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. 2009 Nov 24:1:63-9.

Screening the single nucleotide polymorphisms in patients with internal carotid artery stenosis by oligonucleotide-based custom DNA array

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Screening the single nucleotide polymorphisms in patients with internal carotid artery stenosis by oligonucleotide-based custom DNA array

Kenji Nakai et al. Bioinform Biol Insights. .

Abstract

Early screening of individuals considered to be at risk for severe internal carotid artery (ICA) stenosis is an important strategy for preventing ischemic cerebral stroke. The purpose of this study is to screening candidate single nucleotide polymorphisms (SNPs) associated with severe ICA stenosis using a newly developed oligonucleotide-based custom DNA array. The subjects consisted of 47 controls and 46 patients with severe ICA stenosis (>/=70%) who underwent carotid endarterectomy (CEA). Subjects gave informed consent and we obtained samples of blood and genomic DNA. We studied 8 candidate genes: renin-angiotensin system [angiotensinogen (AGT), angiotensin II receptor type 1 (AGTR1), nitric oxide synthase 3 (NOS3)]; growth factor [hepatocyte growth factor (HGF)]; transgelin (SM22); cytokine [chemokine receptor 2 (CCR2)]; coagulation-fibrinolysis system [5,10-methylenetetrahydrofolate reductase (MTHFR)]; and plasminogen activator inhibitor 1 (PAI-1). Genotyping of candidate SNPs was done with a line probe assay (LiPA) based on an oligonucleotide-based DNA array.

Results: The allele frequency of PAI-1 -1965 delG (odds ratio (OR), 0.3; 95% confidence interval (CI), 0.2-0.6) and MTHFR (OR 1.3, 95% CI, 1.0-1.5) were significantly different between controls and cases with ICA stenosis by Fisher's exact test. Multiple logistic analysis revealed that diabetes mellitus (DM), SNPs in PAI-1 -1965 delG and MTHFR were an independent risk for ICA stenosis. In conclusion, genetic factors of coagulation-fibrinolysis as well as diabetes mellitus (DM) were relevant in ICA stenosis.

Keywords: 10-methylenetetra hydrofolate reductase; atherosclerosis; carotid artery stenosis; plasminogen activator inhibitor 1; 5; polymorphism.

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Figures

Figure 1.
Figure 1.
Schema for genotyping candidate SNPs (analyzed by LiPA) by reverse hybridization. Amplified biotinylated DNA material is hybridized with specific oligonucleotide probes immobilized as parallel lines on membrane-based strips. After hybridization, streptavidin labeled with alkaline phosphatase is added, and binds to any hybrid formed. After washing, incubation with nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (p-toluidine salt) (BCIP) results in formation of a purple/brown precipitate.b.
Figure 2.
Figure 2.
Representative genotyping of 8 SNPs analyzed using oligonucleotide-based DNA array (LiPA).

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