Current Status of Familial LCAT Deficiency in Japan.

Lecithin cholesterol acyltransferase (LCAT) is a lipid-modification enzyme that catalyzes the transfer of the acyl chain from the second position of lecithin to the hydroxyl group of cholesterol (FC) on plasma lipoproteins to form cholesteryl acylester and lysolecithin. Familial LCAT deficiency is an intractable autosomal recessive disorder caused by inherited dysfunction of the LCAT enzyme. The disease appears in two different phenotypes depending on the position of the gene mutation: familial LCAT deficiency (FLD, OMIM 245900) that lacks esterification activity on both HDL and ApoB-containing lipoproteins, and fish-eye disease (FED, OMIM 136120) that lacks activity only on HDL. Impaired metabolism of cholesterol and phospholipids due to LCAT dysfunction results in abnormal concentrations, composition and morphology of plasma lipoproteins and further causes ectopic lipid accumulation and/or abnormal lipid composition in certain tissues/cells, and serious dysfunction and complications in certain organs. Marked reduction of plasma HDL-cholesterol (HDL-C) and corneal opacity are common clinical manifestations of FLD and FED. FLD is also accompanied by anemia, proteinuria and progressive renal failure that eventually requires hemodialysis. Replacement therapy with the LCAT enzyme should prevent progression of serious complications, particularly renal dysfunction and corneal opacity. A clinical research project aiming at gene/cell therapy is currently underway.

Identification and functional analysis of missense mutations in the lecithin cholesterol acyltransferase gene in a Chilean patient with hypoalphalipoproteinemia.

BACKGROUND: Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol in high- and low-density lipoproteins (HDL and LDL). Mutations in LCAT gene causes familial LCAT deficiency, which is characterized by very low plasma HDL-cholesterol levels (Hypoalphalipoproteinemia), corneal opacity and anemia, among other lipid-related traits. Our aim is to evaluate clinical/biochemical features of a Chilean family with a proband showing clinical signs of familial LCAT deficiency, as well as to identify and assess the functional effects of LCAT mutations. METHODS: An adult female proband with hypoalphalipoproteinemia, corneal opacity and mild anemia, as well as her first-degree relatives, were recruited for clinical, biochemical, genetic, in-silico and in-vitro LCAT analysis. Sequencing of exons and intron-exon boundaries was performed to identify mutations. Site-directed mutagenesis was carried out to generate plasmids containing cDNA with wild type or mutant sequences. Such expression vectors were transfected to HEK-239 T cells to asses the effect of LCAT variants in expression, synthesis, secretion and enzyme activity. In-silico prediction analysis and molecular modeling was also used to evaluate the effect of LCAT variants. RESULTS: LCAT sequencing identified rare p.V333 M and p.M404 V missense mutations in compound heterozygous state in the proband, as well the common synonymous p.L363 L variant. LCAT protein was detected in proband's plasma, but with undetectable enzyme activity compared to control relatives. HEK-293 T transfected cells with vector expression plasmids containing either p.M404 V or p.V333 M cDNA showed detectable LCAT protein expression both in supernatants and lysates from cultured cells, but with much lower enzyme activity compared to cells transfected with the wild-type sequence. Bioinformatic analyses also supported a causal role of such rare variations in LCAT lack of function. Additionally, the proband carried the minor allele of the synonymous p.L363 L variant. However, this variant is unlikely to affect the clinical phenotype of the proband given its relatively high frequency in the Chilean population (4%) and its small putative effect on plasma HDL-cholesterol levels. CONCLUSION: Genetic, biochemical, in vitro and in silico analyses indicate that the rare mutations p.M404 V and p.V333 M in LCAT gene lead to suppression of LCAT enzyme activity and cause clinical features of familial LCAT deficiency.

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.

Lipoprotein-X fifty years after its original discovery.

AIMS: To review the formation, catabolism, and the possible atherogenic properties of Lp-X. DATA SYNTHESIS: The conversion of cholesterol to bile acids is regulated by several mechanisms including cholesterol 7 alpha hydroxylase, fibroblast growth factor 19, and farnesoid X receptors. During cholestasis these mechanisms are altered and there is an accumulation of bile acids and cholesterol in plasma. The hypercholesterolemia observed in cholestasis is due to the presence of an anomalous lipoprotein called lipoprotein-X (Lp-X). Lp-X is a lipoprotein rich in phospholipid and free cholesterol present in plasma of patients with cholestasis and, with some variations, in patients with lecithin-cholesterol-acyl-transferase deficiency (LCAT), and after lipid infusion. Lp-X is formed from a bile lipoprotein moving to the blood vessels where it incorporates small quantities of triglycerides, apo-C and esterified cholesterol and becomes a "mature" Lp-X. The activity of the phosphatidilcholine canalicular transporter Mdr2 P-glycoprotein (homologous to the human ABCB4) is essential for LpX appearance, since its suppression abolishes Lp-X formation. However, the concentration of Lp-X in plasma is determined also by the degree of the cholestasis, the residual liver function, and the LCAT deficiency. The Lp-X catabolism seems to be mediated by the reticuloendothelial system and possibly the kidney. CONCLUSIONS: Lp-X might be considered a defense mechanism against the toxic effect of free cholesterol in cholestasis. The frequency of cardiovascular events in patients affected by primary biliary cholangitis, in whom the Lp-X is present in high concentration, are not increased. Further studies could now clarify the remaining open questions on the role of Lp-X in the dyslipidemia of cholestasis.

A proteomic approach to identify novel disease biomarkers in LCAT deficiency.

Genetic LCAT deficiency is a rare recessive autosomal disease due to loss-of-function mutations in the gene coding for the enzyme lecithin:cholesterol acyltransferase (LCAT). Homozygous carriers are characterized by corneal opacity, haemolytic anaemia and renal disease, which represent the first cause of morbidity and mortality in these subjects. Diagnostic and prognostic markers capable of early detecting declining kidney function in these subjects are not available, and the specific serum or urine proteomic signature of LCAT deficient carriers has never been assessed. Taking advantage of a proteomic approach, we performed 2-DE analysis of carriers' plasma and identified proteins present at different concentration in samples from homozygous carriers. Our data confirm the well-known alterations in the concentration of circulating apolipoproteins, with a statistically significant decrease of both apoA-I and apoA-II and a statistically significant increase of apoC-III. Furthermore, we observed increased level of alpha-1-antitrypsin, zinc-alpha-2-glycoprotein and retinol-binding protein 4, and reduced level of clusterin and haptoglobin. Interestingly, only beta but not alpha subunit of haptoglobin is significant reduced in homozygous subjects. Despite the limited sample size, our findings set the basis for assessing the identified protein in a larger population and for correlating their levels with clinical markers of renal function and anaemia. SIGNIFICANCE: This investigation defines the effects of LCAT deficiency on the level of the major plasma proteins in homozygous and heterozygous carriers. Increase for some proteins, with different function, together with a drop for haptoglobin, and specifically for haptoglobin beta chains, are reported for the first time as part of a coherent signature. We are glad to have the opportunity to report our findings on this subject, which is one of the main interests for our research group, when Journal of Proteomics celebrates its 10th anniversary. With its various sections devoted to different areas of research, this journal is a privileged forum for publishing proteomic investigations without restrictions either in sample type or in technical approach. It is as well a privileged forum for reviewing literature data on various topics related to proteomics investigation, as colleagues in our research group have done over the years; by the way, a good share of the reviewed papers were as well reports published in Journal of Proteomics itself. The journal also offers opportunities for focused surveys through thematic issues devoted to a variety of subjects, timely selected for their current relevance in research; it was an honour for colleagues in our group to recently act as editors of one of those. Out of this diverse experience, we express our appreciation for the endeavour of Journal of Proteomics in its first 10 years of life - and wish identical and possibly greater success for the time to come.

Familial lecithin:cholesterol acyltransferase deficiency: First-in-human treatment with enzyme replacement.

BACKGROUND: Humans with familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) have extremely low or undetectable high-density lipoprotein cholesterol (HDL-C) levels and by early adulthood develop many manifestations of the disorder, including corneal opacities, anemia, and renal disease. OBJECTIVE: To determine if infusions of recombinant human LCAT (rhLCAT) could reverse the anemia, halt progression of renal disease, and normalize HDL in FLD. METHODS: rhLCAT (ACP-501) was infused intravenously over 1 hour on 3 occasions in a dose optimization phase (0.3, 3.0, and 9.0 mg/kg), then 3.0 or 9.0 mg/kg every 1 to 2 weeks for 7 months in a maintenance phase. Plasma lipoproteins, lipids, LCAT levels, and several measures of renal function and other clinical labs were monitored. RESULTS: LCAT concentration peaked at the end of each infusion and decreased to near baseline over 7 days. Renal function generally stabilized or improved and the anemia improved. After infusion, HDL-C rapidly increased, peaking near normal in 8 to 12 hours; analysis of HDL particles by various methods all revealed rapid sequential disappearance of preß-HDL and small α-4 HDL and appearance of normal α-HDL. Low-density lipoprotein cholesterol increased more slowly than HDL-C. Of note, triglyceride routinely decreased after meals after infusion, in contrast to the usual postprandial increase in the absence of rhLCAT infusion. CONCLUSIONS: rhLCAT infusions were well tolerated in this first-in-human study in FLD; the anemia improved, as did most parameters related to renal function in spite of advanced disease. Plasma lipids transiently normalized, and there was rapid sequential conversion of small preß-HDL particles to mature spherical α-HDL particles.

Lipoprotein subfractions highly associated with renal damage in familial lecithin:cholesterol acyltransferase deficiency.

OBJECTIVE: In familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD), deposition of abnormal lipoproteins in the renal stroma ultimately leads to renal failure. However, fish-eye disease (FED) does not lead to renal damage although the causative mutations for both FLD and FED lie within the same LCAT gene. This study was performed to identify the lipoproteins important for the development of renal failure in genetically diagnosed FLD in comparison with FED, using high-performance liquid chromatography with a gel filtration column. APPROACH AND RESULTS: Lipoprotein profiles of 9 patients with LCAT deficiency were examined. Four lipoprotein fractions specific to both FLD and FED were identified: (1) large lipoproteins (>80 nm), (2) lipoproteins corresponding to large low-density lipoprotein (LDL), (3) lipoproteins corresponding to small LDL to large high-density lipoprotein, and (4) to small high-density lipoprotein. Contents of cholesteryl ester and triglyceride of the large LDL in FLD (below detection limit and 45.8±3.8%) and FED (20.7±6.4% and 28.0±6.5%) were significantly different, respectively. On in vitro incubation with recombinant LCAT, content of cholesteryl ester in the large LDL in FLD, but not in FED, was significantly increased (to 4.2±1.4%), whereas dysfunctional high-density lipoprotein was diminished in both FLD and FED. CONCLUSIONS: Our novel analytic approach using high-performance liquid chromatography with a gel filtration column identified large LDL and high-density lipoprotein with a composition specific to FLD, but not to FED. The abnormal lipoproteins were sensitive to treatment with recombinant LCAT and thus may play a causal role in the renal pathology of FLD.

A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy.

A kindred affected with fish eye disease (FED) from Oklahoma is reported. Two probands with corneal opacification had mean levels of high-density lipoprotein (HDL) cholesterol (C), apolipoprotein (apo) A-I, and apoA-I in very large alpha-1 HDL particles that were 9%, 17%, and 5% of normal, whereas their parents and 1 sibling had values that were 61%, 77%, and 72% of normal. The probands had no detectable lipoprotein-X, and had mean low-density lipoprotein cholesterol (LDL-C) and triglyceride levels that were elevated. Their mean lecithin cholesterol acyltransferase (LCAT) activities, cholesterol esterification rates, and free cholesterol levels were 8%, 42%, and 258% of normal, whereas their parents and 1 sibling had values that were 55%, 49%, and 114% of normal. The defect was due to 1 common variant in the LCAT gene in exon 1: c101t causing a proline34leucine substitution and a novel mutation c1177t causing a threonine37methionine substitution, with the former variant being found in the father and 1 sibling, and the latter mutation being found in the mother, and both mutations being present in the 2 probands. FED is distinguished from familial LCAT deficiency (FLD) by the lack of anemia, splenomegaly, and renal insufficiency as well as normal or increased LDL-C. Both FLD and FED cases have marked HDL deficiency and corneal opacification, and FED cases may have premature coronary heart disease in contrast to FLD cases. Therapy, using presently available agents, in FED should be to optimize LDL-C levels, and 1 proband responded well to statin therapy. The investigational use of human recombinant LCAT as an enzyme source is ongoing.

Familial LCAT deficiency in a child with nephrotic syndrome.

BACKGROUND: Lecitin cholesterol acyltransferase (LCAT) deficiency comprises a group of rare disorders related to HDL metabolism. These disorders are characterized by ophthalmologic, hematologic, and renal findings. Case diagnosis/treatment: A 15-year-old female who presented with nephrotic syndrome and hypertension was diagnosed with LCAT deficiency by renal biopsy and LCAT enzyme activity. Her edema and hypertension improved with diuretic and antihypertensive therapies. Continued care of her LCAT deficiency is ongoing. CONCLUSION: Although rare, LCAT deficiency should be in the differential diagnosis of nephrotic syndrome in the setting of abnormally low HDL cholesterol levels.

Recombinant human LCAT normalizes plasma lipoprotein profile in LCAT deficiency.

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. Mutations in the LCAT gene leads to two rare disorders, familial LCAT deficiency and fish-eye disease, both characterized by severe hypoalphalipoproteinemia associated with several lipoprotein abnormalities. No specific treatment is presently available for genetic LCAT deficiency. In the present study, recombinant human LCAT was expressed and tested for its ability to correct the lipoprotein profile in LCAT deficient plasma. The results show that rhLCAT efficiently reduces the amount of unesterified cholesterol (-30%) and promotes the production of plasma cholesteryl esters (+210%) in LCAT deficient plasma. rhLCAT induces a marked increase in HDL-C levels (+89%) and induces the maturation of small preß-HDL into alpha-migrating particles. Moreover, the abnormal phospholipid-rich particles migrating in the LDL region were converted in normally sized LDL.

Patients with low HDL-cholesterol caused by mutations in LCAT have increased arterial stiffness.

OBJECTIVE: Carriers of a functional mutation in LCAT, encoding lecithin:cholesterol acyl transferase, are exposed to lifelong low high-density lipoprotein cholesterol (HDL-c) levels. We investigated whether LCAT mutation carriers have increased arterial stiffness as a marker of cardiovascular disease and whether arterial stiffness was associated with carotid wall thickening. METHODS: We assessed 45 carriers of LCAT mutations (mean age ± SD 46 ± 13 yrs) and 45 age-matched controls. Probands referred with established cardiovascular disease were excluded. We measured carotid-fermoral pulse wave velocity (PWV) and carotid artery wall thickening by ultrasound and 3.0 T magnetic resonance imaging. RESULTS: In carriers, HDL-c was lower (32 ± 12 vs. 59 ± 16 mg/dl; p < 0.0001) and triglycerides were higher (median 116 [IQR 80-170] vs. 71 [IQR 53-89] mg/dl; p < 0.001) vs. controls. PWV was higher in carriers vs. controls (7.9 ± 2.0 m/s vs. 7.1 ± 1.6 m/s; p < 0.01). This difference retained significance in multivariate analysis including age, sex, mean arterial pressure and body mass index, and after exclusion of carriers and controls with cardiovascular disease. Both in carriers and controls, PWV was correlated with wall thickening of the carotid arteries as assessed by ultrasound (R 0.50, p < 0.001 for carriers and R 0.36, p < 0.04 for controls) and 3.0 T magnetic resonance imaging (R 0.54, p < 0.001 for carriers and R 0.58, p < 0.001 for controls). CONCLUSION: Pulse wave velocity is increased in LCAT mutation carriers with low HDL-c and is associated with carotid wall thickening.

Very low levels of HDL cholesterol and atherosclerosis, a variable relationship--a review of LCAT deficiency.

A number of epidemiological and clinical studies have demonstrated that plasma high-density lipoprotein (HDL) level is a strong inverse predictor of cardiovascular events. HDL is believed to retard the formation of atherosclerotic lesions by removing excess cholesterol from cells and preventing endothelial dysfunction. Lecithin cholesterol acyltransferase (LCAT) plays a central role in the formation and maturation of HDL, and in the intravascular stage of reverse cholesterol transport: a major mechanism by which HDL modulates the development and progression of atherosclerosis. A defect in LCAT function would be expected to enhance atherosclerosis, by interfering with the reverse cholesterol transport step. As such, one would expect to find more atherosclerosis and cardiovascular events in LCAT-deficient patients. But this relationship is not always evident. In this review, we describe contradictory reports in the literature about cardiovascular risks in this patient population. We discuss the paradoxical finding of severe HDL deficiency and an absence of subclinical atherosclerosis in LCAT-deficient patients, which has been used to reject the hypothesis that HDL level is important in the protection against atherosclerosis. Furthermore, to illustrate this paradoxical finding, we present a case study of one patient, referred for evaluation of global cardiovascular risk in the presence of a low HDL cholesterol level, who was diagnosed with LCAT gene mutations.

Genetic lecithin:cholesterol acyltransferase deficiency and cardiovascular disease.

The lecithin:cholesterol acyltransferase (LCAT) enzyme is responsible for the synthesis of cholesteryl esters in human plasma and plays a critical role in high density lipoprotein (HDL) metabolism. Genetic LCAT deficiency is a rare metabolic disorder characterized by low HDL cholesterol levels. This paper reviews the genetic and biochemical features of LCAT deficiency, highlighting the absence of enhanced preclinical atherosclerosis in carriers, despite the remarkably low HDL cholesterol.

Homozygous lecithin:cholesterol acyltransferase (LCAT) deficiency due to a new loss of function mutation and review of the literature.

BACKGROUND: A case of homozygous familial lecithin:cholesterol acyltransferase (LCAT) deficiency with a novel homozygous LCAT missense mutation (replacement of methionine by arginine at position 293 in the amino acid sequence of the LCAT protein) is reported. METHODS AND RESULTS: The probable diagnosis was suggested by findings of marked high density lipoprotein (HDL) deficiency, corneal opacification, anemia, and renal insufficiency. The diagnosis was confirmed by two dimensional gel electrophoresis of HDL, the measurement of free and esterified cholesterol, and sequencing of the LCAT gene. CONCLUSIONS: In our view the most important aspects of therapy to prevent the kidney disease that these patients develop is careful control of blood pressure and lifestyle measures to optimize non HDL lipoproteins. In the future replacement therapy by gene transfer or other methods may become available.

Proteinuria in early childhood due to familial LCAT deficiency caused by loss of a disulfide bond in lecithin:cholesterol acyl transferase.

INTRODUCTION: Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare recessive disorder of cholesterol metabolism characterized by the absence of high density lipoprotein (HDL) and the triad of corneal opacification, hemolytic anemia and glomerulopathy. PATIENTS: We here report on FLD in three siblings of a kindred of Moroccan descent with HDL deficiency. In all cases (17, 12 and 3 years of age) corneal opacification and proteinuria were observed. In the 17-year-old female proband, anemia with target cells was observed. RESULTS: Homozygosity for a mutation in LCAT resulted in the exchange of cysteine to tyrosine at position 337, disrupting the second disulfide bond in LCAT. LCAT protein and activity were undetectable in the patients' plasma and in media of COS7 cells transfected with an expression vector with mutant LCAT cDNA. Upon treatment with an ACE inhibitor and a thiazide diuretic, proteinuria in the proband decreased from 6g to 2g/24h. CONCLUSION: This is the first report that FLD can cause nephropathy at a very early age.