Potential Gene Therapy for Lecithin-Cholesterol Acyltransferase (LCAT)–Deficient and Hypoalphalipoproteinemic Patients With Adenovirus-Mediated Transfer of Human LCAT Gene
Background Overexpression of human lecithin-cholesterol acyltransferase (LCAT) in transgenic mice results in an increase of the antiatherogenic HDLs.
Methods and Results To investigate the potential use of LCAT for gene therapy, a recombinant adenovirus was constructed in which the human LCAT cDNA was expressed under the control of the human cytomegalovirus immediate/early promoter followed by a chimeric intron (AdCMV human LCAT). Human apolipoprotein (apo) A-I transgenic mice infected with AdCMV human LCAT by intravenous injection accumulated reactive LCAT in the plasma. LCAT activity was increased 201-fold in the plasma of mice infected with 1×109 pfu AdCMV human LCAT, from 45±2 to 9068±812 nmol·mL−1·h−1, in comparison with basal LCAT activity measured in control mice, 5 days after injection. Plasma HDL cholesterol levels rose from 117±12 to 797±48 mg/dL, and plasma human apo A-I concentrations increased from 247±14 to 616±17 mg/dL, in AdCMV human LCAT–infected mice compared with control mice. HDL particles were larger and had a different electrophoretic mobility. Studies of cholesterol efflux by incubation of serum with cholesterol-loaded Fu5AH cells showed that serum from AdCMV human LCAT–infected mice promoted a significantly higher efflux than did that of the controls.
Conclusions These data establish the potential of this approach for treatment of subjects with LCAT gene defects as well as patients with low plasma levels of apo A-I and HDL cholesterol.
Lecithin-cholesterol acyltransferase is a key enzyme in extracellular cholesterol metabolism.1 LCAT catalyzes the synthesis of CEs and lysolecithin from phosphatidylcholine and unesterified cholesterol present in the plasma. LCAT is associated primarily with HDLs and uses the FC of HDL as substrate. The major structural protein of HDL, apo A-I, is the principal activator of the enzyme.2 HDL particles are the main acceptors of cholesterol that desorbs from lipid interfaces. By maintaining an FC concentration gradient between peripheral cells and HDL, LCAT may play a major role in reverse cholesterol transport, a physiological process by which peripheral cell–derived cholesterol excess is transported through plasma to the liver for catabolism.3
The physiological consequences of a lack of cholesterol esterification by LCAT in plasma are profound. They are dramatically illustrated by the physiological changes seen in subjects with FLD or FED, both of which result from LCAT gene defects. Both FED and FLD patients develop corneal opacities and marked hypoalphalipoproteinemia.4 5 6 In addition, patients with FLD may present with renal insufficiency and anemia. Furthermore, premature atherosclerosis has been observed in some of these patients.4 Biochemically, both syndromes are associated with hypoalphalipoproteinemia and high levels of triglycerides. In FLD patients, the LCAT activity is completely absent, whereas some residual LCAT activity is found in patients with FED.6 In both types of patient, abnormal small spherical HDL particles as well as disk-shaped HDL particles are present, suggesting that LCAT plays a major role in the maturation and metabolism of HDL.4
Recently, we first overexpressed human LCAT in transgenic mice, which resulted in an increase in HDL cholesterol concentrations,7 suggesting that LCAT plays a major role in modulating plasma HDL cholesterol levels. High expression of human LCAT in mice resulted in the formation of larger-sized HDL particles that were enriched in cholesteryl esters and phospholipids, consistent with the enhanced cholesterol esterification catalyzed by human LCAT in mouse HDL.7 8 9
In this study, we used somatic cell gene transfer to investigate the role of LCAT in lipoprotein metabolism and its potential use for gene therapy. We found that infection of human apo A-I transgenic mice with a recombinant adenovirus encoding human LCAT transiently increased plasma levels of HDL cholesterol as well as human apo A-I.
Generation of Recombinant Adenoviruses
A cDNA encoding mature human LCAT was recloned from HepG2 cell total RNA by reverse transcription (Pharmacia) and polymerase chain reaction amplification with the oligonucleotide primers 5′-CCC TCG AGG CCA TCG ATG AGG CCT GAC TTT TTC AAT AAA-3′ and 5′-GCG TCG ACA GCT CAG TCC CAG GCC TCA GCA GAG-3′. The LCAT cDNA was inserted upstream of the SV40 early region polyadenylation signal and downstream of the CMV immediate/early promoter-enhancer sequences followed by a chimeric intron composed of a splice donor site from an intron of CMV genome and splice acceptor sequences derived from an immunoglobulin G variable region.10 The recombinant virus was prepared, purified, and titered as described.11 Titers are given as pfu/mL.
The study protocol was approved by the Animal Use Committee of Rhoˆne-Poulenc Rorer. The human apo A-I transgenic mice have been described previously.12 Male mice, 3 months old, were treated by tail-vein injection of purified recombinant adenovirus stocks or virus-free media (mock-infected mice). Blood was taken after infection from the retro-orbital plexus of mice fasted for 4 hours. Plasma was separated by centrifugation at 5000 rpm for 20 minutes at 4°C. At the end, animals were killed and livers were harvested.
Protein and Lipoprotein Analysis
LCAT activity was assayed by use of an exogenous proteoliposome substrate as described by Chen and Albers.13 We considered the level of LCAT to be directly proportional to its activity. Plasma CER was measured as previously described.14 Triglycerides, phospholipids, total cholesterol, and FC were measured with commercially available kits (Boehringer Mannheim). CE values were calculated by subtracting FC from plasma cholesterol concentrations. Lipids in HDL were measured after precipitation of plasma by sodium phosphotungstate with magnesium chloride. Plasma levels of human apo A-I were determined by rocket immunoelectrophoresis. Plasma lipoprotein distribution was assayed by analytical gel filtration chromatography with a Superose 6 HR 10/30 column (Pharmacia).
Size and Electrophoretic Mobility of HDL Particles
The size of plasma human apo A-I–containing lipoproteins was determined by nondenaturing gradient polyacrylamide gel electrophoresis15 followed by a Western blot analysis using specific anti–human apo A-I antibodies.16 Mobilities of plasma lipoproteins were analyzed by agarose gel electrophoresis with the Hydragel system (SEBIA). Blots were analyzed by quantitative scanning densitometry (Hoefer GS-300).17
Total RNA was isolated from homogenized livers by the acid guanidinium thiocyanate–phenol-chloroform method18 and was further purified by an additional precipitation with 1 vol 8 mol/L LiCl. Northern blot hybridizations were performed as described.17 Complete human LCAT cDNA and rat apo E cDNA were used as a probe and positive control, respectively. Both probes were labeled by random priming (Rediprime kit, Amersham).
Fatty Acid Composition
The fatty acid composition of plasma phospholipids, triacylglycerols, and CEs was determined by gas chromatography as described previously.7 Fatty acids were identified by use of commercially available fatty acid methyl esters.
In Vitro Cellular Cholesterol Efflux
Cellular cholesterol efflux studies with the rat Fu5AH hepatoma cells incubated with 2.5% diluted serum or 50 μg/mL of human apo A-I diluted serum were performed as described previously.19
All data are expressed as mean±SEM. Data were evaluated with ANOVA. To establish significance between groups, post hoc Scheffe´'s F test was used.
AdCMV human LCAT–Infected Mice Express Human LCAT
Human apo A-I transgenic mice were injected in the tail vein either with purified recombinant virus encoding human LCAT (1×108, 5×108, or 1×109 pfu), with β-galactosidase (1×109 pfu), or with virus-free solution. Plasma LCAT activities in mice infected with 1×108, 5×108, and 1×109 pfu AdCMV human LCAT were 2-, 72-, and 201-fold higher, respectively, than that of control mice 5 days after injection (Table⇓). Levels of LCAT activity detected in plasma from mock-infected or AdCMV β-galactosidase–infected mice corresponded to basal mouse LCAT activity. By contrast, the endogenous plasma LCAT activity (CER) was increased only 2-fold in the mice infected with 1×109 pfu AdCMV human LCAT compared with control mice 5 days after injection. Interestingly, LCAT activities were still increased 4 weeks after the administration of 5×108 and 1×109 pfu AdCMV human LCAT (1.8- and 3-fold higher, respectively, than the basal LCAT activity).
Northern blotting of liver RNA from AdCMV human LCAT–infected mice 7 days after infection revealed the expression of a single species of RNA (≈3 kb in size) hybridizing to the human LCAT cDNA probe (Fig 1⇓).
Fatty Acid Composition of Plasma Phospholipids, Triacylglycerols, and CEs
The distribution profiles of fatty acids in plasma triacylglycerides and phospholipids were similar in uninfected and infected mice, whereas the distribution profile of fatty acids in plasma CEs clearly differed between control and infected mice. The ratio of palmitic acid to arachidonic acid in plasma CE was 0.7 to 1 in AdCMV β-galactosidase–infected mice or mock-infected mice, whereas the ratio of palmitic acid to arachidonic acid in plasma CE was 2.7 to 1 in mice infected with 1×109 pfu AdCMV human LCAT (7 days after infection).
Effects of Human LCAT Transient Expression on Lipoprotein and Apolipoprotein Plasma Levels
Total cholesterol (Fig 2⇓), HDL cholesterol (Fig 2⇓), and human apo A-I (Fig 3⇓) levels in serum samples from uninfected and infected mice were determined 3, 5, and 7 days after infection. The major lipoprotein metabolism alteration was observed 5 days after injection. Lipid, human apo A-I, and LCAT activity parameters for this time point are summarized in the Table⇑. The following results correspond to the values obtained 5 days after injection.
For mice infected with 1×109 pfu AdCMV human LCAT, plasma HDL cholesterol, total cholesterol, and phospholipid levels were 600%, 500%, and 200% higher, respectively, than that of AdCMV β-galactosidase–infected mice. These changes were associated with increases in both CE and FC, which were 700% and 250% higher, respectively, in the plasma of AdCMV human LCAT–infected mice than in the plasma of AdCMV β-galactosidase–infected mice. Furthermore, the enhanced accumulation of plasma CE led to a significant increase in the CE-to-total cholesterol ratio in HDL. AdCMV human LCAT–infected mice showed an elevation of their plasma human apo A-I concentrations (Fig 3⇑), corresponding to a 2.5-fold increase in human apo A-I levels in comparison with that in AdCMV β-galactosidase–infected mice. The injection of 1×109 pfu AdCMV β-galactosidase corresponding to the higher dose of AdCMV human LCAT in mice revealed an expression of β-galactosidase in 30±5% of hepatocytes 7 days after injection. No major perturbations in the lipoprotein profile were observed in AdCMV β-galactosidase–infected mice.
For mice infected with 5×108 pfu AdCMV human LCAT, which had intermediate levels of LCAT activity (Table⇑), we observed intermediate alteration in circulating lipids. Total and HDL cholesterol, FC, phospholipid, and human apo A-I plasma concentrations were increased by 200%, 260%, 100%, 150%, and 100% of control values, respectively.
For mice infected with 1×108 pfu AdCMV human LCAT, no major variations in lipoprotein metabolism were observed (data not shown).
Four weeks after injection, plasma HDL cholesterol levels were still 1.5-fold higher in mice infected with 1×109 pfu AdCMV human LCAT (226±10 mg/dL) than in AdCMV β-galactosidase–infected and mock-infected mice (148±4 and 149±3 mg/dL, respectively), whereas human apo A-I concentrations fell to basal levels (data not shown).
Cholesterol Distribution in Lipoproteins, HDL Sizes, and Electrophoretic Mobility
Analysis of the lipoprotein profile revealed an accumulation of cholesterol, predominantly in the HDL range, and an increase of HDL particle size for AdCMV human LCAT–infected mice compared with mock-infected mice (Fig 4⇓). Human apo A-I was associated primarily with HDL-sized particles (data not shown).
As previously reported,12 the apo A-I–containing lipoproteins of human apo A-I transgenic mice had a bimodal distribution, with peak sizes of 9.4 and 11 nm (Stokes diameter). The size distribution of the apo A-I–containing lipoproteins was the same for AdCMV β-galactosidase–infected mice (Fig 5⇓). However, the expression of human LCAT induced a major alteration of HDL size distribution. The smaller peak of apo A-I–containing lipoproteins disappeared, whereas two new larger peaks of 13.3 and 14.2 nm were observed (Fig 5⇓).
Plasma lipoproteins were separated by nondenaturing agarose gel electrophoresis. As shown in Fig 6⇓, three distinct HDL subpopulations were observed in the plasma of mice infected with 5×108 pfu AdCMV human LCAT. Two were similar to those of the control (one that migrated in pre-β position and one with an α mobility), and the third had a pre-α mobility not observed in the control. After the administration of 1×109 pfu AdCMV human LCAT, we observed two distinct HDL subpopulations, one with a pre-β mobility and one with a pre-α mobility. Thus, overexpression of human LCAT altered not only HDL sizes but also the charges at the surface of HDL.
Whether human apo A-I was present in the pre-β-, α-, and pre-α-migrating fractions was evaluated by use of anti–human apoA-I monoclonal antibodies. The detected bands were analyzed by scanning densitometry, and human apoA-I distribution was calculated. Control mice had 22±4% of human apoA-I in the pre-β-HDL and the remainder in α-HDL. Mice infected with 1×109 pfu AdCMV human LCAT had 23±5% of their human apoA-I in the pre-β-HDL and the rest in the pre-α-HDL. Therefore, calculated plasma apo A-I concentration in pre-β-HDL corresponded to 63±8 and 150±43 mg/dL for control mice and mice infected with 1×109 pfu AdCMV human LCAT, respectively.
Cholesterol Efflux From Hepatoma Cells
To determine whether expression of human LCAT in human apo A-I transgenic mice would increase the ability of the serum lipoproteins to promote cholesterol efflux, Fu5AH hepatoma cells were incubated for 2 and 4 hours with 2.5% diluted serum from mice infected with 1×109 pfu AdCMV human LCAT, 1×109 pfu AdCMV β-galactosidase, or mock-infected mice (5 days after infection). A significantly higher cholesterol efflux was observed with serum from AdCMV human LCAT–infected mice than with control serum, reaching up to 170% of the cholesterol efflux promoted by serum from AdCMV β-galactosidase–infected mice (Fig 7⇓).
Fu5AH hepatoma cells were also incubated with diluted serum so that the concentration of human apo A-I was the same (50 μg/mL) in the medium for all samples. In this experiment, no statistically significant differences in cholesterol efflux were observed between results obtained with serum from AdCMV human LCAT–infected mice and serum from control mice (data not shown). At 50 μg/mL of human apo A-I, for AdCMV human LCAT–infected mice, serum HDL cholesterol concentration and serum LCAT activity were 65 μg/mL and 736 nmol·mL−1·h−1, respectively, whereas for AdCMV β-galactosidase–infected mice, they were 24 μg/mL and 9 nmol·mL−1·h−1, respectively. Therefore, cholesterol efflux was not the direct consequence of the high levels of LCAT activity or HDL cholesterol but only of the increase of plasma human apo A-I levels induced by human LCAT gene transfer.
This study demonstrates that human apoA-I transgenic mice infected with recombinant adenoviruses encoding human LCAT secrete high levels of the active enzyme in the serum. This secretion is associated with a significant increase in HDL cholesterol as well as in human apoA-I plasma levels. On the basis of these observations, we suggest that LCAT is an important determinant of circulating HDL cholesterol. Transgenic mice overexpressing the human apoA-I gene have several advantages as an animal model for human LCAT study: they have human-like HDL particles in plasma12 and thus provide an excellent means to study the interaction between LCAT and human apoA-I–containing lipoproteins and its consequences on HDL metabolism. Moreover, it has been shown that in vitro activation of LCAT by apo A-I is species-specific.20
Adenovirus-mediated delivery of human LCAT gene to human apoA-I transgenic mice resulted in human LCAT expression. Plasma LCAT activities, measured with an exogenous substrate, were taken as a measure of the mass of active LCAT. LCAT activity increased in a dose-dependent manner, ranging from 2- to 201-fold higher than the basal enzyme activity found in human apoA-I transgenic mice. The endogenous plasma LCAT activity (CER) was also increased but to a lesser extent. This result suggests that in vivo, other factors may limit LCAT activation. In humans, VLDL and LDL provide the major portion of FC for the LCAT reaction, which occurs primarily on the HDL surface.21 Because of the action of LCAT, the surface content of CE increases. Once saturation with CE is reached, the LCAT reaction ceases. Then, CEs are either transferred to VLDL and LDL by the CETP or delivered by HDL to the steroidogenic tissues and the liver.22 The CE content in HDL from AdCMV human LCAT–infected mice was found to be very high and may be closer to the maximum of saturation. Moreover, in mice, a species that lacks CETP, the saturation with CE might occur more rapidly. The slight elevation of the CER relative to the LCAT mass available cannot be explained by the lack of FC in the various lipoprotein subclasses (the FC content was normal or elevated in lipoproteins of AdCMV human LCAT–infected mice). In AdCMV human LCAT–infected mice, we observed a variation in HDL structure; ie, the enlargement and change in the charge of HDL, as well as a variation in the HDL lipid composition. It has been shown that the CER was directly related to HDL size; ie, large HDLs, such as HDL2, competitively inhibit the reaction.23 Thus, several observations may account for the limitation of endogenous cholesterol esterification.
The expression of human LCAT resulted in major changes in the distribution profile of fatty acids in plasma CE, in agreement with our previous study of human LCAT transgenic mice.7 The fatty acids in plasma CE of AdCMV human LCAT–infected mice contained a higher proportion of palmitic acid CE, whereas arachidonic acid CE was decreased. Then, the ratio of palmitic to arachidonic acid in plasma CE was increased in AdCMV human LCAT–infected mice in comparison with that in control mice. Therefore, the fatty acid CE profile in AdCMV human LCAT–infected mice resembles a human profile more closely than a mouse profile. This modification of plasma CE composition is a direct effect of the expression of human LCAT and can be attributed to the substrate and positional specificities of LCAT.24 Several studies have suggested that CE fatty acid composition influences the atherogenic susceptibility. Animal species resistant to atherosclerosis, ie, mouse and rat, have a low palmitic to arachidonic acid CE ratio, whereas humans or animals such as rabbits, which have higher palmitic to arachidonic acid CE ratios, are more susceptible to atherosclerosis. Therefore, the modification of fatty acid composition of plasma CE induced by human LCAT may increase atherogenic risk in the mouse model.
Transient overexpression of human LCAT in human apo A-I transgenic mice produced abnormal HDL that differed in lipid composition, size, and net charge. Lipid perturbations in HDL of AdCMV human LCAT–infected mice included a transient elevation of the CE-to-TC ratio at the peak of human LCAT expression that returned to normality after a few days (data not shown). The elevation of HDL cholesterol and phospholipid levels was higher than that of human apo A-I in AdCMV human LCAT–infected mice, indicating the formation of lipid-rich HDL. A significant increase in the size of HDL particles, which appeared to depend on the level of human LCAT expression, was observed in AdCMV human LCAT–infected mice. This increase in size resulted in the appearance of two new larger human apo A-I–containing particles. Interestingly, these large HDL particles have a size similar to that of HDL found in the plasma of patients with CETP deficiency.25 This suggests that CETP may be necessary for the recycling of human HDL. The increase in HDL size was accompanied by the appearance of particles with pre-α electrophoretic mobility, caused by increased negative charges on HDL. The alteration of the surface charge of HDL particles could be explained by an increase of the neutral lipid core of HDL particles, created by LCAT-mediated accumulation of CE. It has been proposed that the change in charge associated with the neutral lipid core was due to a conformational change in apo A-I molecules, leading to a higher net negative particle charge.26 Moreover, it has been shown that this specific electronegative HDL fraction may be the favorite substrate for the CETP-mediated lipid exchange.27 These data indicate that LCAT plays a key role in regulating HDL metabolism by acting on lipids and apolipoprotein A-I contents in HDL as well as on HDL size and charge.
Mice with high plasma LCAT activities demonstrated a significant increase in plasma total cholesterol levels, resulting mainly from a dramatic elevation of the cholesterol in the HDL fraction. This suggests a specific interaction between human LCAT and human-like HDL, such as that observed in the study of double human apo A-I and human LCAT transgenic mice.9 Moreover, we report here that AdCMV human LCAT–infected mice demonstrated a 2.5-fold increase in plasma human apo A-I level. Such an increase was reported neither for double human apo A-I and human LCAT transgenic mice,9 possibly because the level of human LCAT expression was low, nor in a study of human transgenic mice,8 because of the lack of human apo A-I. Together, these data confirm that human LCAT and human apo A-I interact specifically and that human LCAT strongly affects the plasma levels of human apo A-I–containing lipoproteins.
HDL turnover studies have shown that apo A-I fractional catabolic rate may explain the variability observed in HDL cholesterol or apo A-I levels.28 29 30 Therefore, the increase in apo A-I concentration in AdCMV human LCAT–infected mice is more likely to be attributable to a delayed catabolism of apo A-I, as has been reported for human LCAT transgenic rabbits.31 Data from several laboratories suggest that HDL particle size affects metabolism, with larger particles having a slower catabolism.30 In contrast, a rapid catabolism of HDL and apo A-I has been observed in patients with LCAT deficiency.32 The HDL of these subjects cannot be matured normally by LCAT, and the HDL particles are small.4 Therefore, the delayed catabolism of apo A-I may result from the increased size of HDL in AdCMV human LCAT–infected mice. However, other parameters, such as lipid composition, apo A-I conformation, or charge, may also alter the metabolic regulation of HDL and apo A-I levels.
HDLs are implicated in reverse cholesterol transport.33 The first step of this process consists of the cholesterol efflux from peripheral cells to HDL particles, mediated principally by the pre-β-migrating HDL.34 In the present study, cellular cholesterol efflux was significantly increased after incubation of serum from AdCMV human LCAT–infected mice compared with control serum. This effect appears to be directly related to the higher levels of plasma human apo A-I in AdCMV human LCAT–infected mice compared with control mice, rather than to the high levels of LCAT activity or HDL cholesterol. This is consistent with previous studies showing that apo A-I–containing particles are primarily responsible for serum-stimulated efflux of cell cholesterol35 and that cholesterol esterification does not enhance cholesterol efflux.36 The increase of cholesterol efflux seems to result from the accumulation of large human apo A-I–containing lipoproteins in serum, in agreement with studies showing the effects of large HDL on the cholesterol efflux promotion.37 38
Pre-β-migrating HDL particles are converted by LCAT to α-migrating HDL particles.39 It has been reported that the level of pre-β-HDL/α-HDL ratio was reduced in double human LCAT and human apo A-I transgenic mice expressing a low level of LCAT.9 In this study, a fraction of HDL from mice infected with 1×109 pfu AdCMV human LCAT had a pre-α instead of an α mobility, and the pre-β-HDL/pre-α-HDL ratio was not reduced. Moreover, the net amount of pre-β-HDL was higher in AdCMV human LCAT–infected mice than in control mice. Therefore, a high LCAT activity in plasma resulted in an increase of pre-β-HDL concentration; this HDL subpopulation may play an active role as an interface between cells and lipoproteins. This also implied that LCAT is one factor modulating pre-β-HDL plasma concentration.
Human LCAT gene transfer mediated by adenoviral vector in human apo A-I transgenic mice results in a dramatic elevation of HDL cholesterol and human apo A-I plasma levels and the appearance of large HDL particles, leading to a less atherogenic plasma lipoprotein profile. Furthermore, cellular cholesterol efflux promoted by HDL particles resulting from LCAT overexpression is also increased, leading to a higher efficiency of reverse cholesterol transport. This study demonstrates the utility of adenovirus-mediated gene transfer as an experimental tool for studies of lipoprotein metabolism. Furthermore, this study opens new avenues toward genetic treatment of dyslipoproteinemia, here for LCAT deficiency as well as hypoalphalipoproteinemia, based not only on introducing structural proteins such as apo A-I or apo E or on receptors like the LDL receptor40 but also on plasmatic enzymes that potentiate the role of preexisting lipoprotein particles. Therefore, we currently investigate the potential of gene transfer for hypoalphalipoproteinemia using both adenovirus-expressing human apo A-I and human LCAT. When first-generation adenoviral vectors were used for LCAT gene transfer, plasma LCAT activity and HDL cholesterol levels were still threefold and twofold higher, respectively, 4 weeks after injection and returned to basal levels 6 weeks after injection. Strategies to stabilize foreign gene expression must be developed to prolong the efficacy of therapies involving gene transfer. Histopathological analysis of liver tissue from mice infected with AdCMV human LCAT or AdCMV β-galactosidase revealed inflammation at 7 days that diminished within 6 weeks after injection. To attenuate the expression of immunogenic viral proteins, known to be an important factor limiting the duration of foreign gene expression, new adenoviral vectors, in which the E1 and E4 regions have been deleted, have been generated and provide one potentially promising approach.41
Selected Abbreviations and Acronyms
|CER||=||cholesterol esterification rate|
|CETP||=||cholesteryl ester transfer protein|
|FED||=||fish eye disease|
|FLD||=||familial LCAT deficiency|
This work was supported in part by the BIO/AVENIR program financed by Rhoˆne-Poulenc Rorer SA and the Ministe`re de la Recherche et de l'Enseignement Supe´rieur. We gratefully acknowledge Nathalie Aubailly, Florence Emmanuel, Florence Attenot, Isabelle Viry, Pierre Gallix, Patrick Feydel, Brigitte Lacroix, Patrice Ardouin, Catherine Dengremont, and Catherine De Geite`re for excellent assistance.
- Received April 11, 1996.
- Revision received June 25, 1996.
- Accepted July 5, 1996.
- Copyright © 1996 by American Heart Association
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