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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1382-1391

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1382-1391.)
© 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

Francisco Blanco-Vaca; Shi-Jing Qu; Concha Fiol; Hui-Zhen Fan; Quein Pao; Àfrica Marzal-Casacuberta; John J. Albers; Isabel Hurtado; Vicente Gracia; Xavier Pintó; Tomás Martí; ; Henry J. Pownall

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


*    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 (Arg99->Cys) by transfected COS-6 cells was {approx}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 (Arg99->Cys) 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


*    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.


*    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 1Down; 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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  		Figure 1. A, Genomic structure of the human LCAT gene. Exons are represented by black bars and are numbered. Introns are represented by continuous line between exons. Letters indicate primers used for PCR, and arrows show direction of amplification. Size of amplified fragments are exon 1, 323 bp; exons 2 and 3, 474 bp; exons 4 and 5, 329 bp; and exon 6, 696 bp. B, Primers used for PCR of LCAT exons and exon-intron boundaries of LCAT gene. Restriction sites for EcoRI (GAATTC) and for BamHI (GGATCC) are underlined.

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.


*    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 1Down); 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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  		Table 1. Plasma Lipids and Lipoprotein Profiles of the Proband, Her Daughters, and Unrelated Female Control Subjects

LCAT Activities and Concentrations
Table 2Down 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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  		Table 2. Plasma LCAT Concentration and Activity Toward Exogenous and Endogenous Substrates

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 1AUp and 1BUp, 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 Arg->Cys mutation at this site (Fig 2Down). 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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  		Figure 2. DNA sequence of a region of exon 3 of LCAT gene from FED patient and a normal control patient. Both are antisense strands. Exchange of guanidine for adenine causes replacement of arginine by cysteine in residue 99 of mature LCAT protein.

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 Arg99->Cys mutation (Fig 3ADown). 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 3ADown) and the biochemical analysis (Tables 1Up and 2Up) 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 3BDown).



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  		Figure 3. A, RFLP analysis of exons 2 and 3 of human LCAT gene after treatment with Bgl II revealed a new restriction site that appeared in mutant (Arg99->Cys). Lanes: 1, molecular weight standard; 2, control individual; 3, daughter 1; 4, daughter 2; 5, daughter 3; 6, FED patient; 7, granddaughter. In all lanes, 150 ng of DNA was applied. B, Pedigree of Spanish FED family for Arg99->Cys mutation, determined on the basis of RFLP and biochemical analysis (see Table 1Up). + sign indicates presence of LCAT mutation on an allele; - sign designates presence of a normal LCAT gene. Solid symbols indicate deceased family members; open symbols, living family members. Sister of proband, who also has corneal opacities, is designated by *.

In Vitro Expression and Activity of LCAT (Arg99->Cys)
Site-directed mutagenesis was used to clarify the connection between the presence of FED in our proband and the Arg99->Cys mutation. The cDNA of the human LCAT gene containing the Arg99->Cys mutation was prepared and expressed in COS-6 cells; wild-type LCAT cDNA was expressed as a control. The mass of LCAT (Arg99->Cys) found in the media was 48% of that of the wild-type LCAT found in control cultures (Table 4Down). 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 (Arg99->Cys) activity against LDL was also much lower (3.2% of that of the wild type). Thus, the percentage of specific activity present in LCAT (Arg99->Cys) 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 (Arg99->Cys) 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 (Arg99->Cys) 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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  		Table 4. Concentration and Activity Toward R-HDL and LDL for the Wild-Type LCAT and FED LCAT (Arg99->Cys) Present in the Media of Transfected COS-6 Cells

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 5Down. 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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  		Table 5. Quantification of Lipids in the Cornea of the FED Proband and a Control Subject

There were only minor differences in the fatty acid compositions of the lipids of control and FED corneas (Table 6Down). 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 {approx}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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  		Table 6. Percent Fatty Acid Composition of the Corneal Lipids of the FED Proband and a Control Subject

More notable are the quantitative disparities between the fatty acid compositions of lipids in the cornea and in plasma (Fig 4Down). 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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  		Figure 4. Comparison of fatty acid compositions of phospholipids, triglycerides, and cholesteryl esters in cornea and plasma for FED subjects (top rows) and normal subjects (bottom rows).


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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 1Up). 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 {alpha}-LCAT activity (Table 2Up).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 (Arg99->Cys). 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 ({alpha}-LCAT activity) is not necessarily representative of its reactivity toward pre–ß-HDL.

A novel mutation in the LCAT gene, LCAT Arg99->Cys, was identified in the proband (Fig 2Up). 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 3AUp). 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 Arg99->Cys 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 Arg99->Cys 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 Arg99->Cys mutation is the fifth detailed description of a homozygous mutation of the LCAT gene in an FED patient; the others are Thr123->Ile, Pro10->Leu, Asn131->Asp, and the deletion of Leu300.34 38 49 50 Several compound heterozygous mutations of the LCAT gene have also been described; these are Thr123->Ile and Thr347->Met, Pro10->Gln and Arg135->Gln, and Thr123->Ile and Tyr144->Cys.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, Ala93->Thr and Arg135->Trp, which occur in FLD, are vicinal to Arg99->Cys and Thr123->Ile, 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 {approx}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 5Up). 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.


*    Selected Abbreviations and Acronyms
 
ACAT = acyl-CoA:cholesterol O-acyltransferase
apo = apolipoprotein
FED = fish-eye disease
FLD = familial LCAT deficiency
HDL-C = HDL cholesterol
LCAT = lecithin:cholesterol acyltransferase
PCR = polymerase chain reaction
R-HDL = reassembled HDL
RFLP = restriction fragment length polymorphism
TLC = thin-layer chromatography


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  		Table 3. Percent Composition of Fatty Acids in Cholesteryl Esters, Triglycerides, and Phospholipids in the Plasma of the FED Family and Control Subjects


*    Acknowledgments
 
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.


*    Footnotes
 
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.


*    References
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