- Email: [email protected]
Common Variants in the ATP-binding Cassette Transporter 1 Gene with Decreased HDL-Cholesterol Levels and Coronary Artery Disease
Archives of Medical Research 39 (2008) 735e742
ORIGINAL ARTICLE
Common Variants in the ATP-binding Cassette Transporter 1 Gene with Decreased HDL-Cholesterol Levels and Coronary Artery Disease Alp Burak Catakoglu,a,* Belgin Su¨sleyici Duman,b,* Hilal Kurtog˘lu,c Melike Erso¨z,d Murat Sxener,c Hu¨seyin C ¸ elebi,a Penbe C ¸ ag˘atay,e Vedat Aytekin,c and Saide Aytekinc b
a Department of Cardiology, Florence Nightingale Hospital, Istanbul, Turkey Department of Molecular Biology, Marmara University, Science and Art Faculty, Biology Division, Goztepe-Istanbul, Turkey c Department of Cardiology, Istanbul Bilim University Medical Faculty, Florence Nightingale Hospital, Istanbul, Turkey d Laboratory of Basic Sciences, Istanbul Bilim University Medical Faculty, Istanbul, Turkey e Department of Biostatistics, Istanbul University Cerrahpas xa Medical Faculty, Istanbul, Turkey
Received for publication April 11, 2008; accepted July 16, 2008 (ARCMED-D-08-00152).
Background. Our aim was to determine whether the common variants within the coding sequence of ABCA1 gene affects low plasma high-density lipoprotein cholesterol (HDLC) levels in Turkish patients with coronary artery disease (CAD). The study group was composed of 552 CAD patients, of which 251 had HDL-C levels #40 mg/dL, and 301 had HDL-C levels O40 mg/dL. Methods. PCR-RFLP was used to determine the A2589G and G3456C DNA polymorphisms of the ABCA1 gene. The study group was analyzed for potential clinical predictors of low HDL-C. Results. The GG variant of the ABCA1 gene A2589G polymorphism was found in 3.6% patients within the HDL-C #40 mg/dL group and in 4% of HDL-C levels O40 mg/dL group. Frequency distributions of the A2589G genotypes were not found to differ significantly among groups. The CC genotype of the G3456C polymorphism was found in 6.8% of HDL-C #40 mg/dL group and in 11.6% individuals of the HDL-C levels O40 mg/dL group. Frequency distributions of the G3456G genotypes were not significantly different among groups. The A2589G genotypes were not found to be effective over the analyzed lipid parameters. Among G3456C genotypes, in CAD patients with HDL-C #40 mg/dL the low-density lipoprotein (LDL-C) levels were elevated, whereas HDL-C levels decreased in CC genotype carriers compared to GG and GC. Conclusions. No significant association was found between cardiovascular endpoints and ABCA1 gene A2589G and G3456C genotypes in this study population. Ó 2008 IMSS. Published by Elsevier Inc. Key Words: Coronary artery disease, Low HDL, G3456, A2589G, Cardiovascular endpoints.
Introduction Among the many potential risk factors, high-density lipoprotein cholesterol (HDL-C) is considered to play a major role in coronary artery disease (CAD) (1e5). About 4% of CAD patients have low HDL-C concentrations as an iso*
These authors contributed equally to this work. Address reprint requests to: Assoc. Prof. Belgin Su¨sleyici Duman, Marmara University, Science and Art Faculty, Biology Division, Molecular Biology Department, 34722 Goztepe-Istanbul, Turkey; E-mail: [email protected]
lated finding, and another 25% have low HDL-C concentrations associated with other lipoprotein abnormalities. In the Turkish population, the incidence of low HDL-C was observed in 53% of men and 26% of women (6). HDL-C concentrations are affected by multiple factors that have a complex interaction, including obesity, physical inactivity, cigarette smoking, alcohol consumption, very-low-fat diets and genetic causes. ATP-binding cassette A1 (ABCA1) gene encodes a cholesterol-efflux regulatory protein (CERP) (7e9). Human ABCA1 is a protein of 2201 amino acids characterized
0188-4409/08 $esee front matter. Copyright Ó 2008 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2008.07.008
736
Catakoglu et al./ Archives of Medical Research 39 (2008) 735e742
by two hydrophilic nucleotide binding folds (NBF) containing highly conserved peptide motifs (Walker A and Walker B) typical of many ATPases and two transmembrane hydrophobic domains, each consisting of six membrane-spanning helices (7e10). ABCA1 protein facilitates the efflux of unesterified cholesterol and phospholipids from cells and their transfer to lipid-poor apo AI particles, which are converted into nascent HDL-C. If this efflux is impaired, cholesterol tends to accumulate in the cells in the esterified form, whereas lipid-poor apo AI particles are rapidly removed from the plasma (11,12). Therefore, ABCA1 may be a candidate gene for the determination of plasma HDL-C concentration. Mutations in the gene encoding ATP-binding cassette transporter 1 (ABCA1) gene have been reported in various studies, characterized in patients with decreased HDL-C (13e16). Brousseau et al. reported G3456C variant of the ABCA1 gene to be associated with increased risk for CAD endpoints, suggesting a role of this polymorphism in the premature CAD observed in the general population (17). Kakko et al. demonstrated A2589G polymorphism of the ABCA1 gene to contribute 3% of the variation in plasma HDL-C levels in human, which suggests the minor importance of ABCA1 locus in regulation of HDL-C in the Finn population (18). According to our knowledge, this is the first study in a Turkish population to detect the association of common ABCA1 variants with low HDL-C with CAD. Because only a limited number of studies have investigated common variants and polymorphisms in ABCA1 and their potential association with plasma concentrations of HDL-C (17e19), we aimed to determine whether common variants (A2589G, G3456C) within the coding sequence of ABCA1 gene affect plasma HDL-C levels in CAD patients.
Materials and Methods Study Population Patients who were invited for diagnostic coronary angiography between January 2003 and June 2005 and who had clinically defined CAD were included consecutively and prospectively in this study. A total of 552 patients were enrolled in the study. Mean age of the patients with HDL-C #40 mg/dL was 59.2 10.7 years (188 males, 63 females) and with O40 mg/dL was 62.3 10.1 years (168 males, 133 females). The study group with HDL-C #40 mg/dL was significantly ( p !0.05) younger than the group with HDL-C O40 mg/dL. The study population was analyzed for potential clinical predictors of low HDL-C. Exclusion criteria were normal coronary arteries, age !21 years, severe comorbidity with a life expectancy !12 months, use of fibrates, steroid therapy, hormone replacement and oral contraceptives, cirrhosis and thyroid disorders. CAD was defined as angiographically documented coronary atherosclerosis with any level of stenosis. Hypertension was diagnosed according
to the Joint National Committee seven-guideline criteria; diabetes mellitus was defined according to the American Diabetes Association criteria; hypercholesterolemia and family history of premature CAD were defined according to National Cholesterol Education Programme/Adult Treatment Panel III guideline criteria. Cut-offs for HDL-C group classification were also defined by the ATP III guidelines. All patients gave informed consent, and the study was approved by the local ethics committee. Laboratory Methods Fasting blood was collected from all subjects into 0.1% Na EDTA tubes. Plasma was separated promptly and frozen at 80 C until analysis. Total cholesterol (Total-C), lowdensity lipoprotein cholesterol (LDL-C) and triglycerides (TG) were analyzed by enzymatic procedures (20,21). HDL-C was measured after precipitation of apo-B containing lipoproteins, very-low-density lipoprotein cholesterol (VLDL-C) and LDL-C with dextran-sulfate/Mg solution (22). Plasma apo A-I, apo A2, apo B-100, apo C, and apo E were determined by immunoturbidity assay methods using nephelometry (23). Genotyping of Single Nucleotide Polymorphisms (SNP) at the ABCA1 Locus The A2589G polymorphism (changing isoleucine, amino acid 823 of the ABCA1 protein to methionine) was analyzed with a forced EcoRV restriction site. The mismatching sense primer (50 -CCG ATT ACT GGT TCC AAC CAG AAG AGG AT-30 ) and the anti-sense primer (50 -GAC ATA ATG GCA TGG GAG GAT T-30 ) were supplied by Life Technologies (Gaithersburg, MD) and created a PCR product of 298 bp. PCR reaction were performed on Programmable Thermal Block II (Lab-Line, Melrose Park, IL). Amplification was performed in 7.5 mL containing 30 ng of genonic DNA, 12.5 pmol of each primer, 200 mmol of dNTPs, and 0.15 U of TaqDNA polymerase in a PCR buffer with 1.5 mmol MgCl2. PCR conditions were 95 C for 10 min followed by 39 cycles of 95 C for 30 sec, 60 C for 35 sec and 72 C for 45 sec and final extension at 72 C for 10 min. PCR products were then digested with 1.25 U of the EcoRV restriction enzyme at 37 C for 2 h in the buffer recommended by the manufacturer. Digested PCR products were electrophoresed with 4% agarose gel and visualized by UV light. The A allele was digested into 175and 29-bp fragments, whereas the G allele was not digested. The G3456C polymorphism (changing glutamic acid 1112 to aspartic acid) was analyzed with PflFI restriction site. Sense primer (50 -ATA AGC ATA GCA TAC GGA CAC GGT CCT TTA GTT CTC ACA CAA CAG-30 ) and mismatching anti-sense primer (50 ATC TCC TAC TAC CGA TGG TCA GCG TGA CAC T-30 ) was used to create a 214-bp PCR product. PCR and the digestion conditions PflFI restriction enzyme were similar to that used for the
ATP-binding Cassette Transporter 1 Gene, HDL-Cholesterol and CAD
genotyping of the A2589G polymorphism. The G allele was digested into fragments of 191 and 20 bp and the C allele into 20-, 29- and 132-bp fragments. Statistical Analysis Discrete variables are reported as counts (percentages) and continuous variables as mean SD. To test differences between treatment groups for discrete variables, we used c2 test or Fisher’s exact test, as appropriate. Continuous variables were analyzed by t-test for unpaired samples. When three or more groups were compared, analysis of covariance was used (statin medication was used as covariant in Table 3). Body mass index, hypertension, diabetes mellitus, hypercholesterolemia, active smoking habit, and the A2589G, G3456C variants of ABCA1 gene were selected as potential risk factors for cardiovascular events (myocardial infarction, unstable angina, percutaneous coronary intervention or CABG and stroke) and for peripheral artery disease. Predictors of these events were determined by using multivariate logistic regression analysis. Odds ratios (OR) with two-tailed p values were calculated as a measure of the association of the selected parameters with CAD. Male gender, body mass index, active smoking, hypertension, diabetes mellitus and the A2589G, G3456C variants of ABCA1 gene were selected as potential risk factors for low HDL-C. Predictors of low HDL-C were determined by using multiple logistic regression analysis; p values #0.05 were considered significant. All computations were done using STATA 5.0 statistical software package (Stata Corporation, College Station, TX). Results Baseline clinical characteristics of the whole study population and in patients with HDL-C levels #40 mg/dL and O40 mg/dL are presented in Table 1. Only the HDL-C strata were compared regarding clinical characteristics. Patients with HDL-C #40 mg/dL were younger ( p 5 0.028), male/female ratio was higher ( p 5 0.0001), had lower total cholesterol ( p 5 0.0001), higher VLDL-C and triglyceride levels ( p 5 0.0001) and the number of active smokers was higher ( p 5 0.027) in comparison with patients with HDLC O40 mg/dL. Hypertension, diabetes mellitus and family history of premature CAD percentages were not significantly different among CAD patients with HDL-C levels #40 mg/dL and O40 mg/dL. CAD patients with HDL-C levels #40 mg/dL and O40 mg/dL were similar regarding comorbid conditions such as chronic renal failure, chronic obstructive pulmonary disease, peripheral vascular disease, congestive heart failure and history of stroke. ST segment elevation myocardial infarction and unstable angina as baseline clinical presentation were significantly higher in patients with HDL-C levels #40 mg/dL ( p 5 0.023 and 0.009, respec-
737
tively) whereas they were less in exertional angina ( p 5 0.016) compared to patients with HDL-C O40 mg/dL. History of prior coronary artery bypass graft surgery or PCI was similar between patients with HDL-C levels #40 mg/dL and O40 mg/dL. No significant difference was observed between groups for baseline medications (Table 1). Genotype frequency distributions of the ABCA1 gene A2589G and G3456C polymorphisms for the total study group, patients with HDL #40 mg/dL and O40 mg/dL were compared in Table 2. The sequence variant (GG) of the ABCA1 gene A2589G polymorphism was found in 21 patients (3.8%) within the total population, in nine patients (3.6%) within the HDL-C #40 mg/dL group and in 12 (4%) individuals of the HDL-C levels O40 mg/dL group. Frequency distributions of the A2589G genotypes was not significantly different among groups (c2 5 3.524, p 5 0.172) (Table 2). The ABCA1 A2589G A allelic frequency has been found to be 0.73, G allelic frequency 0.27 in the total population. The sequence variant (CC) of the ABCA1 gene G3456C polymorphism was found in 52 subjects (5.9%) in the total population, 17 subjects (6.8%) of the HDL-C #40 mg/dL group and in 35 (11.6%) individuals of the HDL-C levels O40 mg/dL group. Frequency distributions of the G3456C genotypes was not significantly different among groups (c2 5 4.005, p 5 0.135) (Table 2). For G3456C polymorphism, frequencies of G and C alleles were 0.72 and 0.28, respectively, in the total population. To test whether the ABCA1 gene A2589G and G3456C single nucleotide polymorphisms could be associated with any lipid parameter, we compared serum lipid levels between ABCA1 A2589G and G3456C (Table 3) genotypes for CAD patients with HDL-C #40 mg/dL. Because statin medication may influence levels of cholesterol parameters, this medication was included as a covariate in the statistical analysis. The association analysis was performed both for HDL #40 mg/dL group and in the total population No significant difference was observed for the analyzed lipid variables when A2589G genotypes were compared (Table 3). We investigated gender-specific effects of ABCA1 gene variants; however, we could not find any significant effect on serum lipid parameters. Although only in males, a borderline significant ( p 5 0.066) influence of ABCA1 gene 2589GG genotype on HDL-C levels were found. In detail, male CAD patients with GG genotype had the lowest HDLC levels (35.9 mg/dL) and the AG genotype had the highest HDL-C levels (42 mg/dL) (data not included). LDL-C ( p 5 0.046) and HDL-C ( p 5 0.02) levels were found to differ among ABCA1 gene G3456C genotypes in CAD patients with HDL-C levels #40 mg/dL (Table 3). In detail, LDL-C ( p 5 0.046) levels were found to be elevated, whereas HDL-C levels were found to be decreased in CC genotype carriers when compared to GG and GC genotypes (Table 3) in CAD patients with HDL-C #40 mg/dL. Age, gender, body mass index, waist/hip ratio, HDL-C levels #40 mg/dL, family history for premature CAD,
738
Catakoglu et al./ Archives of Medical Research 39 (2008) 735e742
Table 1. Baseline clinical characteristics Coronary artery disease patients
Age, years Male/Female LDL cholesterol, mg/dL Total cholesterol, mg/dL VLDL cholesterol, mg/dL Triglyceride, mg/dL Hypertension, n (%) Diabetes mellitus, n (%) Type I diabetes mellitus, n (%) BMI, kg/m2 W/H ratio Active smoker, n (%) Family history, n (%) Menopause, n (% among women) Sedentary lifestyle, n (%) COPD, n (%) CRF, n (%) Exertional angina, n (%) ST segment elevation MI, n (%) Unstable angina, n (%) Silent ischemia, n (%) Peripheral vascular disease, n (%) Prior CABG, n (%) Prior MI, n (%) Prior PCI, n (%) Stroke or TIA, n (%) CHF, n (%) Baseline medications, n (%) Beta blocker ACE inhibitor Diuretic Aspirin Statin
Total population (n 5 452)
HDL-C #40 mg/dL (n 5 251)
HDL-C O40 mg/dL (n 5 301)
p value
61.00 10.4 356/196 121.7 1.7 194.8 1.9 32.2 0.9 153.1 3.9 167 (30.3) 73 (13.2) 12 (2.2) 29.8 0.9 0.98 0.02 77 (13.9) 98 (17.8) 57 (10.3) 46 (8.3) 16 (2.9) 3 (0.5) 101 (18.3) 27 (4.9) 51 (9.2) 40 (7.2) 11 (2) 39 (5.4) 64 (11.6) 34 (6.3) 12 (2.2) 16 (2.9)
59.2 10.7 188/63 118.8 2.5 187.0 2.8 36.8 1.5 177.0 6.4 70 (27.9) 38 (15.1) 7 (2.8) 29.92 6.34 1 0.08 47 (18.7) 52 (20.7) 17 (26.9) 21 (8.4) 6 (2.24) 1 (0.4) 35 (19.9) 18 (7.2) 32 (12.7) 13 (5.2) 7 (2.8) 17 (6.9) 39 (15.5) 17 (6.9) 4 (1.6) 11 (4.4)
62.3 10.1 168/133 124.2 2.3 201.3 2.7 28.2 1.1 133.1 4.8 97 (32.2) 35 (11.6) 5 (1.7) 29.68 17.17 0.98 0.15 30 (10) 46 (15.3) 40 (30.1) 25 (8.3) 10 (3.3) 2 (0.7) 66 (21.9) 9 (3) 19 (6.3) 27 (9) 4 (1.3) 13 (4.5) 25 (8.3) 17 (5.6) 8 (2.7) 5 (1.7)
0.028 0.0001 0.106 0.0001 0.0001 0.0001 0.269 0.228 0.366 0.896 0.599 0.027 0.096 0.728 0.979 0.516 0.672 0.016 0.023 0.009 0.087 0.222 0.260 0.008 0.599 0.393 0.058
135 102 35 156 216
(24.5) (18.5) (6.3) (28.3) (39.1)
68 49 19 74 96
(27.1) (19.5) (7.6) (29.5) (38.2)
67 53 16 82 120
(22.3) (17.6) (5.3) (27.2) (39.9)
0.188 0.564 0.279 0.561 0.698
p value corresponds to the comparison between patients with HDL-C #40 mg/dL and O40 mg/dL. ACE, angiotensin converting enzyme; CABG, coronary artery bypass graft; CHF, congestive heart failure; COPD, chronic obstructive pulmonary disease; CRF, chronic renal failure; MI, myocardial infarction; PCI, percutaneous coronary intervention; TIA, transient ischemic attack; BMI, body mass index; W/H, wasit/hip.
diabetes mellitus, smoking, hypercholesterolemia, hypertension, congestive heart failure, ABCA1 A2589G and G3456C genotypes were selected as conventional risk factors to be analyzed for cardiovascular events (myocardial infarction, unstable angina, percutaneous coronary intervention or CABG and stroke) and for peripheral artery disease in multiple logistic regression analysis (Table 4). We were unable to detect any significant association of A2589G and G3456C genotypes with the cardiovascular events analyzed. Discussion This study reports the association of ABCA1 SNPs with HDL-C levels and CAD in 552 Turkish patients. The analysis is based on both categorically classified HDL-C levels (#40 mg/dL and O40 mg/dL) (24) and in the total study
population. Although no clear association is found, the analyzed ABCA1 SNPs are likely to contribute a small increment to HDL-C levels. In the Turkish Adult Risk Factor Study, the mean values for HDL-C were reported to be 37 12 mg/dL in men and 45 13 mg/dL in women (25). The stated levels were 20% lower than those provided by NHANES III for the U.S. Caucasian population (26) and for Germans (27). In our study, HDL-C levels were 43 12 mg/dL for the entire study group (41 11 for males and 47 12 for females). Investigations on the function of ABCA1 have revealed its role in 1) engulfment of apoptotic cells by macrophages (28,29); 2) macrophage interleukin-1b secretion (30); 3) modulation of apolipoprotein-mediated cellular lipid efflux (31); and 4) caveolar processing (32). Due to its diverse functions, ABCA1 is an attractive candidate gene for the modulation of plasma HDL-C
ATP-binding Cassette Transporter 1 Gene, HDL-Cholesterol and CAD
739
Table 2. ABCA1 gene A2589G and G3456C genotype and allele frequencies in coronary artery disease patients with HDL-C #40 mg/dL, HDL-C O40 mg/dL and total population ABCA1 gene A2589G polymorphism
CAD patients with HDL-C #40 mg/dL CAD patients with HDL-C O40 mg/dL Total population
AA, n (%)
AG, n (%)
GG, n (%)
A
G
136 (54.2%) 139 (46.2%) 275 (49.6%)
106 (42.2%) 150 (49.8%) 256 (46.6%)
9 (3.6%) 12 (4%) 21 (3.8%)
0.75 0.71 0.73
0.25 0.29 0.27
ABCA1 gene G3456C polymorphism
CAD patients with HDL-C #40 mg/dL CAD patients with HDL-C O40 mg/dL Total population
GG, n (%)
GC, n (%)
CC, n (%)
G
C
135 (53.8%) 159 (52.8%) 294 (53.3%)
99 (39.4%) 107 (35.5%) 206 (37.3%)
17 (6.8%) 35 (11.6%) 52 (9.4%)
0.74 0.71 0.72
0.26 0.29 0.28
Results are expressed as number (percentage) for genotypes, and allelic frequencies were presented for both polymorphisms. The ABCA1 A2589G genotype and G3456C genotype frequencies of the study groups (CAD patients with HDL-C #40 mg/dL and CAD patients with HDL-C O40 mg/dL) with c2 test were respectively as c2 5 3.524, p 5 0.172 and c2 5 4.005, p 5 0.135.
levels in the general population as well. Rubins et al. have determined the frequencies of three common variants within the coding sequence of ABCA1 (G596A, A2589G and G3456C) in men participating in the Veterans Affairs Cooperative HDL Cholesterol Intervention Trial (VA-HIT), a population comprised of men having low HDL-C and CHD (33). In view of the subject composition of our study, we believed that this would be an ideal population for the identi-
fication of ABCA 1 variants and, ultimately, for associations to be made with plasma lipids and/or CAD endpoints. In the present study we analyzed G3456C and A2589G polymorphisms in the gene encoding ABCA1, a member of the family of ATP-binding cassette transporters (34). Both variants affect amino acids that are either conserved between human and mouse or are very similar as in the case of A2589G (isoleucine and valine, respectively), suggesting that each has an important functional role. The A2589C region is located
Table 3. Serum lipids comparisons by ABCA1 gene A2589G and G3456C genotypes in all coronary artery disease patients and in patients with HDL-C levels #40 mg/dL with covariance analysis ABCA1 gene A2589G polymorphism Patients Total-C LDL-C HDL-C VLDL-C TG
All HDL-C#40 All HDL-C#40 All HDL-C#40 All HDL-C#40 All HDL-C#40
mg/dL mg/dL mg/dL mg/dL mg/dL
AA 194.2 183.1 121.9 115.7 42.3 33.5 32.2 36 153.9 169.8
AG
46.3 (275) 43.2 (136) 39.5 (275) 36.6 (136) 10.9 (275) 4.9 (136) 20.3 (275) 21.4 (136) 91.3 (275) 91.1 (136)
194.7 191.5 120.9 121.9 44.3 33.9 32.0 38.1 151.8 184.6
46.1 (256) 45.9 (106) 39.9 (256) 41.1 (106) 12.2 (256) 4.1 (106) 21.6 (256) 22.2 (106) 93.9 (256) 104.8 (106)
GG 202.4 193.9 128.6 127.8 43.5 30.8 32.5 39.2 158.9 196.4
47.4 (21) 59.1 (9) 39.4 45.0 (9) 13.1 5.8 (9) 25.1 34.9 (9) 123.0 173.9 (9)
p value O0.05 O0.05 O0.05 O0.05 O0.05 O0.05 O0.05 O0.05 O0.05 O0.05
ABCA1 gene G3456C polymorphism Patients Total-C LDL-C HDL-C VLDL-C TG
All HDL-C All HDL-C All HDL-C All HDL-C All HDL-C
#40 mg/dL #40 mg/dL #40 mg/dL #40 mg/dL #40 mg/dL
GG 192.6 180.7 119.8 113.2 42.9 33.3 31.5 36.1 151.5 171.2
46.7 (294) 45.2 (135) 40.3 (294) 39.6 (135) 11.4 (294) 4.8 (135) 18.2 (294) 19.9 (135) 80.9 (294) 81.8 (135)
GC 197.3 194.5 123.8 124.7 43.2 34.3 33.8 38.2 157.8 184.4
47.2 (206) 45.1 (99) 39.5 (206) 37.8 (99) 11.4 (206) 4.2 (99) 24.7 (206) 24.7 (99) 107.7 (206) 117.2 (99)
CC 197.5 193.7 124.6 128.6 45.7 31.2 29.5 36.1 143.2 180.1
38.9 (52) 36.2 (17) 36.0 (52) 33.3 (17) 13.1 (52) 4.9 (17) 21.1 (52) 26.6 (17) 101.9 (52) 132.2 (17)
p value O0.05 O0.05 O0.05 O0.05 O0.05 !0.05 O0.05 O0.05 O0.05 O0.05
Values are mean SD. p values are for A2589G and G3456C genotype effect. Statin medication was included as covariate in the statistical analysis.
Catakoglu et al./ Archives of Medical Research 39 (2008) 735e742
740
Table 4. Risk factors identified to be associated with low HDL cholesterol level by multiple logistic regression analysis All Males BMI Active smoking habit Hypertension DM ABCA1 A2589G (AA) (AG) (GG) ABCA1 G3456C (GG) (GC) (CC)
b
SE
OR
p
1.038 0.003 0.126 0.049 0.384 0.017 0.330 0.347 0.224 0.805 1.029
0.365 0.011 0.239 0.165 0.176 0.363 0.338 0.580 0.319 0.307 0.475
2.823 1.003 0.882 1.050 0.681 0.983 0.719 1.415 1.251 2.237 0.357
0.004 0.747 0.600 0.769 0.030 0.962 0.329 0.550 0.482 0.009 0.030
Multivariate logistic regression model contained male gender, body mass index (BMI), smoking habit, hypertension, diabetes mellitus (DM) and ABCA1 genotype variables. b indicates estimated coefficient; SE, standard error; OR, adjusted odds ratio.
proximal to the sixth subdomain within the first group of transmembranous domains, adjacent to the putative glycosylation site of ABCA1. Amino acid substitution at A2589C region may cause conformational changes around this portion, resulting in altering the state of glycosylation of the transporter and enhancing cholesterol efflux. Harada et al. did not find the A2589C variant to be associated with susceptibility to CAD, although an inverse relationship between plasma HDL-C level and incidence of CAD was shown (35). Other studies suggested that both the A2589C and the variant may be associated with susceptibility to CAD. These discrepancies may be at least partially attributed to environmental or social factors or racial differences. In light of previous reports by Imai et al. (36) and others (19) showing the existence of large interracial differences in the frequency of genetic variations and disease susceptibilities, it is possible that the discrepancy may be caused, at least partially, by methodological factors such as sampling schemes or trial size. Previous studies in other ethnic groups have shown that variations in the coding region of ABCA1 affect plasma lipid level. Wang et al. (19) noted a wide range of allelic frequencies in the A2589G genotype across six ethnic groups (Caucasian, South Asians, Inuit, Africans, Oji-Cree, and Chinese), each group comprising a small number of subjects. They demonstrated that the A2589G variant contributes to increased level of HDL-C only in Canadian Inuits probably due to social or ethnic difference. Kakko et al.18 reported A2589G genotypes contributing to |3% of the variation in plasma HDL-C levels in women and suggested ABCA1 locus as of minor importance for the regulation of HDL-C. In contrast, Brousseau et al. reported A2589G polymorphism not to be significantly associated with plasma HDL-C concentrations in men with low HDL-C levels having CAD (17). Our data, derived from the Turkish population, showed consistent alterations in lipid profile associated with A2589G variant.
Brousseau et al. (17) demonstrated that carriers of the G3456C variant were at increased risk for adverse CHD endpoints relative to noncarriers within the same population. The G3456C polymorphism affects the highly charged linker region of ABCA1, a site rich in potential phosphorylation sites (37). This region is very similar to that of the regulatory domain of another ABC transporter, the cystic fibrosis transmembrane regulator, where mutations responsible for cystic fibrosis have been localized (38). Hence, it is reasonable that this ABCA1 variant may influence function, confirmed by our demonstration of decreased cellular cholesterol efflux in homozygotes for the mutant C allele of this variant. In view of the role of ABCA1 in the modulation of HDL-C concentrations and lipid efflux, we also performed analyses to test for potential associations between each common variant and plasma lipid profiles. Two statistically significant associations between plasma lipids and ABCA1 G3456C variants were observed in our study group. The mutant C allele of the G3456C variant was associated with increased plasma LDL-C and decreased HDL-C concentrations. The G3456C variant of ABCA1 has been shown to be associated with a significantly increased risk for CAD endpoints, which suggests a role of this variant in the premature CAD observed in the Finn population (17). In our study, similar to results of Brousseau et al. (17), we found a significant association of G3456C variant with both HDL-C and LDLC concentrations. However, we were not able to demonstrate a significant risk for CAD endpoints with G3456C variation. The A2589G polymorphism of ABCA1 was not associated with any of the analyzed lipid parameters. One possible explanation for this may be because HDL-C levels are modulated by other genetic and environmental factors (39,40). Direct effects of ABCA1 A2589G polymorphism on HDL-C levels may not be readily apparent. Because our study population consists of 13e17% of patients with type 1 and type 2 diabetes, this is likely to confound HDL-C levels and is one of the restrictions of the study. Another restriction of this study is that the presence of pre- and postmenopausal women will introduce further confounding. There appears to be a large difference in the distribution of different SNP genotypes among ethnic groups. Concerning ABCA1 variants, the M 823 allele has been shown to be common in Japanese (allele frequency 5 0.507). In comparison, the M 823 allele frequency was shown to vary widely among other ethnic groups as follows: Caucasian, 0.079; South Asians, 0.145; Inuit, 0.294; Africans, 0.419; Oji-Cree, 0.690; and Chinese, 0.737, respectively (19). In the present study the 2589G and 3456C allele frequencies were found as 0.270 and 0.281, respectively. The influence of diet and environmental factors on lipid profile may differ widely among study groups. We found a significant association between the G3456C variant of the ABCA1 gene and both serum LDL-C and HDL-C levels in our study population with CAD who have
ATP-binding Cassette Transporter 1 Gene, HDL-Cholesterol and CAD
HDL-C levels #40 mg/dL. However, we could not find any association of these variants with outcomes of CAD. In conclusion, the frequency distributions of neither the A2589G nor the G3456C genotypes were not found to differ significantly among groups. The A2589G genotypes were not found to be effective over the analyzed lipid parameters. In CAD patients with HDL-C #40 mg/dL, among G3456C genotypes the LDL-C levels were elevated, whereas HDL-C levels decreased in CC genotype carriers compared to GG and GC. No significant association was found between cardiovascular events and ABCA1 gene A2589G and G3456C genotypes. We could not suggest a role of A2589G or G3456C variants in the CAD endpoints observed in this population. References 1. Castelli WP, Garrison RJ, Wilson PW, Abbott RD, Kalousdian S, Kannel WB. Incidence of coronary heart disease and lipoprotein cholesterol levels. The Framingham Study. JAMA 1986;256:2835e2838. 2. Kinosian B, Glick H, Garland G. Cholesterol and coronary heart disease: predicting risks by levels and ratios. Ann Intern Med 1994;121:641e647. 3. Onat A, Surdum-Avci G, Senocak M, Ornek E, Go¨zu¨kara Y. Plasma lipids and their interrelationship in Turkish adults. J Epidemiol Community Health 1992;46:470e476. 4. Ridker PM, Glynn RJ, Hennekens CH. C-reactive protein adds to the predictive value of total and HDL cholesterol in determining risk of first myocardial infarction. Circulation 1998;97:2007e2011. 5. Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershatz H, Kannel WB. Prediction of coronary heart disease using risk factor categories. Circulation 1998;97:1837e1847. 6. Mahley RW, Palaog˘lu KE, Atak Z, Dawson-Pepin J, Langlois AM, Cheung V, et al. Turkish Heart Study: lipids, lipoproteins, and apolipoproteins. J Lipid Res 1995;36:839e859. 7. Breckenridge WC, Little JA, Alaupovic P, Wang CS, Kuksis A, Kakis G, et al. Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase. Atherosclerosis 1982;45:161e179. 8. Kuivenhoven JA, de Knijff P, Boer JM, Smalheer HA, Botma GJ, Seidell JC, et al. Heterogeneity at the CETP gene locus. Influence on plasma CETP concentrations and HDL cholesterol levels. Arterioscler Thromb Vasc Biol 1997;17:560e568. 9. Reymer PW, Gagne E, Groenemeyer BE, Zhang H, Forsyth I, Jansen H, et al. A lipoprotein lipase mutation (Asn291Ser) is associated with reduced HDL cholesterol levels in premature atherosclerosis. Nat Genet 1995;10:28e34. 10. Kuivenhoven JA, Pritchard H, Hill J, Frohlich J, Assmann G, Kastelein J. The molecular pathology of lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes. J Lipid Res 1997;38:191e205. 11. Batal R, Tremblay M, Krimbou L, Mamer O, Davignon J, Genest J Jr, et al. Familial HDL deficiency characterized by hypercatabolism of mature apoA-I but not proapoA-I. Arterioscler Thromb Vasc Biol 1998;18:655e664. 12. Murray CJ, Lopez AD. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet 1997;349:1269e1276. 13. Bodzioch M, Orso´ E, Klucken J, Langmann T, Bo¨ttcher A, Diederich W, et al. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 1999;22:347e351. 14. Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, et al. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 1999;22:336e345. 15. Remaley AT, Rust S, Rosier M, Knapper C, Naudin L, Broccardo C, et al. Human ATP-binding cassette transporter 1 (ABC1): genomic
16.
17.
18.
19.
20. 21.
22.
23.
24. 25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
741
organization and identification of the genetic defect in the original Tangier disease kindred. Proc Natl Acad Sci USA 1999;96:12685e12690. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, et al. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 1999;22:352e325. Brousseau ME, Bodzioch M, Schaefer EJ, Goldkamp AL, Kielar D, Probst M, et al. Common variants in the gene encoding ATP-binding cassette transporter 1 in men with low HDL cholesterol levels and coronary heart disease. Atherosclerosis 2001;154:607e611. Kakko S, Kelloniemi J, von Rohr P, Hoeschele I, Tamminen M, Brousseau ME, et al. ATP-binding cassette transporter A1 locus is not a major determinant of HDL-C levels in a population at high risk for coronary heart disease. Atherosclerosis 2003;166:285e290. Wang J, Burnett JR, Near S, Young K, Zinman B, Hanley AJ, et al. Common and rare ABCA1 variants affecting plasma HDL cholesterol. Arterioscler Thromb Vasc Biol 2000;20:1983e1989. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974;20:470e475. Foster LB, Dunn RT. Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method. Clin Chem 1973;19:338e340. Warnick GR, Benderson J, Albers JJ. Dextran sulfate-Mg2þ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem 1982;28:1379e1388. Cheung MC, Albers JJ. The measurement of apolipoprotein A-I and A-II levels in men and women by immunoassay. J Clin Invest 1977; 60:43e50. Rader DJ. Pathophysiology and management of low high-density lipoprotein cholesterol. Am J Cardiol 1999;83:22Fe24F. Onat A, Yıldırım B, Uslu N. Plasma lipoproteins and apolipoproteins in Turkish adults: overall levels, association with other risk parameters, and HDL’s role as a marker of coronary risk in women. Arch Turk Soc Cardiol 1999;27:72e79. Johnson CL, Rifkind BM, Sempos CT, Carroll MD, Bachorik PS, Briefel RR, et al. Declining serum total cholesterol levels among US adults. The National Health and Nutrition Examination Surveys. JAMA 1993;269:3002e3008. Assmann G, Cullen P, Schulte H. The Munster Heart Study (PROCAM). Results of follow-up at 8 years. Eur Heart J 1998;19(Suppl A):A2e11. Tanaka AR, Ikeda Y, Abe-Dohmae S, Arakawa R, Sadanami K, Kidera A, et al. Human ABCA1 contains a large amino-terminal extracellular domain homologous to an epitope of Sjogren’s Syndrome. Biochem Biophys Res Commun 2001;283:1019e1025. Luciani MF, Chimini G. The ATP binding cassette transporter ABC1 is required for the engulfment of corpses generated by apoptotic cell death. EMBO J 1996;15:226e235. Hamon Y, Luciani MF, Becq F, Verrier B, Rubartelli A, Chimini G. Interleukin-1b secretion is impaired by inhibitors of the ATP binding cassette transporter, ABC1. Blood 1997;90:2911e2915. Langmann T, Klucken J, Reil M, Liebisch G, Luciani MF, Chimini G, et al. Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages. Biochem Biophys Res Commun 1999;257:29e33. Orso E, Broccardo C, Kaminski WE, Bo¨ttcher A, Liebisch G, Drobnik W, et al. Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and ABC1-deficient mice. Nat Genet 2000;24:192e196. Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, et al. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. N Engl J Med 1999;341:410e418. Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol 1992;8:67e113.
742
Catakoglu et al./ Archives of Medical Research 39 (2008) 735e742
35. Harada T, Imai Y, Nojiri T, Morita H, Hayashi D, Maemura K, et al. A common Ile 823 Met variant of ATP-binding cassette transporter A1 gene (ABCA1) alters high density lipoprotein cholesterol level in Japanese population. Atherosclerosis 2003;169:105e112. 36. Imai Y, Morita H, Kurihara H, Sugiyama T, Kato N, Ebihara A, et al. Evidence for association between paraoxonase gene polymorphisms and atherosclerotic diseases. Atherosclerosis 2000;149:435e442. 37. Luciani MF, Denizot F, Savary S, Mattei MG, Chimini G. Cloning of two novel ABC transporters mapping on human chromosome 9. Genomics 1994;21:150e159.
38. Welsh MJ, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 1993;73:1251e1254. 39. Heller DA, de Faire U, Pedersen NL, Dahlen G, McClearn GE. Genetic and environmental influences on serum lipid levels in twins. N Engl J Med 1993;328:1150e1156. 40. Schaefer EJ, Lamon-Fava S, Ordovas JM, Cohn SD, Schaefer MM, Castelli WP, et al. Factors associated with low and elevated plasma high density lipoprotein cholesterol and apolipoprotein A-I levels in the Framingham Offspring Study. J Lipid Res 1994;35: 871e882.
ORIGINAL ARTICLE
Common Variants in the ATP-binding Cassette Transporter 1 Gene with Decreased HDL-Cholesterol Levels and Coronary Artery Disease Alp Burak Catakoglu,a,* Belgin Su¨sleyici Duman,b,* Hilal Kurtog˘lu,c Melike Erso¨z,d Murat Sxener,c Hu¨seyin C ¸ elebi,a Penbe C ¸ ag˘atay,e Vedat Aytekin,c and Saide Aytekinc b
a Department of Cardiology, Florence Nightingale Hospital, Istanbul, Turkey Department of Molecular Biology, Marmara University, Science and Art Faculty, Biology Division, Goztepe-Istanbul, Turkey c Department of Cardiology, Istanbul Bilim University Medical Faculty, Florence Nightingale Hospital, Istanbul, Turkey d Laboratory of Basic Sciences, Istanbul Bilim University Medical Faculty, Istanbul, Turkey e Department of Biostatistics, Istanbul University Cerrahpas xa Medical Faculty, Istanbul, Turkey
Received for publication April 11, 2008; accepted July 16, 2008 (ARCMED-D-08-00152).
Background. Our aim was to determine whether the common variants within the coding sequence of ABCA1 gene affects low plasma high-density lipoprotein cholesterol (HDLC) levels in Turkish patients with coronary artery disease (CAD). The study group was composed of 552 CAD patients, of which 251 had HDL-C levels #40 mg/dL, and 301 had HDL-C levels O40 mg/dL. Methods. PCR-RFLP was used to determine the A2589G and G3456C DNA polymorphisms of the ABCA1 gene. The study group was analyzed for potential clinical predictors of low HDL-C. Results. The GG variant of the ABCA1 gene A2589G polymorphism was found in 3.6% patients within the HDL-C #40 mg/dL group and in 4% of HDL-C levels O40 mg/dL group. Frequency distributions of the A2589G genotypes were not found to differ significantly among groups. The CC genotype of the G3456C polymorphism was found in 6.8% of HDL-C #40 mg/dL group and in 11.6% individuals of the HDL-C levels O40 mg/dL group. Frequency distributions of the G3456G genotypes were not significantly different among groups. The A2589G genotypes were not found to be effective over the analyzed lipid parameters. Among G3456C genotypes, in CAD patients with HDL-C #40 mg/dL the low-density lipoprotein (LDL-C) levels were elevated, whereas HDL-C levels decreased in CC genotype carriers compared to GG and GC. Conclusions. No significant association was found between cardiovascular endpoints and ABCA1 gene A2589G and G3456C genotypes in this study population. Ó 2008 IMSS. Published by Elsevier Inc. Key Words: Coronary artery disease, Low HDL, G3456, A2589G, Cardiovascular endpoints.
Introduction Among the many potential risk factors, high-density lipoprotein cholesterol (HDL-C) is considered to play a major role in coronary artery disease (CAD) (1e5). About 4% of CAD patients have low HDL-C concentrations as an iso*
These authors contributed equally to this work. Address reprint requests to: Assoc. Prof. Belgin Su¨sleyici Duman, Marmara University, Science and Art Faculty, Biology Division, Molecular Biology Department, 34722 Goztepe-Istanbul, Turkey; E-mail: [email protected]
lated finding, and another 25% have low HDL-C concentrations associated with other lipoprotein abnormalities. In the Turkish population, the incidence of low HDL-C was observed in 53% of men and 26% of women (6). HDL-C concentrations are affected by multiple factors that have a complex interaction, including obesity, physical inactivity, cigarette smoking, alcohol consumption, very-low-fat diets and genetic causes. ATP-binding cassette A1 (ABCA1) gene encodes a cholesterol-efflux regulatory protein (CERP) (7e9). Human ABCA1 is a protein of 2201 amino acids characterized
0188-4409/08 $esee front matter. Copyright Ó 2008 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2008.07.008
736
Catakoglu et al./ Archives of Medical Research 39 (2008) 735e742
by two hydrophilic nucleotide binding folds (NBF) containing highly conserved peptide motifs (Walker A and Walker B) typical of many ATPases and two transmembrane hydrophobic domains, each consisting of six membrane-spanning helices (7e10). ABCA1 protein facilitates the efflux of unesterified cholesterol and phospholipids from cells and their transfer to lipid-poor apo AI particles, which are converted into nascent HDL-C. If this efflux is impaired, cholesterol tends to accumulate in the cells in the esterified form, whereas lipid-poor apo AI particles are rapidly removed from the plasma (11,12). Therefore, ABCA1 may be a candidate gene for the determination of plasma HDL-C concentration. Mutations in the gene encoding ATP-binding cassette transporter 1 (ABCA1) gene have been reported in various studies, characterized in patients with decreased HDL-C (13e16). Brousseau et al. reported G3456C variant of the ABCA1 gene to be associated with increased risk for CAD endpoints, suggesting a role of this polymorphism in the premature CAD observed in the general population (17). Kakko et al. demonstrated A2589G polymorphism of the ABCA1 gene to contribute 3% of the variation in plasma HDL-C levels in human, which suggests the minor importance of ABCA1 locus in regulation of HDL-C in the Finn population (18). According to our knowledge, this is the first study in a Turkish population to detect the association of common ABCA1 variants with low HDL-C with CAD. Because only a limited number of studies have investigated common variants and polymorphisms in ABCA1 and their potential association with plasma concentrations of HDL-C (17e19), we aimed to determine whether common variants (A2589G, G3456C) within the coding sequence of ABCA1 gene affect plasma HDL-C levels in CAD patients.
Materials and Methods Study Population Patients who were invited for diagnostic coronary angiography between January 2003 and June 2005 and who had clinically defined CAD were included consecutively and prospectively in this study. A total of 552 patients were enrolled in the study. Mean age of the patients with HDL-C #40 mg/dL was 59.2 10.7 years (188 males, 63 females) and with O40 mg/dL was 62.3 10.1 years (168 males, 133 females). The study group with HDL-C #40 mg/dL was significantly ( p !0.05) younger than the group with HDL-C O40 mg/dL. The study population was analyzed for potential clinical predictors of low HDL-C. Exclusion criteria were normal coronary arteries, age !21 years, severe comorbidity with a life expectancy !12 months, use of fibrates, steroid therapy, hormone replacement and oral contraceptives, cirrhosis and thyroid disorders. CAD was defined as angiographically documented coronary atherosclerosis with any level of stenosis. Hypertension was diagnosed according
to the Joint National Committee seven-guideline criteria; diabetes mellitus was defined according to the American Diabetes Association criteria; hypercholesterolemia and family history of premature CAD were defined according to National Cholesterol Education Programme/Adult Treatment Panel III guideline criteria. Cut-offs for HDL-C group classification were also defined by the ATP III guidelines. All patients gave informed consent, and the study was approved by the local ethics committee. Laboratory Methods Fasting blood was collected from all subjects into 0.1% Na EDTA tubes. Plasma was separated promptly and frozen at 80 C until analysis. Total cholesterol (Total-C), lowdensity lipoprotein cholesterol (LDL-C) and triglycerides (TG) were analyzed by enzymatic procedures (20,21). HDL-C was measured after precipitation of apo-B containing lipoproteins, very-low-density lipoprotein cholesterol (VLDL-C) and LDL-C with dextran-sulfate/Mg solution (22). Plasma apo A-I, apo A2, apo B-100, apo C, and apo E were determined by immunoturbidity assay methods using nephelometry (23). Genotyping of Single Nucleotide Polymorphisms (SNP) at the ABCA1 Locus The A2589G polymorphism (changing isoleucine, amino acid 823 of the ABCA1 protein to methionine) was analyzed with a forced EcoRV restriction site. The mismatching sense primer (50 -CCG ATT ACT GGT TCC AAC CAG AAG AGG AT-30 ) and the anti-sense primer (50 -GAC ATA ATG GCA TGG GAG GAT T-30 ) were supplied by Life Technologies (Gaithersburg, MD) and created a PCR product of 298 bp. PCR reaction were performed on Programmable Thermal Block II (Lab-Line, Melrose Park, IL). Amplification was performed in 7.5 mL containing 30 ng of genonic DNA, 12.5 pmol of each primer, 200 mmol of dNTPs, and 0.15 U of TaqDNA polymerase in a PCR buffer with 1.5 mmol MgCl2. PCR conditions were 95 C for 10 min followed by 39 cycles of 95 C for 30 sec, 60 C for 35 sec and 72 C for 45 sec and final extension at 72 C for 10 min. PCR products were then digested with 1.25 U of the EcoRV restriction enzyme at 37 C for 2 h in the buffer recommended by the manufacturer. Digested PCR products were electrophoresed with 4% agarose gel and visualized by UV light. The A allele was digested into 175and 29-bp fragments, whereas the G allele was not digested. The G3456C polymorphism (changing glutamic acid 1112 to aspartic acid) was analyzed with PflFI restriction site. Sense primer (50 -ATA AGC ATA GCA TAC GGA CAC GGT CCT TTA GTT CTC ACA CAA CAG-30 ) and mismatching anti-sense primer (50 ATC TCC TAC TAC CGA TGG TCA GCG TGA CAC T-30 ) was used to create a 214-bp PCR product. PCR and the digestion conditions PflFI restriction enzyme were similar to that used for the
ATP-binding Cassette Transporter 1 Gene, HDL-Cholesterol and CAD
genotyping of the A2589G polymorphism. The G allele was digested into fragments of 191 and 20 bp and the C allele into 20-, 29- and 132-bp fragments. Statistical Analysis Discrete variables are reported as counts (percentages) and continuous variables as mean SD. To test differences between treatment groups for discrete variables, we used c2 test or Fisher’s exact test, as appropriate. Continuous variables were analyzed by t-test for unpaired samples. When three or more groups were compared, analysis of covariance was used (statin medication was used as covariant in Table 3). Body mass index, hypertension, diabetes mellitus, hypercholesterolemia, active smoking habit, and the A2589G, G3456C variants of ABCA1 gene were selected as potential risk factors for cardiovascular events (myocardial infarction, unstable angina, percutaneous coronary intervention or CABG and stroke) and for peripheral artery disease. Predictors of these events were determined by using multivariate logistic regression analysis. Odds ratios (OR) with two-tailed p values were calculated as a measure of the association of the selected parameters with CAD. Male gender, body mass index, active smoking, hypertension, diabetes mellitus and the A2589G, G3456C variants of ABCA1 gene were selected as potential risk factors for low HDL-C. Predictors of low HDL-C were determined by using multiple logistic regression analysis; p values #0.05 were considered significant. All computations were done using STATA 5.0 statistical software package (Stata Corporation, College Station, TX). Results Baseline clinical characteristics of the whole study population and in patients with HDL-C levels #40 mg/dL and O40 mg/dL are presented in Table 1. Only the HDL-C strata were compared regarding clinical characteristics. Patients with HDL-C #40 mg/dL were younger ( p 5 0.028), male/female ratio was higher ( p 5 0.0001), had lower total cholesterol ( p 5 0.0001), higher VLDL-C and triglyceride levels ( p 5 0.0001) and the number of active smokers was higher ( p 5 0.027) in comparison with patients with HDLC O40 mg/dL. Hypertension, diabetes mellitus and family history of premature CAD percentages were not significantly different among CAD patients with HDL-C levels #40 mg/dL and O40 mg/dL. CAD patients with HDL-C levels #40 mg/dL and O40 mg/dL were similar regarding comorbid conditions such as chronic renal failure, chronic obstructive pulmonary disease, peripheral vascular disease, congestive heart failure and history of stroke. ST segment elevation myocardial infarction and unstable angina as baseline clinical presentation were significantly higher in patients with HDL-C levels #40 mg/dL ( p 5 0.023 and 0.009, respec-
737
tively) whereas they were less in exertional angina ( p 5 0.016) compared to patients with HDL-C O40 mg/dL. History of prior coronary artery bypass graft surgery or PCI was similar between patients with HDL-C levels #40 mg/dL and O40 mg/dL. No significant difference was observed between groups for baseline medications (Table 1). Genotype frequency distributions of the ABCA1 gene A2589G and G3456C polymorphisms for the total study group, patients with HDL #40 mg/dL and O40 mg/dL were compared in Table 2. The sequence variant (GG) of the ABCA1 gene A2589G polymorphism was found in 21 patients (3.8%) within the total population, in nine patients (3.6%) within the HDL-C #40 mg/dL group and in 12 (4%) individuals of the HDL-C levels O40 mg/dL group. Frequency distributions of the A2589G genotypes was not significantly different among groups (c2 5 3.524, p 5 0.172) (Table 2). The ABCA1 A2589G A allelic frequency has been found to be 0.73, G allelic frequency 0.27 in the total population. The sequence variant (CC) of the ABCA1 gene G3456C polymorphism was found in 52 subjects (5.9%) in the total population, 17 subjects (6.8%) of the HDL-C #40 mg/dL group and in 35 (11.6%) individuals of the HDL-C levels O40 mg/dL group. Frequency distributions of the G3456C genotypes was not significantly different among groups (c2 5 4.005, p 5 0.135) (Table 2). For G3456C polymorphism, frequencies of G and C alleles were 0.72 and 0.28, respectively, in the total population. To test whether the ABCA1 gene A2589G and G3456C single nucleotide polymorphisms could be associated with any lipid parameter, we compared serum lipid levels between ABCA1 A2589G and G3456C (Table 3) genotypes for CAD patients with HDL-C #40 mg/dL. Because statin medication may influence levels of cholesterol parameters, this medication was included as a covariate in the statistical analysis. The association analysis was performed both for HDL #40 mg/dL group and in the total population No significant difference was observed for the analyzed lipid variables when A2589G genotypes were compared (Table 3). We investigated gender-specific effects of ABCA1 gene variants; however, we could not find any significant effect on serum lipid parameters. Although only in males, a borderline significant ( p 5 0.066) influence of ABCA1 gene 2589GG genotype on HDL-C levels were found. In detail, male CAD patients with GG genotype had the lowest HDLC levels (35.9 mg/dL) and the AG genotype had the highest HDL-C levels (42 mg/dL) (data not included). LDL-C ( p 5 0.046) and HDL-C ( p 5 0.02) levels were found to differ among ABCA1 gene G3456C genotypes in CAD patients with HDL-C levels #40 mg/dL (Table 3). In detail, LDL-C ( p 5 0.046) levels were found to be elevated, whereas HDL-C levels were found to be decreased in CC genotype carriers when compared to GG and GC genotypes (Table 3) in CAD patients with HDL-C #40 mg/dL. Age, gender, body mass index, waist/hip ratio, HDL-C levels #40 mg/dL, family history for premature CAD,
738
Catakoglu et al./ Archives of Medical Research 39 (2008) 735e742
Table 1. Baseline clinical characteristics Coronary artery disease patients
Age, years Male/Female LDL cholesterol, mg/dL Total cholesterol, mg/dL VLDL cholesterol, mg/dL Triglyceride, mg/dL Hypertension, n (%) Diabetes mellitus, n (%) Type I diabetes mellitus, n (%) BMI, kg/m2 W/H ratio Active smoker, n (%) Family history, n (%) Menopause, n (% among women) Sedentary lifestyle, n (%) COPD, n (%) CRF, n (%) Exertional angina, n (%) ST segment elevation MI, n (%) Unstable angina, n (%) Silent ischemia, n (%) Peripheral vascular disease, n (%) Prior CABG, n (%) Prior MI, n (%) Prior PCI, n (%) Stroke or TIA, n (%) CHF, n (%) Baseline medications, n (%) Beta blocker ACE inhibitor Diuretic Aspirin Statin
Total population (n 5 452)
HDL-C #40 mg/dL (n 5 251)
HDL-C O40 mg/dL (n 5 301)
p value
61.00 10.4 356/196 121.7 1.7 194.8 1.9 32.2 0.9 153.1 3.9 167 (30.3) 73 (13.2) 12 (2.2) 29.8 0.9 0.98 0.02 77 (13.9) 98 (17.8) 57 (10.3) 46 (8.3) 16 (2.9) 3 (0.5) 101 (18.3) 27 (4.9) 51 (9.2) 40 (7.2) 11 (2) 39 (5.4) 64 (11.6) 34 (6.3) 12 (2.2) 16 (2.9)
59.2 10.7 188/63 118.8 2.5 187.0 2.8 36.8 1.5 177.0 6.4 70 (27.9) 38 (15.1) 7 (2.8) 29.92 6.34 1 0.08 47 (18.7) 52 (20.7) 17 (26.9) 21 (8.4) 6 (2.24) 1 (0.4) 35 (19.9) 18 (7.2) 32 (12.7) 13 (5.2) 7 (2.8) 17 (6.9) 39 (15.5) 17 (6.9) 4 (1.6) 11 (4.4)
62.3 10.1 168/133 124.2 2.3 201.3 2.7 28.2 1.1 133.1 4.8 97 (32.2) 35 (11.6) 5 (1.7) 29.68 17.17 0.98 0.15 30 (10) 46 (15.3) 40 (30.1) 25 (8.3) 10 (3.3) 2 (0.7) 66 (21.9) 9 (3) 19 (6.3) 27 (9) 4 (1.3) 13 (4.5) 25 (8.3) 17 (5.6) 8 (2.7) 5 (1.7)
0.028 0.0001 0.106 0.0001 0.0001 0.0001 0.269 0.228 0.366 0.896 0.599 0.027 0.096 0.728 0.979 0.516 0.672 0.016 0.023 0.009 0.087 0.222 0.260 0.008 0.599 0.393 0.058
135 102 35 156 216
(24.5) (18.5) (6.3) (28.3) (39.1)
68 49 19 74 96
(27.1) (19.5) (7.6) (29.5) (38.2)
67 53 16 82 120
(22.3) (17.6) (5.3) (27.2) (39.9)
0.188 0.564 0.279 0.561 0.698
p value corresponds to the comparison between patients with HDL-C #40 mg/dL and O40 mg/dL. ACE, angiotensin converting enzyme; CABG, coronary artery bypass graft; CHF, congestive heart failure; COPD, chronic obstructive pulmonary disease; CRF, chronic renal failure; MI, myocardial infarction; PCI, percutaneous coronary intervention; TIA, transient ischemic attack; BMI, body mass index; W/H, wasit/hip.
diabetes mellitus, smoking, hypercholesterolemia, hypertension, congestive heart failure, ABCA1 A2589G and G3456C genotypes were selected as conventional risk factors to be analyzed for cardiovascular events (myocardial infarction, unstable angina, percutaneous coronary intervention or CABG and stroke) and for peripheral artery disease in multiple logistic regression analysis (Table 4). We were unable to detect any significant association of A2589G and G3456C genotypes with the cardiovascular events analyzed. Discussion This study reports the association of ABCA1 SNPs with HDL-C levels and CAD in 552 Turkish patients. The analysis is based on both categorically classified HDL-C levels (#40 mg/dL and O40 mg/dL) (24) and in the total study
population. Although no clear association is found, the analyzed ABCA1 SNPs are likely to contribute a small increment to HDL-C levels. In the Turkish Adult Risk Factor Study, the mean values for HDL-C were reported to be 37 12 mg/dL in men and 45 13 mg/dL in women (25). The stated levels were 20% lower than those provided by NHANES III for the U.S. Caucasian population (26) and for Germans (27). In our study, HDL-C levels were 43 12 mg/dL for the entire study group (41 11 for males and 47 12 for females). Investigations on the function of ABCA1 have revealed its role in 1) engulfment of apoptotic cells by macrophages (28,29); 2) macrophage interleukin-1b secretion (30); 3) modulation of apolipoprotein-mediated cellular lipid efflux (31); and 4) caveolar processing (32). Due to its diverse functions, ABCA1 is an attractive candidate gene for the modulation of plasma HDL-C
ATP-binding Cassette Transporter 1 Gene, HDL-Cholesterol and CAD
739
Table 2. ABCA1 gene A2589G and G3456C genotype and allele frequencies in coronary artery disease patients with HDL-C #40 mg/dL, HDL-C O40 mg/dL and total population ABCA1 gene A2589G polymorphism
CAD patients with HDL-C #40 mg/dL CAD patients with HDL-C O40 mg/dL Total population
AA, n (%)
AG, n (%)
GG, n (%)
A
G
136 (54.2%) 139 (46.2%) 275 (49.6%)
106 (42.2%) 150 (49.8%) 256 (46.6%)
9 (3.6%) 12 (4%) 21 (3.8%)
0.75 0.71 0.73
0.25 0.29 0.27
ABCA1 gene G3456C polymorphism
CAD patients with HDL-C #40 mg/dL CAD patients with HDL-C O40 mg/dL Total population
GG, n (%)
GC, n (%)
CC, n (%)
G
C
135 (53.8%) 159 (52.8%) 294 (53.3%)
99 (39.4%) 107 (35.5%) 206 (37.3%)
17 (6.8%) 35 (11.6%) 52 (9.4%)
0.74 0.71 0.72
0.26 0.29 0.28
Results are expressed as number (percentage) for genotypes, and allelic frequencies were presented for both polymorphisms. The ABCA1 A2589G genotype and G3456C genotype frequencies of the study groups (CAD patients with HDL-C #40 mg/dL and CAD patients with HDL-C O40 mg/dL) with c2 test were respectively as c2 5 3.524, p 5 0.172 and c2 5 4.005, p 5 0.135.
levels in the general population as well. Rubins et al. have determined the frequencies of three common variants within the coding sequence of ABCA1 (G596A, A2589G and G3456C) in men participating in the Veterans Affairs Cooperative HDL Cholesterol Intervention Trial (VA-HIT), a population comprised of men having low HDL-C and CHD (33). In view of the subject composition of our study, we believed that this would be an ideal population for the identi-
fication of ABCA 1 variants and, ultimately, for associations to be made with plasma lipids and/or CAD endpoints. In the present study we analyzed G3456C and A2589G polymorphisms in the gene encoding ABCA1, a member of the family of ATP-binding cassette transporters (34). Both variants affect amino acids that are either conserved between human and mouse or are very similar as in the case of A2589G (isoleucine and valine, respectively), suggesting that each has an important functional role. The A2589C region is located
Table 3. Serum lipids comparisons by ABCA1 gene A2589G and G3456C genotypes in all coronary artery disease patients and in patients with HDL-C levels #40 mg/dL with covariance analysis ABCA1 gene A2589G polymorphism Patients Total-C LDL-C HDL-C VLDL-C TG
All HDL-C#40 All HDL-C#40 All HDL-C#40 All HDL-C#40 All HDL-C#40
mg/dL mg/dL mg/dL mg/dL mg/dL
AA 194.2 183.1 121.9 115.7 42.3 33.5 32.2 36 153.9 169.8
AG
46.3 (275) 43.2 (136) 39.5 (275) 36.6 (136) 10.9 (275) 4.9 (136) 20.3 (275) 21.4 (136) 91.3 (275) 91.1 (136)
194.7 191.5 120.9 121.9 44.3 33.9 32.0 38.1 151.8 184.6
46.1 (256) 45.9 (106) 39.9 (256) 41.1 (106) 12.2 (256) 4.1 (106) 21.6 (256) 22.2 (106) 93.9 (256) 104.8 (106)
GG 202.4 193.9 128.6 127.8 43.5 30.8 32.5 39.2 158.9 196.4
47.4 (21) 59.1 (9) 39.4 45.0 (9) 13.1 5.8 (9) 25.1 34.9 (9) 123.0 173.9 (9)
p value O0.05 O0.05 O0.05 O0.05 O0.05 O0.05 O0.05 O0.05 O0.05 O0.05
ABCA1 gene G3456C polymorphism Patients Total-C LDL-C HDL-C VLDL-C TG
All HDL-C All HDL-C All HDL-C All HDL-C All HDL-C
#40 mg/dL #40 mg/dL #40 mg/dL #40 mg/dL #40 mg/dL
GG 192.6 180.7 119.8 113.2 42.9 33.3 31.5 36.1 151.5 171.2
46.7 (294) 45.2 (135) 40.3 (294) 39.6 (135) 11.4 (294) 4.8 (135) 18.2 (294) 19.9 (135) 80.9 (294) 81.8 (135)
GC 197.3 194.5 123.8 124.7 43.2 34.3 33.8 38.2 157.8 184.4
47.2 (206) 45.1 (99) 39.5 (206) 37.8 (99) 11.4 (206) 4.2 (99) 24.7 (206) 24.7 (99) 107.7 (206) 117.2 (99)
CC 197.5 193.7 124.6 128.6 45.7 31.2 29.5 36.1 143.2 180.1
38.9 (52) 36.2 (17) 36.0 (52) 33.3 (17) 13.1 (52) 4.9 (17) 21.1 (52) 26.6 (17) 101.9 (52) 132.2 (17)
p value O0.05 O0.05 O0.05 O0.05 O0.05 !0.05 O0.05 O0.05 O0.05 O0.05
Values are mean SD. p values are for A2589G and G3456C genotype effect. Statin medication was included as covariate in the statistical analysis.
Catakoglu et al./ Archives of Medical Research 39 (2008) 735e742
740
Table 4. Risk factors identified to be associated with low HDL cholesterol level by multiple logistic regression analysis All Males BMI Active smoking habit Hypertension DM ABCA1 A2589G (AA) (AG) (GG) ABCA1 G3456C (GG) (GC) (CC)
b
SE
OR
p
1.038 0.003 0.126 0.049 0.384 0.017 0.330 0.347 0.224 0.805 1.029
0.365 0.011 0.239 0.165 0.176 0.363 0.338 0.580 0.319 0.307 0.475
2.823 1.003 0.882 1.050 0.681 0.983 0.719 1.415 1.251 2.237 0.357
0.004 0.747 0.600 0.769 0.030 0.962 0.329 0.550 0.482 0.009 0.030
Multivariate logistic regression model contained male gender, body mass index (BMI), smoking habit, hypertension, diabetes mellitus (DM) and ABCA1 genotype variables. b indicates estimated coefficient; SE, standard error; OR, adjusted odds ratio.
proximal to the sixth subdomain within the first group of transmembranous domains, adjacent to the putative glycosylation site of ABCA1. Amino acid substitution at A2589C region may cause conformational changes around this portion, resulting in altering the state of glycosylation of the transporter and enhancing cholesterol efflux. Harada et al. did not find the A2589C variant to be associated with susceptibility to CAD, although an inverse relationship between plasma HDL-C level and incidence of CAD was shown (35). Other studies suggested that both the A2589C and the variant may be associated with susceptibility to CAD. These discrepancies may be at least partially attributed to environmental or social factors or racial differences. In light of previous reports by Imai et al. (36) and others (19) showing the existence of large interracial differences in the frequency of genetic variations and disease susceptibilities, it is possible that the discrepancy may be caused, at least partially, by methodological factors such as sampling schemes or trial size. Previous studies in other ethnic groups have shown that variations in the coding region of ABCA1 affect plasma lipid level. Wang et al. (19) noted a wide range of allelic frequencies in the A2589G genotype across six ethnic groups (Caucasian, South Asians, Inuit, Africans, Oji-Cree, and Chinese), each group comprising a small number of subjects. They demonstrated that the A2589G variant contributes to increased level of HDL-C only in Canadian Inuits probably due to social or ethnic difference. Kakko et al.18 reported A2589G genotypes contributing to |3% of the variation in plasma HDL-C levels in women and suggested ABCA1 locus as of minor importance for the regulation of HDL-C. In contrast, Brousseau et al. reported A2589G polymorphism not to be significantly associated with plasma HDL-C concentrations in men with low HDL-C levels having CAD (17). Our data, derived from the Turkish population, showed consistent alterations in lipid profile associated with A2589G variant.
Brousseau et al. (17) demonstrated that carriers of the G3456C variant were at increased risk for adverse CHD endpoints relative to noncarriers within the same population. The G3456C polymorphism affects the highly charged linker region of ABCA1, a site rich in potential phosphorylation sites (37). This region is very similar to that of the regulatory domain of another ABC transporter, the cystic fibrosis transmembrane regulator, where mutations responsible for cystic fibrosis have been localized (38). Hence, it is reasonable that this ABCA1 variant may influence function, confirmed by our demonstration of decreased cellular cholesterol efflux in homozygotes for the mutant C allele of this variant. In view of the role of ABCA1 in the modulation of HDL-C concentrations and lipid efflux, we also performed analyses to test for potential associations between each common variant and plasma lipid profiles. Two statistically significant associations between plasma lipids and ABCA1 G3456C variants were observed in our study group. The mutant C allele of the G3456C variant was associated with increased plasma LDL-C and decreased HDL-C concentrations. The G3456C variant of ABCA1 has been shown to be associated with a significantly increased risk for CAD endpoints, which suggests a role of this variant in the premature CAD observed in the Finn population (17). In our study, similar to results of Brousseau et al. (17), we found a significant association of G3456C variant with both HDL-C and LDLC concentrations. However, we were not able to demonstrate a significant risk for CAD endpoints with G3456C variation. The A2589G polymorphism of ABCA1 was not associated with any of the analyzed lipid parameters. One possible explanation for this may be because HDL-C levels are modulated by other genetic and environmental factors (39,40). Direct effects of ABCA1 A2589G polymorphism on HDL-C levels may not be readily apparent. Because our study population consists of 13e17% of patients with type 1 and type 2 diabetes, this is likely to confound HDL-C levels and is one of the restrictions of the study. Another restriction of this study is that the presence of pre- and postmenopausal women will introduce further confounding. There appears to be a large difference in the distribution of different SNP genotypes among ethnic groups. Concerning ABCA1 variants, the M 823 allele has been shown to be common in Japanese (allele frequency 5 0.507). In comparison, the M 823 allele frequency was shown to vary widely among other ethnic groups as follows: Caucasian, 0.079; South Asians, 0.145; Inuit, 0.294; Africans, 0.419; Oji-Cree, 0.690; and Chinese, 0.737, respectively (19). In the present study the 2589G and 3456C allele frequencies were found as 0.270 and 0.281, respectively. The influence of diet and environmental factors on lipid profile may differ widely among study groups. We found a significant association between the G3456C variant of the ABCA1 gene and both serum LDL-C and HDL-C levels in our study population with CAD who have
ATP-binding Cassette Transporter 1 Gene, HDL-Cholesterol and CAD
HDL-C levels #40 mg/dL. However, we could not find any association of these variants with outcomes of CAD. In conclusion, the frequency distributions of neither the A2589G nor the G3456C genotypes were not found to differ significantly among groups. The A2589G genotypes were not found to be effective over the analyzed lipid parameters. In CAD patients with HDL-C #40 mg/dL, among G3456C genotypes the LDL-C levels were elevated, whereas HDL-C levels decreased in CC genotype carriers compared to GG and GC. No significant association was found between cardiovascular events and ABCA1 gene A2589G and G3456C genotypes. We could not suggest a role of A2589G or G3456C variants in the CAD endpoints observed in this population. References 1. Castelli WP, Garrison RJ, Wilson PW, Abbott RD, Kalousdian S, Kannel WB. Incidence of coronary heart disease and lipoprotein cholesterol levels. The Framingham Study. JAMA 1986;256:2835e2838. 2. Kinosian B, Glick H, Garland G. Cholesterol and coronary heart disease: predicting risks by levels and ratios. Ann Intern Med 1994;121:641e647. 3. Onat A, Surdum-Avci G, Senocak M, Ornek E, Go¨zu¨kara Y. Plasma lipids and their interrelationship in Turkish adults. J Epidemiol Community Health 1992;46:470e476. 4. Ridker PM, Glynn RJ, Hennekens CH. C-reactive protein adds to the predictive value of total and HDL cholesterol in determining risk of first myocardial infarction. Circulation 1998;97:2007e2011. 5. Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershatz H, Kannel WB. Prediction of coronary heart disease using risk factor categories. Circulation 1998;97:1837e1847. 6. Mahley RW, Palaog˘lu KE, Atak Z, Dawson-Pepin J, Langlois AM, Cheung V, et al. Turkish Heart Study: lipids, lipoproteins, and apolipoproteins. J Lipid Res 1995;36:839e859. 7. Breckenridge WC, Little JA, Alaupovic P, Wang CS, Kuksis A, Kakis G, et al. Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase. Atherosclerosis 1982;45:161e179. 8. Kuivenhoven JA, de Knijff P, Boer JM, Smalheer HA, Botma GJ, Seidell JC, et al. Heterogeneity at the CETP gene locus. Influence on plasma CETP concentrations and HDL cholesterol levels. Arterioscler Thromb Vasc Biol 1997;17:560e568. 9. Reymer PW, Gagne E, Groenemeyer BE, Zhang H, Forsyth I, Jansen H, et al. A lipoprotein lipase mutation (Asn291Ser) is associated with reduced HDL cholesterol levels in premature atherosclerosis. Nat Genet 1995;10:28e34. 10. Kuivenhoven JA, Pritchard H, Hill J, Frohlich J, Assmann G, Kastelein J. The molecular pathology of lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes. J Lipid Res 1997;38:191e205. 11. Batal R, Tremblay M, Krimbou L, Mamer O, Davignon J, Genest J Jr, et al. Familial HDL deficiency characterized by hypercatabolism of mature apoA-I but not proapoA-I. Arterioscler Thromb Vasc Biol 1998;18:655e664. 12. Murray CJ, Lopez AD. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet 1997;349:1269e1276. 13. Bodzioch M, Orso´ E, Klucken J, Langmann T, Bo¨ttcher A, Diederich W, et al. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 1999;22:347e351. 14. Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, et al. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 1999;22:336e345. 15. Remaley AT, Rust S, Rosier M, Knapper C, Naudin L, Broccardo C, et al. Human ATP-binding cassette transporter 1 (ABC1): genomic
16.
17.
18.
19.
20. 21.
22.
23.
24. 25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
741
organization and identification of the genetic defect in the original Tangier disease kindred. Proc Natl Acad Sci USA 1999;96:12685e12690. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, et al. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 1999;22:352e325. Brousseau ME, Bodzioch M, Schaefer EJ, Goldkamp AL, Kielar D, Probst M, et al. Common variants in the gene encoding ATP-binding cassette transporter 1 in men with low HDL cholesterol levels and coronary heart disease. Atherosclerosis 2001;154:607e611. Kakko S, Kelloniemi J, von Rohr P, Hoeschele I, Tamminen M, Brousseau ME, et al. ATP-binding cassette transporter A1 locus is not a major determinant of HDL-C levels in a population at high risk for coronary heart disease. Atherosclerosis 2003;166:285e290. Wang J, Burnett JR, Near S, Young K, Zinman B, Hanley AJ, et al. Common and rare ABCA1 variants affecting plasma HDL cholesterol. Arterioscler Thromb Vasc Biol 2000;20:1983e1989. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974;20:470e475. Foster LB, Dunn RT. Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method. Clin Chem 1973;19:338e340. Warnick GR, Benderson J, Albers JJ. Dextran sulfate-Mg2þ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem 1982;28:1379e1388. Cheung MC, Albers JJ. The measurement of apolipoprotein A-I and A-II levels in men and women by immunoassay. J Clin Invest 1977; 60:43e50. Rader DJ. Pathophysiology and management of low high-density lipoprotein cholesterol. Am J Cardiol 1999;83:22Fe24F. Onat A, Yıldırım B, Uslu N. Plasma lipoproteins and apolipoproteins in Turkish adults: overall levels, association with other risk parameters, and HDL’s role as a marker of coronary risk in women. Arch Turk Soc Cardiol 1999;27:72e79. Johnson CL, Rifkind BM, Sempos CT, Carroll MD, Bachorik PS, Briefel RR, et al. Declining serum total cholesterol levels among US adults. The National Health and Nutrition Examination Surveys. JAMA 1993;269:3002e3008. Assmann G, Cullen P, Schulte H. The Munster Heart Study (PROCAM). Results of follow-up at 8 years. Eur Heart J 1998;19(Suppl A):A2e11. Tanaka AR, Ikeda Y, Abe-Dohmae S, Arakawa R, Sadanami K, Kidera A, et al. Human ABCA1 contains a large amino-terminal extracellular domain homologous to an epitope of Sjogren’s Syndrome. Biochem Biophys Res Commun 2001;283:1019e1025. Luciani MF, Chimini G. The ATP binding cassette transporter ABC1 is required for the engulfment of corpses generated by apoptotic cell death. EMBO J 1996;15:226e235. Hamon Y, Luciani MF, Becq F, Verrier B, Rubartelli A, Chimini G. Interleukin-1b secretion is impaired by inhibitors of the ATP binding cassette transporter, ABC1. Blood 1997;90:2911e2915. Langmann T, Klucken J, Reil M, Liebisch G, Luciani MF, Chimini G, et al. Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages. Biochem Biophys Res Commun 1999;257:29e33. Orso E, Broccardo C, Kaminski WE, Bo¨ttcher A, Liebisch G, Drobnik W, et al. Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and ABC1-deficient mice. Nat Genet 2000;24:192e196. Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, et al. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. N Engl J Med 1999;341:410e418. Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol 1992;8:67e113.
742
Catakoglu et al./ Archives of Medical Research 39 (2008) 735e742
35. Harada T, Imai Y, Nojiri T, Morita H, Hayashi D, Maemura K, et al. A common Ile 823 Met variant of ATP-binding cassette transporter A1 gene (ABCA1) alters high density lipoprotein cholesterol level in Japanese population. Atherosclerosis 2003;169:105e112. 36. Imai Y, Morita H, Kurihara H, Sugiyama T, Kato N, Ebihara A, et al. Evidence for association between paraoxonase gene polymorphisms and atherosclerotic diseases. Atherosclerosis 2000;149:435e442. 37. Luciani MF, Denizot F, Savary S, Mattei MG, Chimini G. Cloning of two novel ABC transporters mapping on human chromosome 9. Genomics 1994;21:150e159.
38. Welsh MJ, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 1993;73:1251e1254. 39. Heller DA, de Faire U, Pedersen NL, Dahlen G, McClearn GE. Genetic and environmental influences on serum lipid levels in twins. N Engl J Med 1993;328:1150e1156. 40. Schaefer EJ, Lamon-Fava S, Ordovas JM, Cohn SD, Schaefer MM, Castelli WP, et al. Factors associated with low and elevated plasma high density lipoprotein cholesterol and apolipoprotein A-I levels in the Framingham Offspring Study. J Lipid Res 1994;35: 871e882.