ICAM‐1 and MKL‐1 polymorphisms impose considerable impacts on coronary heart disease occurrence

Abstract This study was aimed to explore the correlation of intercellular adhesion molecule‐1 (ICAM‐1) K469E and megakaryoblastic leukaemia factor‐1 (MKL‐1) −184C/T polymorphisms with the susceptibility to coronary heart disease (CHD) in the Chinese Han population. 100 CHD patients and 91 healthy people that had no blood connection with each other were enrolled in this case‐control study. ICAM‐1 and MKL‐1 polymorphisms were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) approach. Multiple logistic regression was used to analyse the correlation between polymorphisms of ICAM‐1 and MKL‐1 and CHD susceptibility. Differences of genotype and allele frequencies of the two SNPs between case and control groups were analysed by chi‐square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were indicated relative susceptibility of CHD. The distributions of ICAM‐1 and MKL‐1 polymorphisms in each group conformed to Hardy‐Weinberg equilibrium (HWE). After adjusting for traditional risk factors, the TT genotype frequency of MKL‐1 −184C/T polymorphism was found significantly higher in case group than in control group (P < .05). Meanwhile, T allele frequency increased in case group compared with control group, and the differences had statistical significance (P = .04, OR = 2.34, 95% CI = 1.34‐5.26). Logistic regression analysis in this study proved that smoking, hypertension, diabetes and triglyceride (TG) were all risk factors for CHD ICAM‐1 K469E polymorphism has no association with the onset of CHD. But MKL‐1 −184C/T polymorphism is associated with the risk of CHD and T allele might be a susceptibility factor for CHD.


| Cases and controls
The case group of the study enrolled 100 CHD patients including 58 males and 42 females. The mean age of the patients, who were diagnosed in Cardiology department of The First Affiliated Hospital of Jinzhou Medical University, was 59.4 ± 7.33 years old. Coronary angiography examinations of every patient showed that more than one main coronary artery of the cases had 50% or higher level of stenosis. Patients suffered from cardiomyopathy, haemorrhagic diseases, renal failure and malignant tumours were eliminated. The control group recruited 91 homochronous healthy persons (53 males and 38 females) from the physical examination centre of the same hospital, with a mean age of 60.3 ± 6.79 years old. The controls had no malignant tumours and immune inflammatory diseases, and they were proved without CHD history by electrocardiograph (ECG) examinations. All of the subjects were Chinese Han population, and they had no blood relationship with each other. Informed consents were obtained from each participant. And the study was approved by the

Ethics Committee of The First Affiliated Hospital of Jinzhou Medical
University.

| Blood collection and DNA extraction
We collected 2 mL fasting venous blood of each subject and put the blood into PCR tubes, anticoagulated by EDTA. Genome DNA was extracted from 300 μL whole blood by Biospin Whole Blood Genomic DNA Extraction Kit (Sangon Company) and then stored at −70°C.

| PCR amplifications and genetic typing assay
The genotypes of ICAM-1 K469E and MKL-1 −184C/T polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. Primer sequences for ICAM-1 K469E and MKL-1 −184C/T polymorphisms were designed by Primer Premier 5.0 and synthesized by Sang Biotech (Table 1). The PCR amplifications were performed in a total volume of 10 µL of mixture, containing 0.5 µL forward primer, 0.5 µL reverse primer, 3 µL 10 × Buffer, 1 µL dNTP, 2.5 µL MgCl 2 , 0.5 µL Taq DNA polymerase and 2 µL deionized sterile water. The PCR cycle was as the following: 95°C initial denaturation for 5 minutes; followed by 32 cycles of denaturing at 95°C for 30 seconds, annealing at 60°C for 30 seconds and extension for 30 seconds at 72°C; finally extension at 72°C for 5 minutes.
The amplification products were digested by restriction en-

| Statistical analysis
Data analysis was performed by SPSS.18 statistical software. The genotype and allele frequencies in two groups were compared by χ 2 test. The correlation of CAM-1 and MKL-1 gene polymorphisms with CHD was calculated by odds ratios (ORs) and 95% confidence intervals (95% CIs). Multiple logistic regression was adopted for testing the effects of other CHD risk factors. The differences had statistical significance when P < .05.

Locus
Primer sequence

| Characteristics of study subjects
As shown in Table 2, the differences in age, sex and body mass index (BMI) of the two groups had no statistical significance (P > .05).
But the case group had more smokers and patients with hypertension and diabetes, as well as higher levels of glucose (Glu), serum total cholesterol (TC) and triglyceride (TG) than the control group (P < .05). The differences of high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels between two groups were not statistically significant (P > .05).

| Logistic regression analysis on CHD risk factors
The genotype and allele distributions of ICAM-1 K469E and MKL-1 −184C/T polymorphisms in case and control groups are shown in

| D ISCUSS I ON
Coronary heart disease is a complex disease. Multiple gene muta- The present study discovered that −184C/T SNP of MKL-1 gene was closely related to the onset of CHD, and TT genotype increased the onset risk of such disease. T allele frequency was significantly higher in case group than that in control group, and it was suggested to be a risk factor for CHD. The distributions of TT genotype and T allele in case and control groups still had statically significant differences after logistic regression analysis, which attested the correlation with the onset of CHD. This is consistent with a previous study, and they also found that the -184C > T polymorphism of MKL1 is an important risk factor for CHD in Han nationality of Henan Province.
The homozygosity of T allele is related to the risk of CHD and the severity of stenosis. 25 Many researches on the relationship between ICAM-1 polymorphisms and inflammatory diseases have been reported. 26,27 Correlation analyses on ICAM-1 K469E polymorphism and CHD have been reported as well both in domestic and abroad, 28-30 but the results are inconclusive and contradictory. Liu et al conducted meta-analysis and found that rs5498 polymorphism was related to the reduction of CAD risk in

TA B L E 2 General features comparison between cases and controls
Caucasians, but not Asians, which may be caused by different races. 31 The results of our study illustrated that after being assessed by logistic regression analysis, ICAM-1 K469E polymorphism was found to have no obvious association with CHD in the Chinese population.
Some limitations of our research cannot be ignored. First of all, in the case-control study, our sample is relatively small, it is necessary to expand the sample size to further verify our results. Secondly, our research objects are all Han nationality, the possibility of different genetic background and surroundings caused by ethnic and regional differences might affect experimental results. Therefore, a lager or different population should be taken into account to confirm the results.
In summary, the present study suggested the correlation between MKL-1 −184C/T polymorphism and CHD, and T allele might be a susceptible factor for the disease. Meanwhile, the coexistence of TT genotype with smoking status, hypertension, diabetes or high TG level would significantly increase the morbidity of CHD.

CO N FLI C T O F I NTE R E S T
None. Writing-original draft (equal).

E TH I C A L A PPROVA L
With the approval of The First Affiliated Hospital of Jinzhou Medical University Ethics Committee, written informed consent was obtaining from every subject.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.