A polymorphism in the ABCG1 promoter is functionally associated with coronary artery disease in a Chinese Han population

Atherosclerosis 219 (2011) 648–654

Contents lists available at ScienceDirect

Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

A polymorphism in the ABCG1 promoter is functionally associated with coronary artery disease in a Chinese Han population Yan Xu a , Wei Wang a , Li Zhang a , Li-Ping Qi a , Li-Yun Li a , Lian-Feng Chen a , Quan Fang a , Ai-Min Dang b,∗∗ , Xiao-Wei Yan a,∗ a

Department of Cardiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Shuaifuyuan, Dong Cheng District, Beijing 100730, China b Division of Hypertension, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 167 Beilishi Road, Beijing 100037, China

a r t i c l e

i n f o

Article history: Received 20 November 2010 Received in revised form 9 May 2011 Accepted 22 May 2011 Available online 17 June 2011 Keywords: ABCG1 Coronary artery disease HDL SNP Promoter

a b s t r a c t Objective: In this study, we examine the association of single nucleotide polymorphisms (SNPs) of the human ATP binding cassette transporter G1 (ABCG1) gene with atherosclerotic coronary artery disease (CAD) in a Chinese Han population. Methods: 1021 patients with CAD and 1013 unaffected control subjects were enrolled. PCR-based ligation detection reaction (PCR-LDR) method was used to genotype four SNPs of ABCG1, three (rs2234714, rs2234715 and rs57137919) in the promoter region and one (rs1044317) in the 3 -untranslated region (UTR). Results: The human ABCG1 −367G>A polymorphism (rs57137919) showed a significantly decreased risk for CAD and myocardial infarction (MI) in a dominant model (adjusted OR = 0.73, p = 0.033 for CAD, and adjusted OR = 0.65, p = 0.014 for MI, respectively). The rs57137919 also showed an association with angiographic severity of CAD (multi-vessel vs. single-vessel CAD, adjusted OR = 0.40, p = 0.005). The findings were further supported by luciferase reporter assay, in which the polymorphism impaired reporter gene expression. The ABCG1 −768G>A polymorphism (rs2234714) showed an association with CAD in a recessive model (adjusted OR = 0.64, p = 0.015), but did not demonstrate a functional influence on reporter gene expression in the luciferase reporter assay. Conclusions: The SNP rs57137919 in the ABCG1 promoter region is functionally associated with a reduced risk of CAD in a Chinese Han population. © 2011 Published by Elsevier Ireland Ltd.

1. Introduction Coronary artery disease (CAD) is the most common type of heart disease. The disease causes more than 7 million deaths per year in the world, and remains the leading cause of death worldwide [1]. Despite extensive research efforts have been made, the underlying mechanisms remain not clear. A pathological hallmark of atherosclerotic lesion is the excessive accumulation of cholesterol in macrophages, leading to their transformation into foam cells [2], which is caused by imbalance of cholesterol influx and efflux. High density lipoprotein (HDL) is widely considered to protect against atherosclerosis and CAD through its role in cholesterol efflux, in which accumulated choles-

∗ Corresponding author. Tel.: +86 10 65295060; fax: +86 10 65295060. ∗∗ Co-corresponding author. Tel.: +86 10 88398039; fax: +86 10 88396323. E-mail addresses: [email protected] (A.-M. Dang), xswy [email protected] (X.-W. Yan). 0021-9150/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2011.05.043

terol is removed from vascular macrophages by several cellular cholesterol and phospholipids transporters including ABCA1 and ABCG1 [3]. Therefore fewer macrophages are transformed into foam cells. ABCA1 and ABCG1 both belong to the ABC superfamily, a group of cellular transmembrane proteins that mediate the ATPdependent lipid transport [4]. Previous studies identified both transporters as regulators of cholesterol efflux, although they may have different roles in the process. ABCA1 was showed to transport cholesterol to lipid-depleted apoA-I, forming pre-␤-HDL [5,6], whereas ABCG1 transports cellular cholesterol to the major HDL fractions (HDL3 and HDL2 ) [7,8] and to low density lipoprotein (LDL) [3]. ABCG1 deficiency in mice fed with a high-cholesterol diet caused massive lipid accumulation in multiple tissues [8]. Although ABCG1 plays a critical role in regulating cholesterol efflux and lipid homeostasis, its functional significance in the development of atherosclerosis remains unknown. Out et al. reported that macrophage ABCG1 deficiency led to a moderate increase in atherosclerosis in hyperlipidemic LDLr−/− mice [9,10]. In contrast,

Y. Xu et al. / Atherosclerosis 219 (2011) 648–654

Baldan et al. [11] and Ranalletta et al. [12] reported that ABCG1−/− bone marrow derived cells in LDLr−/− mice reduced atherosclerosis. More recently, Burgess et al. showed that overexpression of human ABCG1 in apoE−/− hyperlipidemic mice did not attenuate atherosclerosis development [13]. Thus, the role of ABCG1 in the development of atherosclerosis remains to be clarified. In a recent genetic association study, Furuyama et al. found that ABCG1 promoter −257T>G polymorphism was associated with an increased risk of CAD in 109 Japanese male patients [14]. This study suggests a possibility that altered ABCG1 expression may contribute to CAD in humans. In this study, we investigate whether the SNPs in the ABCG1 promoter region (rs2234714, rs2234715 and rs57137919) and 3 UTR (rs1044317) are associated with the development of CAD in a Chinese Han population.

2. Materials and methods 2.1. Study population A total of 1021 patients with CAD were recruited from hospitalized patients in Peking Union Medical College Hospital (PUMCH) (Beijing, China) from July 2007 to April 2010. Patients eligible for the study included individuals who survived myocardial infarction (MI) (n = 626) or had angina pectoris and documented coronary stenosis ≥ 50% in a major epicardial artery by coronary angiography. Additionally, angiographic severity of disease was defined as 0-, single- or multi-vessel disease based on the number of involved artery (luminal narrowing ≥ 50%) in the three major coronary arteries [15]. The diagnosis of MI was based on clinical symptoms and electrocardiographic changes, as well as increases in the serum markers including creatinine kinase-MB and troponin T. The diagnosis was confirmed by the presence of a wall-motion abnormality on left ventriculography as well as by identification of the culprit lesions in any of the major coronary arteries by coronary angiography. Subjects with a history of hematologic, neoplastic, renal, liver, or thyroid diseases were excluded. Patients who had congenital heart disease, cardiomyopathy, valvular disease, or autoimmune disease were also excluded. A total of 1013 control subjects were consecutively recruited from Physical Examination Center of PUMCH during the same period when CAD patients were recruited. The unaffected controls were judged to be free of CAD by questionnaires, medical history, clinical examination and electrocardiography. A set of questionnaires that included details of medical history, family history of CAD, drug intake, and cigarette smoking was completed. Blood pressure, body weight and height were recorded, and body mass index was derived from these data. The study protocol was approved by the Ethical and Protocol Review Committee of PUMCH. Each participant provided the informed consent.

2.2. SNP selection and genotyping We identified 10 SNPs through PCR screening for the promoter region and 3 -UTR of the ABCG1 gene in 20 CAD patients and 20 non-CAD controls. Based on the linkage disequilibrium analysis result, we chose rs2234714, rs2234715, rs57137919 and rs1044317 as tag-SNPs for subsequent large-scale genotyping experiments (Supplementary Figure 1). There was no obvious LD between any two SNPs. Genomic DNA was genotyped using PCR-based Ligation Detection Reaction (PCR-LDR) method [16], with ABI Prism 3730XL DNA sequencer according to the manufacturer’s protocol.

649

2.3. Construction of luciferase reporter gene and luciferase activity assay Human ABCG1 promoter constructs were generated by PCR amplification of a 1007-bp fragment of human ABCG1 promoter (−957/+50) from genomic DNA, and subcloned into pGL3-basic vectors with firefly luciferase gene (Promega, WI, USA). Human embryonic kidney (HEK) 293T cells and murine macrophage RAW264.7 cells were cultured in DMEM with 10% FBS (GIBCO). Human macrophage-like THP-1 cells were cultured in RPMI-1640 with 10% FBS, and incubated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) for 3 days for monocytic differentiation before transfection. Cells became attached as macrophages through differentiation. Transfection was conducted in cultured HEK293T cells, RAW264.7 and THP-1 cells respectively using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA), as described previously [17]. The empty vector (pGL3-basic) with no gene insertion was used as a negative control, and pGL3-control vector (Promega) was used as a positive control in the transfection experiments. Thirty-six hours after transfection, luciferase activities were measured using a dual-luciferase reporter (DLR) assay system (Promega) with modulus microplate multimode reader (Turner BioSystems, CA, USA). 2.4. ABCG1 protein expression in CAD patients with different genotypes Three CAD subjects were selected for each group based on the genotype of the rs57137919 (GG, AG or AA). Blood was drawn for monocytes isolation and genotyping. ABCG1 protein expression in macrophages was analyzed by Western blotting using a rabbit monoclonal ABCG1 antibody (abcam, USA). 2.5. Statistical analysis Differences in study characteristics were tested through non-parametric Mann–Whitney U-test for continuous variables. Differences between categorical variables, genotype/allele frequencies and Hardy–Weinberg equilibrium (HWE) were tested by chi-squared analysis or Fisher’s exact test. Independence of associations between SNPs and CAD or MI was assessed after adjustment for other potentially confounding factors (age, gender, hypertension, type 2 diabetes, smoking, HDL-C and TG) using multiple logistic regression analysis and calculating the adjusted odds ratios and their 95% confidence intervals. All data were analyzed using SPSS version 13.0 software package. Results were considered to be statistically significant if bilateral p-values were less than 0.05. The pattern of pair-wise linkage disequilibrium between the selected SNPs was measured by D and r2 using the SHEsis software [18]. Haplotype frequency was determined by means of the algorithms implemented in the PHASE program (version 2.1). Retrospective statistical powers were calculated using Epi InfoTM software (version 3.5.1). The study had more than 80% power to detect the differences between case and control subjects with an OR of less than 1.45 at a significant level of 0.05. More detailed methods, along with supplementary results, are available in the supplementary materials. 3. Results 3.1. Characteristics of the subjects Clinical characteristics of patients with CAD (n = 1021) or MI (n = 626) and normal controls (n = 1013) are summarized in Table 1. Age and gender difference were significant between CAD patients and control subjects. Compared with the control group, more

650

Y. Xu et al. / Atherosclerosis 219 (2011) 648–654

Table 1 Characteristics of the study cohorts.a Characteristic

Controls (n = 1013)

Sex (male/female) Age (years) BMI Hypertension (%) T2DM (%) Smokers (%) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) Glucose (mmol/L) SBP (mm Hg) DBP (mm Hg)

676/337 60 (55; 67) 24.8 (22.9; 27.4) 21.0 7.0 13.4 4.85 (4.33; 5.37) 1.2 (0.89; 1.59) 1.31 (1.11; 1.54) 3.04 (2.55; 3.42) 5.2 (4.8; 5.6) 126 (117; 139.5) 78 (72; 85)

pb vs. controls

Patients CAD (n = 1021)

MI (n = 626)

CAD

MI

750/271 62 (54; 71) 25.5 (23.8; 27.8) 66.7 38.3 56.0 4.26 (3.66; 5.00) 1.44 (1.07; 2.03) 1.07 (0.91; 1.28) 2.46 (2.0; 3.14) 5.6 (5.0; 7.0) 130 (120; 140) 75 (70; 80)

479/147 62(54; 71) 25.6 (23.8; 28.0) 62.9 41.2 58.3 4.17 (3.4; 4.93) 1.44 (1.06; 2.0) 1.05 (0.9; 1.24) 2.43 (1.99; 3.06) 5.7 (5.0; 7.3) 130 (120; 140) 74 (70; 80)

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

BMI, body mass index (kg/m2 ); T2DM, type 2 diabetes mellitus; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. a Median (interquartile range). b p < 0.001 obtained in the comparison between CAD or MI patients and controls using Mann–Whitney U-test. MI was a subgroup of CAD.

patients in CAD or MI groups were smokers, had hypertension and T2DM. The patient group also had significant higher BMI, serum triglyceride levels and fasting glucose levels, but lower levels of HDL-C, LDL-C, total cholesterol and lower diastolic blood pressures, which were possibly related to cholesterol-lowering and antihypertensive medications in this group. The percentages of subjects reportedly taking cholesterol-lowering medications were 51.4% in the patient group, and 2.1% in the control group. The main cholesterol-lowering drugs used by patients were statins and, on average, the medication was taken for >20 months. 3.2. Association of ABCG1 SNPs with CAD Genotype distributions of all studied SNPs in both groups followed HWE (p > 0.05). Associations between all four SNPs and CAD were analyzed through logistic regression analysis after adjusting for age, gender, hypertension, T2DM, smoking, HDL-C and TG as covariates (Table 2). Among 4 SNPs, the rs2234714 and rs57137919 showed significant differences in allele frequency and genotype distribution between CAD patients and control subjects. The minor A allele frequency of the rs2234714 in the CAD patients was significantly lower than that in the control group (p = 0.05 for CAD, and p = 0.0046 for MI), indicating that rs2234714 was associated with a reduced risk for CAD or MI in a recessive model after adjustment (adjusted OR = 0.64, p = 0.015 for CAD and adjusted OR = 0.49, p = 0.001 for MI, respectively). The minor AA genotype for the rs57137919 polymorphism was associated with a significantly reduced risk for CAD (OR = 0.77, 95%CI 0.60–0.97, p = 0.026). The minor A allele frequency of rs57137919 was significantly lower in CAD or MI patients than that in controls (p = 0.006 and p = 0.004, respectively). Further analysis using a dominant model revealed that A allele carriers (AG + AA) appeared to be at a significantly lower risk for CAD or MI when compared with that in GG homozygote (adjusted OR = 0.73, p = 0.033 for CAD and adjusted OR = 0.65, p = 0.014 for MI, respectively) (Table 2). For the rs2234715 and rs1044317 polymorphisms, we found no significant difference on either allele frequency or genotype distribution between CAD patients and controls. Next we examined the relationships of the SNPs rs2234714 and rs57137919 with angiographic severity of CAD by comparing minor A allele frequency between single- and multi-vessel CAD patients. Single- or multi-vessel CAD was defined by the number of luminal narrowing ≥50% in the three major coronary arteries. Our results showed that rs57137919, but not rs2234714, had an association with angiographic severity of CAD. The adjusted OR associated with

the A allele of rs57137919 in multi-vessel CAD patients was 0.40 (p = 0.005) in a dominant model compared with that in single-vessel CAD patients (Table 3). 3.3. ABCG1 SNPs and plasma lipid levels ABCG1 plays an important role in cholesterol efflux and lipid homeostasis. We also examined possible associations of the rs2234714 and rs57137919 polymorphisms with plasma lipid levels. We analyzed individuals who did not take cholesterol-lowering medications and found no significant difference between these two SNPs and plasma lipid levels (HDL-C, LDL-C, TG and TC) (Supplementary Tables 2a and 2b). 3.4. Haplotype analysis The linkage disequilibrium analysis for the four SNPs showed no obvious LD between any of two SNPs (data not shown). We then compared haplotype frequency of ABCG1 between CAD or MI group and controls. Six common haplotypes (frequency > 3%) derived from the four SNPs accounted for about 95% haplotype variations. Among these six common haplotypes, the haplotype AA-A-A (rs2234714-rs2234715-rs57137919-rs1044317) was found to be associated with a decreased risk for MI (7.7% vs. 9.5%), which was a subgroup of CAD patients. The adjusted OR for MI was 0.59 (95%CI 0.37–0.93, p = 0.023). There was no significant association of other haplotypes with CAD or MI (Table 4). In addition, no haplotype of ABCG1 was found to be associated with plasma lipid levels in individuals who were not on cholesterollowering medication (Supplementary Table 2a). 3.5. Effects of ABCG1 polymorphisms on gene promoter activity To examine whether the ABCG1 promoter variations −768G>A (rs2234714) and −367G>A (rs57137919) affect promoter transcriptional activity, we measured luciferase activity of ABCG1 promoter constructs containing either single- or double-mutation. Our results showed that genetic variation −367G>A, but not −768G>A, significantly reduced luciferase gene expression in HEK293T cells. The relative luciferase activity of P-GA construct was significantly lower than that of wild-type P-GG construct. In contrast, the activity of P-AG construct was similar to that of wild-type. The P-AA construct also showed a significantly reduced luciferase gene activity (Fig. 1a). The results were confirmed in another two different cell

Y. Xu et al. / Atherosclerosis 219 (2011) 648–654

651

Fig. 1. (a) Luciferase reporter analysis of different mutant constructs of the ABCG1 promoter. Upper left, schematic diagram for wild-type or mutant promoter constructs at two polymorphism sites. ABCG1 SNPs: rs2234714, −768G>A; rs57137919, −367G>A. All constructs were subcloned into pGL3-basic vectors with firefly luciferase gene. P-GG, −768G/−367G; P-AG, −768A/−367G; P-GA, −768G/−367A; P-AA, −768A/−367A; Upper right, luciferase activity assay for all constructs of ABCG1 promoter in HEK293T cells (open bar), RAW264.7 cells (grey bar) and THP-1 cells (black bar), respectively. Cells were transfected with 1 ␮g of each plasmid construct and 5 ng of phRL-TK control plasimid with Renilla luciferase using lipofectamine 2000. The empty vector (pGL3-basic) with no gene insertion was used as a negative control, and pGL3-control vector (Promega) was used as a positive control. Luciferase activities were measured with dual-luciferase reporter assay system. Firefly luciferase activity was normalized to Renilla luciferase activity, and the activity of empty vector was subtracted from each experimental group to derive the relative luciferase activity. Data represent mean values of three independent experiments performed in quadruplicate (± S.D.). n.s., not statistically significant vs. P-GG in each cell line. *p < 0.01 vs. P-GG in THP-1 cells, † p < 0.01 vs. P-GG in RAW264.7 cells, ‡ p < 0.01 vs. P-GG in HEK293T cells. (b) ABCG1 protein expression in CAD patients with AA/AG/GG genotype of the rs57137919. Peripheral blood monocytes were isolated from patients and differentiated into macrophages using M-CSF for 3 days. Lower left, representative immunoblot of ABCG1 protein expression in macrophages isolated from patients with AA, AG or GG genotype of the rs57137919 (n = 3). GAPDH was used as a loading control. Lower right, ABCG1 protein fold change was quantified by densitometric analysis of Western blots, and normalized by GAPDH. Data were representative of three independent experiments.

lines, RAW264.7 and THP-1 cells, which were more physiologically relevant (Fig. 1a). 3.6. ABCG1 protein expression in macrophages isolated from CAD patients To further examine functionality of the SNPs, we measured ABCG1 protein levels in macrophages isolated from CAD patients with different genotypes for the rs57137919. Our result showed that ABCG1 protein expression was significantly lower in macrophages from patient with AA genotype than that in patients with GG genotype (Fig. 1b). ABCG1 protein expression was also decreased in patients with AG genotype for the rs57137919. 4. Discussion In this study, we performed genetic association analysis on four SNPs in the human ABCG1 gene in 1021 CAD patients and 1013 healthy controls from a Chinese Han population. Our results showed that −367G>A (rs57137919) polymorphism in the promoter region was associated with a reduced risk for CAD. The minor A allele frequency of rs57137919 was even lower in patients with multi-vessel CAD than that in patients with single-vessel CAD, suggesting an association between rs57137919 and the severity of CAD. Consistently, the haplotype analysis showed that the AA-A-A haplotype (rs2234714-rs2234715-rs57137919-rs1044317) was associated with a reduced risk for the disease in the same

population. Furthermore, rs57137919 polymorphism had reduced luciferase reporter gene expression in transfected HEK293T cells, as well as in RAW264.7 and THP-1 cells. To the best of our knowledge, this is the first report of the association between ABCG1 polymorphisms and CAD with a large sample size in a case-control setting. The results of our study suggest that ABCG1 may play a deleterious role in human vascular biology and favor the incidence of CAD in humans. Ten SNPs including two novel SNPs were identified in the ABCG1 promoter region or 3 -UTR in our preliminary genotyping screening. Based on pair-wise linkage analysis, four SNPs (rs2234714, rs2234715, rs57137919, and rs1044317) were found no obvious linkage disequilibrium, and were chosen for large-scale genotyping as tag-SNPs. Our results showed that ABCG1 polymorphism rs57137919 had significantly lower allele frequencies in CAD patients compared with that in control subjects. On the other hand, there was no difference on plasma lipid levels including LDL-C and HDL-C to be associated with the minor A allele of the SNP rs57137919. ABCG1 plays a critical role in mediating cholesterol efflux to HDL [3,8]. Recent studies show that ABCG1 is also involved in macrophage apoptosis and endothelial dysfunction, which both are important in the development of atherosclerosis, suggesting a role of ABCG1 in the disease [19–21]. Given these important findings, the functional significance of ABCG1 in atherosclerosis has been investigated. In mouse models, Baldan et al. [11] and Ranalletta et al. [12] independently reported that ABCG1-deficient bone

0.036 0.076 1.28 (1.02–1.62) 1.20 (0.98–1.48) rs1044317

rs57137919

Dominant model: heterozygotes and rare homozygotes compared with common homozygotes. Recessive model: heterozygotes and common homozygotes compared with rare homozygotes. c p-Values, odds ratios (OR) and 95%CI (95% confidence interval) were from logistic regression analysis adjusted for age, gender, hypertension, T2DM, smoking, HDL-C, TG as covariates between CAD or MI patients and controls. MI was a subgroup of CAD. p-Value <0.05 was shown in bold.

1.36 (1.00–1.86)

0.73 (0.54–0.98) 0.005 0.026 0.67 (0.51–0.89) 0.77 (0.60–0.97)

0.682 1.10 (0.82–1.48) 1.06 (0.82–1.36) rs2234715

a

0.075 1.39 (0.97–1.99)

0.051

0.033 0.65 (0.47–0.92)

0.89 (0.63–1.26) 0.93 (0.69–1.26) 0.517

0.003 0.021 0.70 (0.55–0.89) 0.78 (0.64–0.96)

211 (33.7) 315 (50.3) 100 (16.0) 409 (65.3) 194 (31.0) 23 (3.7) 376 (60.1) 226 (36.1) 24 (3.8) 184 (29.4) 304 (48.6) 138 (22.0) 317 (31.0) 527 (51.6) 177 (17.3) 645 (63.2) 334 (32.7) 42 (4.1) 606 (59.4) 368 (36.0) 47 (4.6) 312 (30.6) 499 (48.9) 210 (20.6) 292 (28.8) 506 (50.0) 215 (21.2) 644 (63.6) 338 (33.4) 31 (3.1) 543 (53.6) 406 (40.1) 64 (6.3) 329 (32.5) 484 (47.8) 200 (19.7) GG AG AA AA AG GG GG AG AA GG AG AA rs2234714

b

0.385 1.42 (0.94–2.13) 1.18 (0.82–1.69)

0.097

0.038 0.206 0.46 (0.22–0.96) 0.68 (0.38–1.24) 0.014

0.956 0.90 (0.37–2.17) 0.98 (0.45–2.11) 0.507 0.649

0.087 0.155 0.73 (0.51–1.05) 0.80 (0.58–1.09)

MI All MI All MI All

CAD patients (%)

MI

OR (95%CI) vs. controls

All

OR (95%CI) vs. controls p vs. controls

Dominant modela

c

Genotype frequency Control (%) Genotype SNP ID

Table 2 Comparison of genotype frequencies of four SNPs in ABCG1 in CAD or MI patients and CAD-free controls.

0.813

0.015 0.49 (0.32–0.76) 0.64 (0.45–0.92)

MI All MI All MI All

OR (95%CI) vs. controls p vs. controls

Recessive modelb

c

0.001

Y. Xu et al. / Atherosclerosis 219 (2011) 648–654

pc vs. controls

652

Table 3 Effects of the rs2234714 or rs57137919 on angiographic severity of CAD.a SNP ID

Effect of the A allele

Odds ratio (95% CI)

rs2234714

Additive modelb Dominant model Recessive model Additive modelb Dominant model Recessive model

0.87 (0.70–1.09) 0.93 (0.67–1.30) 0.72 (0.49–1.06) 0.80 (0.62–1.02) 0.88 (0.64–1.19) 0.40 (0.21–0.75)

rs57137919

p-Value 0.225 0.683 0.095 0.076 0.392 0.005

p-Value < 0.05 is shown in bold. a Logistic regression analysis of the additive, recessive and dominant models on multi-vessel disease compared with single-vessel disease, adjusted for age, gender, hypertension, T2DM, smoking, HDL-C and TG as covariates. b Additive model: per difference in number of rare alleles.

marrow-derived cells in LDLr−/− mice led to decreased atherosclerosis. However, Burgess et al. showed that overexpression of human ABCG1 did not affect atherosclerotic lesion area in apoE−/− mice [13]. Most recently, Tarling et al. found that ABCG1 deficiency in mice caused smaller atherosclerotic lesions with increased numbers of apoptotic macrophages [21]. The reason for the apparent phenotypic differences in these mouse models is not clear and may be due to different genetic background and/or methods of analysis. Genetic association studies in humans have been informative for investigating the role of disease candidate genes. Recently, Abellan et al. found an association between ABCG1 polymorphism rs1893590 and HDL-C levels under postprandial conditions for women in a Spanish population [22]. Plasma HDL-C levels in the AA genotype were lower than that in the AC + CC genotype. In another study, Furuyama et al. has shown an association between ABCG1 polymorphism and CAD risk in a Japanese population [14]. The ABCG1 promoter −257T>G polymorphism, located upstream of exon 1, was associated with the severity of CAD in men. The presence of the G allele conferred an increased risk for multi-vessel CAD compared with that for single-vessel CAD, suggesting that ABCG1 may protect against CAD in humans. On the other hand, several studies in humans provided evidences that ABCG1 may play a distinct role rather than the potential atheroprotective function. Larrede et al. reported that ABCG1 did not promote free cholesterol efflux to HDL in primary human macrophage-foam cells [23]. More recently, Chinetti-Gbaguidi et al. found that the atheroprotective M2 macrophages in human atherosclerotic plaques have a

Table 4 Association of ABCG1 gene haplotypes with CAD or MI. Variables

CAD (n = 1021) Haplotypeb H1 AAAA H2 AAAG H3 AGGA H4 AGGG H5 GAGA H6 GAGG MI (n = 626) Haplotype H1 AAAA H2 AAAG H3 AGGA H4 AGGG H5 GAGA H6 GAGG

Haplotype frequency

OR (95%CI)

pa

Controls

Cases

0.095 0.168 0.036 0.161 0.303 0.234

0.081 0.145 0.045 0.160 0.323 0.245

0.75 (0.51–1.11) 0.94 (0.68–1.30) 1.10 (0.62–1.97) 0.91 (0.67–1.24) 1.52 (1.13–2.03) 0.92 (0.69–1.24)

0.151 0.710 0.746 0.56 0.005 0.584

0.095 0.168 0.036 0.161 0.303 0.234

0.077 0.141 0.047 0.144 0.339 0.25

0.59 (0.37–0.93) 0.92 (0.63–1.34) 1.30 (0.68–2.51) 0.81 (0.56–1.16) 1.72 (1.22–2.42) 0.91 (0.64–1.27)

0.023 0.665 0.426 0.241 0.002 0.565

p-Value < 0.05 is shown in bold. a Age, gender, hypertension, T2DM, smoking, HDL-C and TG were adjusted. MI was a subgroup of CAD. b Loci are arranged in the order rs2234714, rs2234715, rs57137919, rs1044317. Haplotype with frequency less than 3% was pooled and not analyzed.

Y. Xu et al. / Atherosclerosis 219 (2011) 648–654

defective capacity to promote cellular cholesterol efflux because of the reduced expression of ABCA1 and apoE whereas that of ABCG1 was significantly increased in this macrophage population. Their findings suggested that reduced cholesterol efflux capacity of the atheroprotective M2 macrophages in human atherosclerotic plaques might be independent of ABCG1 expression [24]. Thus, the role of ABCG1 in atherosclerosis remains to be elucidated. In our study, three of four SNPs are located in the promoter region upstream of exon 5, which had not been investigated previously in the CAD patients. The human ABCG1 gene consists of 23 exons spanning 98 kb and contains multiple promoters that give rise to several alternative transcripts (Supplementary Figure 1). The promoter region upstream of exon 5 contains potential SP1 sites, NF-␬B binding sites, RXR and sterol response element binding protein (SREBP) binding sites, and is involved in regulating ABCG1 gene transcription. Current evidence suggests that the major transcript of ABCG1 in humans is derived from this promoter [25–27]. Two ABCG1 promoter polymorphisms, rs2234714 and rs57137919, were found to be associated with a reduced risk for CAD in the present study (Table 2). Using online programs (PATCHTM public 1.0), we identified several putative regulatory elements in the promoter region of ABCG1, including RXR and AP-2 binding sites at rs2234714, VDR and GAGA factor binding sites at rs57137919. The two SNPs in the gene promoter could be causative genetic variants, which may alter ABCG1 gene expression. In the luciferase reporter assay, however, the polymorphism rs2234714 did not show any effect on reporter gene expression. We found that promoter construct harboring −768G/−367A, but not −768A/−367G, had a significantly lower transcriptional activity than that of the wild-type construct harboring −768G/−367G in HEK293T cells (Fig. 1a), indicating that the genetic variant rs57137919, but not rs2234714, had an impaired promoter function. The results were also confirmed in two physiologically relevant cell lines, RAW264.7 cells and THP-1 cells. Further examination showed that ABCG1 protein expression was significantly reduced in macrophages isolated from patients with AA genotype for the rs57137919 (Fig. 1b), which was consistent with the reporter gene assay data. Together, our functional data support our hypothesis that the rs57137919 polymorphism may alter ABCG1 gene function and contribute to atherosclerosis and CAD. These data for the first time showed that ABCG1 may favor the incidence of CAD in humans. The polymorphism rs2234714 showed no significant effect in macrophages in the luciferase reporter assay. However, ABCG1 mRNA is expressed in numerous human tissues [7]. To this point, we cannot rule out the possibility that the rs2234714 may be functional in different cell types such as endothelial cells or smooth muscle cells. Alternatively it is also possible that the rs2234714 is in linkage disequilibrium with another causative genetic variant which could be related to CAD. How is the impaired ABCG1 expression linked to a reduced risk of CAD? Given the physiological role of ABCG1 in cholesterol efflux, we would expect that impaired ABCG1 expression may change plasma lipid levels, which consequently contribute to development of CAD. However, we found no apparent association between the ABCG1 SNPs and plasma lipid levels. Consistently, ABCG1 deficiency in mice caused no changes in plasma lipids [8]. Furthermore, Furuyama et al. observed no significant difference in plasma lipid levels in fasting conditions with ABCG1 −257T>G polymorphism in Japanese men. Beside HDL-mediated cholesterol efflux, it is equally possible that reduced ABCG1 expression may increase oxidative-stress-induced macrophage apoptosis, thereby leading to decreased atherosclerotic lesions. Evidences from several recent studies in vitro and in animal models support this hypothesis [19,28,29]. Further investigation is needed to understand the function of the gene.

653

The SNP rs57137919 is also associated with angiographic severity of CAD. The minor A allele frequency is significantly lower in patients with multi-vessel CAD than that in patients with singlevessel CAD. Considering that the SNP had an impaired promoter activity, the data suggest that it may affect the initiation and progression of coronary atherosclerotic lesions. The minor G allele of ABCG1 2424A>G polymorphism (rs1044317) in the 3 -UTR did not appear to be associated with the risk for CAD in our study. Previously, this SNP was investigated for postprandial plasma lipid levels in a Spanish Caucasian population, and shown to have no significant association with the disease [22]. In another study, the SNP was investigated in the German suicide patients, and shown to have a weak association with suicide behavior [30]. The finding was intriguing. It was not clear, however, whether the polymorphism directly altered the function of ABCG1 or, alternatively, was in linkage disequilibrium with another causative genetic variant. At this point, the effect of ABCG1 rs1044317 on atherosclerosis remains unclear, which encourages further independent investigation with large cohorts. The present study has strengths as well as limitations. The large sample size, which is essential for the investigation of mostly modest influence of genetic variants in association study, deserves to be mentioned. The case and control groups were from Chinese Han population, which may help to eliminate false positive association due to population admixture. We also incorporated a haplotype-based analysis across the candidate gene region, which may increase statistic power and reduce the problem of multiple testing. Moreover, the functionality data provide further evidence supporting our hypothesis. With regard to our limitation, we cannot completely rule out the possibility of a false positive finding. It should be mentioned that the observed associations need further replications to avoid spurious associations which are common in genetic association studies. In summary, we report a significant association between ABCG1 gene promoter polymorphism rs57137919 and CAD in a Chinese Han population. The presence of minor A allele conferred a reduced risk for CAD. The genetic variation impaired ABCG1 gene expression in human macrophages. Our findings should be important to help clarifying the role of ABCG1 in atherosclerosis. The precise mechanisms regarding how the promoter variants of the ABCG1 gene affect coronary atherosclerosis need further investigation. Conflict of interest None. Acknowledgements We thank Dr. Qingyu Wu (Cleveland Clinic, USA) for helpful comments on the manuscript; Dr. Yusheng Ma and the technical staff (Generay Biotech, Shanghai) for their technical support on genotyping. We thank all the subjects for participating in this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2011.05.043. References [1] Yusuf S, Reddy S, Ounpuu S, et al. Global burden of cardiovascular diseases. Part I. General considerations, the epidemiologic transition, risk factors, and impact of urbanization. Circulation 2001;104:2746–53. [2] Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340:115–26.

654

Y. Xu et al. / Atherosclerosis 219 (2011) 648–654

[3] Wang N, Lan D, Chen W, et al. ATP-binding cassette transporters G1, G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci USA 2004;101:9774–9. [4] Klein I, Sarkadi B, Varadi A. An inventory of the human ABC proteins. Biochim Biophys Acta 1999;1461:237–62. [5] Fitzgerald ML, Mujawar Z, Tamehiro N. ABC transporters, atherosclerosis and inflammation. Atherosclerosis 2010;211:361–70. [6] Cavelier C, Lorenzi I, Rohrer L, et al. Lipid efflux by the ATP-binding cassette transporters ABCA1 and ABCG1. Biochim Biophys Acta 2006;1761:655–66. [7] Klucken J, Buchler C, Orso E, et al. ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport. Proc Natl Acad Sci USA 2000;97:817–22. [8] Kennedy MA, Barrera GC, Nakamura K, et al. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation. Cell Metab 2005;1:121–31. [9] Out R, Hoekstra M, Hildebrand RB, et al. Macrophage ABCG1 deletion disrupts lipid homeostasis in alveolar macrophages and moderately influences atherosclerotic lesion development in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol 2006;26:2295–300. [10] Lammers B, Out R, Hildebrand RB, et al. Independent protective roles for macrophage Abcg1 and Apoe in the atherosclerotic lesion development. Atherosclerosis 2009;205:420–6. [11] Baldan A, Pei L, Lee R, et al. Impaired development of atherosclerosis in hyperlipidemic Ldlr−/− and ApoE−/− mice transplanted with Abcg1−/− bone marrow. Arterioscler Thromb Vasc Biol 2006;26:2301–7. [12] Ranalletta M, Wang N, Han S, et al. Decreased atherosclerosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1−/− bone marrow. Arterioscler Thromb Vasc Biol 2006;26:2308–15. [13] Burgess B, Naus K, Chan J, et al. Overexpression of human ABCG1 does not affect atherosclerosis in fat-fed ApoE-deficient mice. Arterioscler Thromb Vasc Biol 2008;28:1731–7. [14] Furuyama S, Uehara Y, Zhang B, et al. Genotypic Effect of ABCG1 gene promoter −257T>G polymorphism on coronary artery disease severity in Japanese men. J Atheroscler Thromb 2009;16:194–200. [15] Chen Q, Reis SE, Kammerer CM, et al. Association between the severity of angiographic coronary artery disease and paraoxonase gene polymorphisms in the National Heart, Lung, and Blood Institute-sponsored Women’s Ischemia Syndrome Evaluation (WISE) study. Am J Hum Genet 2003;72:13–22. [16] Barany F. Genetic disease detection DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci USA 1991;88:189–93. [17] Wang W, Liao X, Fukuda K, et al. Corin variant associated with hypertension and cardiac hypertrophy exhibits impaired zymogen activation and natriuretic peptide processing activity. Circ Res 2008;103:502–8.

[18] Shi YY, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res 2005;15:97–8. [19] Yvan-Charvet L, Pagler TA, Seimon TA, et al. ABCA1 and ABCG1 protect against oxidative stress-induced macrophage apoptosis during efferocytosis. Circ Res 2010;106:1861–9. [20] Terasaka N, Yu S, Yvan-Charvet L, et al. ABCG1 and HDL protect against endothelial dysfunction in mice fed a high-cholesterol diet. J Clin Invest 2008;118:3701–13. [21] Tarling EJ, Bojanic DD, Tangirala RK, et al. Impaired development of atherosclerosis in Abcg1−/− Apoe−/− mice: identification of specific oxysterols that both accumulate in Abcg1−/− Apoe−/− tissues and induce apoptosis. Arterioscler Thromb Vasc Biol 2010;30:1174–80. [22] Abellan R, Mansego ML, Martinez-Hervas S, et al. Association of selected ABC gene family single nucleotide polymorphisms with postprandial lipoproteins: results from the population-based Hortega study. Atherosclerosis 2010;211:203–9. [23] Larrede S, Quinn CM, Jessup W, et al. Stimulation of cholesterol efflux by LXR agonists in cholesterol-loaded human macrophages is ABCA1-dependent but ABCG1-independent. Arterioscler Thromb Vasc Biol 2009;29:1930–6. [24] Chinetti-Gbaguidi G, Baron M, Bouhlel MA, et al. Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPAR{gamma} and LXR{alpha} pathways. Circ Res 2011;108:985–95. [25] Langmann T, Porsch-Ozcurumez M, Unkelbach U, et al. Genomic organization and characterization of the promoter of the human ATP-binding cassette transporter-G1 (ABCG1) gene. Biochim Biophys Acta 2000;1494:175–80. [26] Kennedy MA, Venkateswaran A, Tarr PT, et al. Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein. J Biol Chem 2001;276:39438–47. [27] Lorkowski S, Rust S, Engel T, et al. Genomic sequence and structure of the human ABCG1 (ABC8) gene. Biochem Biophys Res Commun 2001;280:121–31. [28] Terasaka N, Wang N, Yvan-Charvet L, et al. High-density lipoprotein protects macrophages from oxidized low-density lipoprotein-induced apoptosis by promoting efflux of 7-ketocholesterol via ABCG1. Proc Natl Acad Sci USA 2007;104:15093–8. [29] Seres L, Cserepes J, Elkind NB, et al. Functional ABCG1 expression induces apoptosis in macrophages and other cell types. Biochim Biophys Acta 2008;1778:2378–87. [30] Gietl A, Giegling I, Hartmann AM, et al. ABCG1 gene variants in suicidal behavior and aggression-related traits. Eur Neuropsychopharmacol 2007;17: 410–6.