Correlation Between Common Genetic Variants and Risk Factors Associated with Prediction of Cardiovascular Diseases in Dyslipidemic Patients

Abstract

Aims:

The aim of the study was to investigate genetic variants predicting cardiovascular events in patients with dyslipidemia and compare its relationship with common risk factors including hyperlipidemia, metabolic syndrome, history of acute myocardial infarction, thrombosis, obesity, and smoking.

Materials and Methods:

Five hundred two individuals divided into six groups corresponding with the risk factors and a control group of normolypidemic patients were analyzed for the presence of eight mutations and polymorphisms (endothelial nitric oxide synthase −786T→C and G894T; lymphotoxin A C804A; angiotensin-converting enzyme [ACE] ins/del; human platelet antigen 1 a/b; beta-fibrinogen −455G→A; apolipoprotein B [ApoB] R3500Q; APOE E2/E3/E4) using the ViennaLab CVD Strip assay.

Results:

ACE deletions are the most frequent genetic variants in risk groups of dyslipidemic patients (from 58% in cardiovascular events to 51% in smokers). We found a strong relationship between genetic variants and risk factors. G894T is significantly associated with smoking (value of odds ratio [OR]=1.62, p=0.04), and ACE deletions are negatively associated with cardiovascular events (OR=0.62, p=0.03).

Conclusion:

Significant associations between genetic variants predicting cardiovascular events and common risk factors in dyslipidemic patients were found.

Introduction

The pathological process leading to atherosclerosis is characterized by endothelial dysfunction, hyperlipidemia, inflammation, and hypertension. Polymorphisms and mutations in genes involved in these processes in combination with an unhealthy lifestyle and metabolic syndrome support thromboembolic events on disrupted atherosclerotic lesions in the vessel wall, the basic mechanism underlying myocardial infarction and ischemic stroke. A single genetic defect rarely exerts a dramatic effect in the development of cardiovascular diseases. Most gene variations contribute to minor effects, and the individual cardiovascular risk is related to a critical accumulation of detrimental polymorphisms acting in synergy with unfavorable environmental factors (e.g., cigarette smoking, dietary fat consumption, hypertension, and physical inactivity) (Nordlie et al., 2005; Wang, 2005; Mayer et al., 2007). Genes and polymorphisms involved in blood homeostasis, coagulation, and atherosclerosis are excellent candidates for prediction of cardiovascular events (Cheng et al., 1998; Duga et al., 2000; Wang, 2005; Visvikis-Siest and Marteau, 2006).

The aim of the study was to investigate polymorphisms and mutations predicting cardiovascular events and the relationship to common environmental risk factors (smoking, metabolic syndrome, arterial thrombosis, obesity, and cardiovascular events, including history of myocardial infarction or acute myocardial infarction) in dyslipidemic patients.

An overview of investigated mutations and polymorphisms associated with cardiovascular risk in patients with various defects of lipid metabolism is given in Table 1.

Table 1.

Overview of Investigated Mutations and Polymorphisms Associated with Cardiovascular Risk

Gene	Mutations/Polymorphisms	Comment	References
eNOS; NOS3	−786 T>C	The C allele causes a higher susceptibility to coronary heart disease.	Nakayama et al., 1999
	894 G>T (Glu298Asp)	The T allele confers an increased risk for premature MI.	Casas et al., 2004
LTA	804 C>A (Thr26Asn)	It is in almost complete linkage with 252A>G; both variants act strongly proinflammatory and are associated with coronary artery disease.	Ozaki et al., 2002
ACE	287 bp insertion/deletion (I/D):	The D allele is associated with the elevated ACE activity and plasma levels and represents a risk factor for MI in elder patients and in smokers.	Cambien et al., 1992
HPA1	L33P (1a/b)	HPA1b is a risk factor for early-onset MI and stroke, particularly in smokers.	Newman et al., 1989Weiss et al., 1996
βFBG	−455 G>A	This confers elevated β-fibrinogen plasma levels and increases risk for premature MI and ischemic stroke.	de Maat et al., 1998Folsom et al., 2001
Apo B	R3500Q	It is a dominant but rare genetic defect causing severe hypercholesterolemia and elevated risk for atherosclerosis.	Tybjaerg-Hansen et al., 1991 and 1998; Tybjaerg-Hansen and Humphries, 1992
ApoE	E2/E3/E4 variants	These are important predictors of the plasma lipid profile, with E2 showing lowest and E4 showing highest LDL and total cholesterol levels; E4 allele is associated with increased susceptibility to early-onset MI, particularly in smokers.	Weisgraber et al., 1981; Eto et al., 1989

eNOS and NOS3, endothelial nitric oxide synthase; LTA, lymphotoxin A; ACE, angiotensin-converting enzyme; HPA1, human platelet antigen 1 (glycoprotein III receptor; integrin β3); βFBG, beta-fibrinogen; ApoB, apolipoprotein B; ApoE, apolipoprotein E, LDL, low-density lipoprotein; MI, myocardial infarction.

Materials and Methods

Patients

We analyzed DNA samples from adult patients already on lipid-lowering therapy (statins and fibrates) suffering from various defects of lipid metabolism and DNA samples from patients with normal values of lipid parameters (S-cholesterol <5 mM, S-triglycerides <1.7 nM, low-density lipoprotein [LDL]-cholesterol <3 mM; serum levels of high-density lipoprotein, apolipoprotein B (ApoB), and lipoprotein (a) ranged within the reference values) taken as control group. The values of lipid parameters in the control group and in patients correlated with age and gender.

Dyslipidemic patients were divided into five groups according to the risk factors (obesity, smoking, thrombotic events [arterial thrombosis], metabolic syndrome, and history of acute myocardial infarction).

Demographic, clinical, and biochemical data and characteristics of cases and controls are listed in Table 2.

Table 2.

Demographic, Clinical, and Biochemical Data and Characteristics of Patients and Controls

	Control group N=63	Obesity^a N=107	Smoking N=108	Metabolic syndrome^b N=57	History of acute myocardial infarction N=144	Thrombosis N=23
Mean age (range)	52 (21-82)	57 (21-88)	57 (30-85)	61 (32-84)	61 (31-85)	63 (48-77)
Males/females	10/53	48/59	42/66	26/31	49/95	9/14
S-Cholesterol (mM)	4.20+0.06	6.00+0.11	6.30+0.11	6.30+0.16	6.20+0.09	6.40+0.16
		p<0.0001	p<0.0001	p<0.0001	p<0.0001	p<0.0001
S-LDL-cholesterol (mM)	2.52+0.06	3.6+0.08	3.64+0.10	3.57+0.13	3.57+0.08	3.45+0.14
		p<0.0001	p<0.0001	p<0.0001	p<0.0001	p<0.0001
S-HDL-cholesterol (mM)	1.51+0.05	1.35+0.04	1.43+0.04	1.39+0.06	1.59+0.03	1.58+0.09
		p=0.019	p=0.38	p=0.06	p=0.93	p=0.74
S-Triacylglycerols (mM)	0.95+0.04	1.76+0.34	1.82+0.25	2.75+0.59	1.57+0.22	1.73+0.31
		p<0.0001	p<0.0001	p<0.0001	p<0.0001	p<0.0001
S-Apolipoprotein B (g/L)	0.75+0.02	1.11+0.02	1.11+0.02	1.14+0.04	1.09+0.02	1.15+0.05
		p<0.0001	p<0.0001	p<0.0001	p<0.0001	p<0.0001
S-Lipoprotein (a) (mg/L)	158+65	166+43	194+50	169+54	168+37	114+92
		p=0.84	p=0.36	p=0.84	p=0.52	p=0.63

Serum levels of all lipid parameters correlated with age and gender. Mann-Whitney test was used to determine the differences between the control group and patients groups. Value of p<0.05 is considered significant.

Obesity is defined as BMI >30 kg/m².

Metabolic syndrome is defined according to the NCEPIII and IDF criteria.

S, serum levels of lipid parameters expressed as median±SEM; AMI, acute myocardial infarction; BMI, body mass index; HDL, high-density lipoprotein.

Methods

We tested DNA samples for common mutations and polymorphisms (ApoB R3500Q, ApoE alleles, beta-fibrinogen [βFBG] −455G>A, human platelet antigen 1 [HPA1] Leu33Pro, angiotensin-converting enzyme (ACE) 287bp insertion/deletion, endothelial nitric oxide synthase [eNOS] −786T>C and 894 G>T [Glu298Asp], and lymphotoxin alpha [LTA] Thr26Asn) using the Vienna Lab CVD StripAssay (CVD Strip Assay-A; Vienna Lab) based on a single multiplex polymerase chain reaction followed by the reverse hybridization of biotinylated amplification products and detection by visible enzymatic color reaction.

Strip Assay Evaluator Software Version 2.4 (ViennaLab) was used for investigation of mutations and polymorphisms and for evaluation of the genotype in the patients.

Statistical analysis

Differences between the control group and the patients groups were tested by the nonparametric Mann-Whitney U test. A value of p<0.05 was considered significant. The Kolmogorov-Smirnov test was used for testing of normality of the distribution.

The parametric one sample t-test and value of odds ratio (OR) with chi-square test were used to determine the association of mutations with risk factors. A value of p<0.05 was considered significant.

Hardy-Weinberg equilibrium and the chi-square test were used to assess the distribution of allelic variants in cases and controls.

Results

We investigated the relationship between allelic frequencies of investigated variants in the control group and risk groups including obesity, smoking, metabolic syndrome, thrombosis, and cardiovascular events. The strongest association was found between the G894T mutation and smoking (OR=1.62, 95% confidence interval [CI]=1.01-2.58, p=0.044) and between ACE deletion and cardiovascular events (OR=0.63, 95% CI=0.41-0.96, p=0.03). Results are summarized in Tables 3 and 4.

Table 3.

Relationship Between Allelic Frequencies in Various Groups of Hyperlipidemic Patients

Mutations and polymorphisms	Control group (n=126)	Obesity (n=214)	Smoking(n=216)	Metabolic syndrome (n=114)	History of acute myocardial infarction (n=288)	Thrombosis (n=46)
eNOS −786 T>C	1.03 (0.5-2.12)	0.77 (0.48-1.22)	0.85 (0.54-1.36)	0.80 (0.46-1.35)	0.75 (0.49-1.17)	1.01 (0.5-2.12)
eNOS G894T	1.27 (0.62-2.0)	1.27 (0.8-2.0)	1.617 (1.01-2.58) p =0.044^a	1.281 (0.75-2.18)	1.42 (0.91-2.21)	1.27 (0.62-2.6)
LTA C804A	1.01 (0.48-2.15)	1.03 (0.63-1.67)	0.84 (0.52-1.36)	0.74 (0.43-1.28)	1.20 (0.75-1.92)	1.02 (0.48-2.15)
βFBG −455G>A	1.07 (0.46-2.50)	0.7 (0.41-1.18)	0.83 (0.48-1.41)	0.98 (0.52-1.82)	0.84 (0.5-1.41)	1.07 (0.45-2.49)
HPA1 L33P	1.05 (0.41-2.68)	1.11 (0.6-2.05)	1.07 (0.58-1.97)	0.94 (0.48-1.88)	0.89 (0.51-1.58)	1.05 (0.41-2.68)
ACE del 287 bp	0.80 (0.41-1.59)	0.76 (0.49-1.18)	0.82 (0.53-1.27)	0.69 (0.1-1.14)	0.63 (0.41-0.96) p =0.03^a	0.80 (0.41-1.56)
ApoB R3500Q+ApoE E4	1.51 (0.41-5.61)	0.80 (0.38-1.46)	0.70 (0.34-1.43)	1.23 (0.49-3.03)	0.63 (0.32-1.25)	0.98 (0.25-3.72)

ApoB and ApoE are investigated together because of the small number of samples in groups. Relative risk is expressed as odds ratio value with 95% confidence interval.

p-Value of <0.05 shows significant association of allelic variants with risk factors. Bold values are statistically significant.

Table 4.

Allelic Frequencies of Mutations and Polymorphisms Interrelated with Risk Factors (Frequencies Are Expressed in %)

Mutations and polymorphisms	Control group (n=126)	Obesity (n=214)	Smoking (n=216)	Thrombosis (n=23)	Metabolic syndrome (n=114)	History of acute myocardial infarction (n=288)	p-Value
eNOS −786 T>C	33	39	37	33	39	40 ^a	p=0.049
G894T	30	30	27	33 ^a	32	30	p=0.016
LTA C804A	29	28	32	28	35 ^a	25	p=0.039
βFBG −455G>A	21	27 ^a	24	20	21	24	p=0.004
HPA1 L33P	14	14	15	15	17 ^a	17 ^a	p=0.021
APOB R3500Q	0	0	0	2 ^a	0	0
ACE deletions	47	54	51	52	56	58 ^a	p=0.017
Apo E4	12	13	15	21 ^a	10	20	p=0.015

Parametric one-sample t-test was used to calculate p-value.

The highest frequency of risk alleles found between all groups and related p-value.

Discussion

Research into lipid metabolism disorders, vascular and inflammatory impairment, and investigation of risky mutations and polymorphism is important, because they play major role in the development of atherosclerosis and cardiovascular diseases, which are the leading cause of mortality in Europe (Sans et al., 1997). The presented study uses a complex multigenic approach involving various mutations and polymorphisms and their relationship with risk factors. We investigated polymorphisms and mutations in genes coding proteins playing major roles in lipid metabolism (ApoB, variants of ApoE), inflammation and endothelial growth (eNOS, LTA, glycoprotein III receptor, beta-fibrinogen), and hypertension (angiotensin-converting enzyme) in relation to various risk factors (metabolic syndrome, smoking, obesity, arterial thrombosis, history of cardiovascular events).

Distribution of all variants except of βFBG −455G→A fills the criteria of Hardy-Weinberg equilibrium. We found relationship between genetic variants, clinical status of the patients, and interrelated risk factors. ACE deletions are the most frequent variants found in all risk groups of dyslipidemic patients. The frequencies vary from 58% in dyslipidemic patients with history of cardiovascular events to 51% for dyslipidemic smokers. This finding correlated with previously published data showing similar results in various populations and risk groups (Sayed-Tabatabaei et al., 2003; Wiwanitkit, 2004; Arias-Vasquez et al., 2005).

−786 T>C and HPA1 L33P variants are predominant in the risk group concerning history of cardiovascular events (40% and 17% of all variants respectively); G894T, ApoB R3500Q, and Apo E4 are prevalent in thrombosis (33%, 21%, and 2% of variants, respectively). The relative high prevalence of ApoB R3500Q and Apo E4 in thrombosis could be explained by the fact that the number of dyslipidemic patients with thrombosis is low (thrombosis occurs only in 23 patients).

Despite similar frequencies in various risk groups, some close relationship between allelic variants and risk factors occurs.

eNOS G894T mutation is significantly associated with smoking (OR=1.617, 95% CI=1.01-2.58, p=0.044). This finding correlates with results of other studies (Lembo et al., 2001; Lee et al., 2006). Although in many variants (G894T in cardiovascular events, metabolic syndrome, obesity and thrombosis, ApoB and Apo E4 in metabolic syndrome), there is a more than 1.2-fold increased odds of a strength association; these associations are not statistically significant at the 5% level. On the other hand, ACE deletion showed negative association with cardiovascular events in dyslipidemic patients (OR=0.63, 95% CI=0.41-0.96, p=0.03). This result confirms the findings of other studies presenting the negative association of genetic variants with coronary arteriosclerosis and cardiovascular diseases in patients treated with statins and fibrates (Marian et al., 2000; Maitland-van der Zee et al., 2002, 2004, 2005, 2007, Militiadous, et al., 2005).

These studies show that statins act as inhibitors of hydroxymethyl glutaryl coenzyme A reductase and then lead to the reduction of serum levels of cholesterol and LDL and a decrease in cardiovascular risk in the presence of risk variants (ACE deletions, APO E4 and −455G>A, cholesterol ester transfer protein (CETP) lipoprotein lipase gene, stromelysin -1) during the long-term follow-up independently of gender. In our study, the OR values for ACE deletions, Apo E4 and −455G>A, were below or slightly above the value of 1 (ranged from OR=0.63 to 1.07), showing negative correlation with risk factors (obesity, smoking, metabolic syndrome, cardiovascular events, and thrombosis), although the associations are not significant except for ACE deletion as shown in Table 3. Dyslipidemic patients included in our study were already on follow-up in lipid-lowering therapy (statins or fibrates). The serum levels of lipid parameters included in the present study were the initial values before the start of therapy.

Dyslipidemia, metabolic syndrome, and venous thrombosis are interrelated and share common risk factors, including obesity, smoking, and diabetes. Testing for presence of polymorphisms and mutations will help in diagnostic and therapeutic medical care of patients at high cardiovascular risk. A complex multigenic approach plays an important role in the investigation and management of dyslipidemic patients.

Footnotes

Disclosure Statement

No competing financial interests exist.

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