Genetic variations of apolipoprotein A5 gene is associated with the risk of coronary artery disease among Chinese in Taiwan

Atherosclerosis 185 (2006) 143–149

Genetic variations of apolipoprotein A5 gene is associated with the risk of coronary artery disease among Chinese in Taiwan Lung-An Hsu, Yu-Lin Ko ∗ , Chi-Jen Chang, Chiao-Feng Hu, Semon Wu, Ming-Sheng Teng, Chun-Li Wang, Wan-Jing Ho, Yu-Shien Ko, Tsu-Shiu Hsu, Ying-Shiung Lee The First Cardiovascular Division, Department of Internal Medicine, Chang Gung Memorial Hospital, No. 199, Tung-Hwa North Road, Taipei, Taiwan Received 10 March 2005; received in revised form 26 May 2005; accepted 31 May 2005 Available online 27 July 2005

Abstract Recently, a T/C polymorphism of the promoter region of the APOA5 gene at position -1131 and a G/T polymorphism at position 553 were found to be associated with increased levels of plasma triglyceride. Triglyceride plays a role in coronary artery disease (CAD), so this case–control study tested for a possible link between these two APOA5 polymorphisms, their common haplotypes and the risk of CAD. The subjects included 211 CAD patients and 677 unrelated controls. A significantly higher level of triglycerides and a lower level of high-density lipoprotein cholesterol (HDL-C) were noted for carriers with -1131C than for non-carriers (P < 0.001 and 0.013, respectively) among controls. Plasma triglyceride levels were significantly higher (P = 0.014) in controls with genotypes that contained the c.553T allele than in homozygotes for the G allele. Subjects homozygous for the wild-type haplotype had significantly lower triglyceride levels and higher HDL-C levels than subjects with all other haplotype pairs. The -1131C homozygous carriers and c.553T heterozygous carriers were found more frequently in 211 patients with CAD than in the 317 age/sex-matched controls (P = 0.008 and 0.023, respectively) in univariate analysis. The significant association between c.553T allele carriers with CAD remained in multivariate regression analysis (OR, 1.79; CI, 1.07–3.00; P = 0.028), after adjustments were made for other risk factors. Notably, haplotype analysis further verified that the APOA5 -1131C and c.553T bi-loci haplotype was significantly overpresented in CAD, as compared to the controls. These results indicate that the variants of APOA5 gene modulate plasma triglyceride and may use them to predict CAD susceptibility in Taiwanese Chinese. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Polymorphisms; APOA5; Triglycerides; High-density lipoprotein cholesterol; Coronary disease

1. Introduction Although the specific role of triglycerides in atherogenesis remains controversial, some epidemiological investigations have demonstrated that, in addition to elevated low-density lipoprotein cholesterol (LDL-C) and reduced high-density lipoprotein cholesterol (HDL-C) levels, elevated triglycerides constitute an independent risk factor of coronary artery disease (CAD) [1]. Triglyceride levels may be altered by a variety of environmental factors, including smoking, obesity, alcohol consumption and exercise. Twin studies have also shown a strong genetic contribution to triglyceride levels [2]. ∗

Corresponding author. Tel.: +886 3 3281200x8162; fax: +886 3 3271192. E-mail address: [email protected] (Y.-L. Ko).

0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.05.031

A novel human apolipoprotein gene (APOA5) located proximal to the APOA1/C3/A4 gene cluster on human 11q23 has been independently identified by human and mouse comparative sequence analysis [3], and by a differential gene expression approach as a gene highly up-regulated in the early phase of liver regeneration [4]. Functional studies in mice indicate that altering the level of APOA5 substantially affected plasma triglyceride levels. Plasma triglyceride concentrations in transgenic mice that overexpressed human APOA5 were one third those in control mice, whereas APOA5 knockout mice had four times as much plasma triglycerides as the controls [3]. The adenovirus-mediated overexpression of APOA5 was associated with markedly decreased (−70%) serum triglyceride levels caused primarily by the reduced triglyceride content of the very-low-density lipoprotein (VLDL) fraction [5].

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In humans, a single nucleotide polymorphism (SNP) -1131T > C in the promoter region of the APOA5 gene has been found to be independently associated with plasma triglyceride levels in several populations of various ethnicities [3,6–11]. Moreover, studies of both Asian and Caucasian populations have demonstrated that this polymorphism was also associated with reduced HDL-C levels in addition to elevated triglyceride levels [7,9–11]. Recently, another polymorphism c.56C > G (S19W) in the coding region of APOA5 gene, which was designated a third haplotype for this gene, was also reported to be significantly associated with high plasma triglyceride levels in African–Americans, Hispanics and Caucasians [12]. More recently, Kao et al. [13] described a novel variant, c.553G > T, which substitutes a cysteine for a glycine residue, and is associated with hypertriglyceridmia in a Taiwanese Chinese population. Taken together, these reports provide important genetic evidence that APOA5 is crucial in the human metabolism of triglycerides. APOA5 polymorphisms may contribute to an increased CAD risk by modulating the level of plasma triglycerides. Therefore, this case–control study examines the associations among -1131T > C, c.56C > G, c.553G > T, the common APOA5 haplotypes defined by them and the risk of CAD. 2. Methods 2.1. Study population Two hundred and eleven patients with CAD and 677 control subjects were enrolled. The demographic details of the CAD patients and control subjects have been described elsewhere [14,15]. Briefly, all of the CAD patients were from Chang Gung Memorial Hospital (Taipei, Taiwan), and were recruited between August 1994 and September 1996. All CAD patients had >50% stenosis in at least one major coronary artery, as documented by coronary angiography. Control subjects were recruited during routine health examination and had no clinical evidence of CAD: (1) no history of typical angina pectoris, (2) no abnormal Q wave or ST-T changes on electrocardiography and (3) negative Master exercise test results. The presence of hypertension, diabetes mellitus, hypercholesterolemia or smoking was determined based on history, previous medical records, current medication and by examination during hospitalization. The study protocol was approved by the hospital ethics committee, and oral informed consent was obtained from all subjects. 2.2. Plasma lipid measurements A 5 ml blood sample was obtained from all subjects following a 12–14-h overnight fast. Total cholesterol and triglyceride concentrations were measured using automatic enzymatic colorimetry. Meanwhile, HDL-C levels were measured enzymatically following phosphotungsten/magnesium precipitation. Finally, LDL-C was calculated using the Friedewald formula. The CAD cases were enrolled between

1994 and 1996 when HDL-C level was not checked routinely in our hospital, so only 112 of 211 cases underwent complete lipid profile examination. 2.3. Genomic DNA extraction and genotyping The genomic DNA was extracted from the peripheral blood leukocytes by the standard method with proteinase K digestion of nuclei. Phenol and chloroform extraction was followed by isopropanol precipitation of the DNA. The genotyping of the three SNPs in this study has been described. This includes the APOA5 -1131T > C [3], S19W [12], c553G > T [13]. 2.4. Statistical analysis The chi-square test was used to examine differences among categorical variables. The clinical characteristics of continuous variables were expressed as mean ± S.D., and were tested using a two-sample t test or ANOVA. Furthermore, a general linear model was applied to analyze the triglyceride and HDL-C levels with respect to predictors of the investigated genotypes and other confounders. Multiple logistic regression analysis was used to evaluate the independent effect of investigated genotypes on the risk of CAD, adjusted for the presence of established risk factors. Haplotype analysis was conducted with the haplotype estimated using the expectation–maximization (EM) algorithm and permutation-based hypothesis testing procedures were performed as previously reported [16]. The significance level was P < 0.05 for the omnibus test, and 0.0125 (0.05/4) for individual haplotype analyzes (four haplotypes for two loci). The extent of disequilibrium was expressed in terms of D = D/Dmax [17]. Variables were logarithmically transformed before statistical analysis to meet a normality assumption.

3. Results 3.1. Characteristics of subjects Table 1 lists the characteristics of the cases and control subjects. The frequencies of classical risk factors for CAD, such as hypertension, diabetes mellitus and smoking in the CAD patients were significantly higher than in the control subjects. Additionally, we stratified the controls by age and sex, and randomly selected 317 controls to match approximately the age and sex distribution of the CAD group for the following case–control comparison. 3.2. Univariate analysis of APOA5 genotypes and haplotypes in controls Among the 677 healthy controls, the allele frequency of APOA5 -1131C was 29.9%, and the allele frequency

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Table 1 Demographic variables and laboratory data of 211 CAD patients, 677 controls and 317 age/sex-matched controls

Age (years) Gender (male/female (% male)) Smokers (%) Hypertension (%) Diabetes (%) Body mass index (kg/m2 ) Cholesterol (mg/dl) HDL-cholesterol (mg/dl) LDL-cholesterol (mg/dl) Triglycerides (mg/dl)

CAD (n = 211)

Controls (n = 677)

Age/sex-matched controls (n = 317)

61.6 ± 8.9 161/50 (76.3) 56.9 45.0 30.3 25.2 ± 3.8 207 ± 45 (n = 190) 41 ± 12 (n = 112) 135 ± 40 (n = 112) 195 ± 203 (n = 187)

55.1 ± 9.8*

61.0 ± 9.3 242/75 (76.3) 49.8 26.8* 9.1* 24.8 ± 3.8 204 ± 40 51 ± 15* 125 ± 36* 146 ± 117*

366/311 (54.1%)* 37.3* 21.4* 7.2* 24.8 ± 3.7 204 ± 40 53 ± 15* 123 ± 34* 146 ± 136*

Triglycerides were logarithmically transformed before statistical testing to meet the assumption of normal distribution, however, untransformed data are shown. * Denotes P < 0.05 compared with CAD group. Table 2 APOA5 -1131T/C, 553G/T genotypes and lipid profiles for 677 control individuals Variables

Number Total C (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) TG (mg/dl) BMI (kg/m2 )

P

-1131T/C TT

TC

CC

GG

318 202 ± 123 ± 54 ± 129 ± 25.1 ±

313 206 ± 41 125 ± 37 52 ± 15 150 ± 93 24.6 ± 3.4

46 209 ± 117 ± 48 ± 234 ± 24.2 ±

579 205 ± 124 ± 53 ± 142 ± 24.9 ±

36 31 19 107 3.8

55 37 12 354 4.0

P

553G > T

0.370 0.305 0.013 <0.001 0.134

GT 98 200 ± 117 ± 51 ± 170 ± 24.5 ±

40 34 14 130 3.7

37 34 16 164 3.7

0.315 0.064 0.384 0.014 0.296

Triglycerides were logarithmically transformed before statistical testing to meet the assumption of normal distribution, however, untransformed data are shown.

of APOA5 553T was 7.2%. The genotype frequencies for APOA5 -1131T > C polymorphisms were in Hardy–Weinberg equilibrium (HWE). However, a slight departure from HWE was detected for c.553G > T (χ2 = 4.354, P = 0.039, d.f. = 1), which remained after careful assessment of the data. Statistical significance was demonstrated for linkage disequilibrium (LD) between the two rare alleles of APOA5 G553T and -1131T > C in control subjects (D/Dmax = 0.93, P < 0.001). Only two control subjects were heterozygous for APOA5 S19W, whereas no CAD subject was. Therefore, S19W was excluded from haplotype analysis. Our results indicated a strong association between APOA5 -1131C allele and elevated triglyceride levels (Table 2, P < 0.001). TC heterozygotes had significantly higher triglyceride levels (150 ± 93 mg/dl) than TT homozygous subjects (129 ± 107 mg/dl), and the CC homozygotes had the highest triglyceride levels (234 ± 354 mg/dl). In contrast, a significant trend towards lower HDL-C levels was also

demonstrated for carriers of the C allele (Table 2, P = 0.013). Triglyceride levels were significantly higher in subjects with genotypes containing the minor allele of the c.553G > T compared to the homozygotes for the major allele (Table 2, P = 0.014). There was also a trend that HDL-C levels were lower in subjects with genotypes containing the minor allele of the c.553G > T but the differences did not reach significance. Four haplotypes derived from the APOA5 -1131T > C and c.553G > T polymorphisms and their estimated frequencies were as follows: haplotype*A (TG) = 69.5%, haplotype*B (CG) = 22.4%, haplotype*C (TT) = 0.3% and haplotype*D (CT) = 7.8%. Both rare alleles of these two polymorphisms were in significantly strong LD, so subjects with the haplotype*C were few (only four subjects were haplotype pair *A*C carriers). Therefore, subjects who carried haplotype pair *A*A were compared only with individuals with the haplotype pair *A*B, *B*B or *B*D. As shown in Table 3, subjects with the wild-type haplotype

Table 3 APOA5 -1131T/C, 553G/T diplotypes and lipid profiles for 677 control individuals Variables

Number Total C (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) TG (mg/dl) BMI (kg/m2 )

P

Genotype APOA5*A*A

APOA5*A*B

APOA5*B*B

APOA5*B*D

314 202 ± 123 ± 54 ± 126 ± 25.1 ±

239 208 ± 128 ± 52 ± 149 ± 24.7 ±

26 207 ± 111 ± 50 ± 234 ± 24.0 ±

20 211 ± 124 ± 47 ± 236 ± 24.6 ±

35 31 15 106 3.8

43 38 14 93 3.5

63 3 13 390 2.9

43 41 11 311 5.1

0.246 0.062 0.035 <0.001 0.317

Triglycerides were logarithmically transformed before statistical testing to meet the assumption of normal distribution, however, untransformed data are shown.

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Table 4 Distribution of genotypes and alleles for APOA5 -1131T/C and 553G/T in 211 cases and 317 age/sex-matched controls Polymorphism

-1131T/C

553G/T

Genotype

Cases Controls

Cases Controls

TT

TC

CC

104 (49.3%) 145 (45.7%)

83 (39.3%) 156 (49.2%)

24 (11.4%) 16 (5.0%)

GG

TG

TT

166 (78.7%) 276 (87.1%)

44 (20.9%) 41 (12.9%)

1 (0.5%)

pair *A*A exhibited significantly lower triglyceride levels and higher HDL-C levels than those with all other haplotype pairs. Subjects who carried more haplotype*B and *D had significantly higher triglyceride levels and lower HDL-C levels than those homozygous for the haplotype*A. When CAD cases were also included in the same haplotype analysis, this trend became even stronger for triglyceride levels (135 ± 105 mg/dl for *A*A (n = 404), 157 ± 140 mg/dl for *A*B (n = 286), 249 ± 350 mg/dl for *B*B (n = 35) and 300 ± 373 mg/dl for *B*D (n = 30), P < 0.001).

P

Minor allele frequency (%)

P

0.008

31 29.7

0.630

0.023

10.9 6.5

0.010

3.4. Case–control association studies with individual SNPs The allele and genotype frequencies of the two APOA5 variants of 211 established CAD cases and 317 age/sexmatched controls were compared. Although the frequency of APOA5 -1131CC homozygotes differed significantly between the cases and the controls (11.4% versus 5.0%, P = 0.008, Table 4), no significant difference was observed between the APOA5 -1131C allele frequencies of the cases and the controls. In contrast, significant differences were observed between the APOA5 c.553T allele frequencies of the cases and the controls (10.9% versus 6.5%, P = 0.010, Table 4), and between their APOA5 c.553G > T heterogygote frequencies (20.9% versus 12.9%, P = 0.023, Table 4). After adjustment for common cardiovascular risk factors, including age, gender, hypertension, smoking, diabetes, BMI and APOA5 -1131T > C genotypes, the APOA5 c.553T carriers remained significantly associated with increased risk of CAD (OR, 1.79; 95% CI, 1.07–3.00; P = 0.028, Table 5). In contrast, the addition of c.553T to the model resulted in a loss of the statistical significance of the relationship between the homozygote carriers of the -1131C allele and CAD (P = 0.115, Table 5). Although the lipid profile for the CAD cases was incomplete (of 211, 187 (89%) cases had triglyceride levels data), the association was also analyzed after adjusting cholesterol, LDL-C, HDL-C and triglyceride levels. Notably, adding lipid profile to the model did not change the result (Table 5), indicating that the influence of APOA5 c.553G > T polymorphism on the susceptibility of CAD is probably independent of these variables.

3.3. Multivariate linear regression model with triglyceride, HDL-C as dependent variables in controls Multivariate linear regression analyzes were applied to determine whether the APOA5 -1131T > C, 553G > T or APOA5 haplotype independently predicts triglyceride and HDL-C levels. Homozygotes of the major allele or common haplotype were compared to all other genotypes or diplotypes. Multiple linear regression analysis demonstrated that the -1131T > C, c.553G > T and APOA5 haplotype remained independent predictors of triglyceride levels, when age, sex, smoking, diabetes mellitus and body mass index (BMI) are taken into account (P < 0.001, P = 0.001 and P < 0.001, respectively). Furthermore, the -1131T > C and APOA5 haplotype remained independent predictors of HDL-C levels following adjustment of the indicated variables (P = 0.002 and 0.006, respectively). The addition of triglyceride to the model resulted in a loss of statistical significance of the relationships between the -1131T > C, APOA5 haplotype and HDL-C (P = 0.664 and 0.594, respectively).

Table 5 Multiple logistic regression analysis for the association between APOA5 genotypes and risk for CAD Model 1

APOA5 -1131T/C (CC vs. CT + TT) APOA5 c.553G/T (GT + TT vs. GG)

Model 2

Odds ratio (95% CI)

P

2.26 (1.12, 4.53) 1.11 (0.41, 3.03)a

0.022 0.837

Odds ratio (95% CI)

2.01 (1.23, 3.30) 2.13 (1.12, 4.03)a

Model 3 P

0.006 0.021

Odds ratio (95% CI)

P

1.80 (0.87, 3.72) 0.86 (0.31, 2.41)a

0.115 0.776

1.79 (1.07, 3.00) 2.17 (1.13, 4.18)a

0.028 0.021

Model 1 variables included age, gender, hypertension, smoking, diabetes, body mass index and APOA5 -1131 T > C. Model 2 variables included age, gender, hypertension, smoking, diabetes, body mass index and APAO5 c.553G > T. Model 3 variables included age, gender, hypertension, smoking, diabetes, body mass index, APOA5 -1131T > C and c.553G > T. a Lipid profile (cholesterol, LDL-C, HDL-C, triglyceride levels) also included.

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Table 6 Estimates of haplotype frequency of APOA5 gene in 211 cases and 317 age/sex-matched controls and significance levels of comparison from permutation tests Haplotype -1131

553

T C T C

G G T T

Log(ln) Likelihoods * a

Overall (n = 528)

Cases (n = 211)

Controls (n = 317)

χ2

P*

0.696 0.222 0.002 0.080

0.6960 0.201 0.0020 0.109

0.699 0.236 0.004 0.061

0.110 1.769 1.821 8.162

0.410 0.100 0.151 0.001

−663.5

−279.8

−378.5

10.496a

0.006

P-values based on 1000 permutations. Likelihood ratio test statistical values for omnibus test.

3.5. Association between APOA5 haplotype and CAD Haplotype frequencies were separately estimated for 211 cases and 317 age/sex-matched controls using an expectation–maximization algorithm. Table 6 displays the results of a 2-loci estimated haplotype frequency analysis for the APOA5 genes in the case and control groups. The individual haplotype analyzes identified haplotype -1131C/c.553T with significantly higher frequency in the CAD than in the controls at a level of significance, P = 0.001 (<0.0125, Bonferroni-corrected significance level = 0.05/4). The omnibus haplotype profile test was significant (χ2 = 10.496, P = 0.006), indicating that the overall haplotype frequency profiles differed between the CAD cases and the controls.

4. Discussion This study analyzed the APOA5 gene variants for triglyceride levels in a sample of Taiwanese Chinese and the subsequent association between the APOA5 gene polymorphisms or the haplotype profile and angiographically determined CAD. Both univariate and multivariate analyzes showed that the genetic variants of the APOA5 gene, including the 1131T > C, c553G > T and the haplotypes derived from them, significantly influence triglyceride levels. Furthermore, a significant association was demonstrated by a case-controlled study between the c.553G > T polymorphism and the risk of angiographically defined CAD in both univariate and multivariate analyzes. A significant association between bi-loci APOA5 gene polymorphisms and CAD in a Taiwanese Chinese population was verified by permutation-based haplotype analysis. Consistent with previous reports, this study demonstrated a significant association between the APOA5 -1131T > C polymorphism and plasma triglyceride and HDL-C levels. The C-allele frequency for our sample population (30%) is similar to that reported for Chinese in China [8] and Singapore [9] and for Japanese [6,7], but higher than that reported for Caucasian populations [3,18]. As in previous reports, the carriers of the APOA5 -1131C had triglyceride levels that were approximately 25% higher than those in non-carriers

in the control group of this population, indicating that this is also an important genetic determinant of triglyceride levels in Taiwanese Chinese. The results of previous studies for the claim that APOA5 -1131C carriers bear increased risk of CAD are inconsistent [19–25]. This study shows that homozygous carriers of APOA5 -1131C allele were associated with increased risk of CAD, which finding agrees with Bi et al. [21] study of another Chinese population. This polymorphism is located in the promoter region of the APOA5 gene, so -1131C may affect the transcriptional activity of the APOA5 gene. Another possibility is that an unknown functional mutation may be present at, or adjacent to the APOA5 gene locus, which is in LD with the -1131T > C polymorphism, giving rises to the inter-individual triglyceride and HDL-C variation, and thus contributes to the risk of CAD. The -3A > G, which is in strong LD with -1131T > C and is located in the Kozak sequence preceding the predicted translation start codon, potentially affects the rate of APOA5 translation and could be a candidate [12]. However, no functional study has supported the claim that either of these two polymorphisms is a functional variant. The S19W mutation, which changes hydrophilic serine to hydrophobic tryptophan within the APOA5 signal peptide, is another potential candidate [23,25]. However, S19W mutation is much rarer in the Chinese population than in the Caucasian population, according to the data herein and those from other studies [9]. Accordingly, whether c.553G > T, which was in strong LD with APOA5*2 haplotype defined by Pinnacchio and causes a substitution of a cysteine for a glycine, as described by Kao et al. [13] in another Taiwanese Chinese population, is associated with the risk of CAD is examined. This study reproduced Kao’s finding that c.553G > T is associated with hypertriglyceridemia in Taiwanese, using both univariate and multivariate analyzes. This study also demonstrated that both APOA5 -1131C and APOA5 -1131C/c553T haplotype were associated with reduced plasma HDL-C levels, perhaps through influencing serum triglyceride levels, because the association between APOA5 genotypes/haplotypes and HDL-C levels was lost when the triglyceride level was included in the analysis. Moreover, the carriers of c.553T allele were significantly associated with an increased risk of CAD. The differences remained significant either after the use of stringent Bonferroni correction for multiple tests (2 SNPs, P < 0.025) or

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adjustment for the other traditional risk factors. Notably, haplotype analysis was used to confirm further that the APOA5 -1131C/c553T haplotype was significantly overpresented in CAD group, as compared to the control group. Whether c553G > T is associated with an increased risk of CAD through atherogenic lipid profile or APOA5 function per se, remains unclear. Although APOA5 -1131C, c.553T alleles and CT haplotype have been demonstrated to be associated with higher triglyceride and lower HDL-C levels in the control group, this study is limited by the fact that only half of the CAD cases’ complete lipid profile is available. Therefore, triglyceride and HDL-C levels could only be partially included in the multivariate logistic regression analysis of the risk of CAD. However, the findings that APOA5 polymorphism is associated with increased risk of CAD independently of other traditional risk factors including triglyceride are similar to those of Szalai et al. [20], who proposed that this association might result from LD with APOC3 -482T allele and through insulin resistance conferred by this haplotype (APOC3 -482T/APOA5 -1131C). In contrast, although APOA5 -1131C is in LD with APOC3 -482T as shown in other studies, APOA5 c.553 > T is not in LD with APOC3 -482T in this Taiwanese population (data not shown). Furthermore, APOA5 c553T was not associated with plasma APOC3 levels in Kao et al. [13] study. In Bi et al. [21] study, the association between -1131T > C and the risk of CAD in the Chinese population remained significant after adjustment for the APOC3 -482 variant. Moreover, c553T mutation occurs in an evolutionary conserved region of the APOA5 protein, suggesting that it is a causative mutation [26]. Taken together, these results indicate that the association between APOA5 polymorphisms and CAD is independent of APOC3 polymorphisms in this Taiwanese population. Another possible mechanism is that variation in the plasma concentration or function of APOA5 as determined by APOA5 c553G > T may result in the differences of HDL composition as reflected by changes in the fractional esterification rate in apoB-depleted lipoproteins (FERHDL ), which has been documented to be an independent predictor of CAD [27] and was recently reported to be significantly modulated by APOA5 -1131T > C [19]. The pathogenic mechanism for the relationship between hypertriglyceridemia and CAD remains uncertain. Recent research reveals that triglyceride-rich particles may directly damage the endothelium by increasing oxidative stress [28]. Possible mechanisms of the hypotriglyceridemic effect of APOA5 are that APOA5 intracellularly inhibits VLDL assembly [26], or activates lipoprotein lipase to increase lipolysis and VLDL clearance [29,30]. Recently, Jang et al. [31] showed that -1131C carriers exhibited reduced clearance of postprandial triglyceride-rich lipoproteins, along with higher oxidative stress with increased serum dense LDL, C-reactive protein and urinary 8-epi-prostaglandin F2␣ levels. They also exhibit more lymphocyte damage. Lai et al. [22] demonstrated that APOA5 -1131T > C and S19W and haplotype*2 and *3, defined by them, respectively, were associated with the VLDL-intermediate, remnant-like particles (RLP) and

triglyceride levels. Female -1131C carriers were significantly associated with a higher risk of CAD in the Framingham population. Therefore, a larger, prospective and longitudinal study that stratifies patients according to plasma triglycerides, HDL-C, VLDL, LDL, RLP, apolipoprotein B and oxidative stress marker levels would be necessary to elucidate more clearly the underlying mechanisms by which APOA5 c553G > T affects the risk of CAD in Chinese and other ethnic populations. In conclusion, this case–control study demonstrated that APOA5 -1131C and c.553T carriers are associated with raised plasma triglycerides, and that c.553T and -1131C/553T haplotype carriers are associated with increased risk of CAD in Taiwanese Chinese. This observation suggests that the APOA5 polymorphisms/haplotype could be used as predictors for CAD in hypertriglyceridemia subjects among Taiwanese Chinese in the future.

Acknowledgement The authors would like to thank Dr. Chia-Ti Tsai of the National Taiwan University Hospital for his kind help in the haplotype analysis.

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