Deciphering the role of V200A and N291S mutations leading to LPL deficiency.

BACKGROUND AND AIMS: Type I hyperlipoproteinemia is an autosomal recessive disorder of lipoprotein metabolism caused by mutations in the LPL gene, with an estimated prevalence in the general population of 1 in a million. In this work, we studied the molecular mechanism of two known mutations in the LPL gene in ex vivo and in vitro experiments and also the effect of two splice site mutations in ex vivo experiments. METHODS: Two patients with hypertriglyceridemia were selected from the Lipid Clinic in Vienna. The first patient was compound heterozygote for c.680T > C (exon 5; p.V200A) and c.1139+1G > A (intron 7 splice site). The second patient was compound heterozygote for c.953A > G (exon 6; p.N291S) and c.1019-3C > A (intron 6 splice site). The LPL gene was sequenced and post-heparin plasma samples (ex vivo) were used to test LPL activity. In vitro experiments were performed in HEK 293T/17 cells transiently transfected with wild type or mutant LPL plasmids. Cell lysate and media were used to evaluate LPL production, secretion, activity and dimerization by Western blot analysis and LPL enzymatic assay, respectively. RESULTS: Our data show that in both patients, LPL activity is absent. V200A is a mutation that alters LPL secretion and activity whereas the N291S mutation affects LPL activity, but both mutations do not affect dimerization. The effect of these mutations in patients is more severe since they have splice site mutations on the other allele. CONCLUSIONS: We characterized these LPL mutations at the molecular level showing that are pathogenic.

Lipoprotein Lipase Deficiency Impairs Bone Marrow Myelopoiesis and Reduces Circulating Monocyte Levels.

OBJECTIVE: Tissue macrophages induce and perpetuate proinflammatory responses, thereby promoting metabolic and cardiovascular disease. Lipoprotein lipase (LpL), the rate-limiting enzyme in blood triglyceride catabolism, is expressed by macrophages in atherosclerotic plaques. We questioned whether LpL, which is also expressed in the bone marrow (BM), affects circulating white blood cells and BM proliferation and modulates macrophage retention within the artery. APPROACH AND RESULTS: We characterized blood and tissue leukocytes and inflammatory molecules in transgenic LpL knockout mice rescued from lethal hypertriglyceridemia within 18 hours of life by muscle-specific LpL expression (MCKL0 mice). LpL-deficient mice had ≈40% reduction in blood white blood cell, neutrophils, and total and inflammatory monocytes (Ly6C/Ghi). LpL deficiency also significantly decreased expression of BM macrophage-associated markers (F4/80 and TNF-α [tumor necrosis factor α]), master transcription factors (PU.1 and C/EBPα), and colony-stimulating factors (CSFs) and their receptors, which are required for monocyte and monocyte precursor proliferation and differentiation. As a result, differentiation of macrophages from BM-derived monocyte progenitors and monocytes was decreased in MCKL0 mice. Furthermore, although LpL deficiency was associated with reduced BM uptake and accumulation of triglyceride-rich particles and macrophage CSF-macrophage CSF receptor binding, triglyceride lipolysis products (eg, linoleic acid) stimulated expression of macrophage CSF and macrophage CSF receptor in BM-derived macrophage precursor cells. Arterial macrophage numbers decreased after heparin-mediated LpL cell dissociation and by genetic knockout of arterial LpL. Reconstitution of LpL-expressing BM replenished aortic macrophage density. CONCLUSIONS: LpL regulates peripheral leukocyte levels and affects BM monocyte progenitor differentiation and aortic macrophage accumulation.

Molecular analysis of three known and one novel LPL variants in patients with type I hyperlipoproteinemia.

BACKGROUND AND AIMS: Type I hyperlipoproteinemia, also known as familial chylomicronemia syndrome (FCS), is a rare autosomal recessive disorder caused by variants in LPL, APOC2, APOA5, LMF1 or GPIHBP1 genes. The aim of this study was to identify novel variants in the LPL gene causing lipoprotein lipase deficiency and to understand the molecular mechanisms. METHODS AND RESULTS: A total of 3 individuals with severe hypertriglyceridemia and recurrent pancreatitis were selected from the Lipid Clinic at Sahlgrenska University Hospital and LPL was sequenced. In vitro experiments were performed in human embryonic kidney 293T/17 (HEK293T/17) cells transiently transfected with wild type or mutant LPL plasmids. Cell lysates and media were used to analyze LPL synthesis and secretion. Media were used to measure LPL activity. Patient 1 was compound heterozygous for three known variants: c.337T > C (W113R), c.644G > A (G215E) and c.1211T > G (M404R); patient 2 was heterozygous for the known variant c.658A > C (S220R) while patient 3 was homozygous for a novel variant in the exon 5 c.679G > T (V227F). All the LPL variants identified were loss-of-function variants and resulted in a substantial reduction in the secretion of LPL protein. CONCLUSION: We characterized at the molecular level three known and one novel LPL variants causing type I hyperlipoproteinemia showing that all these variants are pathogenic.

The role of registries in rare genetic lipid disorders: Review and introduction of the first global registry in lipoprotein lipase deficiency.

A good understanding of the natural history of rare genetic lipid disorders is a pre-requisite for successful patient management. Disease registries have been helpful in this regard. Lipoprotein Lipase Deficiency (LPLD) is a rare, autosomal-recessive lipid disorder characterized by severe hypertriglyceridemia and a very high risk for recurrent acute pancreatitis, however, only limited data are available on its natural course. Alipogene tiparvovec (Glybera®) is the first gene therapy to receive Marketing Authorization in the European Union; GENIALL (GENetherapy In the MAnagement of Lipoprotein Lipase Deficiency), a 15-year registry focusing on LPLD was launched in 2014 as part of its Risk Management Plan. The aim of this publication is to introduce the GENIALL Registry within a structured literature review of registries in rare genetic lipid disorders. A total of 11 relevant initiatives/registries were identified (homozygous Familial Hypercholesterolemia (hoFH) [n = 5]; LPLD [n = 1]; Lysosomal Acid Lipase Deficiency [LALD, n = 1], detection of mutations in genetic lipid disorders [n = 4]). Besides one product registry in hoFH and the LALD registry, all other initiatives are local or country-specific. GENIALL is the first global prospective registry in LPLD that will collect physician and patient generated data on the natural course of LPLD, as well as long-term outcomes of gene therapy. CONCLUSION: There is a limited number of international initiatives focusing on the natural course of specific rare genetic lipid disorders. The GENIALL LPLD Registry could be the first step towards a future broader global initiative that collects data related to familial chylomicronemia syndrome and their underlying genetic causes.

Identification and characterization of two novel mutations in the LPL gene causing type I hyperlipoproteinemia.

BACKGROUND: Type 1 hyperlipoproteinemia is a rare autosomal recessive disorder most often caused by mutations in the lipoprotein lipase (LPL) gene resulting in severe hypertriglyceridemia and pancreatitis. OBJECTIVES: The aim of this study was to identify novel mutations in the LPL gene causing type 1 hyperlipoproteinemia and to understand the molecular mechanisms underlying the severe hypertriglyceridemia. METHODS: Three patients presenting classical features of type 1 hyperlipoproteinemia were recruited for DNA sequencing of the LPL gene. Pre-heparin and post-heparin plasma of patients were used for protein detection analysis and functional test. Furthermore, in vitro experiments were performed in HEK293 cells. Protein synthesis and secretion were analyzed in lysate and medium fraction, respectively, whereas medium fraction was used for functional assay. RESULTS: We identified two novel mutations in the LPL gene causing type 1 hyperlipoproteinemia: a two base pair deletion (c.765_766delAG) resulting in a frameshift at position 256 of the protein (p.G256TfsX26) and a nucleotide substitution (c.1211 T > G) resulting in a methionine to arginine substitution (p.M404 R). LPL protein and activity were not detected in pre-heparin or post-heparin plasma of the patient with p.G256TfsX26 mutation or in the medium of HEK293 cells over-expressing recombinant p.G256TfsX26 LPL. A relatively small amount of LPL p.M404 R was detected in both pre-heparin and post-heparin plasma and in the medium of the cells, whereas no LPL activity was detected. CONCLUSIONS: We conclude that these two novel mutations cause type 1 hyperlipoproteinemia by inducing a loss or reduction in LPL secretion accompanied by a loss of LPL enzymatic activity.

Pathogenic classification of LPL gene variants reported to be associated with LPL deficiency.

BACKGROUND: Lipoprotein lipase (LPL) deficiency is a serious lipid disorder of severe hypertriglyceridemia (SHTG) with chylomicronemia. A large number of variants in the LPL gene have been reported but their influence on LPL activity and SHTG has not been completely analyzed. Gaining insight into the deleterious effect of the mutations is clinically essential. METHODS: We used gene sequencing followed by in-vivo/in-vitro and in-silico tools for classification. We classified 125 rare LPL mutations in 33 subjects thought to have LPL deficiency and in 314 subjects selected for very SHTG. RESULTS: Of the 33 patients thought to have LPL deficiency, only 13 were homozygous or compound heterozygous for deleterious mutations in the LPL gene. Among the 314 very SHTG patients, 3 were compound heterozygous for pathogenic mutants. In a third group of 51,467 subjects, from a general population, carriers of common variants, Asp9Asn and Asn291Ser, were associated with mild increase in triglyceride levels (11%-35%). CONCLUSION: In total, 39% of patients clinically diagnosed as LPL deficient had 2 deleterious variants. Three patients selected for very SHTG had LPL deficiency. The deleterious mutations associated with LPL deficiency will assist in the diagnosis and selection of patients as candidates for the presently approved LPL gene therapy.

Spectrum of mutations of the LPL gene identified in Italy in patients with severe hypertriglyceridemia.

BACKGROUND: Monogenic hypertriglyceridemia (HTG) may result from mutations in some genes which impair the intravascular lipolysis of triglyceride (TG)-rich lipoproteins mediated by the enzyme Lipoprotein lipase (LPL). Mutations in the LPL gene are the most frequent cause of monogenic HTG (familial chylomicronemia) with recessive transmission. METHODS: The LPL gene was resequenced in 149 patients with severe HTG (TG > 10 mmol/L) and 106 patients with moderate HTG (TG > 4.5 and <10 mmol/L) referred to tertiary Lipid Clinics in Italy. RESULTS: In the group of severe HTG, 26 patients (17.4%) were homozygotes, 9 patients (6%) were compound heterozygotes and 15 patients (10%) were simple heterozygotes for rare LPL gene variants. Single or multiple episodes of pancreatitis were recorded in 24 (48%) of these patients. There was no difference in plasma TG concentration between patients with or without a positive history of pancreatitis. Among moderate HTG patients, six patients (5.6%) were heterozygotes for rare LPL variants; two of them had suffered from pancreatitis. Overall 36 rare LPL variants were found, 15 of which not reported previously. Systematic analysis of close relatives of mutation carriers led to the identification of 44 simple heterozygotes (plasma TG 3.2 ± 4.1 mmol/L), none of whom had a positive history of pancreatitis. CONCLUSIONS: The prevalence of rare LPL variants in patients with severe or moderate HTG, referred to tertiary lipid clinics, was 50/149 (33.5%) and 6/106 (5.6%), respectively. Systematic analysis of relatives of mutation carriers is an efficient way to identify heterozygotes who may develop severe HTG.

GPIHBP1 missense mutations often cause multimerization of GPIHBP1 and thereby prevent lipoprotein lipase binding.

RATIONALE: GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1 missense mutations that interfere with LPL binding cause familial chylomicronemia. OBJECTIVE: We sought to understand mechanisms by which GPIHBP1 mutations prevent LPL binding and lead to chylomicronemia. METHODS AND RESULTS: We expressed mutant forms of GPIHBP1 in Chinese hamster ovary cells, rat and human endothelial cells, and Drosophila S2 cells. In each expression system, mutation of cysteines in GPIHBP1's Ly6 domain (including mutants identified in patients with chylomicronemia) led to the formation of disulfide-linked dimers and multimers. GPIHBP1 dimerization/multimerization was not unique to cysteine mutations; mutations in other amino acid residues, including several associated with chylomicronemia, also led to protein dimerization/multimerization. The loss of GPIHBP1 monomers is relevant to the pathogenesis of chylomicronemia because only GPIHBP1 monomers-and not dimers or multimers-are capable of binding LPL. One GPIHBP1 mutant, GPIHBP1-W109S, had distinctive properties. GPIHBP1-W109S lacked the ability to bind LPL but had a reduced propensity for forming dimers or multimers, suggesting that W109 might play a more direct role in binding LPL. In support of that idea, replacing W109 with any of 8 other amino acids abolished LPL binding-and often did so without promoting the formation of dimers and multimers. CONCLUSIONS: Many amino acid substitutions in GPIHBP1's Ly6 domain that abolish LPL binding lead to protein dimerization/multimerization. Dimerization/multimerization is relevant to disease pathogenesis, given that only GPIHBP1 monomers are capable of binding LPL.

Gestational hyperlipidemia and acute pancreatitis with underlying partial lipoprotein lipase deficiency and apolipoprotein E3/E2 genotype.

We report the case of a patient who experienced extreme recurrent gestational hyperlipidemia. She was diagnosed with partial lipoprotein lipase (LPL) deficiency but without an associated LPL gene mutation in the presence of the apolipoprotein E3/2 genotype. This is the first reported case of extreme gestational hyperlipidemia with a partial LPL deficiency in the absence of an LPL gene mutation and the apolipoprotein E 3/2 genotype. She was managed with strict dietary control and medicated with omega-3 acid ethyl esters. A patient with extreme hyperlipidemia that is limited to the gestational period should be considered partially LPL-deficient. Extreme instances of hyperlipidemia increase the risk of acute pancreatitis, and the effect of parturition on declining plasma lipid levels can be immediate and dramatic. Therefore, decisions regarding the timing and route of delivery with extreme gestational hyperlipidemia are critical and should be made carefully.

Lipoprotein lipase deficiency in an infant.

Patients with isolated hypertriglyceridemia usually present with recurrent abdominal pain, pancreatitis, eruptive xanthomas, lipemia retinalis and hepatosplenomegaly. We describe the diagnosis and treatment of an infant with severe hypertriglyceridemia. The child was found to be heterozygous for two novel mutations in the lipoprotein lipase gene.

Review of the clinical development of alipogene tiparvovec gene therapy for lipoprotein lipase deficiency.

Alipogene tiparvovec (AAV1-LPL(S447X)) gene therapy is developed to prevent complications and decrease the clinical morbidity of lipoprotein lipase deficiency (LPLD). LPLD is an autosomal recessive disease associated with severe hypertriglyceridemia (hyperTG), severe chylomicronaemia, and low HDL. Acute pancreatitis, the most frequent serious clinical LPLD complication, is a complex and heterogeneous inflammatory condition having many causes including hyperTG and chylomicronaemia. In many patients, low fat diet and currently available lipid lowering drugs are ineffective to prevent hyperTG or pancreatitis in LPLD. The clinical development program of alipogene tiparvovec includes observational studies as well as phase I/II and II/III clinical trials. Pooled data are collected on safety and efficacy issues, including the incidence of pancreatitis.

AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells.

In a clinical trial for adeno-associated virus serotype 1 (AAV-1)-mediated gene transfer to muscle for lipoprotein lipase (LPL) deficiency, 1 subject from the high-dose cohort experienced a transient increase in the muscle enzyme creatine phosphokinase (CPK) 4 weeks after gene transfer. Simultaneously, after an initial downward trend consistent with expression of LPL, plasma triglyceride levels returned to baseline. We characterized B- and T-cell responses to the vector and the transgene product in the subjects enrolled in this study. IFN-gamma enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining assays performed on peripheral blood mononuclear cells (PBMCs) from the subject who experienced the CPK elevation showed the activation of capsid-specific CD4(+) and CD8(+) T cells. Four of 8 subjects had detectable T-cell responses to capsid with dose-dependent kinetics of appearance. Subjects with detectable T-cell responses to capsid also had higher anti-AAV-1 IgG3 antibody titer. No subject developed B- or T-cell responses to the LPL transgene product. These findings suggest that T-cell responses directed to the AAV-1 capsid are dose-dependent. Whether they also limit the duration of expression of the transgene at higher doses is unclear, and will require additional analyses at later time points.

Some things just have to be done in vivo: GPIHBP1, caloric delivery, and the generation of remnant lipoproteins.

The recent discovery of a dysfunctional mutation of GPIHBP1 in a man with chylomicronemia implicates this protein in human physiology. GPIHBP1 can be placed in the larger context of other molecular participants in chylomicron docking and hydrolysis on microvascular endothelium, caloric delivery, and remnant lipoprotein generation. Critical questions include the regulation--and dysregulation--of these processes in states of overnutrition, underexertion, obesity, insulin resistance, and diabetes.

Chylomicronemia with a mutant GPIHBP1 (Q115P) that cannot bind lipoprotein lipase.

OBJECTIVE: GPIHBP1 is an endothelial cell protein that binds lipoprotein lipase (LPL) and chylomicrons. Because GPIHBP1 deficiency causes chylomicronemia in mice, we sought to determine whether some cases of chylomicronemia in humans could be attributable to defective GPIHBP1 proteins. METHODS AND RESULTS: Patients with severe hypertriglyceridemia (n=60, with plasma triglycerides above the 95th percentile for age and gender) were screened for mutations in GPIHBP1. A homozygous GPIHBP1 mutation (c.344A>C) that changed a highly conserved glutamine at residue 115 to a proline (p.Q115P) was identified in a 33-year-old male with lifelong chylomicronemia. The patient had failure-to-thrive as a child but had no history of pancreatitis. He had no mutations in LPL, APOA5, or APOC2. The Q115P substitution did not affect the ability of GPIHBP1 to reach the cell surface. However, unlike wild-type GPIHBP1, GPIHBP1-Q115P lacked the ability to bind LPL or chylomicrons (d < 1.006 g/mL lipoproteins from Gpihbp1(-/-) mice). Mouse GPIHBP1 with the corresponding mutation (Q114P) also could not bind LPL. CONCLUSIONS: A homozygous missense mutation in GPIHBP1 (Q115P) was identified in a patient with chylomicronemia. The mutation eliminated the ability of GPIHBP1 to bind LPL and chylomicrons, strongly suggesting that it caused the patient's chylomicronemia.

Lipoprotein lipase deficiency is associated with elevated acylation stimulating protein plasma levels.

Acylation stimulating protein (ASP, C3adesArg) is an adipose tissue derived hormone that stimulates triglyceride (TG) synthesis. ASP stimulates lipoprotein lipase (LPL) activity by relieving feedback inhibition caused by fatty acids (FA). The present study examines plasma ASP and lipids in male and female LPL-deficient subjects primarily with the P207L mutation, common in the population of Quebec, Canada. We evaluated the fasting and postprandial states of LPL heterozygotes and fasting levels in LPL homozygotes. Homozygotes displayed increased ASP (58-175% increase, P < 0.05-0.01), reduced HDL-cholesterol (64-75% decrease, P < 0.0001), and elevated levels of TG (19-38-fold, P < 0.0001) versus control (CTL) subjects. LPL heterozygotes with normal fasting TG (1.3-1.9 mmol/l) displayed increased ASP (101-137% increase, P < 0.05-0.01) and delayed TG clearance after a fatload; glucose levels remained similar to controls. Hypertriglyceridemics with no known LPL mutation also had increased ASP levels (63-192% increase, P < 0.001). High-TG LPL heterozygotes were administered a fatload before and after fibrate treatment. The treatment reduced fasting and postprandial plasma ASP, TG, and FA levels without changing insulin or glucose levels. ASP enhances adipose tissue fatty-acid trapping following a meal; however in LPL deficiency, high ASP levels are coupled with delayed lipid clearance.

Familial lipoprotein lipase deficiency caused by known (G188E) and novel (W394X) LPL gene mutations.

Lipoprotein lipase (LPL) is the key enzyme in the catabolism of triglyceride-rich lipoproteins in the circulation. Familial LPL deficiency is characterized by hypertriglyceridaemia and absence of LPL activity. We report a case of LPL deficiency in a 43-year-old woman, who initially presented in childhood with chylomicronaemia syndrome. At that time, her plasma triglyceride concentration was approximately 30 mmol/L and post-heparin lipolytic activity was very low. In addition to having the known missense mutation LPL G188E, the patient was also found to have a novel nonsense mutation in exon 8, namely LPL W394X. The novel substitution in exon 8 (c.1262G > A) predicts a truncated protein product of 393 amino acids that lacks the carboxylterminal 12% of the mature LPL. Trp(394) is part of a cluster of exposed tryptophan residues in the carboxyl-terminal domain of LPL important for binding lipid substrate. Of 11 members from her three-generation family, three were heterozygotes for G188E (mean plasma triglyceride, 3.5 +/- 2.0 mmol/L), whereas six were heterozygotes for W394X (triglyceride, 4.3 +/- 1.8 mmol/L). In summary, we describe a case of familial LPL deficiency caused by compound heterozygosity for known (G188E) and novel (W394X) LPL gene mutations.