© 1997 American Heart Association, Inc.
Articles |
Molecular Basis of Fish-Eye Disease in a Patient From Spain
Characterization of a Novel Mutation in the LCAT Gene and Lipid Analysis of the Cornea
From the Department of Medicine, Baylor College of Medicine and Methodist Hospital, Houston, Tex (F.B.-V., S.-J.Q., H.-Z.F., Q.P., H.J.P.); Servei de Bioquímica and Institut de Recerca, Hospital de la Santa Creu I Sant Pau, and Departament de Bioquímica I Biologia Molecular, Universitat Autònoma de Barcelona, Spain (F.B.-V., À.M.-C.); Serveis d'Oftalmologia, Medicina Interna i Recerca Experimental, Residencia Sanitària i Universitària Prínceps d'Espanya, L'Hospitalet de Llobregat, Barcelona, Spain (C.F., I.H., V.G., X.P., T.M.); and Department of Medicine, Northwest Lipid Research Laboratories, University of Washington, Seattle (J.J.A.). The first two authors contributed equally to this study.
Correspondence to Henry J. Pownall, Baylor College of Medicine and Methodist Hospital, Department of Medicine, 6565 Fannin MS A601, Houston, TX 77030. E-mail hpownall{at}bcm.tmc.edu
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Abstract |
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Abstract The genetic and biochemical basis of fish-eye disease (FED) was investigated in a 63-year-old female proband with low plasma HDL cholesterol. Analyses of corneal and plasma lipids of the proband were consistent with impaired lecithin:cholesterol acyltransferase (LCAT) activity. Free cholesterol and phospholipid levels were elevated relative to control values, whereas cholesteryl ester levels were greatly reduced. Fatty acid compositions of corneal lipids from the proband and control subjects differ from the respective fatty acid compositions of their plasma lipids. This suggests that the metabolic pathways and acyl chain specificities for phospholipid, cholesteryl ester, and triglyceride metabolism within the cornea are distinct from those of plasma. Sequencing of the LCAT gene from the proband revealed a novel mutation at nucleotide 399, corresponding to an Arg99
Cys substitution.
Secretion of LCAT (Arg99Cys) by transfected COS-6 cells
was 
50% of that of the wild type, but its specific activity against
reassembled HDL was 93% lower than that of wild-type LCAT. The
specific activities of wild-type and LCAT (Arg99Cys)
against LDL were reduced similarly, suggesting that the appearance of
the FED phenotype does not require enhanced activity against
LDL. Our data support the hypothesis that FED is a partial LCAT
deficiency in which poor esterification in specific types of HDL
particles may contribute to the appearance of the corneal opacities.
Key Words: lipoproteins • HDL • lecithin acyltransferase deficiency • cholesteryl esters • corneal opacities • arteriosclerosis
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Introduction |
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According to prospective population studies, the inverse correlation between plasma HDL-C concentrations and coronary atherosclerosis suggests a protective role for HDL.1 2 The antiatherogenic role of HDL has also been demonstrated directly by intravenous injection of HDL into cholesterol-fed rabbits3 and by overexpression of its major apolipoprotein, apo A-I, in transgenic mice.4 5 6 Although much remains to be learned about this subject, it is widely thought that reverse cholesterol transport may account for part or all of the antiatherogenic action attributed to HDL.
LCAT (EC 2.3.1.43) is a key enzyme in reverse cholesterol transport, the process by which cholesterol is mobilized from peripheral tissue to the liver. The plasma HDLs are the initial acceptors of cholesterol from the cell membranes of peripheral tissue. LCAT is important in this process because its activity, which converts cholesterol to its esterified form, maintains the cholesterol gradient that drives the spontaneous transfer of cellular cholesterol to the plasma HDL.7 8 9 Subsequently, the esterified cholesterol is transferred to the apo B–containing lipoproteins, which are removed by hepatic LDL receptors.
Natural mutations of LCAT are associated with certain abnormalities in plasma lipid profiles, such as hypoalphalipoproteinemia and an increased ratio of free to esterified cholesterol.10 11 Clinically, these mutations are sometimes expressed as FLD and FED.10 11 FLD is characterized by nephropathy, anemia, and corneal opacities. The plasma cholesterol levels of FLD patients are abnormally low, and nearly all of the cholesterol in every lipoprotein subclass is unesterified. In contrast, corneal opacities are the only clinical manifestation of FED,10 11 and the ratios of free to esterified cholesterol in the apo B–containing plasma lipoproteins of FED patients are normal. Thus, in FED, the abnormal elevations of plasma free cholesterol are due entirely to elevations within HDL.10 11
FLD and FED are very rare disorders. Nevertheless, studies of these diseases are interesting because they may shed additional light on the roles of HDL and LCAT in reverse cholesterol transport. Paradoxically, even though a high level of HDL-C is a negative risk factor for coronary artery disease, neither FLD nor FED is clearly associated with premature atherosclerosis. Other areas that remain to be clarified include the role of cholesterol esterification in the prevention of corneal opacities and the association of two phenotypes, FLD and FED, with mutations in the LCAT gene. In this article, we describe a novel homozygous mutation in the LCAT gene of an FED patient, the lipid composition of a cornea that was removed from the patient during transplantation, and the biochemical properties of the mutant LCAT expressed in COS-6 cells.
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Methods |
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Description of the Proband
The proband, a 63-year-old woman from Barcelona, Spain, consulted one of us (T.M.) in 1990 because of massive corneal opacities that were first treated by unilateral corneal transplantation. Electron microscopy of the cornea revealed membranous electron-dense deposits localized in the stroma; these deposits were similar to those reported in FLD and FED.10 11 12 The patient had no symptoms of coronary atherosclerosis. The results of tests of liver and renal function were normal, as were most of the routine biochemical and hematological tests. The one exception was a markedly low plasma concentration of HDL-C. There was no family history of consanguineous marriages, but the patient's only sister also exhibited corneal opacities. Three daughters and a granddaughter of the proband agreed to participate in the study; her sister would not agree to participate. In 1994, the proband underwent corneal transplantation of the second eye, and the cornea was collected for lipid analysis. After an overnight fast, control plasma from a group of five women from Barcelona was collected.
Lipoprotein Isolation and Analysis
Lipoproteins were isolated by
ultracentrifugation.13 Plasma
concentrations of cholesterol, free
cholesterol, triglycerides, apo A-I, and apo
B-100 were determined with commercial kits (Boehringer
Mannheim). The cholesteryl ester contents of plasma and lipoprotein
fractions were calculated from the difference between the values for
free cholesterol and those for total
cholesterol. HDL2-C and HDL3-C were
separated by selective precipitation with dextran sulfate magnesium
salt.14
LCAT Concentration, LCAT Activity, and Cholesterol
Molar Esterification Rate in Plasma
The endogenous esterification rate was measured with
the lipoproteins of the whole plasma as a substrate by the method of
Dobiasova.15 16 The results were expressed as the
fractional esterification rate and the molar esterification rate. LCAT
activity was determined by use of model R-HDL that contained
1-palmitoyl-2-oleoyl-sn-3-phosphocholine,
[3H]cholesterol, and apo A-I as a
substrate.17 LCAT reactivity toward LDL labeled with
[3H]cholesterol was measured by a
modification of our previously reported method.18
Typically, 120 µL of LDL (0.87 mg/mL apo B-100) was incubated with 25
µL of plasma or cell culture media at 37°C for 5 hours. Plasma LCAT
concentrations were measured by a radioimmunoassay as previously
reported.19
Fatty Acid Analysis of Plasma
Lipids from 0.5 mL of plasma were extracted with a mixture of
isopropanol:heptane:sulfuric acid (4:1:0.1, vol:vol:vol), dried with
nitrogen, reconstituted with benzene, and separated by TLC in silica
gel plates with a mixture of isooctane:ether:acetic acid (3:1:0.08,
vol:vol:vol) as a solvent. Phospholipids, triglycerides,
cholesteryl esters, and free cholesterol were visualized
under an ultraviolet lamp after the TLC plates were sprayed with 0.5%
2',7'-dichlorofluorescein in ethanol. The bands containing
fatty acid esters were removed and saponified by incubation with 2 mL
of a mixture containing methanol and 0.5N methanolic NaOH (1:1,
vol:vol) plus boron trifluoride for 30 minutes at 100°C. The
acyl esters were extracted with petroleum ether and saturated NaCl
(1.6:1, vol:vol), dried under a stream of nitrogen, redissolved in
isooctane, and subjected to gas chromatography.
Lipid Analysis of the Cornea
A cornea of the proband and a control cornea from an unrelated
25-year-old woman affected by keratoconus were lyophilized. Lipids were
then extracted with chloroform:methanol (2:1, vol:vol), dried with
nitrogen, reconstituted with benzene, and applied to TLC plates. Zones
of the plate corresponding to free cholesterol, cholesteryl
esters, triglycerides, and phospholipids were identified,
removed from the plate, extracted with chloroform:methanol (2:1,
vol:vol), and dried under a stream of nitrogen. The extracts were
divided into two aliquots. The smaller aliquot was used to quantify
each lipid, and the other was used to determine the fatty acid
composition. Lipids were quantified with commercial enzymatic kits
(Boehringer Mannheim), and the fatty acid analyses were
performed as described above.
Oligonucleotide Primers, PCR, and Gene
Sequencing
DNAs were isolated from white blood cells by treatment with
proteinase K (20 mg/mL), extraction with phenol and phenol-chloroform
(1:1, vol:vol), and precipitation with 95% ethanol. The pellets of DNA
were resuspended in Tris-EDTA buffer (pH 7.5) and stored at 4°C. To
obtain fragments that contained all exons and exon-intron boundaries of
the LCAT gene, a Cyclone Plus synthesizer was used to synthesize four
pairs of oligonucleotides according to the reported
sequence (Fig 1
; References 20 and 2120 21 ). They were
purified by Oligo-Pak synthetic oligonucleotide
purification columns (MilliGen/Biosearch). LCAT gene amplification was
carried out by PCR using a PCR reagent kit and Taq DNA
polymerase (Perkin-Elmer Cetus).22 In each reaction, 1
µg of genomic DNA and 100 pmol of each primer were added to the
100-µL reaction mixtures. The mixtures were subjected to 35 cycles of
PCR (denaturation at 95°C for 1 minute, annealing at 55°C for 1
minute, and polymerization at 72°C for 3 minutes). The amplified DNA
was digested with EcoRI and BamHI (Promega) and
subjected to 1.2% agarose gel electrophoresis. After staining with 0.5
µg/mL ethidium bromide, the desired band was visualized under
ultraviolet light and removed. The DNA fragments were removed from the
gel by electroelution23 and subcloned into M13 mp18 or M13
mp19 vectors previously digested by EcoRI and
BamHI.24 DNA sequencing was performed by the
dideoxynucleotide method25 using a kit
(Sequenase version 2.0 DNA, United States Biochemical Corp).
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RFLP Analysis
Restriction enzyme map analysis showed that the mutation
identified in codon 99 (exon 3) should produce a new restriction site
for Bgl II. Exons 2 and 3 of the LCAT gene were subjected to
PCR as described above, using genomic DNA from the proband, several of
her relatives, and an unrelated control subject. The amplified
fragments were purified by electroelution and incubated with 10 U/µg
DNA of Bgl II (Bethesda Research Laboratories) for 2 hours
at 37°C. Then the DNA was subjected to agarose gel electrophoresis,
and the gel was stained with ethidium bromide.
Mutagenesis In Vitro and Expression in COS-6 Cells
Mutant clones were constructed as previously
described.26 27 An antisense
oligonucleotide
(5'-GCCAGGGACGCAGATCTGGAC-3') that included the change
found in codon 99 of the mature LCAT protein in the FED patient was
synthesized. The wild-type and mutant cDNAs were subcloned into the
eukaryotic expression vector pSG5 and transformed into
Escherichia coli AG1. The selected clones were prepared in
larger quantities and used for transfection into COS-6 cells. Flasks
containing only COS-6 cells or COS-6 cells plus pSG5 plasmids were used
as controls. After 48 hours of incubation, the media were collected and
immediately tested for LCAT activity. No LCAT appeared in the media of
untransfected cells or in the cells transfected with only pSG5 plasmid.
The appearance of LCAT protein in the media was confirmed by Western
blot analysis, and the LCAT concentration was determined by a
solid-phase immunoassay.26 27 The activity of the LCAT
secreted into the media was measured with R-HDL16 or
[3H]cholesterol-labeled LDL.
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Results |
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Lipid and Lipoprotein Profile
The initial biochemical study of the plasma from the proband and her relatives was consistent with the diagnosis of FED (Table 1
); elevated concentrations of free
cholesterol were found in the plasma and HDL of the proband
but not in her LDL. The
hypertriglyceridemia that was observed in
the proband is also common in FED patients. Observations of corneal
opacities in the proband's sister and low plasma levels of HDL-C and
apo A-I among her three daughters suggest that the FED was
inherited.
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LCAT Activities and Concentrations
Table 2 shows the plasma LCAT concentrations
and activities of the FED family. The concentration of LCAT in the
plasma of the proband was 31% to 50% lower than those of her
daughters. Although the plasma LCAT levels of the control subjects were
not determined, the LCAT concentration of the proband was only 49% of
that of another group of female control subjects previously
reported.28 Plasma from the proband contained less LCAT
activity toward R-HDL (26% of control) and was less reactive toward
labeled LDL than plasma from her daughters (19% to 32% of the
daughters' LCAT reactivity). The specific activity toward R-HDL for
the proband's LCAT was 42% of the value found for previously studied
control subjects,29 thus providing further support for the
existence of a primary defect in the LCAT protein. With the plasma of
the proband as the source of enzyme and substrate, the molar
esterification rate of cholesterol was 45% of that of
control subjects. Typically, the esterification rates in FED plasma are
between 50% and 100% of those of the control subjects; in contrast,
the plasma esterification rates for FLD patients are usually much lower
and sometimes approach zero.10 11 The plasma
esterification rates for the daughters of the proband were similar to
those of the control subjects. However, the specific activity of the
LCAT from daughter number 3 was similar to that of the proband, which
implies that the different lipoprotein profile was mainly because the
plasma LCAT concentration of that daughter was twice that of the
proband. Collective consideration of LCAT analyses of the
proband and her family suggests but does not prove an inherited
mutation of the LCAT gene.
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Fatty Acid Composition of the Plasma
Fatty acid analysis of the plasma of the FED family
revealed differences in the compositions of the cholesteryl esters,
triglycerides, and phospholipids. The dominant fatty acid
among the cholesteryl esters and triglycerides of the
plasma from both the proband and a control group of women from the same
city was linoleate, which represented about 50% of the
total. The cholesteryl esters, triglycerides, and
phospholipids from the proband were slightly lower in palmitate but
higher in oleate than those of control subjects. In contrast, the fatty
acid analyses of the plasma lipids of the granddaughter were
similar to those of control subjects. Moreover, the fatty acid
compositions for our control subjects were similar to those from
another Spanish study in Madrid.30
LCAT Gene Analysis
Using the PCR primers shown in Fig 1A
and 1B, we amplified,
subcloned into M13 vectors, and sequenced all exons and exon-intron
boundaries of the LCAT gene from the proband. Only one difference was
found between the sequences of LCAT DNA from the proband and control
genomic DNA: the LCAT gene from the proband contained a substitution of
adenine for guanine in the antisense codon corresponding to amino acid
number 99 of the mature LCAT protein. The wild-type sequence was
5'-CAGATCCGCCTC-3'; that of the FED LCAT was
5'-CAGATCTGCCTC-3'. This change in DNA sequence corresponds
to an ArgCys mutation at this site (Fig 2). The same
results were obtained by four different sequence analyses of
DNA from different colonies of E coli containing exons 2 and
3 of LCAT genes from the patient and a control subject.
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RFLP Analysis
The restriction analysis tested for the appearance of a
new restriction site for Bgl II in the amplified exons 2 and
3 of the LCAT gene (AvGATCT). If a new site for Bgl II was
present, the 474-bp band corresponding to exons 2 and 3 should have
been converted to a 334-bp band and a 140-bp band. The presence of two
bands of these sizes in the DNA analysis for the FED patient
showed homozygosity for the Arg99Cys mutation (Fig 3A). The appearance of bands measuring 474, 334, and 140
bp in the DNA analyses for the three daughters indicated that
they were heterozygous for the same mutation, whereas the absence of
the 334-bp and the 140-bp bands in the DNA analyses for the
control subject and the granddaughter indicated normal LCAT genes. The
results of RFLP (Fig 3A) and the biochemical analysis (Tables 1 and 2) are consistent in determining which family members are
homozygous for the FED mutation, which are heterozygous, and which do
not carry the FED LCAT gene. Taking the available data into account, we
were able to construct a family pedigree (Fig 3B).
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In Vitro Expression and Activity of LCAT
(Arg99Cys)
Site-directed mutagenesis was used to clarify the connection
between the presence of FED in our proband and the
Arg99Cys mutation. The cDNA of the human LCAT gene
containing the Arg99Cys mutation was prepared and
expressed in COS-6 cells; wild-type LCAT cDNA was expressed as a
control. The mass of LCAT (Arg99Cys) found in the media
was 48% of that of the wild-type LCAT found in control cultures (Table 4). With R-HDL as the substrate, the
activity of the media from cells transfected with the mutant LCAT cDNA
was only 3.3% of that of the media from the wild type. Similarly, the
LCAT (Arg99Cys) activity against LDL was also much lower
(3.2% of that of the wild type). Thus, the percentage of specific
activity present in LCAT (Arg99Cys) was
approximately the same for LDL and R-HDL (6.8% and 6.9% of that of
the wild type, respectively). This result contrasts with the specific
activities of the proband's plasma LCAT toward R-HDL and LDL, which
were 42% and 43% of the values found for the control subjects,
respectively. Thus, the specific activity of the LCAT
(Arg99Cys) in the media is less than one sixth of that
found in the plasma of the proband. The reason for this difference is
unclear. However, the presence of LCAT protein in the media of the
cells transfected with LCAT (Arg99Cys) was confirmed by
a Western blot. The immunoreactive protein detected by this method had
the expected apparent molecular weight of plasma LCAT and was similar
in intensity to that of the wild-type LCAT. Furthermore, this band was
not seen in the control cell media (data not shown).
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Lipid Composition of the Proband's Cornea
The results of the lipid analyses performed on corneas
from the FED proband and from a control subject are shown in Table 5. Total cholesterol and free
cholesterol levels in the FED cornea were 3.1- and 4.2-fold
higher, respectively, than those of the control cornea, whereas the
level of cholesteryl esters in the FED cornea was 2.7-fold lower than
that of the control cornea. Thus, free cholesterol
represents 96% of the total cholesterol in the FED
cornea but only 71% of the cholesterol in the control
cornea. In contrast, corneal phospholipid levels were elevated 6-fold
in the FED patient with respect to that of the control. The
triglyceride contents of both corneas were too low for
quantification.
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There were only minor differences in the fatty acid compositions of the
lipids of control and FED corneas (Table 6). For both
the control and the FED corneas, palmitate, stearate, and oleate
appeared in >80% of the phospholipids; however, the stearate content
of the control cornea was higher than that of the FED cornea. The
cholesteryl ester compositions of the control and FED corneas
correlated with those of the phospholipids
(r2=.96 and .91, respectively). The
fatty acid compositions of the triglycerides from the
control and FED corneas were very similar, with palmitate, stearate,
and oleate making up 90% of the total. The fatty acid compositions
of the triglycerides also correlated with those of the
phospholipids (r2=.87 and .86 for
control and FED corneas, respectively).
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More notable are the quantitative disparities between the fatty acid
compositions of lipids in the cornea and in plasma (Fig 4). In the control and FED corneas, the palmitate,
stearate, and arachidonate contents of the phospholipids
were higher than those of the plasma. This difference was most profound
for stearate in the control subject. Conversely, oleate and linoleate
esters of phospholipids were lower in the cornea than in plasma; most
of this difference was due to reduced levels of linoleate. Compared
with the plasma, the triglycerides of the control and FED
corneas were enriched in palmitate and stearate and deficient in oleate
and linoleate. This difference between plasma and corneal
triglycerides was greater for the FED subject. Cholesteryl
esters of palmitate and stearate were much higher in the cornea than in
the plasma, whereas oleate and linoleate were lower.
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Discussion |
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Biochemical and Molecular Basis of the FED Phenotype
Although FLD and FED share some characteristics, FED is distinguished from FLD by the appearance of corneal opacities later in life and the absence of any other clinical symptoms. Biochemically, FLD differs from FED by its vanishingly low plasma concentrations of cholesteryl esters.18 31 On the basis of these distinguishing criteria, we diagnosed the proband as having FED.
The concentration of HDL-C in the proband's plasma is the highest of
the eight FED families that have been reported in detail to
date.32 33 34 35 36 37 38 Comparison of the relative amounts of
HDL2 cholesterol and HDL3
cholesterol suggests that HDL3, which is
smaller and denser, is more abundant in the proband (Table 1). Thus, as
in FLD,39 the HDLs from the proband are enriched with
small particles. Of the known FED subjects, our proband also had the
highest percentage (relative to control subjects) of LCAT activity
toward R-HDL, also known as 
-LCAT activity (Table 2).32 33 34 35 36 37 38 LCAT specific activity toward R-HDL was low (42%
of that of the control) but higher than that of most other reported
cases of FED.32 33 34 35 36 37 38
The magnitude of residual LCAT activity toward R-HDL and LDL was
similar in both the LCAT of the proband's plasma and the
transfected LCAT (Arg99Cys). This finding, similar to
that of another FED patient reported recently,38 supports
the idea that FED is not determined solely by a very low activity of
LCAT toward HDL and an increased activity toward LDL (also known as
ß-LCAT activity).40 Moreover, in vitro tests of various
LCAT mutants associated with FLD and FED by site-directed mutagenesis
have not revealed a clear-cut relationship between activity and the
phenotype.18 30 We believe that the current
pathophysiological knowledge would justify the
classification of FED as a partial LCAT deficiency in which (1) there
is low LCAT activity toward HDL and normal or high activity toward LDL
or (2) there is low but significant activity of LCAT toward both LDL
and HDL.17 29 37 As a partial LCAT deficiency, FED could
occur through one of the following mechanisms: (1) impaired binding or
reactivity toward all or specific subclasses of HDL and LDL, which
would result in low LCAT specific activity toward one or more
lipoprotein classes, or (2) alterations of the synthesis of LCAT, its
intracellular processing, or its secretion to the plasma, which may
result in a phenotype of low concentration of LCAT in plasma
with normal specific activity toward both HDL and LDL. Of course, FED
may originate from the combination of more than one of these
mechanisms.
It has been clearly demonstrated that the FED phenotype can
appear even when the LCAT activity is substantially conserved.
Moreover, it is possible that there is a threshold of LCAT activity
that is sufficient to prevent alterations in red blood cells and kidney
function but insufficient to avert the appearance of corneal opacities.
Conversely, at least some FED cases could result mainly from a
defective function of LCAT upon a minor fraction of small
pre–ß-migrating HDL, which are the initial acceptors of cell-derived
cholesterol.41 42 43 44 45 It has been proposed that
this minor fraction of HDL is critical for reverse
cholesterol transport in the cornea,46 whereas
its function in other tissues could be replaced by other types of HDL
particles or even other lipoproteins. It is important to note that the
size of R-HDL is similar to that of HDL2
particles17 ; thus, the activity of FED LCAT usually
measured (-LCAT activity) is not necessarily
representative of its reactivity toward
pre–ß-HDL.
A novel mutation in the LCAT gene, LCAT Arg99Cys,
was identified in the proband (Fig 2). Analysis of a new
restriction site created by the mutation in the proband and her family
demonstrated that the proband is homozygous for this mutation (Fig 3A).
The proband was not aware of any consanguineous marriages in her
family. Because of the low HDL-C found in the plasma of the
heterozygotes, it is possible that RFLP analysis of exons 2 and
3 of the LCAT gene using Bgl II could define the molecular
basis of some cases of inherited hypoalphalipoproteinemia found in the
general population.
Collective consideration of the RFLP analysis and the in vitro
mutagenesis results, which show lower than normal LCAT secretion and
specific activity, strongly supports the hypothesis that an inherited
Arg99Cys mutation is the cause of FED in the proband.
However, the mechanism by which this mutation in the LCAT protein
affects secretion and activity is not clear.
Arg99 is part of a sequence of eight amino acids bounded by
prolines. Because this sequence is conserved in mouse and rat
LCAT,47 48 it may be essential for LCAT structure and/or
activity. The substitution of a cysteine for an arginine is not an
insignificant change. This mutation substitutes a basic hydrophilic
residue for a smaller, neutral one that could change the charge and/or
the conformation of the region surrounding the mutation. Furthermore,
cysteine residues have a distinct chemistry that includes the formation
of internal and intramolecular disulfide links that could greatly
reduce LCAT activity. It is also unclear why there is such a large
difference between the specific activity of the Arg99Cys
LCAT found in plasma and in the cell media. Different disulfide linkage
patterns in the LCAT derived from the proband's plasma and in the cell
media could be associated with differences in activity.
This report of the Arg99Cys mutation is the fifth
detailed description of a homozygous mutation of the LCAT gene in an
FED patient; the others are Thr123Ile,
Pro10Leu, Asn131Asp, and the deletion of
Leu300.34 38 49 50 Several compound
heterozygous mutations of the LCAT gene have also been described; these
are Thr123Ile and Thr347Met,
Pro10Gln and Arg135Gln, and
Thr123Ile and
Tyr144Cys.31 51 52 Other mutations that are
associated with FED have been reported in abstracts.53 54
Thus, at the level of the molecular structure, FED is a
heterogeneous disease. This structural
heterogeneity could explain the tremendous variations
in the activity of plasma LCAT and the severity of the attendant
hypoalphalipoproteinemia that are seen in different FED patients.
Comparison of the locations of mutations for FLD with those for
FED shows that neither phenotype is associated exclusively with
a distinct region of the gene and that mutations giving rise to FLD and
FED can occur in similar regions of the gene. For example,
Ala93Thr and Arg135Trp, which occur in
FLD, are vicinal to Arg99Cys and
Thr123Ile, which occur in FED. In contrast with the
finding that relatively high residual LCAT activities, at least those
measured with R-HDL, are insufficient to preclude the appearance of
FED,38 relatively small amounts of LCAT activity are
sufficient to prevent FLD. The occurrence of one allele associated
with FLD in homozygotic subjects with a normal allele or one that
codes for FED in the homozygotes averts the appearance of the FLD
phenotype.32 51 53
LCAT Impairment and Corneal Opacities
Lipoprotein disorders are frequently associated with
alterations in the morphology and lipid composition of the cornea. The
corneal arcus appears in several types of
dyslipoproteinemias,55 and corneal opacities are found in
several HDL deficiencies, including Tangier disease, FLD, and
FED.10 11 12 46 55 In FED, corneal opacities are the only
clinical manifestation of the disease.10 11 Because the
corneal opacities are associated with HDL deficiencies and primary or
secondary impaired LCAT activity, reverse cholesterol
transport is thought to be an essential part of lipid homeostasis in
the eye. It is likely that the avascular anatomy of the cornea
may make it particularly vulnerable to impaired cholesterol
transport.12 46 55
Structural analysis of the cornea in FED patients has shown
electron-dense deposits accumulating mainly in the
stroma.55 Our data are the first direct evidence that the
accumulation of free cholesterol and phospholipids also
occurs in a cornea affected by FED. As in FLD,12 the
distribution of lipids among lipid classes in the proband's cornea is
consistent with impaired LCAT activity. Concentrations of
cholesterol and phospholipids, which are the substrates for
LCAT, are elevated 3.1- and 6.1-fold above control levels.
Conversely, the product of the LCAT reaction, cholesteryl esters,
is reduced by more than half. Given that the elevations of free
cholesterol and phospholipids are the major compositional
differences that distinguish the cornea of the FED patient from that of
the control, it is probable that the excess of these two lipids forms
the membranous deposits found in the corneas of patients with FED.
The normalized ratios of cholesteryl esters in the cornea to those in
plasma are similar for both the FED patient and the control subject,
even though the amounts of cholesteryl ester in each of these
compartments are lower in the proband than in the control subject
(Table 5). In contrast, the normalized ratios of free
cholesterol and phospholipids in the cornea to those in
plasma are much higher in the proband than in the control subject. The
preferential accumulation of free cholesterol and
phospholipids but not cholesteryl esters in the cornea of the proband
requires that the former two lipids preferentially enter into the
cornea, or alternatively, that they exit the cornea at a slower than
normal rate. The lack of preferential accumulation of cholesteryl
esters in the proband suggests that the flux of cholesteryl esters
between the cornea and the plasma compartment may be normal and that
the products but not the substrates of the LCAT reaction can exit
the cornea. Therefore, in the cornea, LCAT is essential for
cholesterol and phospholipid homeostasis. It is likely that
in FED and other HDL deficiencies, the corneal opacities are due to
defects in the removal of these lipids from the
cornea.10 11 12 55
Conversely, our fatty acid analyses indicate that the cornea is also the site of a lipid metabolism that is exclusive of the plasma compartment. Previous studies showed that the cholesteryl esters of the cornea in FLD were rich in linoleate.12 However, the fatty acid compositions of the plasma lipids and the phospholipids and triglycerides of the cornea were not reported. Our data, which include the first complete fatty acid profile of the plasma and corneal lipids of an FED patient, provide additional insight into the distinctive lipid metabolism of the cornea. If the plasma and corneal lipids were metabolized via the same machinery, they would exhibit similar fatty acid compositions. Our data show that the fatty acid compositions of all lipid classes in the cornea are different from those in plasma. This result suggests that the metabolic pathways and acyl chain specificities for phospholipid, cholesteryl ester, and triglyceride metabolism within the cornea are distinct from those in the plasma compartment. This interpretation is in agreement with the concept of inadequate LCAT-mediated removal of free cholesterol and phospholipids as the cause of lipid accumulation in the FED cornea.
The source of the differences between the fatty acid compositions of lipids from the FED and normal corneas is difficult to identify. There are two known sources of cholesteryl esters: LCAT and ACAT. The primary substrate for ACAT is oleoyl coenzyme A, and in the total absence of LCAT activity, the fatty acid compositions of cholesteryl esters would be expected to reflect this specificity. Although the corneal levels of cholesteryl oleate in the proband were twice those of the control subject, they were not the major esters of cholesterol for control or FED corneas. Therefore, the fatty acid analysis of the cholesteryl esters provides some support for ACAT as the source of corneal esters. It is also unlikely that LCAT is the source of cholesteryl esters; LCAT is severely impaired in FED, and in vitro studies of LCAT specificity have shown that cholesteryl stearate, which is a major corneal lipid, is formed at a very low rate.56 Two possible explanations remain. There may be a third mechanism for cholesteryl ester formation in the cornea that is independent of ACAT and LCAT12 57 58 ; alternatively, the cornea may be unusually rich in coenzyme A esters of palmitate. The latter conclusion is consistent with the remainder of the fatty acid data, which show that all three lipid classes are rich in stearate and palmitate.
Finally, a comparison of the fatty acid compositions of the plasma lipids from the proband with those of control subjects reveals an elevation of palmitate and a reduction in oleate. This appears to be associated with the occurrence of FED because the values for the daughters, who are obligate heterozygotes, fall between those of the proband and the control subjects. Moreover, the fatty acid compositions of the plasma lipids of the granddaughter, whom genetic analysis shows to have two normal genes, is similar to that of control subjects. We know of no mechanism by which impaired LCAT activity could lead to these changes in the plasma lipid compositions.
LCAT Deficiencies and Risk of Atherosclerosis
Given the putative role of LCAT in reverse
cholesterol transport, it is curious that FED and other HDL
deficiencies are not associated with premature
atherosclerosis. One reason for the lack of increased
risk of atherosclerosis in patients with FLD and FED
may be that, although decreased, the reverse cholesterol
transport does function in those patients.59 60 This could
be due, at least in part, to reverse cholesterol transport
mediated through particles containing apo E and
LDL.44 45 61 Furthermore, the low plasma levels of LDL-C
and lipoprotein(a) in FED could also reduce the risk of
atherosclerosis in FED and FLD
patients.50 62 Additional studies of FLD and FED in
transgenic models could provide greater insight into the
antiatherogenic role of LCAT.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the National Institutes of Health (HL-30914, HL-27341, and HL-30086), the Welch Foundation (Q906), Fundació August Pí i Sunyer, and Fondo de Investigación Sanitaria (FIS 90/293 and FIS94/1304, Spain). Dr Blanco-Vaca was supported in part by a fellowship from the Fondo de Investigación Sanitaria 91/5175. Dr Marzal-Casacuberta was supported by the Comissionat per a Universitats i Recerca, Generalitat de Catalunya (Spain). Our thanks to Joanne Jeter and Brook Watts for editorial assistance.
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Footnotes |
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Portions of this work were presented at the XI Symposium on Atherosclerosis, Montreal, Canada, 1994, and published in abstract form (Atherosclerosis. 1994;109:199).
Received March 4, 1996; accepted November 18, 1996.
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References |
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