Clinical and Experimental Pharmacology and Physiology (2009) 36, 567–570
EFFECT OF R219K POLYMORPHISM OF THE ABCA1 GENE
Blackwell
ABCA1
J Li et al.polymorphism
Publishing Asia
and HDL-C and CHD
ON THE LIPID-LOWERING EFFECT OF PRAVASTATIN IN CHINESE
PATIENTS WITH CORONARY HEART DISEASE
Jia Li, Lan-Feng Wang, Zhu-Qin Li and Wei Pan
Department of Cardiac Care Unit, The First Affiliated Hospital of Harbin Medical University, Harbin, China
SUMMARY
1. In the present study, we investigated the effects of the
R219K polymorphism of the ATP-binding cassette transporter
A1 (ABCA1) gene on serum lipid levels and the response to statin
therapy in Chinese patients with coronary heart disease (CHD).
2. The study population consisted of 365 patients with CHD
and 246 control subjects without signs or symptoms of CHD.
Patients with CHD were treated with 20 mg/day pravastatin.
Fasting serum lipids were determined before and after 12 weeks
of treatment. Genotyping was performed by polymerase chain
reaction–restriction fragment length polymorphism (PCR-RFLP).
3. The R219K polymorphism of the ABCA1 gene was not sig-
nificantly associated with CHD (P > 0.05). Compared with
controls, patients with the RR genotype had significantly higher
serum triglyceride levels and lower high-density lipoprotein–
cholesterol (HDL-C) levels than those with the KK genotype
(P < 0.05). In addition, the effects of pravastatin in increasing
HDL-C levels were significantly greater in patients with the KK
genotype compared with those with the RR genotype (P < 0.05).
4. In conclusion, the R219K polymorphism of ABCA1 was
associated with altered lipoprotein levels and the R219K variant
significantly modulated the HDL-C response to pravastatin in
Chinese patients with CHD.
Key words: ATP-binding cassette transporter A1 (ABCA1),
coronary heart disease, genetic polymorphism, 3-hydroxy-3-
methylglutaryl coenzyme A reducatse inhibitor.
INTRODUCTION
Coronary heart disease (CHD) is one of the leading causes of mortality
and morbidity worldwide. High-density lipoprotein–cholesterol
(HDL-C) is a known inverse predictor of coronary heart disease and
is thus a potential therapeutic target.1,2 The atheroprotective effect
of HDL-C is largely attributed to the ability of HDL to mediate the
efflux of excess cholesterol from peripheral macrophages back to
the liver, a process known as reverse cholesterol transport.3 The first
Correspondence: Lan-Feng Wang, Department of Cardiac Care Unit, The
First Affiliated Hospital of Harbin Medical University, Harbin Medical Uni-
versity, 23 Youzheng St., Nangang District, Harbin 150001, PR China.
Email: wlfeng@hotmail.com
Received 12 August 2008; revision 7 October 2008; accepted 7 October
2008.
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Asia Pty Ltd
doi: 10.1111/j.1440-1681.2008.05119.
step in this process is mediated by ATP-binding cassette transporter
A1 (ABCA1), a member of the ATP-binding cassette family that
plays a major role in cholesterol homeostasis and HDL metabolism.4
Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase that decrease cholesterol
biosynthesis and are widely used for cholesterol-lowering therapy
and the prevention of atherosclerosis-related events. Statins impair
cholesterol biosynthesis by inhibiting the activity of HMG-CoA
reductase, the rate-limiting step in cholesterol synthesis. This both
decreases circulating lipoproteins and increases their hepatic uptake
by upregulating low-density lipoprotein (LDL) receptors. The overall
lipid-lowering effect of statins includes increased uptake and
degradation of LDL, inhibition of LDL oxidation, a reduction in
cholesterol accumulation and esterification and decreased lipoprotein
secretion and cholesterol synthesis.5–7
Single nucleotide polymorphisms are essential tools for studying
the pathophysiology of complex human diseases.8,9 A recent study
reported that lower plasma levels of HDL-C due to heterozygosity
for loss-of-function mutations in ABCA1 were not associated with
an increased risk of ischaemic heart disease.10 Some studies have
suggested that ABCA1 gene polymorphisms affect serum lipid
levels.11,12 The results of these studies prompted us to explore the
clinical significance of ABCA1 gene polymorphisms in lipid metabolism.
The aim of the present study was to examine the effects of the R219K
polymorphism of ABCA1 on serum lipid levels and responses to
statin therapy in Chinese patients with CHD.
METHODS
Subjects and study protocol
Three hundred and sixty-five unrelated CHD patients who underwent coronary
angiography were enrolled in the study (189 women, 176 men; age range
58–76 years). Written informed consent was obtained from each subject after
a full explanation of the study, which was approved by the Ethics Committee
of Harbin Medical University, had been provided. Coronary arteriograms
were interpreted by two or more independent, experienced cardiologists; over
50% stenosis in at least one major vessel was diagnosed as CHD according
to the criteria of American Heart Association.13 Multivessel disease was
diagnosed when at least two major coronary arteries were affected on
angiography. All subjects were of Chinese ancestry and were not first- or
second-degree relatives. None of the subjects had taken HMG-CoA reductase
inhibitors previously and all strictly abstained from smoking, alcohol and
caffeine during treatment. Subjects with liver or renal failure, dropsical
nephritis, diabetes mellitus, diagnosed myocardial infarction or liver or kidney
transplantation were excluded from the study. To rule out the potential
confounding effect of concomitant medications on plasma lipid concentrations,
patients who had taken any other lipid-lowering drugs in the previous
568 J Li et al.

Table 1 Anthropometric characteristics in controls and patients with Table 2 Distribution frequency of genotypes and alleles of the ABCA1 gene
coronary heart disease between patients with coronary heart disease and controls

Control group CHD group Group n Genotype frequency (%) Allele frequency (%)

n 246 365 RR RK KK R K
Mean (±SD) age (years) 61 ± 13 63 ± 14
No. smokers (%) 52 (21.1) 147* (40.3) Control 246 92 (37.4) 116 (47.1) 38 (15.5) 300 (61.0) 192 (39.0)
No. alcohol abusers† (%) 13 (5.3) 29 (7.9) CHD 365 140 (38.3) 170 (46.6) 55 (15.1) 450 (61.7) 280 (38.3)
No. undertaking physical exercise (%) 139 (56.5) 176 (48.2)
CHD, coronary heart disease.
*P < 0.01 compared with the control group.
†
The definition of alcohol abuse used in the present study was that of World
Health Organization (http://www.dryoutnow.com/alcohol-info/Alcohol-
Abuse.html), specifically the consumption of more than 21 units of alcohol/ Table 3 Plasma lipid concentrations before and after treatment with
week for men and 14 units of alcohol/week for women. pravastatin
CHD, coronary heart disease.
Control group CHD group (n = 365)
(n = 246)
2 months were also excluded from the study. The control population consisted Before treatment After treatment
of 246 healthy subjects (113 men, 133 women; age range 51–75 years) who
attended a routine health check at the medical examination centre. All CHD TG (mmol/L) 1.26 ± 0.13 1.75 ± 0.26§ 1.24 ± 0.19‡
patients were administered pravastatin 20 mg once daily at bedtime. TC (mmol/L) 4.65 ± 0.56 5.12 ± 0.51§ 4.21 ± 0.55‡
HDL-C (mmol/L) 1.31 ± 0.21 1.08 ± 0.15§ 1.25 ± 0.18‡
LDL-C (mmol/L) 2.68 ± 0.34 3.12 ± 0.27§ 2.21 ± 0.21‡
Measurement of plasma lipid levels VLDL-C (mmol/L) 0.87 ± 0.14 1.09 ± 0.13§ 0.72 ± 0.07‡
Plasma concentrations of total cholesterol (TC), HDL-C and triglyceride
(TG) were determined by standard, automated methods (model 747; Hitachi, Values are the mean±SD. §P < 0.01 compared with the control group;
Tokyo, Japan). Low-density lipoprotein–cholesterol was calculated by the
‡
P < 0.01 compared with before treatment.
Friedewald formula14 and very low-density lipoprotein–cholesterol (VLDL-C) CHD, coronary heart disease; TC, total cholesterol; TG, triglyceride;
was derived from TC after subtracting LDL-C and HDL-C. HDL-C, high density lipoprotein–cholesterol; LDL-C, low density lipoprotein–
cholesterol; VLDL-C, very low density lipoprotein–cholesterol.

Genotyping of the R219K polymorphism of the

ABCA1 gene
Genomic DNA was extracted from peripheral blood leucocytes. The R219K
polymorphism of the ABCA1 gene was determined using polymerase
chain reaction (PCR), followed by restriction fragment length polymorphism
(RFLP) with primers and restriction enzymes as described by Saleheen
et al.15
Statistical analysis
Data are presented as the mean±SD. Hardy–Weinberg equilibrium was
assessed with 2 test of goodness-of-fit in the study sample. Paired Student’s
t-test was performed to compare lipid concentrations before and after
pravastatin treatment. Unpaired Student’s t-test was used to compare differ-
ences in the degree of reduction of plasma lipid concentrations between the
two ABCA1 genotype groups. A two-sided test with type error level () set
at 5% was used in all statistical analyses. Two-tailed P < 0.05 was considered
statistically significant. Statistical analyses were performed with spss version
13.0 (SPSS, Chicago, IL, USA).
RESULTS
Comparison of anthropometric characteristics between
the CHD and control groups
Table 1 summarizes the anthropometric characteristics of the CHD
patients and control subjects. All patients had at least one risk factor
for coronary artery disease (they were smokers and/or alcohol abusers
and/or did not exercise regularly). Coronary heart disease was
strongly associated with smoking (CHD patients vs controls:
P < 0.01).
Relationship between the distribution frequency of
genotypes and alleles of the ABCA1 gene
The genotype frequency of the R219K polymorphism of the ABCA1
gene in CHD patients was 38.3% RR homozygotes, 46.7% RK
heterozygotes and 15.1% KK homozygotes The genotype frequency
for this polymorphism was found to be in Hardy–Weinberg equilibrium.
The frequency of the R allele was determined to be 61.7%. The
relationship between the distribution frequency of genotypes and
alleles of ABCA1 is given in Table 2. The genotype and allele
frequencies of the R219K polymorphism of the ABCA1 gene were
similar between control subjects and CHD patients (P > 0.05).
Effects of pravastatin on serum lipid levels
Compared with controls, patients with CHD had significantly higher
serum concentrations of TG, TC, LDL-C and VLDL-C and lower
HDL-C levels (P < 0.01). Pravastatin treatment significantly improved
the lipid profile of patients with CHD (P < 0.01; Table 3).
Effect of the R219K polymorphism of the ABCA1 gene on
serum lipid levels
Patients with the RR genotype had significantly higher serum TG
levels and lower HDL-C levels than those with the KK genotype
(P < 0.05). There were no significant differences in TC, LDL-C and
VLDL-C concentrations among patients in the three genotype
groups prior to treatment (Table 4).

© 2009 The Authors

Journal compilation © 2009 Blackwell Publishing Asia Pty Ltd
 ABCA1 polymorphism and HDL-C and CHD 569

Table 4 Effect of ABCA1 polymorphisms on the response to pravastatin treatment for 12 weeks in patients with coronary heart disease

Week RR (n = 140) RK (n = 170) KK (n = 55)

Mean±SD % Change Mean±SD % Change Mean±SD % Change

TG (mmol/L)
0 1.81 ± 0.29 28 ± 3 1.73 ± 0.26 28 ± 3 1.69 ± 0.21* 28 ± 3
12 1.26 ± 0.21 1.23 ± 0.17 1.23 ± 0.19
TC (mmol/L)
0 5.12 ± 0.51 18 ± 3 5.11 ± 0.48 19 ± 3 5.13 ± 0.52 19 ± 4
12 4.22 ± 0.53 4.21 ± 0.52 4.21 ± 0.59
HDL-C (mmol/L)
0 1.06 ± 0.15 14 ± 2 1.09 ± 0.13 14 ± 2 1.12 ± 0.17* 19 ± 3*
12 1.22 ± 0.18 1.24 ± 0.15 1.35 ± 0.16
LDL-C (mmol/L)
0 3.13 ± 0.26 29 ± 4 3.11 ± 0.24 29 ± 4 3.12 ± 0.28 28 ± 4
12 2.21 ± 0.21 2.20 ± 0.19 2.24 ± 0.23
VLDL-C (mmol/L)
0 1.10 ± 0.13 35 ± 4 1.09 ± 0.11 34 ± 4 1.09 ± 0.12 34 ± 4
12 0.71 ± 0.06 0.73 ± 0.09 0.72 ± 0.05

Values are the mean±SD. *P < 0.05 compared with the RR genotype.
TC, total cholesterol; TG, triglyceride; HDL-C, high density lipoprotein–cholesterol; LDL-C, low density lipoprotein–cholesterol; VLDL-C, very low
density lipoprotein–cholesterol.

Effect of the R219K polymorphism of the ABCA1 gene loop and many potential loss of function-induced variants have been
on the effectiveness of pravastatin treatment in patients proposed.23,24 It has been proposed that the substitution of an arginine
with CHD by a lysine at residue 219 may alter the conformation of the extracellular
domain of the ABCA1 protein, enhance its interaction with apoAI
To investigate the effect of the R219K polymorphism on serum lipid
and increase the efficiency of phospholipid and cholesterol transfer
levels, we determined the relationship between ABCA1 genotype
from intracellular stores to the plasma membrane.3,25 Cholesterol
and plasma lipid changes after pravastatin treatment. Patients with
ester transfer protein activity results in the equilibration of the core
the KK and RR genotype exhibited mean increases in HDL-C from
components of lipoprotein particles.26 Increased ABCA1 activity has
baseline of 18.9 ± 2.6 and 14.1 ± 2.3%, resepctively (P < 0.05 for
been suggested to alter intracellular lipid transport, possibly resulting
RR genotype vs KK genotype). Conversely, there was no significant
in increased HDL-C levels.27,28 There is a possibility that the genetic
difference between the three groups in terms of changes in plasma
polymorphism is merely a genetic marker that exhibits significant
concentrations of LDL-C, VLDL-C, TG and TC (Table 4).
linkage disequilibrium with other nearby genetic variations that
functionally determine HDL-C levels.
DISCUSSION In the present study, there was no association between ABCA1
genotypes and CHD. However, we found that the R219K polymorphism
The ABCA1 protein plays a key role in the first steps of the reverse
of the ABCA1 gene did affect the action of pravastatin on plasma
cholesterol transport pathway by mediating lipid efflux from
HDL-C levels. This is the first report demonstrating that the effect
macrophages.4 Positional cloning has shown that mutations in
of statin therapy on plasma HDL-C levels is modified by ABCA1
ABCA1 within chromosome 9q31 are responsible for Tangier disease
in Chinese patients with CHD. The results show that the mean
and familial HDL deficiency.16,17 Thereafter, several studies established
percentage increase in HDL-C in patients who were homozygous
that ABCA1 regulates cellular cholesterol efflux and facilitates lipid
for KK was higher than that in patients who were homozygous for
binding to apolipoprotein A (apo A), which is the core protein in
RR. Thus, the present study shows that the HDL-raising effect of
HDL particles. Many studies have investigated the role of the
pravastatin could be affected by the R219K polymorphism of
ABCA1 gene in determining plasma lipid and apolipoprotein
ABCA1 gene. The present study provides new, clinically relevant
levels.13,18–22 Accumulating evidence indicates that the R219K
information regarding the genetic determinants modulating the
polymorphism is associated with coronary artery disease, athero-
responses of CHD patients to pravastatin and the potential underlying
sclerosis and HDL-C levels.13,18–22 In the present study, we examined
mechanisms. Further studies with larger sample sizes are needed to
the effects of the R219K polymorphism of the ABCA1 gene on the
confirm these findings.
serum lipid profile, as well as on the response to pravastatin, in
Chinese patients with CHD. We found no association between the
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