely to be involved in multifactorial hyperchylomicronemia. Partial LMF1 defects could be associated with intermittent phenotype as described for p.Gly172Arg homozygous and p.Trp464Ter heterozygous carriers. Show less

The heterogeneity and mechanisms of multifactorial chylomicronemia (MCM) remain poorly understood. To gain new insights, post heparin lipolysis measured at 60 min (PHLA60), in addition to the more commonly used 10 min (PHLA10), was assessed in patients with history of MCM. 62 consecutive MCM patients were studied. The evaluation included LPL, APOC2, APOA5, GPIHBP1, LMF1 and APOE gene sequencing, as well as pre- and post-heparin injection biochemical analysis, including lipid profiles, determination of apolipoprotein B, B-48, CII, CIII, lipoprotein lipase (LPL) concentrations (LPLC0, LPLC10 and LPLC60) and post-heparin LPL activity (PHLA10 and PHLA60). In controls, PHLA60 did not differ from PHLA10, while in MCM patients, PHLA60 was significantly lower than PHLA10 (p<0.001). PHLA60 showed a bimodal distribution in MCM patients (p=0.03). One subgroup exhibited PHLA60 similar to controls, with persistent lipoprotein remodeling and, paradoxically, the highest basal plasma TG concentration. APOE ε4 was over-represented compared to the European population (p<0.05) and Apo CIII/Apo B ratio was increased (p<0.01). The other subgroup exhibited low PHLA60 (p<0.001) compared to both controls and the other MCM subgroup with a lipoprotein profile consistent with fast and transient remodeling. LMF1 p. Arg364Gln was over-represented compared to the European population (p<0.05). The study showed that PHLA60 identifies a subgroup of MCM with a defect in lipolysability and/or hepatic clearance of triglycerides-rich lipoproteins, and a larger one with a defect in LPL availability. These findings provide new insights into the heterogeneity of MCM and might contribute to adjust treatment targeting. Show less

APOC3 is a major regulator of triglycerides metabolism. Several APOC3 variants are associated with hypertriglyceridemia (HTG). Our aim was to establish the potential regulation of APOC3 3'UTR variants associated with HTG by liver or intestinal miRNAs. We sequenced APOC3 3'UTR in 100 type 2 diabetic (TD2) patients with severe HTG (TG > 15 mmol/L) (HTG group) compared to 100 normotriglyceridemic patients (NTG group). We performed in silico studies to identify potential loss of miRNA binding induced by APOC3 3'UTR variants. We also performed in vitro studies to test the functionality of miRNA/APOC3 variants interactions: APOC3 3'UTR plasmids coupled with a firefly luciferase reporter were transfected in HepG2, HuH-7 and Caco-2 cells. We identified only two variants: SstI (rs5128) and BbvI (rs5225) in APOC3 3'UTR in the 2 groups of patients. Only the SstI-S2 rare allele was significantly associated with HTG (allele frequency 19,5% in HTG group vs. 9,5% in NTG group, p = 0.0045). In silico studies predicted a potential loss in the binding of 5 miRNAs induced by the S2 variant. These 5 miRNAs are all endogenously expressed in human liver and intestine, as well as in the cell models studied. However, in vitro, the S2 variant did not modulate APOC3 3'UTR reporter gene expression in HepG2, HuH-7 and Caco-2 cells. Our results do not confirm the hypothesis of a direct regulation of the APOC3 SstI variant by hepatic or intestinal miRNAs. Show less

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. Show less