Combined Hepatic Lipase -514C/T and Cholesteryl Ester Transfer Protein I405V Polymorphisms Are Associated with the Risk of Coronary Artery Disease

Abstract

Hepatic lipase (LIPC) and cholesteryl ester transfer protein (CETP) are important components of high-density lipoprotein (HDL) metabolism and reverse cholesterol transport. Therefore, their genes are promising candidate genes for cardiovascular disease. The aim of the present study was to investigate whether combined LIPC -514C/T and CETP I405V polymorphisms correlate with angiographically documented coronary artery disease (CAD). Genotyping was performed in 317 patients who underwent clinically indicated coronary angiography. The patients were classified with significantly diseased arteries if one or more coronary arteries had a stenosis >50% and with minimally diseased arteries if there was no significant stenosis (<40%) in any artery. There were no significant associations of individual polymorphisms with the risk of significant CAD. In a multivariate logistic regression analysis including cardiovascular risk factors, simultaneous presence of both LIPC -514T and CETP 405V alleles was an independent predictor of significantly diseased arteries (odds ratio = 2.04; p = 0.022). This association was not significant in women with combined genotype who had the highest HDL-cholesterol. In conclusion, the combined T allele of LIPC -514C/T and V allele of CETP I405V are associated with the risk of CAD. Further, the higher HDL-cholesterol and female gender may reduce the effect of combined genotype on CAD risk.

Introduction

LIPC is a glycoprotein that plays a major role in remodeling HDL by hydrolyzing the triglycerides (TGs) and phospholipids of TG-rich HDL, transferring them into the small, dense HDL (Bensadoun and Berryman, 1996). Low LIPC activity has been shown to be associated with higher HDL-cholesterol (HDL-C) levels (Perret et al., 2002). The less common T allele of LIPC -514C/T polymorphism (rs1800588) in the promoter region has been associated with low LIPC activity and higher plasma HDL-C levels (Cohen et al., 1999). However, the relevance of this single-nucleotide polymorphism (SNP) for coronary heart disease (CHD) remains controversial (Cohen et al., 1999; Fan et al., 2001; Andersen et al., 2003). The frequency of T allele varies widely in different regions of the world and among ethnic groups, ranging from 55% in a Canadian ethnic population (Hegele et al., 1999) to 17% in one of the Caucasian samples (Guerra et al., 1997).

The CETP, a hydrophobic glycoprotein, mediates the transfer of cholesteryl esters from HDL to apolipoprotein-B-containing particles. The critical role of CETP in lipoprotein metabolism has been confirmed by high concentrations of HDL-C observed in patients with genetic CETP deficiency (Inazu et al., 1990). CETP I405V is a very common SNP (rs5882) arising from A→G transition in exon 14 which leads to a missense mutation with the substitution of valine for isoleucine at codon 405 (Boekholdt et al., 2004). The frequency of the 405V allele in African-Americans is 0.611 and 0.318 in Caucasians (Thompson et al., 2005). Previous studies of the CETP I405V polymorphism have shown no association with the risk of CHD or lipid parameters (Pallaud et al., 2001) or higher HDL-C levels and elevated risk of CHD for the carriers of mutated alleles (Bruce et al., 1998a). Despite intense study, it is still unclear whether, how, and under which circumstances this CETP gene variation affects CHD risk status (Borggreve et al., 2006).

It has been suggested that the various SNPs may interact with one another in metabolic networks to produce an overall effect that could not, therefore, be predicted from the effects of any one SNP in isolation (Zivkovic and German, 2007). Therefore, the so-called genetic burden analysis is increasingly used. This approach considers polymorphisms in different genes in critical pathways together and investigates whether potential interaction effects can be observed (Anderson and Carlquist, 2003). A good example of such analyses is a recent study by Muendlein et al. (2008), who evaluate the role of lipoprotein metabolism controlling gene polymorphisms in coronary artery disease (CAD). They showed synergistic effects (odds ratio [OR] = 3.99; p = 0.03) of the Apoε3/ε2/ε4, the CETP Taq1B, and the ApoC3 -482C/T polymorphisms on their association with CAD.

According to the recent findings, simultaneous presence of CETP Taq1B and LIPC -514C/T variants results in an increased risk for CAD (van Acker et al., 2008). This suggests that combined reduction in both CETP and LIPC activities due to other SNPs may also lead to increased risk of CAD. To test this hypothesis, the influence of LIPC -514C/T and CETP I405V polymorphisms and the combined effect of -514T and 405V alleles in the genetic risk for developing CAD were investigated.

Materials and Methods

Subjects

Subjects were recruited from a patient population scheduled for diagnostic coronary angiography at Madani Hospital in Tabriz between February 1, 2007 and September 30, 2008. In the present study, 317 patients (219 men and 98 women) were enrolled. Exclusion criteria were age >75 years, a previous hospital admission related to cardiovascular disease, current lipid-lowering medication, and a previous diagnosis of angina, diabetes, or any other chronic illnesses. Exclusion of persons with diabetes mellitus was based on self-reported diabetes as well as on fasting serum glucose >125 mg/dL. Venous blood samples were taken after 12-14 h fasting in the morning before angiography. Blood was centrifuged and serum supernatant was deep frozen for later analysis. The study was approved by the ethics committee of Tabriz University (Medical Sciences), and all patients gave written informed consent.

Angiographic analysis

All patients underwent coronary angiography; minimally diseased arteries were defined as those with all stenoses <40% and significantly diseased arteries (SDA) as those with at least one stenosis >50% or occlusions.

Laboratory analysis

Serum total cholesterol, TG, and HDL-C were determined using standard enzymatic procedures. Apolipoprotein-A-I and apolipoprotein-B concentrations were measured by immunoturbidometric methods (DiaSys Diagnostics, Holzheim, Germany). Low-density lipoprotein-cholesterol concentrations were calculated using the Friedewald formula (Friedewald et al., 1972).

Genetic analyses were performed on genomic DNA isolated from leukocytes. Genotyping for the LIPC -514C/T polymorphism was performed by NlaIII digestion. The I405V polymorphism can be analyzed by a mutagenesis primer that creates an RsaI restriction site. DNA fragments were amplified by polymerase chain reaction, using the same conditions as previously described (van't Hooft et al., 2000; Padmaja et al., 2007). The sequences of primers used for the amplifications were as follows: LIPC -514C/T primer, forward 5′-GGATCACCTCTCAATGGGTC-3′, reverse 5′-ACCTGGTTTCAGGCTTTGTC-3′; and CETP I405V primer, forward 5′-GCAGAACAGTACTGGCCAAGCAGCG-3′, reverse 5′-GCGGTGATCATTGACTG CAGGAAGCTCTGTA-3′.

Data analyses

The level of significance between groups was calculated according to the t-test for continuous variables and χ² test for categorical variables. The values of the TG were converted by logarithmic transformation to normalize distribution of the data. Analysis of covariance for other risk factors was used to test the independence of associations between the lipid parameters as outcome variables, and genotypes as independent variables. Logistic regression analysis was applied to evaluate whether the LIPC and CETP polymorphisms and their genotype combination predict the risk of CAD. A p-value of <0.05 was considered statistically significant. All analyses were carried out using SPSS for windows version 11.0 (SPSS, Chicago, IL).

Results

Population characteristics

The main demographic and clinical data of both groups of patients are presented in Table 1. Patients with angiographically significant coronary atherosclerosis were 3.5 years older than patients without coronary atherosclerosis (p = 0.001). The prevalence rate of male sex was also significantly higher in patients with significant CAD. Therefore, the analyses of the other parameters were performed after adjustment for age and sex. The two groups also differed significantly by the prevalence of smoking. There were no significant differences between the two groups for serum lipid, lipoprotein, and CETP concentration after adjustment for age and sex.

Table 1.

Clinical Characteristics of Patients with Minimal Versus Severe Coronary Artery Disease

	MDA (n = 139)	SDA (n = 178)	p
Age, year	51.6 ± 8.8	55.3 ± 9.4	0.001
Gender, women (%)	61 (44%)	37 (21%)	0.001
Body mass index, kg/m²	26.9 ± 4.1	27.2 ± 4.1	0.57
Smokers, %	34 (25%)	77 (43%)	0.001
Hypertension, %	34 (24%)	59 (33%)	0.09
Family history, %	18 (13%)	20 (11%)	0.59

Values are means ± SD or percentage with condition. The patients were classified as having SDA if one or more coronary arteries had a stenosis >50% and as having MDA if there was no significant stenosis (<40%) in any artery.

MDA, minimally diseased arteries; SDA, significantly diseased arteries; SD, standard deviation.

In the studied population, genotype and allele frequency were in Hardy-Weinberg equilibrium (p = nonsignificant). The frequencies of -514T and 405V alleles in the studied population were 0.21 and 0.37, respectively. Table 2 shows the distribution of the individual LIPC -514C/T and CETP I405V genotypes with respect to the presence of significant coronary atherosclerosis. There were no significant differences in the genotype distribution and allele frequency between the CAD patients and control subjects.

Table 2.

Metabolic Parameters of Patients with Minimal Versus Severe Coronary Artery Disease

	MDA (n = 139)	SDA (n = 178)	p
Cholesterol, mg/dL	170 ± 40	176 ± 42	0.27
TG, mg/dL	162 (111-216)	170 (121-216)	0.48
HDL-C, mg/dL	38 ± 11	37 ± 9	0.41
LDL-C, mg/dL	94 ± 34	102 ± 36	0.09
Apo-A-I, mg/dL	124 ± 21	121 ± 20	0.14
Apo-B, mg/dL	103 ± 26	104 ± 28	0.79

Values are means ± SD, or if variables did not exhibit normal distribution, medians with 25 and 75 percentiles in brackets. All data were adjusted for age and gender. The patients were classified as having SDA if one or more coronary arteries had a stenosis >50% and as having MDA if there was no significant stenosis (<40%) in any artery.

TG, triglyceride; HDL-C, high-density lipoprotein-cholesterol; LDL-C, Low-density lipoprotein-cholesterol; Apo-A-I, apolipoprotein-A-I; Apo-B, apolipoprotein-B.

Genotype effect on CAD risk

The associations of individual and combined genotypes with the presence of significant CAD are presented in Table 3. No significant associations of individual polymorphisms with SDA were observed. The risk of SDA was significantly increased in carriers of both LIPC -514T and the CETP 405V alleles (OR = 1.86; p = 0.041). Multiple logistic regression analyses that included conventional risk factors (Table 3) also showed that simultaneous presence of both LIPC -514T and CETP 405V alleles was an independent predictor of SDA (OR = 2.04; p = 0.022). When analyzed separately in men and women, we still found no association between individual genotypes and significant CAD in both gender. The association between the combined genotype and SDA was no longer significant in women (OR = 1.34; p = 0.621), whereas the association in men (OR = 2.21; p = 0.042) remained significant (Table 3).

Table 3.

Genotype Distributions in Patients with Minimal Versus Severe Coronary Artery Disease

Gene	MDA (n = 139)	SDA (n = 178)	p
LIPC -514C/T
CC	93 (66.9%)	110 (61.8%)	0.429
CT	37 (26.6%)	59 (33.1%)
TT	9 (6.5%)	9 (5.1%)
CETP I405V
II	59 (42.4%)	71 (39.9%)	0.736
IV	61 (43.9%)	80 (44.9%)
VV	19 (13.7%)	27 (15.2%)

p-Values were calculated by χ² tests. The patients were classified as having SDA if one or more coronary arteries had a stenosis >50% and as having MDA if there was no significant stenosis (<40%) in any artery.

LIPC, hepatic lipase; CETP, cholesteryl ester transfer protein.

Serum lipid and lipoprotein levels

The association of individual and combined genotype with significant CAD was examined after adjustment for age, sex, body mass index, hypertension, and smoking (Table 4). The LIPC -514C/T polymorphism was statistically associated with serum lipids, having higher HDL-C and lower TG concentrations as compared with CC homozygotes (p < 0.05). Since the number of the TT genotype of LIPC and VV genotype of CETP was low, the heterozygotes and homozygotes were combined. Subjects simultaneously having the T allele and V allele were considered as subjects having mutated alleles (LIPC -514CT/TT and CETP 405 IV/VV). Other subjects were classified as subjects having none or one of the mutated alleles (other genotype combinations). No significant association between serum lipids and simultaneous presence of both T allele and V allele was observed (Table 5). When men and women were analyzed separately, women carrying both mutated alleles had a significant increase in their HDL-C and decrease in TG compared with women having none or one mutated allele of both SNPs (Table 6). When combinations were compared among each other, there was also a significantly higher HDL-C in female patients with both mutated alleles than in females with the other three combined genotypes (p = 0.041). This effect was not observed in male patients (Fig. 1).

FIG. 1.

Comparison of high-density lipoprotein-cholesterol (HDL-C) levels according to cholesteryl ester transfer protein (CETP) I405V and hepatic lipase (LIPC) -514C/T genotypes. p-Values were derived from analysis of covariance adjusting for age, sex, body mass index, hypertension, and smoking.

Table 4.

Logistic Regression Analysis of Individual and Combined Genotypes with Respect to the Presence of Significant Stenosis as Dependent Variable

	Univariate			Multivariate ^a
	OR	95% CI	p	OR	95% CI	p
Individual genotypes
LIPC -514 CT/TT
All subjects	1.14	0.72-1.80	0.59	1.28	0.79-2.08	0.31
Men	1.22	0.49-2.99	0.67	1.06	0.58-1.90	0.86
Women	1.77	0.75-4.18	0.19	1.48	0.59-3.72	0.41
CETP 405 IV/VV
All subjects	1.11	0.71-1.75	0.65	1.22	0.76-1.94	0.41
Men	1.45	0.83-2.54	0.19	1.55	0.87-2.75	0.14
Women	0.77	0.29-1.53	0.33	0.69	0.28-1.68	0.42
Combined genotype
LIPC -514 CT/TT and CETP 405 IV/VV
All subjects	1.86	1.04-3.47	0.04	2.04	1.12-3.74	0.02
Men	2.01	0.96-4.23	0.06	2.21	1.03-4.71	0.04
Women	1.41	0.50-3.96	0.51	1.34	0.43-4.17	0.62

Values are 95% CI, the OR, and the level of significance p-value, which show the association between genotypes and the existence of coronary artery disease. The patients were classified as having SDA if one or more coronary arteries had a stenosis >50% and as having MDA if there was no significant stenosis (<40%) in any artery.

Adjusted for age, sex, body mass index, hypertension, smoking, TG, HDL-C and LDL-C, apolipoprotein-A-I, and apolipoprotein-B.

CI, confidence interval; OR, odds ratio.

Table 5.

Metabolic Parameter According to Individual and Combined Genotypes

	LIPC -514C/T		CETP I405V		Combined genotypes
	CC	CT/TT	II	IV/VV	CT/TT and IV/VV	All other genotype combinations
n	202	115	130	187	64	253
Cholesterol, mg/dL	174 ± 42	172 ± 42	169 ± 41	176 ± 42	173 ± 40	174 ± 42
TG, mg/dL	185 ± 80	167 ± 90^a	177 ± 83	180 ± 85	169 ± 99	181 ± 80
HDL-C, mg/dL	37 ± 9	40 ± 12^a	37 ± 9	38 ± 11	40 ± 14	37 ± 10
LDL-C, mg/dL	100 ± 36	97 ± 34	94 ± 35	101 ± 35	99 ± 31	99 ± 36
Apo-A-I, mg/dL	122 ± 19	123 ± 22	123 ± 19	122 ± 22	124 ± 25	123 ± 19
Apo-B, mg/dL	105 ± 27	102 ± 30	103 ± 30	104 ± 26	100 ± 28	105 ± 28

Values are means ± SD. p-Values are derived from ANCOVA adjusting for age, sex, body mass index, hypertension, and smoking. TG values were log-transformed before ANCOVA.

p-Value < 0.05 versus corresponding controls.

ANCOVA, analysis of covariance.

	LIPC -514C/T	CETP I405V	Combined genotypes
Men
n	136	83	91	128	46	173
Cholesterol, mg/dL	168 ± 38	167 ± 42	161 ± 37	172 ± 41	173 ± 43	166 ± 39
TG, mg/dL	185 ± 84	172 ± 98	177 ± 90	182 ± 89	178 ± 109	180 ± 84
HDL-C, mg/dL	35 ± 8	38 ± 10^a	36 ± 9	37 ± 10	37 ± 11	36 ± 9
LDL-C, mg/dL	96 ± 33	94 ± 33	89 ± 31	99 ± 34^a	100 ± 32	94 ± 33
Apo-A-I, mg/dL	120 ± 19	119 ± 20	121 ± 18	119 ± 20	119 ± 22	120 ± 18
Apo-B, mg/dL	103 ± 26	100 ± 31	99 ± 28	104 ± 28	103 ± 32	102 ± 27
CETP, μg/mL	2.00 ± 0.77	2.00 ± 0.69	2.10 ± 0.74	1.90 ± 0.73	1.95 ± 0.73	2.00 ± 0.75
Women
n	66	32	39	59	18	79
Cholesterol, mg/dL	187 ± 47	185 ± 38	188 ± 43	186 ± 45	174 ± 33	188 ± 45
TG, mg/dL	185 ± 74	155 ± 62^a	178 ± 65	174 ± 75	144 ± 66^a	183 ± 71
HDL-C, mg/dL	40 ± 11	43 ± 15^a	38 ± 11	43 ± 13^a	47 ± 16^a	40 ± 11
LDL-C, mg/dL	109 ± 42	104 ± 37	107 ± 41	108 ± 39	98 ± 28	109 ± 41
Apo-A-I, mg/dL	128 ± 21	135 ± 25	129 ± 20	132 ± 24	138 ± 30	129 ± 20
Apo-B, mg/dL	109 ± 28	106 ± 29	113 ± 33	104 ± 25	93 ± 16^a	111 ± 29
CETP, μg/mL	2.61 ± 1.05	2.41 ± 0.77	2.97 ± 0.83	2.25 ± 1.01	2.00 ± 0.74	2.62 ± 1.01

Table 6.
Metabolic Parameters in Men and Women According to Individual and Combined Genotypes

LIPC -514C/T CETP I405V Combined genotypes

CC CT/TT II IV/VV CT/TT and IV/VV All other genotype combinations

Men

n 136 83 91 128 46 173

Cholesterol, mg/dL 168 ± 38 167 ± 42 161 ± 37 172 ± 41 173 ± 43 166 ± 39

TG, mg/dL 185 ± 84 172 ± 98 177 ± 90 182 ± 89 178 ± 109 180 ± 84

HDL-C, mg/dL 35 ± 8 38 ± 10^a 36 ± 9 37 ± 10 37 ± 11 36 ± 9

LDL-C, mg/dL 96 ± 33 94 ± 33 89 ± 31 99 ± 34^a 100 ± 32 94 ± 33

Apo-A-I, mg/dL 120 ± 19 119 ± 20 121 ± 18 119 ± 20 119 ± 22 120 ± 18

Apo-B, mg/dL 103 ± 26 100 ± 31 99 ± 28 104 ± 28 103 ± 32 102 ± 27

CETP, μg/mL 2.00 ± 0.77 2.00 ± 0.69 2.10 ± 0.74 1.90 ± 0.73 1.95 ± 0.73 2.00 ± 0.75

Women

n 66 32 39 59 18 79

Cholesterol, mg/dL 187 ± 47 185 ± 38 188 ± 43 186 ± 45 174 ± 33 188 ± 45

TG, mg/dL 185 ± 74 155 ± 62^a 178 ± 65 174 ± 75 144 ± 66^a 183 ± 71

HDL-C, mg/dL 40 ± 11 43 ± 15^a 38 ± 11 43 ± 13^a 47 ± 16^a 40 ± 11

LDL-C, mg/dL 109 ± 42 104 ± 37 107 ± 41 108 ± 39 98 ± 28 109 ± 41

Apo-A-I, mg/dL 128 ± 21 135 ± 25 129 ± 20 132 ± 24 138 ± 30 129 ± 20

Apo-B, mg/dL 109 ± 28 106 ± 29 113 ± 33 104 ± 25 93 ± 16^a 111 ± 29

CETP, μg/mL 2.61 ± 1.05 2.41 ± 0.77 2.97 ± 0.83 2.25 ± 1.01 2.00 ± 0.74 2.62 ± 1.01

Values are means ± SD. p-Values are derived from ANCOVA adjusting for age, sex, body mass index, hypertension, and smoking. TG values were log-transformed before ANCOVA.

a
p-Values < 0.05 versus corresponding control.

Discussion

Study of LIPC -514C/T and CETP I405V polymorphisms that affect CETP and LIPC activity can provide a better understanding of the role of CETP and LIPC in lipid metabolism and cardiovascular disease. For the first time, we examined whether combined LIPC -514C/T and CETP I405V polymorphisms are associated with angiographically significant CAD.

In the prospective cohort Rotterdam Elderly study, despite an increase in HDL-C levels, combined LIPC -514C/T and CETP I405V genotype did not affect myocardial infarction risk (Isaacs et al., 2007). However, the results of the present study showed that simultaneous presence of rare alleles of these common polymorphisms is associated with increased incidence of stenosis >50%. This association was independent of conventional risk factors. The base rate of risk factors may vary significantly across different populations and this fact can affect the association analysis. This study focused only on cases with no history of heart disease and with no interfering medication, and it accurately selected the control group by angiography to exclude any significant coronary disease. In line with our findings, van Acker et al. (2008) also showed that combined LIPC -514C/T and CETP Taq1B genotype were associated increased risk of CAD.

The pattern of serum lipids found in the carriers of the T allele agree with previous studies that affirm that a LIPC -514C/T polymorphism is associated with high HDL-C and lower TG levels (Boekholdt et al., 2004). Although in the present study no significant relationships were found between the combined genotype and any of the serum lipids, when we investigated the difference in HDL-C and TG concentrations only in the females, the mean differences were greater than that as whole. Moreover, the HDL-C concentration was significantly higher in the female patients with CT or TT genotype than those with the CC genotype if they were V carriers of the CETP gene. These findings are consistent with those of some investigators who have reported that the associations of the individual -514C/T and I405V with higher HDL-C are gender specific and only significant in certain subgroups (Guerra et al., 1997; Bruce et al., 1998b; Couture et al., 2000). Similarly, Isaac et al. (2007) in a population-based cohort study reported stronger effects of combined LIPC -514C/T and CETP I405V genotypes in women than in men (0.3 mM, p = 0.03 and 0.12 mM, p = 0.13, respectively). The different effects of the combined polymorphism on HDL-C levels of men and women may be related to a gender-specific expression of the polymorphic alleles. Further, the rate-limiting factors determining plasma HDL-C concentrations may differ depending on gender, as suggested previously (Guerra et al., 1997).

The present study does not clarify the mechanism by which the combined polymorphism adversely affects coronary atherosclerosis risk status. It can be explained by the effect of each polymorphism on the cholesterol transfer system (Hirano et al., 1995; Dugi et al., 2001). One explanation for the difference between men and women in the effect of the combined genotype on the risk of CAD may be relative to their difference in serum lipids. It appears that the HDL-C-additive raising effect of low CETP and low LIPC alleles is limited to females. In epidemiologic studies, increased HDL-C is protective against the development of atherosclerotic disease. Therefore, it is possible that higher HDL-C may interfere with the relationship between the combined genotype and CAD risk and that the female gender may reduce the effect of the combined genotype on CHD risk status. This hypothesis may be useful for further study on the effects of these polymorphisms. On the other hand, it has been shown that the effect of both CETP I405V and LIPC -514C/T polymorphisms on CHD risk, like HDL levels, is restricted to conditions of hypertriglyceridemia (Bruce et al., 1998b; Kakko et al., 2000; Jansen et al., 2002; Darabi et al., 2009). The results of this study showed that in women with combined genotype who had the lowest TG level, there was no significant association between the prevalence of SDA and the genotype. One possible explanation for this is that there is also an excess in adverse effect of the combined CETP and LIPC gene variants on CAD at high TG levels.

In conclusion, the present results add novel insight to the importance of RCT in the development of atherosclerosis based on genetic variability of a combination of genes involved in RCT regulation. A carrier trait of a combination of LIPC -514C/T and CETP I405V, rather than each polymorphism individually, was associated with the risk of CAD by using a multivariate logistic regression and a more favorable lipid profile in female gender interfered with this association.

Footnotes

Acknowledgments

This work was supported by grants from the Drug Applied Research Center (53/86) and Biotechnology Research Center (85122) at Tabriz University (Medical Sciences).

Author Disclosure Statement

No competing financial interests exist.

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