Clusters of differentiation 14 (CD14) receptor is a mediator of the inflammatory response by recognition of lipopolysaccharide (LPS), a major component of the outer cell wall of Gram-negative bacteria [
The CD14 gene promoter contains a single nucleotide polymorphism, and cytosine (C) to thymine (T) transition polymorphism at position −159 may influence the expression of CD14 [
In this study, we focus on normal healthy Korean population and aim to elucidate the association of risk factors for atherosclerosis and
Healthy patients were enrolled from a teaching hospital of the Catholic University of Medicine, St. Vincent’s Hospital, from March 2009 to February 2010. They were asymptomatic examinees of regular health screening with a simple symptom questionnaire at the Health Promotion Center of the same hospital and had endoscopic examinations for free nationwide gastric cancer screening in a Korean adult population. They had no history of hypertension, diabetes, cardiac disease, or any other chronic illness. Individuals with conditions that might have substantial effects on our study results (e.g., serum creatinine >2.5 mg/dL, total bilirubin >3.0 mg/dL), pregnant women, patients with psychiatric diseases, and patients who did not sign a consent form were excluded. Individuals with grossly severe gastric atrophy or ulceration or who were taking specific gastrointestinal medication, NSAIDs, or any other drugs were also excluded. Alcohol drinking was defined as consumption of at least 20 g alcohol/day and up to three times/week. Smoking was defined as current smoker.
Height, weight, and body mass indices (BMI, kg/m2) were measured using the Inbody 720 (Body Composition Analysis, Biospace Co.). The waist circumference was measured with the individual standing upright, with their top raised and under an exhalation state, at the middle area between the lowest area of the number 12 costa and the highest iliac crest, by an experienced person using a ruler with units of 0.1 cm. For systolic and diastolic blood pressure measurements, the subjects took sufficient rest while sitting down over a 5-minute period, and they were measured once with an automatic hemadynamometer.
After fasting for more than 12 hours, serum samples were collected from each patient’s upper arm. Fasting blood sugar (FBS), aspartate transaminase (AST), alanine aminotransferase (ALT), total cholesterol, triglyceride, HDL cholesterol, high-sensitivity CRP (hs-CRP), lipoprotein a (Lp (a)), free T4, thyroid stimulating hormone (TSH), and ferritin level were measured. Blood samples (10 mL) were obtained from all subjects and collected in a test tube containing EDTA or heparin.
In enrolled patients, two biopsy specimens were taken during upper gastrointestinal endoscopy from greater curvature side of the midantrum and corpus for histology. The diagnosis of
Genomic DNA was acquired from buffy-coat leukocytes using the AccuPrep Genomic DNA Extraction Kit (BIONEER CORPORATION, Daejeon, Republic of Korea). The promoter polymorphism at −159 of CD14 gene was evaluated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The PCRs were set up using i-Star Taq DNA polymerase (iNtRON BIOTECHNOLOGY, Seoul, Republic of Korea). In brief, we amplified the CD14 gene promoter region using the forward primer 5′-ATCATCCTTTTCCCACACC-3′ and the reverse primer 5′-AACTCTTCGGCTGCCTCT-3′. In a total reaction volume of 10
Statistical analysis was conducted using SPSS version 16.0 software. Data was expressed as means ± standard deviation. Allele and genotype frequencies were compared via chi-squared tests. The normality in the distributions was assessed using normal probability plots. ANOVA or Student’s
Informed consent was obtained from all patients, and the study was approved by the Institutional Review Board of the Catholic University of Korea (VC08TISI0082).
A total of 266 healthy adults (170 males and 96 females) were enrolled in this study. The mean age of the subjects was
Digestion of the PCR products for the promoter polymorphism at −159C/T of the CD14 gene yielded bands of 295 bp in TT homozygotes, 140 and 155 bp in CC homozygotes, and all 3 bands (140, 155, and 295 bp) in heterozygotes (Figure
Detection of the −159C/T polymorphism of CD14 gene by using PCR-RFLP. Digestion of the PCR products yielded bands of 295 bp in TT homozygotes, 140 and 155 bp in CC homozygotes, and all 3 bands (140, 155, and 295 bp) in heterozygotes.
Between specific genotypes of CD14 gene and the risk factors of atherosclerosis, BMI, waist circumference, systolic blood pressure, diastolic blood pressure, fasting blood sugar, total cholesterol, hs-CRP, and Lp (a), no significant differences were observed. Also, no significant differences of liver function test, white blood cell, thyroid hormone, thyroid-stimulating hormone, and ferritin level existed according to the specific genotypes. However, as for HDL cholesterol level, there were significant differences among the three genotypes (
Risk factors of atherosclerosis according to CD 14 −159 genotypes.
CC | CT | TT |
| |
---|---|---|---|---|
Age |
|
|
|
0.75 |
Sex (male : female) | 11 : 10 | 80 : 40 | 79 : 46 | 0.44 |
Smoking | 12 | 50 | 42 | 0.09 |
Alcohol drinking | 14 | 72 | 68 | 0.47 |
|
14 | 65 | 54 | 0.06 |
Waist circumference (cm) |
|
|
|
0.58 |
Body mass index (Kg/m2) |
|
|
|
0.94 |
SBP (mmHg) |
|
|
|
0.88 |
DBP (mmHg) |
|
|
|
0.78 |
Hemoglobin (g/dL) |
|
|
|
0.72 |
ESR |
|
|
|
0.76 |
AST (IU/L) |
|
|
|
0.33 |
ALT (IU/L) |
|
|
|
0.41 |
rGTP (IU/L) |
|
|
|
0.31 |
FBS (mg/dL) |
|
|
|
0.45 |
Uric acid (mg/dL) |
|
|
|
0.48 |
Triglycerides (mg/dL) | 105.38 |
|
|
0.08 |
Total cholesterol (mg/dL) |
|
|
|
0.13 |
HDL cholesterol (mg/dL) | 52.67 |
|
|
<0.01* |
hs-CRP (mg/dL) |
|
|
|
0.92 |
Lp (a) |
|
|
|
0.35 |
Free T4 (ng/dL) |
|
|
|
0.23 |
TSH ( |
|
|
|
0.20 |
Ferritin (ng/mL) |
|
|
|
0.87 |
All values are presented as mean
When the subjects were divided by gender, the risk of atherosclerosis according to CD14 polymorphisms was evaluated. In male, there were no differences between specific genotypes of CD14 gene and the risk factors of atherosclerosis (Table
Risk factors for atherosclerosis according to male gender.
CC | CT | TT |
| |
---|---|---|---|---|
Age |
|
|
|
0.29 |
Waist circumference (cm) |
|
|
|
0.64 |
Body mass index (Kg/m2) |
|
|
|
0.82 |
SBP (mmHg) |
|
|
|
0.67 |
DBP (mmHg) |
|
|
|
0.46 |
Hemoglobin (g/dL) |
|
|
|
0.46 |
ESR |
|
|
|
0.60 |
AST (IU/L) |
|
|
|
0.70 |
ALT (IU/L) |
|
|
|
0.68 |
rGTP (IU/L) |
|
|
|
0.38 |
FBS (mg/dL) |
|
|
|
0.82 |
Uric acid (mg/dL) |
|
|
|
0.95 |
Triglycerides (mg/dL) |
|
|
|
0.09 |
Total cholesterol (mg/dL) |
|
|
|
0.51 |
HDL cholesterol (mg/dL) |
|
|
|
0.51 |
hs-CRP (mg/dL) |
|
|
|
0.30 |
Lp (a) |
|
|
|
0.41 |
Free T4 (ng/dL) |
|
|
|
0.45 |
TSH ( |
|
|
|
0.50 |
Ferritin (ng/mL) |
|
|
|
0.84 |
All values are presented as mean
SBP: systolic blood pressure, DBP: diastolic blood pressure, ESR: erythrocyte sedimentation rate, AST: aspartate transaminase, ALT: alanine aminotransferase, FBS: fasting blood sugar, hs-CRP: high-sensitivity C-reactive protein, Lp (a): lipoprotein a, and TSH: thyroid stimulating hormone.
Risk factors for atherosclerosis according to female gender.
CC | CT | TT |
| |
---|---|---|---|---|
Age |
|
|
|
0.93 |
Waist circumference (cm) |
|
|
|
0.79 |
Body mass index (Kg/m2) |
|
|
|
0.83 |
SBP (mmHg) |
|
|
|
0.65 |
DBP (mmHg) |
|
|
|
0.77 |
Hemoglobin (g/dL) |
|
|
|
0.82 |
ESR |
|
|
|
0.96 |
AST (IU/L) |
|
|
|
0.65 |
ALT (IU/L) |
|
|
|
0.96 |
rGTP (IU/L) |
|
|
|
0.59 |
FBS (mg/dL) |
|
|
|
0.85 |
Uric acid (mg/dL) |
|
|
|
0.29 |
Triglycerides (mg/dL) |
|
|
|
0.97 |
Total cholesterol (mg/dL) |
|
|
|
0.18 |
HDL cholesterol (mg/dL) |
|
|
|
0.05* |
hs-CRP (mg/dL) |
|
|
|
0.35 |
Lp (a) |
|
|
|
0.46 |
Free T4 (ng/dL) |
|
|
|
0.10 |
TSH ( |
|
|
|
0.05* |
Ferritin (ng/mL) |
|
|
|
0.42 |
All values are presented as mean
SBP: systolic blood pressure, DBP: diastolic blood pressure, ESR: erythrocyte sedimentation rate, AST: aspartate transaminase, ALT: alanine aminotransferase, FBS: fasting blood sugar, hs-CRP: high-sensitivity C-reactive protein, Lp (a): lipoprotein a, and TSH: thyroid stimulating hormone.
Total of 133 subjects (50.0%) had associated
The frequencies of the CC, CT, and TT genotype with
Risk factors for atherosclerosis according to CD 14 −159 genotypes in subjects with
CC | CT | TT |
| |
---|---|---|---|---|
Age |
|
|
|
0.38 |
Waist circumference (cm) |
|
|
|
0.42 |
Body mass index (Kg/m2) |
|
|
|
0.73 |
SBP (mmHg) |
|
|
|
0.82 |
DBP (mmHg) |
|
|
|
0.64 |
Hemoglobin (g/dL) |
|
|
|
0.39 |
ESR |
|
|
|
0.83 |
AST (IU/L) |
|
|
|
0.33 |
ALT (IU/L) |
|
|
|
0.21 |
rGTP (IU/L) |
|
|
|
0.31 |
FBS (mg/dL) |
|
|
|
0.78 |
Uric acid (mg/dL) |
|
|
|
0.68 |
Triglycerides (mg/dL) |
|
|
|
0.18 |
Total cholesterol (mg/dL) |
|
|
|
0.72 |
HDL cholesterol (mg/dL) |
|
|
|
0.20 |
hs-CRP (mg/dL) |
|
|
|
0.59 |
Lp (a) |
|
|
|
0.88 |
Free T4 (ng/dL) |
|
|
|
0.16 |
TSH ( |
|
|
|
0.13 |
Ferritin (ng/mL) |
|
|
|
0.26 |
All values are presented as mean
SBP: systolic blood pressure, DBP: diastolic blood pressure, ESR: erythrocyte sedimentation rate, AST: aspartate transaminase, ALT: alanine aminotransferase, FBS: fasting blood sugar, hs-CRP: high-sensitivity C-reactive protein, Lp (a): lipoprotein a, and TSH: thyroid stimulating hormone.
Chronic
Previously, it has been reported that acute myocardial infarction/atherosclerosis are more closely related to CD14 −159TT homozygotes [
Although this study includes small size of the study population, it might be difficult to figure out the association between genetic polymorphism and disease phenotype. Also, we did not consider risk factors such as exercise activity and diet pattern. Especially, these are the important modifiable and preventable risk factors for cardiovascular disease. Our result showed that significant difference of HDL cholesterol level was pronounced in female population, because of the possibility that these contributing factors for atherosclerosis were excluded in female. In our results, there was significant difference in TSH level among the three genotypes. Although the high level of serum TSH level is associated with multivessel disease, it was not the determinant of cardiovascular disease in patients with normal range of thyroid function [
In the last two decades, epidemiologic studies have demonstrated that atherosclerosis is associated with several infectious pathogens, including
To date, there are limited data regarding the probability of cardiovascular diseases according to CD14 genetic polymorphisms in Korean population. This provides a clue that the high risk individuals of cardiovascular diseases are discriminated using genetic analysis. In conclusion, the promoter polymorphism at −159C/T of the CD14 gene is positively associated with the risk factor of atherosclerosis in healthy Korean population. Broader studies will be required before any relatively concrete conclusions can be drawn.
There is no conflict of interests.