Seung Ho Hong

Genotype-specific Influence on Nitric Oxide Synthase Gene Expression, Protein Concentrations, and Enzyme Activity in Cultured Human Endothelial Cells

BACKGROUND The results of studies on the association of ecNOS polymorphisms and vascular diseases are inconsistent. To explore the nature of this interaction in the absence of confounding factors, such as smoking, we measured ecNOS mRNA, protein, and enzyme activity in cultured human umbilical vein endothelial cells (HUVECs) with and without ecNOS polymorphisms. METHODS We identified a T(-786)-->C polymorphism in the promoter region, the intron 4 variable number of tandem repeats (VNTR), the E298A polymorphism in exon 7, and the G10-T polymorphism in intron 23 of the ecNOS gene in the DNA from 43 human umbilical cords. We measured ecNOS and GAPDH mRNA from the cultured HUVECs by reverse transcription-PCR and ecNOS protein and enzyme activity by Western blotting (as ratio to positive control band) and by determining the conversion of [(3)H]arginine to [(3)H]citrulline, respectively. RESULTS The T(-786)-->C polymorphism showed the same allelic distribution as the intron 4 VNTR. Mean (SD) ecNOS protein from the cultured HUVECs was significantly lower in the 4a/4b genotype [0.84 (1.23); n = 9] of the intron 4 VNTR than in the 4b/4b genotype [2.14 (2.26); n = 34; P = 0.0300]. The enzyme activity was also significantly lower in the 4a/4b genotype [0.84 (0.21) pmol.min(-1).mg protein(-1); n = 9] than in the 4b/4b genotype [1.07 (0.31) pmol.min(-1).mg protein(-1); n = 34; P = 0.0197]. CONCLUSIONS ecNOS gene expression, protein concentrations, and enzyme activity are genotype-dependent in HUVECs. The intron 4 VNTR has a consistent influence that may be mediated by the T(-786)-->C polymorphism in the promoter region.

Association Between VEGF Polymorphisms and Homocysteine Levels in Patients With Ischemic Stroke and Silent Brain Infarction

Background and Purpose— Vascular endothelial growth factor (VEGF) plays a role in atherosclerosis-related diseases such as cerebrovascular or cardiovascular diseases. However, the effect of VEGF -2578C>A, -1154G>A, -634G>C, and 936C>T polymorphisms on the susceptibility to stroke and silent brain infarction has not been reported. Methods— Using polymerase chain reaction-amplified DNA, VEGF polymorphisms were analyzed in 615 patients with ischemic stroke, 376 patients with silent brain infarction, and 494 control subjects. Results— The AA and CC+CA (C allele bearing) genotype frequencies of the -2578C>A polymorphism and the CT+TT (T allele-bearing) genotype frequency of the 936C>T polymorphism were significantly different between the stroke and control groups (false discovery rate-adjusted probability values of 0.016, 0.044, and 0.044, respectively). When stratified by the size of the occluded vessel, the VEGF polymorphisms were associated with patients with multiple small-artery occlusions. Several haplotypes of the VEGF polymorphisms were significantly different between the control and stroke groups. With respect to silent brain infarction, the difference in the frequency of the -634G>C polymorphism between the GC+CC (C allele-bearing) genotype and the controls was marginally significant (false discovery rate-adjusted probability value of 0.056). On the other hand, the -634G>C and 936C>T polymorphisms were associated with plasma homocysteine levels of patients with multiple or single small-artery occlusions, respectively. Conclusions— This study suggests that VEGF polymorphisms and haplotypes are possible genetic determinants for the risk of ischemic stroke, particularly in patients with multiple small-artery occlusions. However, VEGF polymorphisms had only a weak association with plasma homocysteine levels in the Korean population.

Association between HaeIII polymorphism of scavenger receptor class B type I gene and plasma HDL-cholesterol concentration.

Background: Evidence has recently been found for significant associations between genetic variation within the scavenger receptor class B type I gene (SR-BI), plasma lipids and anthropometric measurements in healthy Caucasians. The present case-control study was conducted to determine whether there is an association between three polymorphisms identified by the restriction endonucleases HaeIII, AluI and ApaI of SR-BI and coronary artery disease (CAD) in Korean subjects. Methods: DNA was extracted from 137 subjects with CAD and 124 age-matched controls; it was amplified using the polymerase chain reaction. Individual alleles at each of the three polymorphic sites were identified by digestion with the appropriate restriction enzyme. Results: Only a single allele was identified at the AluI and ApaI polymorphic sites. The frequency of the common (+) allele at the HaeIII polymorphic site was higher in CAD patients than in the controls (P = 0·001). The concentrations of plasma HDL-cholesterol and apolipoprotein AI also varied significantly among HaeIII genotypes in the CAD patients. The common (+) allele of the HaeIII polymorphism was associated with a lower body mass index in female controls. Conclusions: Allele frequencies of the AluI and ApaI polymorphisms in this study were different to those in a Caucasian population studied previously, suggesting a difference in the genetic background. Further comparative studies of SR-BI polymorphism in other racial or ethnic groups should therefore prove to be of value.

Analysis of multiple single nucleotide polymorphisms of candidate genes related to coronary heart disease susceptibility by using support vector machines

Abstract Coronary heart disease (CHD) is a complex genetic disease involving gene-environment interaction. Many association studies between single nucleotide polymorphisms (SNPs) of candidate genes and CHD have been reported. We have applied a new method to analyze such relationships using support vector machines (SVMs), which is one of the methods for artificial neuronal network. We assumed that common haplotype implicit in genotypes will differ between cases and controls, and that this will allow SVM-derived patterns to be classifiable according to subject genotypes. Fourteen SNPs of ten candidate genes in 86 CHD patients and 119 controls were investigated. Genotypes were transformed to a numerical vector by giving scores based on difference between the genotypes of each subject and the reference genotypes, which represent the healthy normal population. Overall classification accuracy by SVMs was 64.4% with a receiver operating characteristic (ROC) area of 0.639. By conventional analysis using the χ2 test, the association between CHD and the SNP of the scavenger receptor B1 gene was most significant in terms of allele frequencies in cases vs. controls (p = 0.0001). In conclusion, we suggest that the application of SVMs for association studies of SNPs in candidate genes shows considerable promise and that further work could be usefully performed upon the estimation of CHD susceptibility in individuals of high risk.

ACE I/D polymorphism in Korean patients with ischemic stroke and silent brain infarction

Objectives –  Angiotensin‐converting enzyme (ACE) polymorphism may play a role in stroke and silent brain infarction (SBI) susceptibility, but the results among the populations studied to date have not been consistent. Thus, we investigated the association between ACE genotypes and ischemic stroke and SBI in Korean patients.

Association between Tumor Necrosis Factor-Alpha (–308G→A and –238G→A) Polymorphisms and Homocysteine Levels in Patients with Ischemic Strokes and Silent Brain Infarctions

Background and Purpose: The aims of this study were to evaluate the role of tumor necrosis factor-α (TNF-α) polymorphisms in patients susceptible to ischemic stroke and silent brain infarction (SBI), and to determine the relationship between TNF-α polymorphisms and plasma total homocysteine (tHcy) levels. Methods: We studied 237 patients with ischemic stroke, 257 patients with SBIs, and 216 control subjects. For control subjects, we selected healthy individuals matched for gender and age from those individuals who came to our hospital for health examinations. The TNF-α–308G→A and –238G→A genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism method. Results: The frequency of the TNF-α–308G→A polymorphism was significantly different between the patients with ischemic stroke and the control group (GG vs. GA+AA; adjusted odds ratio, AOR, 0.50; 95% CI 0.255–0.989). By subgroup analysis, when tHcy levels were stratified into high (>10.80 µmol/l), moderate (8.21–10.80 µmol/l), and low levels (<8.21 µmol/l), the frequency of the TNF-α–308GA+AA genotype in the highest tertile group was higher than in the lowest tertile group (AOR 2.46; 95% CI 1.063–5.699). However, the relationship between SBI susceptibility and polymorphisms of TNF-αwas not established. The tHcy levels were significantly and inversely correlated with folate levels in the TNF-α–308GG and TNF-α–238GG genotypes in the ischemic stroke, SBI, and control groups (p< 0.05). Conclusions: Our results suggest that the TNF-α–308G→A polymorphism is responsible for susceptibility to ischemic stroke and is associated with high tHcy levels in Koreans.

Genetic Variations of Cholesterol Ester Transfer Protein Gene in Koreans

Abstract An absence of cholesterol ester transfer protein (CETP, protein; CETP, gene) results in an increase of the apolipoprotein AI levels and a decrease in the low density lipoprotein (LDL) levels. Thus, the CETP polymorphism is important in the assessment of risk of atherosclerosis. This study was conducted to elucidate the genotype distributions of the CETP polymorphism and association with plasma lipid levels in Koreans. The genotypes of the TaqI A and B polymorphic loci were associated with plasma triglyceride levels in the control and coronary artery disease (CAD) groups. There was linkage disequilibrium between TaqI A and B loci in the control group (X2= 5.58, p < 0.05). Association studies of the CETP polymorphism have been carried out mainly with Caucasian populations; however, the results have not been consistent among different populations. A possible explanation for this diversity among populations may be differences in genetic backgrounds, which may be more important than environmental factors. We discuss the reasons for the incompatibility of the CETP polymorphism among populations.

Gene–environment interactions between methylenetetrahydrofolate reductase (MTHFR) 677C>T and metabolic syndrome for the prevalence of ischemic stroke in Koreans

Although metabolic syndrome (MS) is recognized as a risk factor for ischemic stroke, little is known about genetic variants that confer susceptibility to ischemic stroke among individuals with or without MS. This study was completed to investigate whether the methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism is associated with MS as a risk factor for ischemic stroke in the Korean population. The prevalence of MS was significantly higher in ischemic stroke patients than controls (adjusted odds ratio [AOR]=1.420; 95% confidence interval [CI]=1.017-1.982, P=0.040). MS prevalence was also significantly different between patients with subtypes of small-artery occlusion (AOR=1.707; 95% CI=1.081-2.695, P=0.022) and large-artery occlusion (AOR=1.661; 95% CI=1.089-2.534, P=0.019) versus controls. The frequencies of the MTHFR 677 TT genotype (AOR=3.001; 95% CI=1.487-6.057, P=0.002) and CT genotype (AOR=1.772; 95% CI=1.053-2.983, P=0.031) of the MS group, and for the CC genotype in those without MS were significantly different between the ischemic stroke patients and controls. The MTHFR 677C>T polymorphism was associated with a higher risk of MS among ischemic stroke patients in the Korean population.

Genotype distribution of the mutations in the coagulation factor V gene in the Korean population: Absence of its association with coronary artery disease

Mutations in the factor V gene are major risk markers for venous thrombosis. Several factors for blood coagulation have been related with cardiovascular disease. I investigated genotype distribution for three mutations (G1691A, A2379G and G2391 A) of the factor V gene in the Korean population. Genotype frequencies were examined by polymerase chain reaction in 135 patients with coronary artery disease (CAD) and 116 healthy subjects. For the G1691A mutation (factor V Leiden), no mutation was detected in either group. Allele frequencies of A2379G and G2391A mutations were not significantly different between CAD patients and controls. Non‐Caucasian populations have a considerably lower factor V Leiden allele frequency than Caucasian populations. Thus, it may be due to differences in the genetic background as well as environmental factors.

Association of the vascular endothelial growth factor (VEGF-1154G>A) polymorphism in patients with colorectal cancer

Angiogenesis plays a pivotal role in the development of colon cancer during the promotion and metastasis of tumor growth. Vascular endothelial growth factor (VEGF) is known to be a potent angiogenic factor. This hospital-based case-control study was carried out to decide where there existed an association between the VEGF-1154G>A polymorphism and the susceptibility to colon cancer. DNA samples taken from 278 colon cancer patients and 226 healthy controls were studied using with real-time PCR for VEGF-1154G>A polymorphism. Genotype frequencies of the VEGF-1154G>A polymorphism were significantly different between patient and control groups (adjusted OR=2.735, 95% CI=1.243−6.015 for AA vs. GG genotype). In addition, upon stratification by gender and age, the frequencies of the A allele-bearing genotypes significantly increased the risk for development of colon cancer in men and patients younger than 55 years (in men, adjusted OR=3.375, 95% CI=1.062−10.717, and in <55 years, adjusted OR=4.908, 95% CI=1.294−18.617). Also, upon stratification of patients with proximal and distal colon cancer individually, the association only showed these significant patterns in distal colon cancer. This study provides evidence that VEGF-1154G>A polymorphism, at least in Koreans, might be associated with risks of the colon cancer, particularly in males.