IVS4-14 A/G and IVS4-73 C/T Polymorphisms in OLR1 Gene in Patients with Ischemic Cerebrovascular Diseases

Abstract

Introduction: Oxidized low-density lipoprotein (ox-LDL) plays a key role in the processes of atherogenesis, the major cause of myocardial infarction. Increased levels of ox-LDL relate to plaque instability in human coronary atherosclerotic lesions. Moreover, ox-LDL levels show a direct correlation to the severity of coronary syndromes. Most of these effects are mediated by the interaction of ox-LDL with its major receptor, named LOX-1, that is encoded by OLR1 gene. Aims and Methods: In the present study, we examined the prevalence of OLR1 gene polymorphisms, IVS4-14 A/G and IVS4-73 C/T, which regulate the expression of LOXIN, in patients with ischemic cerebrovascular diseases (ICVD). We studied 43 consecutive patients (males = 19; females = 24) aged 26 to 65 years. All the patients were from the same geographical area. They were affected by ICVD. The control group comprised 69 healthy blood donors, with age and sex comparable to those of the patients. Results: The distribution of G/G genotype and A/G genotype was statistically significant between patients and controls (χ² = 5.87, p = 0.01 and χ² = 4.33, p = 0.04, respectively). Conclusion: These preliminary data would suggest that in ICVD patients the LOX-1 isoform that induces internalization of ox-LDL is more frequent and a cascade of events responsible for endothelial dysfunction and injury. LOX-1 might play a fundamental role in the initiation and progression of atherosclerosis and have a significant role in the pathogenesis of ICVD. Therefore, the patients with G homozygosity for IVS4-14 polymorphism and T homozygosity for IVS4-73 polymorphism have higher risk to develop ICVD. Future studies are warranted to assess whether the analysis of polymorphisms may be useful for the clinical approach to evaluate risk factors for atherosclerosis and related disorders.

Introduction

Oxidized low-density lipoprotein (ox-LDL) plays a key role in the processes of atherogenesis, a major cause of myocardial infarction (Ehara et al., 2002). Atherosclerosis is responsible for most acute ischemic manifestations, including stroke (Alexandrov and Alagona, 2008).

Ox-LDL elicits endothelial dysfunction by reducing the expression of constitutive nitric oxide synthesis and enhancing expression of adhesion molecules on the endothelium, which facilitates leukocyte adhesion to the intimae (Mehta and Li, 2002). In addition, ox-LDL is involved in inducing smooth muscle cell migration and proliferation and is avidly ingested by macrophages, resulting in the formation of foam cells (Chen et al., 2002).

Increased levels of ox-LDL relate to plaque instability in human coronary atherosclerotic lesions. Moreover, ox-LDL levels show a direct correlation to the severity of coronary syndromes (Ehara et al., 2001). Most of these effects are mediated by the interaction of ox-LDL with its major receptor, named LOX-1, a membrane protein that belongs to the C-type lectin family and is expressed in vivo in endothelial cells, macrophages, smooth muscle cells, and platelets. This receptor is encoded by OLR1 gene, consists of four domains, a short N-terminal cytoplasmasmic, a transmembrane, a connecting neck, and a lectin-like domain at the C terminus, which binds ox-LDL (Sawamura et al., 1997). Expression of OLR1 gene is upregulated by ox-LDL itself, angiotensin II, free radicals, and inflammatory cytokines, such as tumor necrosis factor alpha and shear stress (Li et al., 1999).

Association studies have implicated OLR1 gene variants in acute myocardial infarction susceptibility. In particular seven different single-nucleotide polymorphisms were identified, six of them located within intron 4 (IVS4-27 G>C, IVS4-73 C>T, IVS4-14 A>G), intron 5 (IVS5-70 A>G, IVS5-27 G>T), and 3′UTR (188 C>T), comprised in a linkage disequilibrium block, strongly associated with the elevated risk of coronary artery disease (Chen et al., 2003; Mango et al., 2003). All polymorphisms comprised within the disequilibrium block of OLR1 gene regulate the expression of a new functional splicing isoform, named LOXIN, which lacks exon 5, encoding part of the C-terminus lectin-like domain, responsible for the link to ox-LDL. Interestingly, macrophages from subjects expressing LOXIN resulted in fewer cells undergoing apoptosis on ox-LDL induction. It was demonstrated that the new splicing isoform is protective against acute myocardial infarction (Mango et al., 2005).

In the present study, we examined the prevalence of OLR1 gene polymorphisms, IVS4-14 A/G and IVS4-73 C/T, which regulate the expression of LOXIN, in patients with ischemic cerebrovascular diseases (ICVDs).

Materials and Methods

Patients

We studied 43 consecutive patients (males = 19; females = 24) aged 26 to 65 years, enrolled in the Department of Neurology of the Second Medical School of Naples. All patients were from the same geographical area and were affected by ICVD. No selected patients were affected by aneurysms or cardioembolic diseases. The diagnosis of ICVD was confirmed using the World Health Organization definition stroke: an acute disturbance of focal or global cerebral function with symptoms lasting longer than 24 h or leading to death with presumably no other reasons than of vascular origin (World Health Organization MONICA Project Principal Investigators, 1998).

The control group comprised 69 healthy blood donors, with age and sex comparable to those of the patients. They underwent a detailed clinical history and only patients free of cerebrovascular and cardiovascular diseases were enrolled.

Genotyping

For genotype analysis, peripheral blood from each patient and control was collected in ethylenediaminetetraacetic acid-containing tube. Genomic DNA was prepared from leucocytes using a Wizard Genomic DNA Purification Kit (Wizard genomic DNA Purification Kit; Promega Corporation, Madison, WI). For genotyping of IVS4-14 and IVS4-73 polymorphisms, a 263-bp fragment, containing the A to G and C to T transition at intron 4 of OLR1 gene, was amplified by polymerase chain reaction (PCR), as previously reported (Mango et al., 2003). All PCR amplifications were carried out using a DNA thermal cycler (Primus 96 plus; MWG-Biotech, Ebersber, Germany). Negative and positive controls were assessed during analysis. All PCR products were sequenced on an ABI model 3100 automated sequencer (Applied Biosystems, Foster City, CA) using BigDye terminator cycle sequencing reaction Kit (Applied Biosystems).

Statistical analysis

The data were expressed as mean ± standard deviation, unless otherwise stated. Comparison between the groups was performed using t-test for unpaired data. Frequencies were compared using χ² test with Fisher's correction, when appropriate. Significance was assumed with p-values less then 0.05. Hardy-Weinberg equilibrium was tested using the standard χ² test comparing the expected and actual allele frequencies.

Results

The distribution of AA genotype and allele frequencies of IVS4-14 A/G polymorphism was not significantly different among the groups. Particularly, AA genotype frequencies were 25.6% and 26.1% for subjects with ICVD and controls, respectively (Table 1).

Table 1.

Distribution of Genotypes and Allele Frequencies of IVS4-14 A/G Polymorphism in Ischemic Cerebrovascular Disease Patient and Control Group

	Patients (n = 43) (%)	Controls (n = 69) (%)	p	χ²	Odds ratio
Genotype
GG	17/43 (39.5)	13/69 (18.8)	0.01	5.78	2.82
AG	15/43 (34.9)	38/69 (55.1)	0.04	4.33	0.44
AA	11/43 (25.6)	18/69 (26.1)	ns	-	0.97
Allele frequencies			ns	2.38	1.53
G	49/86 (57.0)	64/138 (46.4)
A	37/86 (43.0)	74/138 (53.6)

In linkage disequilibrium with IVS4-73 C/T.

In contrast, the IVS4-14 GG genotype was considerably overrepresented in patients (χ² = 5.87, p = 0.01). Mainly, the frequency of the GG genotype in patients and controls was 39.5% and 18.8%, respectively (p = 0.01, χ² = 5.78).

The IVS4-14 AG genotype was overrepresented in controls (χ² = 4.33, p = 0.04) and in particular the frequency of the AG genotype in patients and controls was 34.9% and 55.1%, respectively. No significant difference was found in allele frequency between the groups for IVS4-14 polymorphism. In particular, the frequencies of G allele for IVS4-14 A/G polymorphism and T allele for IVS4-73 C/T polymorphism, in linkage disequilibrium, in patients and controls were 57% and 46.4%, respectively. Moreover, the frequencies of A allele and C allele were 43% in patients and 53.6% in the controls.

Discussion

Ischemic stroke is a significant cause of death throughout the world. ICVD is a heterogeneous syndrome and the atherosclerotic plaque represents its most important risk factor. There are multiple conditions associated with elevated risk of atherosclerosis, including high plasma levels of low-density lipoprotein-cholesterol (LDL-cholesterol), smoking, diabetes mellitus, hypertension, sedentary lifestyle, diet, and several other conditions (Lusis, 2000). Among these, high plasma levels of LDL-cholesterol are perhaps the most important risk factor for atherosclerosis.

Several studies have reported that ox-LDL is more effective proatherosclerotic stimulus than native-LDL. Ox-LDL elicits endothelial dysfunction, a key step in the initiation of the atherosclerosis, favoring generation of reactive oxygen species, inhibition of nitric oxide synthesis, and enhancement of monocyte adhesion to activated endothelial cells (Mehta et al., 2006). Most of these effects are mediated by interaction of ox-LDL with its major receptor lectin-like ox-LDL receptor-1 (LOX-1); this receptor is encoded by OLR1 gene (Chen et al., 2002).

All polymorphisms, comprised within the disequilibrium block of OLR1 gene, regulate the expression of a new functional splicing isoform named LOXIN, which lacks exon 5, encoding part of the C-terminus lectin-like domain, responsible for the link to ox-LDL (Mango et al., 2005).

We examined in patients affected with ICVD the prevalence of OLR1 gene polymorphisms, IVS4-14 A/G and IVS4-73 C/T, comprised within disequilibrium block.

Our data show that the distribution of the GG and TT genotype in patients is statistically significant (p = 0.01). The G allele determines expression of LOX-1 protein with lectin-like domain, responsible for the binding, internalization, and degradation of ox-LDL, in endothelial cells. On the contrary, the A allele determines the expression of LOXIN protein that, lacking lectin-like domain, cannot bind and internalize ox-LDL (Mango et al., 2005).

Previous studies demonstrated that polymorphisms in OLR1 gene were associated with coronary atherosclerosis and acute myocardial infarction (Mango et al., 2003; Chen et al., 2003; Tatsuguchi et al., 2003) and LOXIN isoform led to a protective effect versus atherosclerosis and myocardial infarction (Biocca et al., 2008).

These preliminary data would suggest that in ICVD patients, the LOX-1 isoform that induces internalization of ox-LDL and a cascade of events responsible for endothelial dysfunction and injury is more frequent. LOX-1 might play a fundamental role in the initiation and progression of atherosclerosis and have a significant role in the pathogenesis of ICVD. Therefore, the patients with G homozygosity for IVS4-14 polymorphism and T homozygosity for IVS4-73 polymorphism have higher risk to develop ICVD. Our study on the prevalence of these OLR1 gene polymorphisms may provide, particularly in younger individuals, opportunities for more targeted interventions for prevention and optimized drug therapy on the basis of each patient's genetic constitution. In fact, in young subjects the occurrence of ICVD is particularly dramatic because of its heavy social and economical burden.

Future studies are warranted to assess whether the analysis of polymorphisms may be useful for the clinical approach to evaluate risk factors for atherosclerosis and related disorders.

Footnotes

Disclosure Statement

No competing financial interests exist.

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