Original Contributions |
From the Departments of Internal Medicine (W.H., J.S., A.M., H.H., M.K., T.K., K.A.) and Biochemistry (K.M.), Fukuoka University School of Medicine, Fukuoka; and the Department of Internal Medicine, Oita Prefectural Hospital, Oita (H.N., K.Y.), Japan.
Correspondence to Jun Sasaki, MD, Department of Internal Medicine, School of Medicine, Fukuoka University, 451, 7-chome Nanakuma, Jonan-ku, Fukuoka 81480, Japan. E-mail mm034505{at}msat.fukuoka-u.ac.jp
| Abstract |
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50% of normal control. Sequence
analysis of polymerase chain reactionamplified DNA of the
proband's apoA-I gene showed a homozygous T-to-A transition resulting
in the substitution of Val 156 with Glu (apoA-I Oita). Direct
sequencing of samples obtained from other family members showed that
the brother was homozygous, whereas the son was a heterozygous carrier
of apoA-I Oita. The heterozygote for apo A-I Oita showed nearly 60% of
normal apoA-I and normal HDL cholesterol levels. In vivo
turnover studies in rabbits demonstrated that the variant apoA-I was
rapidly cleared from plasma compared with normal human apoA-I. Our data
suggest that the Val156Glu substitution is associated with apoA-I and
HDL deficiency, partial LCAT deficiency, and corneal opacities and that
Val156 of apoA-I may play an important role in apoA-I function.
Key Words: HDL deficiency apolipoprotein variant apoA-I Japanese corneal opacities
| Introduction |
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-helixes
involving amino acid residues between 137 and 186, which are critical
for both activation of LCAT and enhancing cholesterol
efflux from peripheral cells.
More than 10 apoA-I gene defects of either a deletion or insertion
leading to HDL deficiency have been
reported.5 6 7 8 9 10 11 12 13 14 15 However, in subjects with HDL
deficiency, CAD is not always present. In addition,
40
heterozygous structural apoA-I variants with a single amino acid
substitution have been characterized.2 16 17 18 19 20 21
However, most of these variants do not affect HDL concentration except
for nine that are associated with reduced plasma apoA-I and HDL
cholesterol levels.17 18 19 20 21 22 23 24 25 In this
article we report a novel, homozygous, missense point mutation in the
apoA-I gene that is associated with apoA-I and HDL deficiencies,
corneal opacities, and CAD.
| Methods |
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Lipid, Apolipoprotein, and Lipoprotein Analyses
Blood samples were collected in tubes containing disodium EDTA
after a 12-hour fast. Plasma concentrations of cholesterol
and TG were determined by an enzymatic method. Apolipoprotein
concentrations were measured by single radial immunodiffusion, and
plasma lipoproteins were separated by sequential
ultracentrifugation.5 The
following density fractions were obtained: VLDL
(d<1.006 g/mL), LDL (d=1.019 to 1.063
g/mL), HDL2 (d=1.063 to 1.125
g/mL), and HDL3 (d=1.125 to 1.21
g/mL). Fractionation of plasma lipoproteins by Superose 6HR 10/30 gel
chromatography was performed with a fast protein liquid
chromatography system (Pharmacia Fine Chemicals). The
column matrix was equilibrated with PBS containing 1 mmol/L EDTA
and 0.02% NaN3 (pH 7.4). Fifty 0.5-mL fractions
were collected at a flow rate of 0.5 mL/min. Electrophoresis of
lipoproteins from the different plasma density fractions was performed
by 12.5% SDS-PAGE, and the distribution of apoA-I was visualized with
an ECL Western blotting kit (Amersham Life Science). Control samples
for comparing plasma concentrations of lipids, lipoproteins, LCAT
activity, and LCAT mass were obtained from healthy volunteers matched
for age and sex. Informed consent was obtained from all control
subjects.
Electrophoresis and Immunoblotting
IEF gel electrophoresis on a pH gradient from 4 to 6 using
plasma samples from the proband and his family was performed as
described previously.26 In brief, 1 µL plasma
was incubated at room temperature for 1 hour with 30 µL of 10
mmol/L Tris HCl (pH 8.2) containing 1% sodium decyl sulfate, 2%
ampholytes (pH 4 to 6), and 10% ß-mercaptoethanol. The mini-slab gel
system (Biometra-Multigel G-44, Biometra) with a 7.2%
polyacrylamide gel containing 5 mol/L urea and 2% ampholytes
(pH 4 to 6) was used for IEF. A two-dimensional gel electrophoresis of
plasma, consisting of IEF followed by SDS-PAGE, was performed as
described previously.11 The apoA-I bands were
identified by immunoblots with a polyclonal rabbit
anti-human apoA-I antiserum.
Isolation of ApoA-I
HDLs (d=1.063 to 1.21 g/mL) were isolated from a
50- mL plasma sample from the proband and from a normal, healthy
subject by preparative ultracentrifugation,
delipidated, and then solubilized in deionized
water.26 Delipidated HDL was passed through a gel
chromatography column on Sephacryl S-200. Variant
apoA-I and normal apoA-I were isolated by Immobiline IEF, with a pH
gradient ranging from 5 to 6 as described
previously.26
Iodination of ApoA-I
Labeling of normal human apoA-I and apoA-I Oita with
Na[125I] (Du PontNew England Nuclear) was
carried out according to the iodine monochloride method of
McFarlane.27 The labeled apoA-I was dialyzed at
4°C for 40 hours against 0.15 mol/L NaCl containing 0.3 mmol/L
EDTA, pH 7.4. The specific activity of labeled apoA-I ranged from 350
to 450 counts per minute per nanogram protein, and 99% of the
radioactivity in labeled apoA-I could be precipitated by
trichloroacetic acid. Before the turnover studies, labeled apoA-I was
passed through a 0.45- µm filter with the addition of bovine
albumin (fatty acid free, Sigma Chemical Co) to a final
concentration of 10 mg/mL and stored for 1 to 2 days at 4°C.
In Vivo Turnover Study of ApoA-I
Male Japanese White rabbits (body weight, 2.5 to 3.0 kg) were
purchased from Kyudo Co, Fukuoka, Japan. The animals were housed
individually in a 12-hour/12-hour light/dark cycle environment and
provided with a commercial diet and water ad libitum. The experimental
protocol was approved by the Ethics Review Committee for Animal
Experimentation of our institution. The procedures of in vivo turnover
studies were performed essentially as described
previously.28 In brief, 2 days before and
throughout the entire turnover study, five drops of saturated KI were
added each day to the drinking water. Twenty-five µCi of
125I-labeled apoA-I in 1 mL saline with 10 mg/mL bovine
albumin was injected into each animal via the marginal ear vein. Blood
samples were collected via the marginal ear vein at 5 minutes and at 3,
6, 9, and 24 hours after injection and then once a day for 6 days. The
FCR was calculated as described by Matthews.29
The clearance of each labeled apoA-I was measured in four different
rabbits.
Haplotype Analysis
We determined the apoE genotype by using
HhaI digestion of the apoE gene after amplification by
PCR as reported by our laboratory.30 ApoA-I
MspI and apoC-III SstI
polymorphisms were performed as previously
reported.31
Nondenaturing Gradient PAGE
HDL particle sizes were determined by nondenaturing gradient
PAGE on 4% to 30% gels (Pharmacia LKB
Biotechnology).32 Plasma with a
d<1.21g/mL was obtained by
ultracentrifugation at 100 000 rpm for 2 hours
(Beckman TL-100 ultracentrifuge). This was followed by the
addition of 20 µg protein to the gel. Protein was visualized with
0.05% Coomassie brilliant blue G-250, and then the gel was scanned
with a personal densitometer SI (Molecular Dynamics Inc). The gel was
calibrated with a standard protein mixture (high-molecular-weight
calibration kit, Pharmacia LKB Biotechnology). HDL particles size was
calculated from the calibration curve of the peak migration distance
versus that of the standard.
CER, LCAT Activity, and LCAT Mass Measurements
CER indicates the esterification of endogenous
cholesterol, which was determined by the method of Stokke
and Norum.33 LCAT activity was measured by using
a proteoliposome as a substrate.34 LCAT mass was
kindly assayed by Prof John J. Albers, University of Washington,
Department of Medicine, Northwest Lipid Research Laboratories, Seattle,
Wash.35
DNA Amplification by PCR
Genomic DNA was isolated from 120 µL peripheral
blood as described in the Genomix kit manual (Talent srI). Ten
microliters of this extract was used as a template for PCR.
Oligonucleotide primers for the apoA-I gene were
synthesized on the basis of published data (model 380B DNA synthesizer,
Applied Biosystems). The primers for the apoA-I gene are listed in
Table 1
. PCR was performed according to
the protocol published by the manufacturer with the use of a Perkin
ElmerCetus thermal cycler. Amplification was performed by initial
denaturation at 96°C for 10 minutes, followed by 30 cycles at 96°C
for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute, and a
final extension for 7 minutes at 72°C. A reaction volume of 100 µL
contained 2.5 U Taq polymerase (Perkin ElmerCetus),
150 ng genomic DNA, 20 pmol of each primer, and 200 µmol of each
dNTP in 10x reaction buffer containing 0.1 mol/L Tris HCl, pH 8.3, 0.5
mol/L KCl, 15 mmol/L MgCl2, and 0.1%
(wt/vol) gelatin, as described previously.26
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DNA Sequence Analysis
The PCR-amplified DNA products were purified with a
GeneClean kit (Bio101), and purified DNA fragments were ligated to the
pT7Blue-T vector (Novagen). Twelve clones of both strands in opposite
directions were sequenced by the dideoxy chain- termination method
using Sequenase (US Biochemical).37 The DNA
fragment around the mutation in exon 4 of apoA-I was amplified and
subjected to direct sequencing by using a DNA sequencing kit (dye
terminator cycle sequencing ready reaction, Perkin Elmer) with an ABI
373 DNA sequencer.
Statistical Analysis
Data are expressed as mean±SD. Differences in FCR in the
turnover study were evaluated for statistical significance by
Student's t test. A value of P <.05
denoted statistical significance.
| Results |
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Lipid, Apolipoprotein, and Lipoprotein Profiles
The concentrations of lipids, lipoproteins, and apolipoproteins in
members of the family with apoA-I Oita are shown in Tables 2
and 3
.
The concentrations of total cholesterol, TG, and LDL
cholesterol in the proband were within normal levels. The
free cholesteroltototal cholesterol ratio
in the proband was slightly higher in the total, VLDL, and LDL
fractions but threefold higher in the HDL fraction than those of
nonaffected family members, indicating that esterification of
cholesterol was reduced mainly in the HDL fraction (Table 4
). The proband and his brother had
<10% and <15%, respectively, of normal apoA-I and HDL
cholesterol levels. The proband's son had nearly 60% of
normal apoA-I and HDL cholesterol levels, whereas these two
parameters were normal in other family members. Although
cholesterol concentrations in both
HDL2 and HDL3 subfractions
were markedly reduced, the degree of reduction in the
HDL2 subfraction was higher than that in
HDL3 in either homozygotes or heterozygotes.
Plasma lipoproteins separated by gel chromatography
from the proband showed a rearrangement of TGs among plasma
lipoproteins (Fig 3
). The TG of the
proband was distributed mainly in the LDL fraction instead of in VLDL,
a characteristic similar to that of normal control subjects. The
cholesterol level in the HDL fraction was markedly reduced,
whereas TG was only slightly decreased. The distribution of apoA-I was
shifted to a higher density fraction of HDL (Fig 3
). ApoC-II and
apoC-III were at moderately low levels in the homozygote, whereas apoB
and apoE concentrations were within the normal range. ApoE
genotype was E3/E3 in all family members tested in the
present study.
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Plasma CER, LCAT Activity, and LCAT Mass
Analysis of CER, LCAT activity, and LCAT mass showed that
these parameters were
50% lower in homozygous apoA-I
Oita carriers than in normal control subjects but were not different
from those in heterozygous carriers (Table 5
). These data indicate that the partial
reductions in LCAT activity and mass in this case may be secondary to a
Val156Glu substitution in apoA-I rather than a defect in the LCAT gene
itself.
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Electrophoresis and Immunoblots
IEF of plasma at a pH range of 4 to 6 followed by
immunoblotting showed no apoA-I-4 band in the proband
(II 2) or his brother (II 1) but rather showed apoA-I-3 and apoA-I-5
bands. In contrast, a plasma sample from the proband's son (III 1)
showed apoA-I-2, apoA-I-4, and faint apoA-I-3 and apoA-I-5 bands (Fig 4
). Two-dimensional electrophoresis (IEF
and SDS) followed by immunoblotting showed that the
molecular weights of apoA-I-3 and apoA-I-5 bands of the proband were
similar to that of normal apoA-I (data not shown). The nondenaturing
gradient gel electrophoresis of HDL particles followed by scanning with
a personal densitometer SI is shown in Fig 5
. In the homozygous proband (II 2), the
HDL fraction was hardly observed. In the heterozygote (III 1), HDL
particle size was smaller than that in normal control subjects.
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Haplotype Analysis
The results of apoC-III SstI, apoA-I
MspI, and apoA-I promoter (-75G/A) polymorphisms
are shown in Table 3
. Haplotypes S1S1, M1M1,
and AA were observed in both the proband (II 2)
and his brother (II 1). The distribution pattern of these three markers
in the apoA-I Oita family was compatible with that of an apoA-I Oita
mutant allele in the pedigree.
In Vivo ApoA-I Kinetic Study
Plasma decay curves of 125I-labeled normal
and variant apoA-I in rabbits are shown in Fig 6
. The turnover of125I-labeled apoA-I Oita was markedly faster than that
of 125I-labeled normal human apoA-I. The mean FCR
of 125I-labeled apoA-I Oita was more than double
that of 125I-labeled normal human apoA-I (Table 6
).
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| Discussion |
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60% of the control values in the heterozygote. We also observed a
gene- dosage effect of the apoA-I Oita mutant allele on apoA-I and
HDL cholesterol levels in this family.
More than 10 cases of homozygous apoA-I defects caused by a nonsense
mutation, a deletion, and an insertion have been reported to be
associated with apoA-I and HDL deficiency. The clinical manifestation
in these cases was variable and included premature
atherosclerosis, xanthomatosis, and corneal opacities.
Premature CAD was clearly reported in only four
cases.6 7 8 13 In contrast,
40 structural
apoA-I variants have been reported, although most of them are not
associated with changes in HDL level or premature CAD except for nine
cases.17 18 19 20 21 22 23 24 25 The reason for this phenomenon
remains unclear, but it may be partly related to the fact that almost
all patients with apoA-I variants characterized so far have been
heterozygotes. Thus, these apoA-I variants have little dominant effect
on lipoproteins metabolism in vivo. To our knowledge, a
homozygous, missense point mutation in the apoA-I gene that is
associated with clinical manifestations has not yet been reported.
ApoA-I (Glu136Lys) was the first reported case of homozygosity for a
structural apoA-I variant.38 However, this apoA-I
variant was not correlated with either HDL level or any clinical
complication. Therefore, apoA-I (Val156Glu) Oita is the first well-
documented report correlating homozygosity for an apoA-I variant with
reduced HDL concentrations.
The mechanisms by which the apoA-I (Val156Glu) substitution in
homozygosity influences HDL metabolism and results in
clinical complications are probably complex. In vitro experiments with
monoclonal antibodies and mutagenized apo A-I suggest that residues 143
to 164 of apoA-I are involved in the LCAT activation
process.1 39 A naturally occurring mutation,
apoA-I Seattle, with a deletion from Glu146 to Arg160, in
heterozygosity was reported to be associated with a reduction in apoA-I
and HDL cholesterol levels to below 15% of control
values.11 Four of six reported heterozygosities
for apoA-I variants in this domain with a dominant effect on HDL
metabolism are associated with reduced LCAT cofactor
activity or LCAT activity per se. ApoA-I (Pro143Arg) Giessen has been
isolated from plasma, and LCAT cofactor activity of the variant has
been demonstrated to be
60% to 70% of that in control
apoA-I.22 In another study, isolated apoA-I
(Pro165Arg) from the proband's plasma was reconstituted into
artificial discoidal HDL. The apoA-I (Pro165Arg) variant exhibited
30% to 70% of LCAT cofactor activity compared with that of normal
apoA-I.40 Low LCAT activity was also reported in
apoA-I (Arg151Cys) Paris and apoA-I (Arg160Leu)
Oslo.20 21 These data indicate the important role
of residues 143 to 164 in apoA-I function, particularly the LCAT
activation process. The reduced CER, LCAT activity, and LCAT mass but
normal LCAT specific activity observed in the apoA-I Oita homozygote
indicates a partial LCAT deficiency. The reduced LCAT activity in the
apoA-I Oita homozygote is consistent with the elevated free
cholesteroltototal cholesterol ratio
observed in the HDL fraction. Because the substitution of Val for Glu
at residue 156 is positioned within residues 143 to 164 of apoA-I,
partial LCAT deficiency in the present case is secondary to this
substitution and in part is the cause of low plasma HDL levels observed
in patients with apoA-I Oita.
Recently, the central amphipathic
-helixes have been identified as
the functional domains for cholesterol efflux. Monoclonal
antibodies with epitopes recognizing amino acid residues 149 to 186 of
apoA-I reduce the binding of reconstituted HDL to Hela cells and of
cholesterol efflux from adipocytes.41
On the other hand, the naturally occurring heterozygous apoA-I
(Pro165Arg) variant failed to promote cellular cholesterol
efflux in murine adipocytes and peritoneal
macrophages.42 These in vitro studies
suggest that central amphipathic
-helixes of apoA-I contain several
discontinuous, specific regions responsible for the efflux of cellular
cholesterol, probably by selectively interacting with the
cell surface binding sites. Because apoA-I Oita is located within
epitopes of monoclonal antibodies that inhibit cellular
cholesterol efflux by apoA-I, apoA-I Oita may be associated
with impaired efflux of cellular cholesterol. Hence, low
levels of HDL cholesterol in the apoA-I Oita proband may be
associated with impaired cholesterol efflux from cells.
Brinton et al43 have recently demonstrated that
the level of plasma apoA-I in humans is mainly determined by the FCR of
apoA-I. More recently, Miettinen and coworkers19
showed that the presence of apoA-I (Leu159Arg) Fin was associated with
markedly reduced concentrations of plasma apoA-I and HDL
cholesterol and increased catabolism of apoA-I in affected
heterozygous subjects. They speculated that the moderate
hypertriglyceridemia in affected subjects
with apoA-I (Leu159Arg) Fin may have contributed to the increased
apoA-I FCR and markedly reduced concentration of serum HDL
cholesterol. Increased catabolism of apoA-I was also
observed in apoA-I (Arg173Cys) Milano, apoA-I (Arg26Gly) Iowa, apoA-I
(Lys107
0), and in subjects with low HDL cholesterol
concentrations.43 44 45 46 Turnover studies of
isolated apoA-I Milano showed an increased catabolism of the variant
apoA-I due to heterodimerization or homodimerization of mutant apoA-I.
On the other hand, apoA-I Iowa was shifted to a higher density HDL
fraction (HDL3 and d>1.21 g/mL),
whereas the turnover of apoA-I Iowa was faster than that of normal
apoA-I in the high- density HDL fraction. In the present case, the
distribution of the proband's apoA-I was shifted to a higher density
lipoprotein fraction, and gradient gel electrophoresis of HDL particles
indicated that HDL particle size was smaller in the apoA-I Oita
heterozygote than in normal control subjects. We isolated variant
apoA-I Oita from the plasma of the proband and studied its turnover
rate in rabbits. The turnover study demonstrated that apoA-I Oita has
an accelerated catabolic rate in rabbits compared with normal human
apoA-I. The shift of apoA-I to a higher density HDL fraction in the
proband with apoA-I Oita may be related to faster catabolism of apoA-I,
which may contribute to apoA-I deficiency and low HDL
cholesterol levels in our patient.
In the present study, plasma TG of the apoA-I Oita proband was observed mainly in the LDL fraction. A high TG content in the LDL fraction was also observed in Tangier disease and in subjects homozygous for apoA-I (Glu136Lys).38 47 Enrichment of TG in the LDL fraction indicates an imbalance of lipid exchange with HDL.
A common genetic polymorphism (-75 G/A) in the apoA-I promoter region was found in the apoA-I Oita proband (II 2) as well as his brother (II 1) and son (III 1). Although previous studies about the impact of the -75 G- to- A mutation in the apoA-I promoter region on HDL cholesterol or apoA-I level are controversial, more recent investigations in genetically homogeneous populations have indicated that the G- to- A mutation does not have a significant direct effect on plasma HDL cholesterol or apoA-I level.48 49 Thus, the presence of the -75 G/A polymorphism in the apoA-I Oita family is probably not related to low plasma HDL cholesterol levels in this case.
In conclusion, we analyzed a novel, homozygous, missense point mutation in the apoA-I gene. The Val156Glu substitution in exon 4 of the apoA-I gene in both alleles has a serious impact on HDL metabolism, leading to apoA-I and HDL deficiencies, partial LCAT deficiency, and corneal opacities. The proband also presented with clinical features of arteriosclerosis affecting the coronary, carotid, and vertebral arteries. However, the proband's elder brother, who was homozygous for apoA-I Oita, did not have any symptoms suggestive of CAD. Moreover, the proband had other coronary risk factors apart from his apoA-I deficiency, such as age, smoking, and obesity. Because this family study was limited, the impact of this disorder on coronary atherosclerosis remains unknown at present. Further large- scale family studies are necessary to answer this issue. Nevertheless, the unique case presented in this study provides a good opportunity to understand the structure-function relationship between apoA-I and HDL metabolism.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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Received July 22, 1997; accepted November 4, 1997.
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