Characterization of two novel pathogenic variants at compound heterozygous status in lipase maturation factor 1 gene causing severe hypertriglyceridemia.

BACKGROUND: Severe hypertriglyceridemia is a rare disease characterized by triglyceride levels higher than 1000 mg/dL (11.3 mmol/L) and acute pancreatitis. The disease is caused by pathogenic variants in genes encoding lipoprotein lipase (LPL), apolipoprotein A5, apolipoprotein C2, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1, and lipase maturation factor 1 (LMF1). OBJECTIVE: We aim to identify the genetic cause of severe hypertriglyceridemia and characterize the new variants in a patient with severe hypertriglyceridemia. METHODS: The proband was a male showing severe hypertriglyceridemia (triglycerides 1416 mg/dL, 16.0 mmol/L); proband's relatives were also screened. Genetic screening included direct sequencing of the above genes and identification of large rearrangements in the LPL gene. Functional characterization of mutant LMF1 variants was performed by complementing LPL maturation in transfected LMF1-deficient mouse fibroblasts. RESULTS: The proband and his affected brother were compound heterozygotes for variants in the LMF1 gene never identified as causative of severe hypertriglyceridemia c.[157delC;1351C>T];[410C>T], p.[(Arg53Glyfs*5)];[(Ser137Leu)]. Functional analysis demonstrated that the p.(Arg53Glyfs*5) truncation completely abolished and the p.(Ser137Leu) missense variant dramatically diminished the lipase maturation activity of LMF1. CONCLUSIONS: In addition to a novel truncating variant, we describe for the first time a missense variant functionally demonstrated affecting the lipase maturation function of LMF1. This is the first case in which compound heterozygous variants in LMF1 were functionally demonstrated as causative of severe hypertriglyceridemia.

Hepatic Tm6sf2 overexpression affects cellular ApoB-trafficking, plasma lipid levels, hepatic steatosis and atherosclerosis.

The human transmembrane 6 superfamily member 2 (TM6SF2) gene has been implicated in plasma lipoprotein metabolism, alcoholic and non-alcoholic fatty liver disease and myocardial infarction in multiple genome-wide association studies. To investigate the role of Tm6sf2 in metabolic homeostasis, we generated mice with elevated expression using adeno-associated virus (AAV)-mediated gene delivery. Hepatic overexpression of mouse Tm6sf2 resulted in phenotypes previously observed in Tm6sf2-deficient mice including reduced plasma lipid levels, diminished hepatic triglycerides secretion and increased hepatosteatosis. Furthermore, increased hepatic Tm6sf2 expression protected against the development of atherosclerosis in LDL-receptor/ApoB48-deficient mice. In cultured human hepatocytes, Tm6sf2 overexpression reduced apolipoprotein B secretion and resulted in its accumulation within the endoplasmic reticulum (ER) suggesting impaired ER-to-Golgi trafficking of pre-very low-density lipoprotein (VLDL) particles. Analysis of two metabolic trait-associated coding polymorphisms in the human TM6SF2 gene (rs58542926 and rs187429064) revealed that both variants impact TM6SF2 expression by affecting the rate of protein turnover. These data demonstrate that rs58542926 (E167K) and rs187429064 (L156P) are functional variants and suggest that they influence metabolic traits through altered TM6SF2 protein stability. Taken together, our results indicate that cellular Tm6sf2 level is an important determinant of VLDL metabolism and further implicate TM6SF2 as a causative gene underlying metabolic disease and trait associations at the 19p13.11 locus.

Embryonic viability, lipase deficiency, hypertriglyceridemia and neonatal lethality in a novel LMF1-deficient mouse model.

BACKGROUND: Lipase Maturation Factor 1 (LMF1) is an ER-chaperone involved in the post-translational maturation and catalytic activation of vascular lipases including lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). Mutations in LMF1 are associated with lipase deficiency and severe hypertriglyceridemia indicating the critical role of LMF1 in plasma lipid homeostasis. The currently available mouse model of LMF1 deficiency is based on a naturally occurring truncating mutation, combined lipase deficiency (cld), which may represent a hypomorphic allele. Thus, development of LMF1-null mice is needed to explore the phenotypic consequences of complete LMF1 deficiency. FINDINGS: In situ hybridization and qPCR analysis in the normal mouse embryo revealed ubiquitous and high-level LMF1 expression. To investigate if LMF1 was required for embryonic viability, a novel mouse model based on a null-allele of LMF1 was generated and characterized. LMF1-/- progeny were born at Mendelian ratios and exhibited combined lipase deficiency, hypertriglyceridemia and neonatal lethality. CONCLUSION: Our results raise the possibility of a previously unrecognized role for LMF1 in embryonic development, but indicate that LMF1 is dispensable for the viability of mouse embryo. The novel mouse model developed in this study will be useful to investigate the full phenotypic spectrum of LMF1 deficiency.

Lipase maturation factor 1 (lmf1) is induced by endoplasmic reticulum stress through activating transcription factor 6α (Atf6α) signaling.

Lipase maturation factor 1 (Lmf1) is a critical determinant of plasma lipid metabolism, as demonstrated by severe hypertriglyceridemia associated with its mutations in mice and human subjects. Lmf1 is a chaperone localized to the endoplasmic reticulum (ER) and required for the post-translational maturation and activation of several vascular lipases. Despite its importance in plasma lipid homeostasis, the regulation of Lmf1 remains unexplored. We report here that Lmf1 expression is induced by ER stress in various cell lines and in tunicamycin (TM)-injected mice. Using genetic deficiencies in mouse embryonic fibroblasts and mouse liver, we identified the Atf6α arm of the unfolded protein response as being responsible for the up-regulation of Lmf1 in ER stress. Experiments with luciferase reporter constructs indicated that ER stress activates the Lmf1 promoter through a GC-rich DNA sequence 264 bp upstream of the transcriptional start site. We demonstrated that Atf6α is sufficient to induce the Lmf1 promoter in the absence of ER stress, and this effect is mediated by the TM-responsive cis-regulatory element. Conversely, Atf6α deficiency induced by genetic ablation or a dominant-negative form of Atf6α abolished TM stimulation of the Lmf1 promoter. In conclusion, our results indicate that Lmf1 is an unfolded protein response target gene, and Atf6α signaling is sufficient and necessary for activation of the Lmf1 promoter. Importantly, the induction of Lmf1 by ER stress appears to be a general phenomenon not restricted to lipase-expressing cells, which suggests a lipase-independent cellular role for this protein in ER homeostasis.