Articles |
0) Associated With Reduced HDL Cholesterol and LpA-I:A-II Deficiency
From the Third Department of Medicine (M.T.-K., J.K., S.L., M.-R.T.), University of Helsinki, and the Department of Biochemistry (Z.Q., S.E., C.E.), National Public Health Institute, Helsinki, Finland.
Correspondence to Prof Marja-Riitta Taskinen, MD, Department of Medicine, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland.
| Abstract |
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0), caused by a 3-bp deletion of
nucleotides 1396 through 1398 in exon 4 of the apoA-I gene.
These subjects (n=10) were heterozygous for this mutation. The mean
serum HDL cholesterol concentration (26.7±9.7 mg/dL) of
affected family members was 36% lower than that of unaffected family
members (P<.05). Mean serum apoA-I and apoA-II
concentrations in heterozygotes were reduced by 18% and 22%,
respectively, compared with normal family members (P<.05).
In heterozygotes the mean concentration of lipoprotein containing both
apoA-I and apoA-II (LpA-I:A-II) was 31% lower than in those with
normal apoA-I (P<.001), while the mean level of
lipoproteins containing apoA-I without apoA-II was similar in the two
groups. HDL density-gradient ultracentrifugation
showed a lack of HDL2 and small dense
HDL3 in heterozygotes compared with unaffected family
members. The HDL particle size distribution, as analyzed by
nondenaturing gradient gel electrophoresis of heterozygotes, revealed
one major peak at 8.0 to 9.7 nm, a minor peak at 7.8 to 8.5 nm, and an
absence of HDL2b and HDL2a peaks.
These latter peaks were observed in unaffected family members. Serum
levels of LDL cholesterol, triglycerides, VLDL,
IDL, and LDL subclasses were similar in the two groups. However, in
heterozygotes the cholesterol-to-triglyceride
ratios in VLDL2, LDL1,
LDL3, HDL2b,
HDL2a, and HDL3a were 8% to 54%
lower than in unaffected family members (P<.05).
Cholesteryl ester transfer protein activity in heterozygotes was
reduced by 25% compared with unaffected family members
(P<.05), while the plasma lecithin:cholesterol
acyltransferase (LCAT) activity did not differ between heterozygotes
and unaffected family members. The ability of isolated variant apoA-I
to serve as a cofactor for LCAT in vitro did not differ from that of
normal apoA-I. Our data are consistent with the concept that a
low HDL cholesterol level in subjects heterozygous for the
apoA-IHelsinki mutation (Lys107
0) having
normal LCAT activity is a consequence of decreased concentration of
LpA-I:A-II particles and of a smaller size and reduced
cholesterol content of HDL particles.
Key Words: apoA-I gene mutation HDL deficiency lecithin:cholesterol acyltransferase CETP coronary heart disease
| Introduction |
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Some HDL-deficient syndromes, such as Tangier disease or familial apoA-I/apoC-III or apoA-I/apoC-III/apoA-IV deficiency states, are characterized by premature coronary artery disease.20 21 22 23 In contrast, apoA-I variants, including apoA-IMilano, apoA-IMarburg, and apoA-IIowa, have not been associated with premature CHD.24 25 26
In this article we describe a deletion mutation in the human apoA-I gene associated with low serum HDL-C and decreased apoA-I and apoA-II concentrations in a kindred with a high incidence of CHD. We have characterized the association of this apoA-I variant on different lipoproteins and their subclasses, on lipoprotein particle size and density distribution, and on the activities of the major enzymes involved in lipoprotein metabolism.
| Methods |
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The proband's family was from southern Finland. Altogether 25 relatives participated in the study. Her mother (K.K.) was in excellent physical health and was not taking any medication. Her father had received medication for anginal symptoms for 3 years and died of cerebral hemorrhage at the age of 69 years. The father had five brothers and three sisters, of whom only one sister (A.A.S.) was still alive and in good health at age 85 years. The medical records of the proband's father, four uncles, and two aunts revealed that one aunt was diagnosed as having ischemic heart disease based on symptoms and electrocardiographic findings. She received medication for CHD symptoms and died suddenly at age 48 years. The other aunt had died at the age of 66 years of heart failure after she had three prior admissions to HUCH for acute MI. An uncle of the proband had died of his fourth acute MI at HUCH at age 54 years. The autopsy confirmed coronary atherosclerosis. One uncle had died of MI at age 63 years. The autopsy confirmed substantial atherosclerotic changes in the coronary arteries and abdominal aorta. According to medical records two other uncles died of CHD at 63 and 66 years, respectively. The latter uncle suffered his first MI at age 53 years. No HDL-C values for the proband's aunts or uncles were available in their medical records. In addition, three of the proband's cousins had died of CHD between the ages of 46 and 62 years, one of them after receiving a heart transplantation at HUCH. His HDL-C level (measured on several occasions) was below the normal range (0.80 mmol/L [31 mg/dL]).
The proband's older sister (K.A.P.) and two brothers (R.O.K. and
K.K.K.) had no history of cardiovascular disease and
were healthy as assessed by physical examination and laboratory tests.
The proband's two sons (M.J.V. and K.P.V.) and her brothers' sons
(R.K. and J.K.) were also healthy. In addition, 14 cousins participated
in the study. Two of them (T.M.A. and O.E.O.) had suffered an MI, but
the others had no clinical signs or symptoms of CHD. Two unaffected
subjects (P.E.K. and R.E.K.) were diagnosed as having
noninsulin-dependent diabetes mellitus according to the results of
laboratory tests. A pedigree of the family is presented in Fig 1
. The experimental protocol was approved by the Ethical
Committee of the Third Department of Medicine of Helsinki
University.
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| Methods |
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Amplification of DNA
Oligodeoxynucleotide primers used in
amplification and sequencing were chosen according to the published
apoA-I gene structure28 and synthesized on an Applied
Biosystems model 381A DNA synthesizer. The sequences and positions of
these primers are shown in Table 1
. Six fragments of the
apoA-I gene of the subjects were amplified by using PCR with genomic
DNA as a template. The PCR was performed in a reaction mixture
containing 1 µmol/L of primers, 0.2 mmol/L each dATP, dCTP, dGTP, and
dTTP, 50 mmol/L Tris-HCl (pH 8.8), 1.5 mmol/L MgCl2,
15 mmol/L (NH4)2SO4, 0.1%
Triton X-100, 0.1 mg/mL gelatin, and 2.5 U Taq DNA
polymerase (Promega Corp) in a final volume of 100 µL. The reaction
conditions on a programmable heating block (MJ Research Inc) were as
follows: 95°C for 1 minute, 58°C to 63°C for 1 minute, and 72°C
for 2.5 minutes, 30 cycles.
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SSCP Analysis
Six fragments of the apoA-I gene of the subjects were amplified
by PCR following the reaction conditions described above, and 2 to 3
µL [
-32P]dATP or dCTP (3000 Ci/mmol/L; Amersham) was
added to each tube. The PCR product was diluted 1:1.5 with 0.1%
sodium dodecyl sulfate and 10 mmol/L EDTA, denatured, and
loaded on neutral 5% polyacrylamide gel for mobility-shift
electrophoresis.29 Autoradiography was
performed by using an intensifying screen with Kodak X-omat film at
room temperature for 24 to 72 hours.
Direct Sequencing
The PCR products were purified by Qiaex (Diagen GmbH) and
directly sequenced with the dideoxy chain-termination reaction by using
a sequence kit (US Biochemical Corp). For sequencing of the fourth
exon, an additional primer, No. 13, which is located 76
nucleotides 5' of the mutation point, was used.
Solid-Phase Minisequencing
The solid-phase minisequencing and the preparation of the
batches of pooled leukocyte DNA were performed as described by
Syvänen et al.30 The 127-bp fragment located 111
nucleotides downstream of the 5' end of the apoA-I gene
exon 4 was amplified for minisequencing. The PCR reaction mixture and
the conditions were as mentioned above except that the primers used
were 2 µmol/L biotinylated primer No. 14 and 10 µmol/L primer No.
8, and the annealing temperature was 60°C. The detection-reaction
mixture, which consisted of 0.2 µmol/L detection primer No. 15 and
0.4 µmol/L [3H]dTTP (34 Ci/mmol; Amersham) to detect
the mutation as well as 1 U Taq DNA polymerase in 50 µL
PCR buffer, was added to each well. The radioactivity was measured in a
liquid scintillation counter (1210 Ultrobeta, LKB).
ApoA-I Isoform Analysis
The apoA-I isoforms of the subjects were determined by
isoelectric focusing and immunoblotting.31
Lipids and Lipoproteins
Blood samples were obtained in the morning after a 12-hour fast.
Serum lipoproteins were isolated by sequential
ultracentrifugation in a Beckman L8-70
ultracentrifuge (Beckman Instruments) with a Beckman 50 TI
rotor.32
Density-Gradient Ultracentrifugation of
Lipoproteins
Density-gradient ultracentrifugation of VLDL
and IDL was performed in a Beckman L8-70 ultracentrifuge
with an SW40 TI swinging-bucket rotor with centrifuge tubes by
using a 2-mL VLDL and IDL preparation isolated by sequential
ultracentrifugation and the method described
previously.33
The LDL subfractions LDL1, LDL2, and LDL3 were separated by density-gradient ultracentrifugation in a Beckman SW40 TI swinging-bucket rotor from the LDL sample obtained by sequential ultracentrifugation. The method used is slightly modified from that described by Griffin et al34 for plasma samples. Briefly, the discontinuous NaBr gradient was prepared by layering, from bottom to top, 0.5 mL d=1.1900 g/mL, 1.2 mL sample solution (0.9 mL LDL obtained from sequential ultracentrifugation in 0.3 mL NaBr, d=1.5350 g/mL), 1.5 mL d=1.0630 g/mL, 1.5 mL d=1.0560 g/mL, 1.5 mL d=1.0450 g/mL, 2.0 mL d=1.0340 g/mL, 2.0 mL d=1.0240 g/mL, and 0.7 mL d=1.0190 g/mL. The tubes were centrifuged in a Beckman L8-70 ultracentrifuge at 40 000 rpm for 24 hours at 23°C, and the rotor was allowed to stop without braking.
After ultracentrifugation the tubes were discharged from the top by using a Beckman Recovery System, Perfusor V (B. Braun) infusion pump and Maxidens solvent (Nyegaard & Co A/S). The protein-absorbance profiles of the tubes were monitored with an absorbance meter (Pharmacia), and the density gradient was controlled by using a DMA 46 density meter (Anton Paar). Three 1.5-mL fractions were collected: LDL1 (d=1.024 to 1.0330 g/mL), LDL2 (d=1.0330 to 1.042 g/mL), and LDL3 (d=1.0420 to 1.0550 g/mL).
The HDL density-gradient ultracentrifugation method was based on the method described by Groot et al35 except that first all apoB-containing lipoproteins (ie, VLDL, IDL, and LDL) were removed from the serum samples by ultracentrifugation. A serum sample of 2.0 mL in 0.3 mL NaBr solution (d=1.5350 g/mL) was overlaid with 0.7 mL NaBr solution (d=1.0600 g/mL) by using Beckman 1/2x2 polycarbonate centrifuge tubes. After ultracentrifugation in a Beckman Optima TL ultracentrifuge with a Beckman TLA 100.3 rotor at 100 000 rpm for 5 hours at 20°C, the thin yellowish supernatant layer containing VLDL, IDL, and LDL was removed by aspiration. The density of the infranatant was increased by adding 1.0 g dry NaBr, and a 2.0-mL volume of this sample solvent was transferred by pipette into the bottom of a Beckman Ultraclear 9/16x33/4 tube. The discontinuous gradient above the sample was then prepared by layering NaBr solution in the following order: 1.5 mL d=1.2500 g/mL, 6.7 mL d=1.2200 g/mL, and 2.0 mL distilled water. After ultracentrifugation in a Beckman L8-70 ultracentrifuge with an SW40 TI swinging-bucket rotor at 40 000 rpm for 18 hours at 20°C, the tubes were emptied as described for LDL separation. Five fractions of 1.3 mL each, representing HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c, were collected.
LDL Gradient Gel Electrophoresis
Nondenaturing polyacrylamide gradient gel
electrophoresis of LDL was performed on serum samples with 2% to 16%
gels (Pharmacia) by using the method of Nichols et
al.36 The gels were stained with Sudan Black B lipid
stain.37 After the gels were destained and scanned, the
particle diameter of the major LDL peak was defined by comparing the
mobility of the sample with that of reference LDL.37
HDL Gradient Gel Electrophoresis
The HDL gradient gel electrophoresis was
performed36 with Pharmacia 4% to 30% gels. Serum was
adjusted to d=1.2100 g/mL with NaBr solution
(d=1.335 g/mL) and ultracentrifuged in a Beckman
L8-70 ultracentrifuge at 35 000 rpm for 65 hours at 4°C
in a Beckman 50 TI rotor. The supernatant (1 mL) was recovered by tube
slicing and mixed with 0.25 mL of 40% sucrose. Using commercially
available gels (Pharmacia) and a sample volume of 10 µL, the gradient
gel electrophoresis was run in Gel Electrophoresis
Apparatus GE 2/4 (Pharmacia) for 3000 Volt-hours in a
buffer containing 80 mmol/L borate, 90 mmol/L Tris-HCl, 3 mmol/L EDTA,
and 3 mmol/L NaN3, pH 8.35. The gels were fixed with
10% sulfosalicylic acid, stained with 3.5% Coomassie G-250 brilliant
blue, destained with 5% acetic acid, and scanned at 595 nm by using a
computer-assisted Cliniscan 2 (Beckman Instruments). Particle diameters
of HDL fractions were assessed by comparing the mobility of the sample
with the mobility of molecular-weight calibration proteins
(Pharmacia).
Measurement of CETP and LCAT Activity
LDL and HDL isolated from the plasma of healthy donors were
dialyzed extensively against 10 mmol/L Tris HCl, pH 7.4, containing 150
mmol/L NaCl, 1 mmol/L EDTA, and 0.1 g/L NaN3. The LDL was
labeled with cholesteryl(1-14C)oleate (Amersham) by using
the lipid-dispersion method described by Morton and
Zilversmit.38 The labeled LDL was
ultracentrifuged again at 40 000 rpm for 24 hours at
20°C in an SW40 TI rotor. The measurement of CETP activity was
accomplished by the method of Groener et al.39 The results
were calculated by measuring the CETP activity of plasma samples
divided by the CETP activity of the reference sample in the same
assay40 and are expressed in arbitrary units. The LCAT
activity was measured41 by using proteoliposome substrates
containing either control apoA-I or apoA-I
(Lys107
0).
Measurement of LpA-I and LpA-I:A-II Concentrations
The concentration of LpA-I particles was measured by
electroimmunoassay (Sebia) as described by Parra et al.42
The concentration of LpA-I:A-II was calculated by subtracting the LpA-I
concentration from the serum apoA-I concentration. The interassay
variation for LpA-I particle measurements was 7.3%.
Analytical Methods
Cholesterol, free cholesterol,
phospholipid, and triglyceride levels were analyzed
by using enzymatic colorimetric methods with a Cobas
Mira analyzer (Hoffmann-La Roche) and reagent kits (for
cholesterol and triglycerides, Nos. 0715166 and
0722138, Hoffmann-La Roche; for free cholesterol, No.
310328, Boehringer Mannheim GmbH Diagnostica; and
for phospholipids, No. 990-54009, Wako Chemicals GmbH). Serum apoA-I,
apoA-II, and apoB concentrations were measured by using an
immunoturbidometric assay (Orion Diagnostica). The serum
concentrations of apolipoproteins C-II and C-III were determined by
using immunochemical agarose plates according to the instructions of
the manufacturer (Daiichi Pure Chemicals Co). The normal ranges of
apoC-II are 3.5±1.2 and 3.3±1.2 mg/dL and those of apoC-III are
7.6±2.2 and 7.3±3.7 mg/dL for men and women, respectively. Blood
glucose concentration was measured by using the glucose oxidase method
(Auto-Analyzer, Technicon). Glycosylated hemoglobin (normal
range, 4% to 6%) was measured by using a Diamat Analyzer
System (BioRad, Clinical Division). Serum insulin levels were measured
by using a radioimmunoassay after precipitation with polyethylene
glycol with the Phadeseph insulin radioimmunoassay kit (Pharmacia).
Statistical Analyses
The significances of differences between affected and unaffected
subjects were assessed by Mann-Whitney nonparametric tests
using BMDP statistical software (University of
California). Pearson's correlation coefficient analysis was
used to estimate the relationship between variables.
| Results |
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Direct sequencing of the coding region of the apoA-I gene revealed that
the proband and the nine other affected family members (Fig 1
) were
heterozygous for a 3-bp deletion in exon 4 from nucleotides
1396 through 1398, removing the codon (AAG) for Lys107 of
the mature apolipoprotein. Exon 4 of the apoA-I gene contains six
66-bp-long homologous tandem repeats that code for six 22-amino acid
segments.28 The 3-bp deletion described above occurred
within repeat number one. The rest of the apoA-I gene sequence was
normal.
The 3-bp deletion in the apoA-I DNA of the proband and affected family members was further confirmed by using solid-phase minisequencing, in which a detection primer, annealing immediately adjacent to the mutation site, is elongated by a DNA polymerase with a single dNTP corresponding to the nucleotide at the site of the mutation. To identify the normal sequence (TGAAGAA) and the sequence including the 3-bp deletion (TGAAGAC), [3H]dTTP is included in one minisequencing reaction and [3H]dGTP in another reaction. The ratio between the incorporated nucleotides directly reflects the ratio between the two sequences present in the PCR product.
DNA samples from the proband (A.U.T.), her two brothers (K.K.K. and
R.O.K.), and her son (K.P.V.) as well as DNA samples from two
unaffected family members (K.K. and K.A.P.) and two unrelated control
subjects were analyzed by solid-phase minisequencing. The
presence of the mutation resulted in a significant incorporation of
[3H]GTP, leading to a T/G ratio of about 1, while this
ratio in unaffected family members and control subjects was >10 (Table 2
).
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To determine the prevalence of the mutation apoA-I
(Lys107
0) in the Finnish population, solid-phase
minisequencing of pooled DNA from Finnish subjects was performed. Three
separate pools containing DNA from 921, 189, and 180 individuals,
respectively, were analyzed. In these population samples no
subjects with the apoA-I (Lys107
0) mutation could be
identified (Table 2
).
Serum Lipoprotein and Apolipoprotein Levels and Enzyme
Activities
The clinical characteristics and lipid values measured in subjects
of the pedigree are presented in Table 3![]()
. The
mean serum HDL-C concentration of the affected family members was
clearly below the normal range and was reduced by 36% compared with
the mean HDL-C value of the unaffected family members. Total serum
cholesterol, LDL cholesterol, and
triglyceride concentrations were similar in apoA-I
(Lys107
0)affected and unaffected subjects. The proband
had moderately increased serum triglycerides, while the
rest of the apoA-I (Lys107
0) heterozygotes were
normotriglyceridemic.43 The
fasting blood glucose, glycosylated hemoglobin, and serum insulin
values were within the normal ranges for all affected subjects (data
not shown).
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The mean serum apoA-I concentration of heterozygotes was reduced by
18.3% and the apoA-II concentration by 22.2% compared with unaffected
family members (P<.05 and P<.01, respectively,
Table 3![]()
). The mean apoC-II and apoC-III concentrations of heterozygotes
were within the normal ranges and were similar to unaffected family
members (data not shown).
The mean plasma LCAT activity in carriers of the mutant apoA-I gene did
not differ from the LCAT activity of unaffected family members. In
vitro no defect was observed in the LCAT activation properties of the
isolated mutant apoA-I. However, the mean CETP activity was reduced by
25% in affected family members compared with unaffected family members
(P<.05, Table 3![]()
). The mean CETP activity of the unaffected
family members was similar to the level measured in our laboratory for
healthy control subjects.44
Characteristics of HDL
The two most prominent characteristics of the HDL density
distribution profiles of the affected subjects were the total absence
of HDL2 and the shift of the HDL3
subfraction toward a higher density compared with the profiles of
unaffected family members (Fig 4
). The percentages of
HDL found in the five subfractions HDL2b,
HDL2a, HDL3a,
HDL3b, and HDL3c were 5.7%, 7.3%,
18.1%, 33.7%, and 35.2%, respectively, for affected family members
compared with 9.4% (P<.01), 12.1% (P<.001),
19.4% (NS), 29.1% (P<.001), and 29.9%
(P<.05) in unaffected family members (Table 4
). The data indicate that in heterozygotes the HDL
density distribution is altered, with more than two thirds of the HDL
particles being in the two most dense HDL subfractions,
HDL3b and HDL3c.
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The HDL particle size distribution was determined by using gradient gel
electrophoresis (Fig 5
). The HDL particle size
distribution profiles of all the affected family members were
characterized by a predominance of the HDL3 subclass. In
the subjects with the apoA-I (Lys107
0) variant one major
peak appeared at 8.0 to 9.7 nm and a minor peak at 7.8 to 8.5 nm. In
unaffected family members the HDL particle profile was more
polydisperse and consisted of two to five peaks with similar mobility.
The major peak appeared at 8.0 to 8.9 nm and a minor peak between 7.8
to 8.0 nm, and most unaffected subjects also displayed one or two
prominent peaks at 9.3 to 11.5 nm. The results indicate that the apoA-I
(Lys107
0) variant is associated with a very low level of
the HDL2b subclass, similar to subjects with
apoA-IMilano.45
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The mean concentrations of LpA-I particles were comparable in affected
and unaffected family members, but the concentration of LpA-I:A-II
particles was 31% lower in affected than in normal family members
(P<.001; Table 3![]()
).
Characteristics of VLDL, IDL, and LDL
The density distribution curves of VLDL, IDL, and LDL did not show
any characteristic features or specific particle subpopulations in
affected compared with unaffected family members (data not shown).
In all 10 subjects with the apoA-I (Lys107
0) deletion
the LDL particle size distribution revealed one major peak without
clearly distinguishable minor peaks (data not shown). The
computer-calculated particle diameter of the major LDL peak was 25.7 nm
for both the apoA-I (Lys107
0) carriers and noncarriers.
The presence of the apoA-I (Lys107
0) deletion did not
seem to affect the LDL particle size distribution. In all family
members the particle diameter of the major LDL peak correlated
negatively with the serum triglyceride concentration
(r2=.5809, P<.001), serum
insulin level (r2=.2464,
P<.05), and VLDL1,
VLDL2, and IDL concentrations
(r2=.4268,
r2=.5466, and
r2=.3084, respectively,
P
.05). A positive correlation was observed between the
particle diameter of the major LDL peak and the concentrations of the
HDL2b (r2=.1773),
HDL2a (r2=.2758),
HDL3a (r2=.3128), and
HDL3b (r2=.2227) subfractions
(all P<.05).
Composition of Lipoprotein Subclasses
In the HDL2b subfraction of affected family
members the percentage of triglycerides was increased by
63%, and in HDL2a and HDL3a the
percentages of cholesterol were decreased by 15% and 14%,
respectively (both P
.05). The most significant
characteristic of the HDL composition of subjects with the apoA-I
(Lys107
0) mutation was the low
cholesterol-to-triglyceride ratio in the
largest HDL subclasses (Table 4
). In HDL2b,
HDL2a, and HDL3a subfractions the
cholesterol-to-triglyceride ratio was reduced
by 54%, 39%, and 35%, respectively (P
.05), compared
with unaffected family members. Compositions of the two most dense HDL
subfractions, HDL3b and HDL3c, were
comparable in affected and unaffected subjects. Similarly, in
subclasses of LDL and VLDL the heterozygotes tended to have a
moderately increased content of triglycerides and a lower
cholesterol-to-triglyceride ratio, with a
significant difference in LDL1,
LDL3, and VLDL2
(P<.05; Table 4
).
| Discussion |
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The mean HDL-C concentration of affected family members was reduced by
one third compared with unaffected family members, while mean total and
LDL cholesterol concentrations were similar in affected and
unaffected subjects. An increased incidence of
hypertriglyceridemia is typical of some
apoA-I variants,25 46 47 but the serum
triglyceride concentrations of all the apoA-I
(Lys107
0) heterozygotes except one were normal. The
proband had moderately increased serum triglycerides,
possibly as a consequence of obesity and low lipoprotein lipase
activity.48 49 Concomitant moderate
hypertriglyceridemia explains the
proband's exceptionally low HDL-C concentration compared with the
HDL-C levels of other affected family members.50
Heterozygous subjects had mean serum apoA-I and apoA-II concentrations that were reduced by a similar extent. The mean concentrations of LpA-I particles were the same in affected and unaffected subjects, while affected family members had a 31% lower mean concentration of LpA-I:A-II particles than unaffected family members. Puchois et al16 and Barkia et al51 have shown that reduced concentrations of LpA-I but not LpA-I:A-II are associated with coronary artery disease, and in vitro studies support the assumption that LpA-I is cardioprotective.16 51 In contrast, other studies have shown that LpA-I and LpA-I:A-II are reduced in coronary artery disease or CHD patients to a similar degree.17 18 19 52
An inverse correlation between the HDL-C level and the fractional
catabolic rate of apoA-I and apoA-II has been
demonstrated,53 54 55 56 and in heterozygous
apoA-IMilano (Arg173
Cys) and
apoA-IIowa (Gly26
Arg) subjects both the
mutant and the normal apoA-I are catabolized at an increased
rate.26 57 In addition, apoA-II levels are decreased in
subjects with apoA-I (Arg173
Cys) or apoA-I
(Gly26
Arg), which suggests that apoA-II may be more
rapidly catabolized in these subjects as well.26 However,
apoA-II kinetics have not been studied in subjects with apoA-I
variants, and no data have been reported on the metabolism
of the two HDL particle populations in apoA-Ivariant subjects. On the
basis of the normal concentration of LpA-I particles and the decreased
concentration of LpA-I:A-II particles in apoA-I
(Lys107
0) Helsinki heterozygotes, we hypothesize that
this mutation primarily affects the metabolism of HDL
particles containing both apoA-I and apoA-II. Our preliminary data from
in vivo turnover studies in two apoA-I (Lys107
0)
heterozygotes show an increase in the fractional catabolic rate of both
apoA-I and apoA-II (Tilly-Kiesi et al, unpublished data, 1995).
Several characteristic features of the HDL of heterozygotes were observed in comparison with the HDL of unaffected family members. Density-gradient ultracentrifugation studies demonstrated that the heterozygotes lacked HDL2, and more than two thirds of their total HDL was found in subfractions HDL3b and HDL3c, which have the highest density and the lowest cholesterol/protein ratio.2 3 These data are consistent with the observation that these subjects have a greater percentage reduction in serum HDL-C levels than in apoA-I and apoA-II concentrations. HDL particle size distribution studies revealed that heterozygotes lacked HDL peaks in the particle size range >9.7 nm, indicating a deficiency of larger HDL particles.58
The LDL subclass concentrations, density distribution, and particle size of heterozygotes resembled those of unaffected family members. The concentrations and density distribution of VLDL and IDL were also similar in affected and unaffected family members. Lipoprotein lipase and hepatic lipase, which are related to the conversion of VLDL to IDL and LDL, are also involved in the conversion of large LDL to smaller LDL particles.59 60 61 62 Similar mean postheparin lipoprotein lipase and hepatic lipase activities were found in affected and unaffected family members (data not shown), consistent with the similarity of VLDL, IDL, and LDL subclass distributions in these two groups.
In Tangier homozygotes and in subjects homozygous for the apoA-I
(Glu136
Lys) mutation, the content of
triglycerides in LDL is increased.63 64 In
heterozygotes the cholesterol-to-triglyceride
ratio was significantly reduced in VLDL2,
LDL1, LDL3,
HDL2b, HDL2a, and
HDL3a, and in all other lipoprotein subclasses a
similar trend was noted. An imbalance in the amounts of donor (HDL) and
acceptor (VLDL, LDL) particles for cholesteryl ester transfer may
account for the diminished
cholesterol-to-triglyceride ratio in the plasma
lipoproteins of heterozygotes.65 66 The reduced CETP
activity of heterozygotes compared with unaffected family members may
be a compensatory alteration to increase the
HDL2/HDL3 ratio and to correct
the HDL composition.67
ApoA-I is an important activator of the LCAT
enzyme.68 69 As summarized in Table 5
,
partial LCAT deficiency due to reduced LCAT mass or to reduced ability
of the variant apoA-I to activate LCAT is associated with
apoA-I variants such as apoA-I (Cys173
Arg), apoA-I
(Gua202
0), apoA-I
(Glu146
Arg160) deletion, apoA-I
(Lys107
0), and apoA-I
(Pro143
Arg).47 70 71 72 73 We isolated mutant
apoA-I (Lys107
0) from our proband and studied the
interaction of mutant apoA-I with LCAT in vitro. No defect in the LCAT
activating properties of the mutant apoA-I was observed. In addition,
the mean plasma LCAT activity of affected subjects was similar to that
found in unaffected subjects.
|
HDL deficiency due to Tangier disease or apoA-I/apoC-III or
apoA-I/apoC-III/apoA-IV deficiency syndromes has been associated with
premature CHD.20 21 22 23 In addition, apoA-I gene mutations
causing a truncated apoA-I protein (Gln84
stop) and an
absence of apoA-I (Q[-2]X) are associated with xanthomas and
premature CHD.74 89 The majority of apoA-I variants have
not been associated with low HDL-C levels or premature CHD. Of the 23
reported apoA-I variants that we reviewed (Table 5
), only seven are
clearly associated with reduced plasma HDL-C and apoA-I concentrations,
and the mean age of the 69 subjects was 33 years, a very low age to
make accurate estimations of any association with CHD. Interestingly,
in three apoA-I variants causing HDL deficiency the ability of variant
apoA-I to activate LCAT and/or the plasma concentration of LCAT
is markedly reduced.47 71 73 In contrast, apoA-I
(Lys107
0) heterozygotes had HDL deficiency and normal
LCAT activity. It should be noted that markedly premature CHD has not
been observed in LCAT deficiency or fish-eye disease despite strikingly
low levels of HDL-C.12 Therefore, normal LCAT activity may
be necessary for low HDL-C to be a CHD risk factor.
In kindred studies it is hard to actually assess associations of mutations with CHD because of the limited number of subjects and the strong association of CHD with age and gender. Pedigree analysis indicated that the proband received the mutation from her father. Medical-record review revealed that the father and six of his siblings had CHD; in four of these siblings CHD was present before the age of 55 years. These six siblings died of CHD, while the father died of cerebral hemorrhage. The mean age of death in these seven subjects at 61 years is earlier than that observed in the normal population. The one remaining sibling of the father was an unaffected subject and healthy at age 84 years. Among the seven heterozygotes (mean age, 56 years) of the proband and her generation, two (T.M.A.) and (O.E.O.) had CHD at age 65 and 63 years, respectively, without any other risk factors except for a low HDL-C level. In addition, three other subjects of this generation, including T.M.A.'s sister and brother, had died of CHD at the ages of 46, 55, and 62 years. It was not possible to determine whether they were heterozygotes. None of the unaffected family members had symptoms or signs of CHD.
The combined data indicate that the apoA-IHelsinki mutation
(Lys107
0) results in decreased serum HDL-C, apoA-I,
apoA-II, and LpA-I:A-II levels, reduced CETP activity, normal lipolytic
enzyme and LCAT activities, and may be associated with premature
CHD.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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Received September 14, 1994; accepted June 20, 1995.
| References |
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