Familial LCAT Deficiency Patients Research Articles

Rescue of Familial Lecithin:Cholesterol Acyltranferase Deficiency Mutations with an Allosteric Activator.

Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and α/β-hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. SIGNIFICANCE STATEMENT: Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in high density lipoprotein particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study, we show that recently discovered small molecule activators can rescue function in LCAT-deficient variants when the mutations occur in the lid and cap domains of the enzyme.

Correction of Familial LCAT Deficiency by AAV-hLCAT Prevents Renal Injury and Atherosclerosis in Hamsters-Brief Report.

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Progression of chronic kidney disease in familial LCAT deficiency: a follow-up of the Italian cohort

Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the LCAT gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.

Lecithin:cholesterol acyltransferase: symposium on 50 years of biomedical research from its discovery to latest findings

LCAT converts free cholesterol to cholesteryl esters in the process of reverse cholesterol transport. Familial LCAT deficiency (FLD) is a genetic disease that was first described by Kaare R. Norum and Egil Gjone in 1967. This report is a summary from a 2017 symposium where Dr. Norum recounted the history of FLD and leading experts on LCAT shared their results. The Tesmer laboratory shared structural findings on LCAT and the close homolog, lysosomal phospholipase A2. Results from studies of FLD patients in Finland, Brazil, Norway, and Italy were presented, as well as the status of a patient registry. Drs. Kuivenhoven and Calabresi presented data from carriers of genetic mutations suggesting that FLD does not necessarily accelerate atherosclerosis. Dr. Ng shared that LCAT-null mice were protected from diet-induced obesity, insulin resistance, and nonalcoholic fatty liver disease. Dr. Zhou presented multiple innovations for increasing LCAT activity for therapeutic purposes, whereas Dr. Remaley showed results from treatment of an FLD patient with recombinant human LCAT (rhLCAT). Dr. Karathanasis showed that rhLCAT infusion in mice stimulates cholesterol efflux and suggested that it could also enhance cholesterol efflux from macrophages. While the role of LCAT in atherosclerosis remains elusive, the consensus is that a continued study of both the enzyme and disease will lead toward better treatments for patients with heart disease and FLD.

Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.

Finding a very rare mutation in non-Caucasian LCAT patients from Southwest Asia for the first time.

Lecithin cholesterol acyltransferase (LCAT) deficiency is an autosomal recessive disorder occurred by different mutations in the LCAT gene that cause two extremely rare syndromes including familial LCAT deficiency (FLD) and fish-eye disease (FED). Unlike FED in FLD renal failure is the most important defect due to deposition of abnormal lipoproteins in the renal stroma. In this study, FLD patients from the North of Iran were investigated for mutations in the LCAT gene. Eight patients with corneal opacification and renal defect were analyzed for mutations in the LCAT gene by PCR sequencing. Sequencing analysis revealed a missense pathogenic variation c.301 G>A in exon 2 of LCAT gene in all patients changing the amino acid aspartate to asparagine at the conserved position of amino acid 101 of LCAT protein. In this study, a very rare variation was reported for the first time in this part of the world. Investigation of a larger number of LCAT patients in different parts of Iran can provide availability of mutations panel that is useful for phenotype prediction and also prenatal diagnosis programming in families with a previous history of the disease.

Molecular basis for activation of lecithin:cholesterol acyltransferase by a compound that increases HDL cholesterol.

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients.

Crystal Structure of LCAT Bound to a Small Molecule Allosteric Activator Reveals its Active Conformation

There is an inverse relationship between risk for coronary heart disease (CHD) and the level of high density lipoprotein cholesterol (HDL‐C). As CHD is one of the leading causes of death worldwide, there is great interest in mechanisms to raise HDL‐C for treatment of CHD. Lecithin:cholesterol acyltransferase (LCAT) catalyzes the transfer of an acyl chain from phosphatidylcholine to cholesterol to create a cholesteryl ester (CE) in the process of reverse cholesterol transport. The formation of the more hydrophobic CE leads to the maturation of HDL from discoidal to spherical and promotes the clearance of cholesterol from arterial plaques. Mutations in LCAT are known to cause two genetic diseases: fish eye disease (FED) and familial LCAT deficiency (FLD). Individuals with either FED or FLD have low HDL‐C and corneal opacities, while FLD patients ultimately develop renal failure. Recombinant LCAT and LCAT‐activating compounds are being investigated as treatments for both CHD and FLD. However, only recently has any structural information on LCAT been available to guide development of these therapies. Based on recent crystal structures and hydrogen deuterium exchange data, it is now apparent that LCAT has a dynamic lid that is able to protect the LCAT active site from solvent. This lid likely plays a critical role in activation of LCAT by apolipoprotein A‐I, the major protein component of HDLs. We report on a new structure of LCAT bound to an LCAT‐activating compound as well as an acyl intermediate state mimic, isopropyl dodecylfluorophosphonate. The combination of these two ligands promotes a lid‐open conformation. We confirmed that the compound binds to an allosteric site via site directed mutants that both eliminate binding and increase the thermal stability of LCAT. These studies pave the way for the design of rational drugs or improved biotherapeutics that can be used to treat CHD and FLD and augment reverse cholesterol transport. The new structure also allows for a better understanding of the acyl binding site of LCAT and its catalytic mechanism.Support or Funding InformationThis work was supported by US National Institutes of Health (NIH) grants HL122416 and Postdoctoral Fellowship F32HL131288 (K.A.M.).

Structural and Biochemical Studies on the Activation Mechanism of Lecithin:Cholesterol Acyltransferase

Coronary heart disease (CHD) is the leading cause of death in the United States and is strongly associated with low levels of high density lipoprotein‐cholesterol (HDL‐C), thus there is significant clinical interest in methods that raise HDL‐C in order to treat and prevent CHD. Lecithin:cholesterol acyltransferase (LCAT) esterifies cholesterol on high density lipoprotein (HDL), which promotes HDL maturation and reverse cholesterol transport, the process by which cholesterol is transported from arterial plaques to the liver. Mutations in LCAT are known to cause two genetic diseases. Fish eye disease (FED) is characterized by partial loss of LCAT activity, whereas familial LCAT deficiency (FLD) is characterized by complete loss of LCAT. Individuals with either FED or FLD have low HDL‐C levels and corneal opacities, while FLD patients ultimately develop renal failure. Consequently, administration of recombinant LCAT and LCAT‐activating compounds are being investigated as treatments to increase HDL in both CHD and FLD, and have already shown promise in animal models. However, improvement of these therapeutics is limited due to a lack of structural information on LCAT and its interactions with HDL. LCAT is 50% identical to lysosomal phospholipase A2 (LPLA2). We previously solved a 1.84 Å structure of LPLA2, as well as a low‐resolution structure of LCAT (8.7 Å), both of which are in an active “lid open” conformation. In addition, structures of LPLA2 bound to inhibitors that mimic substrate binding led to models for how LPLA2 and LCAT bind to phospholipids and subsequently transfer an acyl group to the appropriate acceptor. Here we present a new “closed” crystal structure of LCAT at 3.3 Å wherein a retractable lid appears to block substrate access to the active site. Interestingly, the lid interacts with residues on the catalytic core that are mutated in LCAT genetic disease and are hypothesized to be important for activation by apolipoprotein A‐I (ApoA‐I), the major protein component of HDL particles. We therefore hypothesized that ApoA‐I binding will compete with the lid and thereby stimulate LCAT activity. We have probed the role of the lid by examining disease‐causing and lid mutations for esterase activity on a soluble substrate, binding to synthetic HDLs, and transacylase activity on HDLs. Overall, our results confirm the importance of the retractable lid and its observed intramolecular contacts in proper HDL binding and cholesterol esterification. Overall, these studies are expected to guide the design of more potent small molecule LCAT activators as well as more catalytically efficient recombinant LCAT that could be used for enzyme replacement therapy.Support or Funding InformationThis work was supported by US National Institutes of Health (NIH) grant F037815, an American Heart Association (AHA) Postdoctoral Fellowship 15POST24870001 (K.A.M.), and a University of Michigan Chemical Biology Postdoctoral Fellowship (A.G.).

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

Characterization of a new LCAT mutation causing familial LCAT deficiency (FLD) and the role of APOE as a modifier gene of the FLD phenotype

Familial LCAT deficiency (FLD) is a disease characterized by a defect in the enzyme lecithin:cholesterol acyltransferase (LCAT) resulting in low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers, presenting classical signs of FLD, were shown to be homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 and causes a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differs markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT−/− genotype associated with an APOE ɛ2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

Metabolism of oxidized phosphatidylcholines formed in oxidized low density lipoprotein by lecithin-cholesterol acyltransferase.

The possible involvement of lecithin-cholesterol acyltransferase (LCAT) in the metabolism of oxidized phosphatidylcholine (PC) in plasma was investigated. A variety of oxidized products are formed from PC following oxidation of low density lipoproteins (LDL). A significant increase in LDL oxidation levels in patients with familial LCAT deficiency (FLD) has been previously demonstrated by a sensitive sandwich ELISA for oxidized LDL using the monoclonal antibody DLH3 which recognizes oxidized products of PC. In the present study, we found that LCAT produces various metabolites from oxidized PC and that oxidized PC molecules in LDL particles serve as substrates. When the neutral lipid fraction was separated by TLC after the incubation of oxidized 1-palmitoyl-2-[1-14C]linoleoyl PC with human plasma, a number of radioactive bands were formed in addition to cholesteryl ester. These products were not formed from native 1-palmitoyl-2-[1-14C]linoleoyl PC. Plasma from FLD patients also failed to form the additional products from oxidized PC. The addition of dithio-bis(nitrobenzoate) (DTNB), an LCAT inhibitor, or the inactivation of LCAT activity by treating the plasma at 56 degrees C for 30 min abolished the generation of these products from oxidized PC. The activity was recovered in the high density lipoprotein (HDL) fraction but not in the LDL fraction separated from normal plasma. When 1-palmitoyl-2-[1-14C](9-oxononanoyl) PC and 1-stearoyl-2-[1-14C](5-oxovaleroyl)PC, PC oxidation products that contain short chain aldehydes, were incubated with human plasma, radioactive products in the neutral lipid fraction were observed on TLC. LDL containing oxidized PC was measured by sandwich ELISA using an anti-apolipoprotein B antibody and DLH3. The reconstituted oxidized PC-LDL particles were found to have lost their ability to bind DLH3 upon incubation with HDL, while the reactivity of the reconstituted oxidized PC-LDL remained unchanged in the presence of DTNB. These results suggest that LCAT is capable of metabolizing a variety of oxidized products of PC and preventing the accumulation of oxidized PC in circulating LDL particles.

Receptor mediated uptake of apo B and apo E rich lipoproteins by human glomerular epithelial cells

Various pathological disorders are accompanied by the deposition of lipids into glomerular cells. To gain insight into these disorders, it is essential to know if glomerular cells possess lipoprotein receptors. We therefore characterized the activity of lipoprotein receptors in cultured epithelial cells of the human glomerulus. Podocytes were chosen as they are directly exposed to lipoproteins in pathological states like in glomerular proteinuria (such as, nephrotic syndrome). Isolated human glomeruli (purity greater than 95%) were incubated in buffered RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum at 37 degrees C and 5% CO2. Outgrowing cells were vimentin and keratin positive. Monolayer cultures of human glomerular epithelial cells upon incubation in lipoprotein deficient serum for 48 hours expressed a receptor-dependent uptake of lipoproteins. These cells showed about 10% of the maximal capacity for LDL uptake as compared to fibroblasts; however, the Km values for binding, internalization and degradation were similar in the cultures of glomerular epithelial cells and fibroblasts. The Km values for degradation of LDL, chylomicron remnants, beta-VLDL from cholesterol-fed rabbits and VLDL from familial LCAT-deficiency patients were 14.2, 4.9, 2.9, 4.5 micrograms protein/ml medium, respectively, for glomerular epithelial cells. The avid uptake of 125I-labeled apo E-containing lipoproteins was further substantiated by their poor displacement by a 25-fold excess of unlabeled LDL and their ability to down regulate the apo B,E receptor activity. LDL as well as beta-VLDL were able to suppress the incorporation of 14C acetate into sterols and to stimulate 3H-cholesterylester formation. These experiments show that cultured glomerular epithelial cells express lipoprotein receptor activity. Plasma concentrations of apo E-containing lipoproteins are increased in certain renal diseases (such as, nephrotic syndrome); these lipoproteins could be rapidly removed by glomerular epithelial cells and lead to lipid deposition in glomeruli.