© 1995 American Heart Association, Inc.
Articles |
Two Different Allelic Mutations in a Finnish Family With Lecithin:Cholesterol Acyltransferase Deficiency
From the Institute of Biotechnology (H.M.) and Department of Medicine (H.M., H.G., T.A.M., K.K.), University of Helsinki, Helsinki, Finland, and the Orion Corp (I.U.), Orion-Farmos, Orion Research, Helsinki, Finland.
Correspondence to Helena Miettinen, MD, Institute of Biotechnology, PO Box 45, Valimotie 7, SF-00014, University of Helsinki, Helsinki, Finland.
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Abstract Lecithin:cholesterol acyltransferase (LCAT) deficiency is a genetic disorder associated with low levels of serum HDL cholesterol. The proband of the Finnish LCAT-deficient family had corneal opacities, proteinuria, anemia with stomatocytosis, low serum HDL cholesterol (0.27 mmol/L), and low LCAT activity. Sequence analysis of his LCAT gene revealed compound heterozygosity for two different mutations: a C insertion in exon 1 between nucleotides 932 and 937 and a C-to-T point mutation in exon 6 at position 4976. The C insertion in exon 1 is predicted to result in premature termination and a truncated polypeptide containing only 16 amino acids. The C-to-T point mutation in exon 6 substitutes cysteine for arginine at residue 399. The functional significance of the Arg399
Cys
mutation was examined by expressing the mutated and wild-type LCAT
cDNAs in COS cells. COS cells transfected with mutated and wild-type
cDNAs showed comparable levels of mature LCAT mRNA. However, LCAT
activity in the cell media of COS cells transfected with the mutant
LCAT cDNA was significantly lower than that of COS cells transfected
with the wild-type cDNA (1.4% versus 12.0% cholesterol esterified,
respectively). A polymerase chain reaction–based duplex assay, in
which both mutations can be detected simultaneously, was used for
preliminary screening of Finnish subjects with serum HDL levels below
0.9 mmol/L; two additional individuals heterozygous for the
Arg399Cys mutation were identified. In conclusion, two
different allelic mutations in the LCAT gene have been identified in a
Finnish family, a C insertion between nucleotides 932 and 937 and a
C-to-T transversion at position 4976, and a convenient polymerase chain
reaction–based assay suitable for regional population screening and
differential diagnosis of low serum HDL was developed.
Key Words: cholesterol esterification • fish-eye disease • lecithin:cholesterol acyltransferase • HDL • stomatocytosis
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Introduction |
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Although low serum HDL cholesterol (HDL-C) concentration is an independent risk factor for coronary artery disease (CAD),1 there are many genetic disorders that are associated with markedly reduced serum HDL-C level in which CAD is frequently lacking.2 One of these disorders is lecithin:cholesterol acyltransferase (LCAT) deficiency.
LCAT is an enzyme that catalyzes the transfer of the sn-2 acyl group from lecithin to the 3-hydroxyl group of cholesterol, thus creating cholesterol esters and lysolecithin.3 This reaction is considered crucial for reverse cholesterol transport. Esterification of cholesterol creates a concentration gradient and flux of free cholesterol from the cells to HDL. The biochemical and physiological details of the enzyme have been reviewed.4 5
LCAT deficiency is a rare, recessively inherited disorder first
described in 1967.6 Phenotypically and genotypically the
disease is heterogeneous.7 8 Phenotypically, LCAT
deficiency has been classified both as fish-eye disease and classic
familial LCAT deficiency. Fish-eye disease, discovered in
1979,9 is characterized by massive corneal opacities,
marked reduction of serum HDL-C level, and selective inability of LCAT
to esterify HDL (
-LCAT activity),10 11 12 while plasma
cholesterol esterification rate is almost normal. Typical findings in
classic familial LCAT deficiency are corneal opacities, normochromic
anemia, and proteinuria due to glomerulosclerosis.13 Serum
HDL-C level is very low, and LCAT activity on both HDL and
apolipoprotein (apo) B–containing lipoproteins (- and ß-activity)
is virtually absent. Biochemically these two LCAT deficiencies have
been distinguished by the measurement of plasma cholesterol
esterification rate and HDL-associated esterification activity
(-activity). However, Klein et al14 report on a patient
with the clinical and biochemical features of fish-eye disease but
normal -LCAT activity, indicating that fish-eye disease and classic
LCAT deficiency could be phenotypically different expressions of the
same disease.
Sequence analysis of the LCAT gene of both familial LCAT deficiency and fish-eye disease patients has revealed several different mutations.7 8 14 15 16 17 18 19 20 Detailed clinical and biochemical data are available for the first known Finnish LCAT-deficient family.21 The purpose of the present study was to investigate the underlying DNA alterations in this family. In this article we report two allelic mutations found in the LCAT gene of the proband, one located in exon 1 and the other in exon 6. The functional significance of the exon 6 mutation was examined by expressing it in COS cells. For convenient screening of these mutations we set up a polymerase chain reaction (PCR)–based assay in which both mutations can be detected simultaneously.
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Patients
The study group consisted of the five-member Finnish LCAT-deficient family21 extended by three other family members: two sons (6 and 10 years) of the proband and a son (32 years) of the proband's sister (Fig 1
).
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Based on the clinical and laboratory findings of the first five family members,21 two siblings (family members 2 and 5) were diagnosed as homozygous and one sibling (member 4) as heterozygous for LCAT deficiency. Family members 2 and 5 had severely impaired cholesterol esterification, extremely low LCAT activity (4% to 6% of normal), corneal opacities, stomatocytes in the peripheral blood, normocytic and normochromic anemia, and very low serum HDL-C levels. In addition, member 5 had mild proteinuria, but serum creatinine and albumin concentrations were normal; no other family member had any evidence of kidney disease. Member 4 had stomatocytes and about half-normal LCAT activity without lipid abnormalities. He had been a heavy smoker for the last 50 years and was on atenolol for elevated blood pressure and chest pain on exertion. None of the other family members presented any clinical signs of CAD. The mother had died at the age of 94 years from complications of femoral neck fracture. She was not anemic nor did she have stomatocytes in her peripheral blood, but her serum HDL-C level was diminished. The father had died in 1958 at the age of 66 from complications of urinary tract infection due to prostate hypertrophy. According to old hospital records, he had been anemic and had mild proteinuria prior to urinary tract infection, but there was no information available about his lipid profile. Three new family members included in this study had normal serum lipid profiles, were not anemic, and had no stomatocytes in their peripheral blood.
Measurement of Serum Lipids and Serum LCAT Activity
Serum lipid and lipoprotein assays were performed21
and the activity of LCAT in serum was measured according to the
technique of Chen and Albers.22
Southern Blot Analysis
DNA was isolated from 20 mL of venous blood.23 DNA
(10 µg) was digested with the restriction enzymes Sac I,
HindIII, Pvu II, and Taq I (New
England Biolabs), fractionated by gel electrophoresis on a 0.6%
agarose gel, and transferred to nylon filters (Hybond N, Amersham
International plc) by using standard techniques.24 The
filters were hybridized with a 32P-labeled human LCAT cDNA
probe, LCAT 14 A (a kind gift from Dr S. Humphries, The Charing Cross
Sunley Research Center, London, England).
DNA Amplification and Sequencing
The sequences, biotinylation, and positions of the synthesized
primers are illustrated in Fig 2. The nucleotide
numbering follows that presented by McLean et al.25
Primer A for sequencing of exon 1 and primer O for sequencing of exon 6
were biotinylated. Genomic DNA (100 ng) was amplified in a 50-µL
mixture containing 50 pmol of each primer, 0.2 mmol/L each of dATP,
dCTP, dGTP, and dTTP, 1.5 mmol/L MgCl2 mmol/L KCl, 10
mmol/L Tris-HCL, pH 9.0, 0.1% Triton X-100, and 2 IU Taq
DNA polymerase (Promega Corp) using a programmable thermal cycler.
Thirty cycles were performed consisting of denaturation at 95°C,
annealing at 53°C to 68°C, and extension at 72°C for 1 minute
each, followed by a final extension for 10 minutes at 72°C and
cooling to 4°C.
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Direct DNA sequencing of PCR-amplified fragments was performed by the dideoxynucleotide chain termination method26 using a commercial sequencing kit (Sequenase 2.0, United States Biochemical Corp). Exons 1 and 6 were sequenced by a solid-phase sequencing method27 using streptavidin-coated magnetic beads (Dynabeads M 280 Streptavidin, DYNAL AS) for the separation of DNA strands.
In Vitro Mutagenesis and Translation of LCAT cDNA
The C-to-T point mutation at position 4976 of the LCAT gene was
introduced to a full-length human LCAT cDNA probe by using
oligonucleotide-directed mutagenesis. LCAT cDNA in the vector pUC19 was
released with the restriction enzymes BamHI and
EcoRI and subcloned to pGem3. The mutagenic oligonucleotide
carrying the mismatched base and a unique PpuMI restriction
site was synthesized (5'-GATGCAGGGGGACCCTGGCAGTAGGCA-3').
PCR was performed by using the mutagenic primer and a reverse primer
(5'-AGTCACGTGACCTCCTG GCA-3'), creating a 328-bp mutated fragment
carrying unique PpuMI and Dra III restriction
sites. The mutated fragment was digested with PpuMI and
Dra III, purified via electrophoresis, and ligated to
PpuMI–Dra III–digested wild-type LCAT cDNA in
pGem3. DNA sequences of the mutated LCAT cDNA pGem3 constructs were
confirmed by dideoxynucleotide sequencing.
In vitro transcription and translation of the wild-type and mutated LCAT cDNAs were performed by using a commercial kit (TnT-coupled reticulocyte lysate system, Promega) in both the absence and presence of microsomal membranes (canine pancreatic microsomal membranes, Promega) following the instructions provided by the manufacturer. 35S-labeled translation products were analyzed on a sodium dodecyl sulfate (SDS)/9% polyacrylamide gel.
Transient Transfection of COS Cells
Wild-type and mutated LCAT cDNA pGem3 constructs were digested
with the restriction enzymes Xho I and BamHI (New
England Biolabs), and the released LCAT cDNAs were subcloned to an
expression vector, pSVL SV40 (Pharmacia LKB Biotechnology Inc),
carrying the SV40 late promoter. COS-7 (monkey kidney) cells were grown
in Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Life
Technologies Inc) supplemented with penicillin, glutamine, glucose
(4500 mg/L), and 10% fetal calf serum (FCS) on 60-mm culture dishes.
Subconfluent COS-7 cells were rinsed once with serum-free medium
(OPTI-MEM, GIBCO BRL) and transfected with both wild-type and mutated
LCAT cDNAs in pSVL SV40 and with the transfection vector pSVL SV40
alone using the liposome (Lipofectin/Lipofectamine reagents, GIBCO BRL)
transfection method. Transfection was performed by using 5 µg DNA and
following the manufacturer's transfection protocol for each liposome
reagent. All transfections were performed in triplicate. After
incubating the cells for 5 hours with serum-free transfection medium
(OPTI-MEM), 1 mL DMEM containing 20% FCS was added to the cells, and
incubation was continued for an additional 12 hours. Cell culture media
were then replaced with serum-free media (OPTI-MEM) and harvested after
48 hours. Supernatants were kept at -70°C until LCAT activity assays
were performed. Cells were collected by centrifugation, resuspended in
0.25 mol/L Tris, pH 7.5, and lysed by freezing in carbon ice/ethanol
followed by melting in a 37°C water bath three times. Intracellular
extracts were collected after centrifugation and stored at 4°C until
DNA slot blot hybridization analysis was performed.
Determination of LCAT Activity
LCAT activity in the cell media was measured according to the
method of Chen and Albers22 using exogenous
proteoliposomes as substrate. LCAT activity was determined as the
percentage of [3H]cholesterol esters synthesized from the
proteoliposome substrate containing [3H]cholesterol
(Amersham), egg lecithin (Sigma Chemical Co), and apoA-I. Cell culture
medium (350 µL) was used to initiate each reaction; the reactions
were continued for 30 minutes at 37°C and stopped by adding 8 mL
chloroform-methanol (2:1) and 3 mL 0.5% NaCl. The labeled cholesterol
esters were separated from free cholesterol by thin-layer
chromatography. Values were expressed as percent
[3H]cholesterol esterified per 30 minutes.
Transfection efficacy was controlled by slot blotting intracellular DNA from each cell-culture dish along with the control standard DNA to nylon filters.28 Filters were hybridized with a 32P-labeled pSVL SV40 plasmid, and the radioactivity of each slot was counted by liquid scintillation counting. The activity of the slot indicated the amount of plasmid DNA present in the cell extract, and the level of LCAT activity was normalized to the amount of transferred plasmid DNA.
Northern Blot Analysis
Total COS cell RNA was isolated 72 hours posttransfection by the
guanidine-isothiocyanate method.29 RNA samples (5 to 10
µg) were electrophoresed in a formaldehyde–agarose gel mixture and
transferred to nylon filter (Hybond N, Amersham) using standard
techniques. A human LCAT cDNA 14 A probe was labeled with digoxigenin
using a commercial kit (DIG DNA labeling kit, Boehringer Mannheim
GmbH). Filters were prehybridized in 0.25 mol/L
Na2HPO4, 1 mmol/L EDTA, 20% SDS, and
0.5% blocking reagent (Boehringer Mannheim) at 68°C for 60 minutes
and hybridized with the digoxigenin-labeled human LCAT cDNA 14 A probe
at 68°C for 12 hours. Filters were washed at 68°C with 0.1xSSC and
1% SDS three times for 20 minutes each followed by detection of the
hybridized DNA using a commercial technique (DIG luminescent detection
kit for nucleic acids, Boehringer Mannheim).
Duplex PCR Assay for the Two LCAT Mutations
A PCR-based assay was developed for simultaneous detection of
both the exon 1 C insertion and exon 6 Arg399Cys
mutations. The duplex assay was based on the principle of using a
mismatch primer in the detection of a point mutation.30
PCR primers are illustrated in Fig 2
. PCR was performed by using
primers N and O (without biotinylation) together with a mismatch primer
P* (5'-TGG CTC CTC AAT GTG CTC TTC CCC TC-3') and primer B.
Primers N and O amplify a 191-bp fragment of exon 6 and primers P* and
B a 120-bp fragment of exon 1. In exon 6 the C-to-T mutation destroys
an Aci I cutting site. The mismatch in primer P* designed
for exon 1 eliminates an Aci I cutting site from the normal
sequence but leaves it intact in the mutated one. PCR was performed for
32 cycles at 95°C, 58°C, and 72°C for 1 minute each under the
conditions described above. PCR products were digested with
Aci I (New England Biolabs), size-fractionated on a 12%
polyacrylamide gel, and stained with ethidium bromide for
visualization.
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Results |
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Identification of the LCAT Gene Mutations
Clinical and biochemical data of the family members are summarized in Table 1
. Clinical findings and serum lipid assays
suggested that family members 2 and 5 (proband) were homozygous for
LCAT deficiency and that member 4 was a heterozygous carrier. Only the
proband had mild proteinuria which, during a follow-up of more than 2
years, has not worsened.
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Southern blot analyses did not show any major rearrangements in the LCAT gene of the proband (data not shown). All six exons and exon-intron junctions of his LCAT gene were then amplified and sequenced. Comparison of the sequencing data to the reported wild-type LCAT gene sequence25 revealed two mutations in the LCAT gene of the proband.
In exon 1 a C insertion (Fig 3) results in the formation
of seven consecutive C nucleotides instead of the normal six at
nucleotides 932 through 937. This mutation causes a translational frame
shift and is predicted to result in premature termination at codon 17.
The mutated LCAT polypeptide, if ever present in the cells,
contains only 16 amino acids, out of which residues 11 through 16
differ from the normal LCAT sequence. In exon 6 at position 4976 there
is a C-to-T point mutation (Fig 4) that substitutes
cysteine for arginine at residue 399.
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Family member 2 was also identified as a compound heterozygote for both
mutations. In addition, family members 1, 6, and 8 were heterozygous
for the C insertion, and members 4 and 7 were heterozygous for the
C-to-T mutation. Family member 3 did not have these mutations in his
DNA. The inheritance pattern of the two mutations demonstrates that
they are present in different alleles of the LCAT gene (Fig 1).
A Duplex PCR Assay for the Two Mutations
For the simultaneous detection of both the exon 1 C insertion and
exon 6 C-to-T mutations we set up a simple PCR assay that was used in
confirming the results of the sequence analysis (Fig 5). In this assay, fragments of exon 6 (191 bp) and exon
1 (120 bp) carrying the mutant nucleotides were amplified by PCR and
then digested with Aci I. DNA fragments were
size-fractionated on a 12% polyacrylamide gel and stained with
ethidium bromide. Digestion of the normal 191-bp exon 6 PCR product
gives fragments of 41, 43, and 107 bp (43- and 41-bp fragments are not
shown in Fig 5). As the C-to-T mutation destroys the other
Aci I restriction site normally present in exon 6, the
mutant allele gives a band of 150 bp in addition to the normal 107-bp
band, confirming that the patient is heterozygous for this mutation.
The mismatch in primer P* used in amplifying exon 1 destroys an
Aci I cleavage site in the normal sequence but leaves it
intact in the mutated one. A 95-bp fragment derived from the mutant
gene is formed together with the normal undigestable 120-bp fragment,
confirming that the patient is also heterozygous for this mutation. The
sizes of amplified PCR products allow identification of all possible
allele combinations (Fig 5).
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In our preliminary studies we tested the versatility of the duplex PCR assay for population screening. After screening approximately 150 unrelated Finnish subjects who had serum HDL-C levels below 0.9 mmol/L, we found two additional heterozygous carriers of the C-to-T exon 6 point mutation. Further screening studies and detailed characterization of the affected families are in progress.
In Vitro Translation and Expression of the LCAT cDNA With the Exon
6 C-to-T Mutation
Wild-type and C4976T mutated LCAT cDNAs in pGem3
were transcribed and translated in vitro using the rabbit reticulocyte
system. In the absence of microsomes this method generates only
nonglycosylated proteins, whereas in the presence of microsomes signal
peptide cleavage and core glycosylation occur. In the absence of
microsomes both wild-type and C4976T mutated LCAT cDNAs
encoded the synthesis of 35S-labeled proteins with an
apparent molecular weight of 46 kD, which is similar to the expected
size of a 440–amino acid LCAT protein31 (Fig 6). Wild-type and mutated LCAT cDNAs transcribed and
translated in the presence of microsomes directed synthesis of protein
products with a molecular weight of 
55 to 58 kD (Fig 6). Wild-type
and mutant cDNAs generated approximately comparable levels of protein
products both in the absence and presence of microsomes.
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Wild-type and C4976T LCAT cDNAs were expressed in COS
cells using the expression vector pSVL SV40. LCAT activity in the cell
media of COS cells transfected with the C4976T LCAT cDNA
was significantly lower than that of COS cells transfected with the
wild-type LCAT cDNA (1.4% versus 12.0% cholesterol esterified per 30
minutes, respectively). The media of the COS cells transfected with the
expression vector pSVL SV40 alone did not present any measurable
LCAT activity.
Northern blot hybridization analysis of the COS cells transfected
either with the wild-type or C4976T mutant LCAT cDNA
showed comparable levels of mature 1.6-kb LCAT mRNA (Fig 7), whereas no detectable LCAT mRNA was present in
the cells transfected with the expression vector pSVL SV40 alone.
Whether bands with slower mobility in the Northern blots
represent unprocessed LCAT mRNA is currently not known.
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We have identified two LCAT mutations in a Finnish family with LCAT deficiency: a C insertion in exon 1 between nucleotides 932 and 937 and a C-to-T transversion at position 4976 substituting cysteine for arginine at residue 399. Mutations were detected by sequence analysis and confirmed by using a duplex PCR assay. The proband and his sister were found to be compound heterozygotes for both defects. They had typical clinical findings for LCAT deficiency: corneal opacities, anemia, and very low serum HDL-C. Low cholesterol esterification percentage in all lipoprotein classes and low cholesterol esterification rate indicated that both LCAT
- and
ß-activity were impaired. Heterozygotes for either the C insertion or
the C-to-T mutation, however, had normal serum total cholesterol and
HDL-C esterification percentages and only slightly, if at all, reduced
HDL-C levels (Table 1). Thus, the heterozygotes cannot be easily
differentiated from the healthy control subjects by the routine serum
total and free cholesterol determination methods.
Not only the proband but also his affected sister and his brother, both
of whom were heterozygous for the Arg399Cys mutation,
had stomatocytes in their peripheral blood. This is not a typical
finding in LCAT deficiency, although there are few observations on
stomatocytes in patients with LCAT deficiency.32 33
However, target cells and mild hemolysis are usually present due to
structural, functional, and compositional changes in erythrocyte
membranes.34 In the present study, family members
heterozygous for the C insertion mutation only did not present with
stomatocytosis. However, heterozygosity only for the C-to-T mutation
caused stomatocytosis in subject 4 but not in subject 8, possibly due
to young age.
A mutation similar to the one reported here, ie, an extra cytosine within the stretch of six consecutive cytosines at nucleotides 932 through 937 in exon 1 of the LCAT gene, has been reported in a Japanese patient who was homozygous for the mutation.15 It is not possible to conclude whether the mutation reported in the present study and that found in Japan actually represent identical mutations or whether the cytosine inserted occupies a different position. Since there is no known racial relationship between the Finns and Japanese, it is most unlikely that the Finnish family and the Japanese one would carry a mutant allele originating from the same ancestor. As the C insertion in exon 1 generates an early stop codon, the 16–amino acid polypeptide product should be devoid of any enzyme activity, a conclusion supported by the clinical and biochemical data of the homozygous Japanese patient.15
The Arg399Cys mutation due to a single base substitution
in exon 6 has not been reported. The functional significance of this
mutation was examined by expressing it in COS cells. Northern blot
analysis of the transfected COS cells showed comparable levels of
mature LCAT mRNA, indicating that the defect does not affect
transcription or mRNA stability. However, the activity level of the
mutant LCAT expressed in vitro was significantly lower than that of the
wild-type one, although the mutated enzyme clearly presented some
enzyme activity compared with the negative controls (Table 2). This indicates that either the enzyme synthesized is
defective or its mass is low. Mutation could disrupt posttranslational
processing and lead to defective secretion or increased catabolism of
an altered protein, as has been postulated with the LCAT
Leu300Del mutation.14 Since no LCAT
antibody was available for us, we could not determine the LCAT mass in
the cell media. On the basis of enzyme activity determinations (Table 2), we propose that some enzyme mass is present. This assumption is
supported by the in vitro transcription and translation studies that
show that the mutated protein is synthesized in comparable levels with
the wild-type protein and that both proteins are core glycosylated in
vitro. Further support for this idea comes from the fact that the
plasma esterification rate of the proband was approximately 14% of
that of the normal subject.21 All functional enzyme
present in his plasma should originate from the LCAT gene with the
Arg399Cys mutation since the other allele can apparently
produce only a truncated polypeptide with 16 amino acids.
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The LCAT protein has four potential N-glycosylation sites,
and on the basis of homology studies with other serine-dependent
esterases the postulated active site of the enzyme is located around
Ser181.25 35 No mutations involving the
proposed functional areas of the gene have been reported. Although many
mutations have been found in exon 6,7 8 14 17 19 36 which
encodes almost half the LCAT protein, the Arg399Cys
mutation seems to be closest to the carboxy terminus of the enzyme. The
exact function of this domain of the enzyme is currently unknown.
Replacement of the positively charged arginine by cysteine may change
the secondary structure and folding of the protein. Wild-type LCAT
contains six cysteines, of which Cys31 and
Cys184 are free. The four remaining cysteines form
disulfide links, one being located between Cys50 and
Cys74 and the other between Cys313 and
Cys356.35 Francone and Fielding37
have shown by site-directed mutagenesis that free cysteine residues are
not essential for cholesterol ester synthesis. Furthermore, by using
the same principle, Qu et al38 have shown that replacement
of any of the four cysteines forming disulfide links by glycine results
in abolished or greatly diminished LCAT activity. We propose that the
additional cysteine at residue 399 may disturb normal disulfide link
formation, thus affecting the stability and/or activity of the
enzyme.
There are many rare genetic disorders associated with a marked
reduction of serum HDL-C level, including apoA-I, apoA-I/C-III, and
apoA-I/C-III/A-IV deficiencies, Tangier disease, HDL deficiency with
planar xanthomas, and LCAT deficiency.39 Although low
serum HDL-C is a risk factor for CAD, LCAT deficiency has not been
consistently associated with CAD. Some patients affected with LCAT
deficiency do suffer from CAD but it is not clear if these cases can be
accounted for by selection bias.40 However,
hypoalphalipoproteinemia, in which patients have HDL-C levels below the
10th percentile, is common and is associated with increased risk for
CAD. According to Genest et al,41 42 isolated familial
hypoalphalipoproteinemia can be found in 4% of CAD patients.
Differential diagnosis of LCAT deficiency is therefore important for
the evaluation of CAD risk in patients with low HDL-C levels.
Homozygous LCAT deficiency patients are easier to distinguish,
especially when typical findings such as corneal opacities, anemia, or
proteinuria are present, but heterozygotes are less likely to be
diagnosed due to lack of clinical symptoms and only modestly, if at
all, reduced plasma cholesterol esterification (Table 1).
No population screening studies of LCAT deficiency have yet been
performed, probably because of problems arising from the phenotypic and
genotypic heterogeneity of the disease. In a geographically isolated
area in Norway the frequency of heterozygous carriers has been
estimated to be as high as 4%.13 At the current stage of
our screening studies in the Finnish population, a unique genetic
isolate with its own variety of inherited diseases,43 we
have found two additional heterozygous carriers of the
Arg399Cys mutation in two apparently unrelated families.
It remains to be seen whether this LCAT gene mutation is enriched among
the Finns similar to the two LDL receptor gene deletions that account
for most of the cases of familial hypercholesterolemia in
Finland.44
The PCR-based duplex assay described in the present study may prove to be a useful tool in population screening for LCAT deficiency and in differential diagnosis of individuals with low serum HDL-C concentrations.
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Acknowledgments |
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This study was supported by grants from the Medical Council of the Finnish Academy, the Sigrid Juselius Foundation, and the University of Helsinki. Kaija Kettunen, Eeva Gustafsson, and Leena Kaipiainen provided excellent technical assistance. We thank Dr Steve Humphries for the gift of the LCAT 14 A cDNA probe.
Received August 15, 1994; accepted January 11, 1995.
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References |
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Klein H-G, Santamarina-Fojo S, Duverger N, Clerc M, Dumon M-F,
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