BR Biomedical Reports 2049-9434 2049-9442 D.A. Spandidos 10.3892/br.2014.384 br-03-01-0110 Articles A lack of association between the CRP rs2794520 polymorphism and coronary artery disease LIANJIANGFANG 1 * LIJUNXING 1 2 * DAIDONGJUN 2 * FANGPEILIANG 1 2 ZHOUJIANQING 1 DUANSHIWEI 2 Ningbo Medical Center, Lihuili Hospital, School of Medicine, Ningbo University, Ningbo, Zhejiang 315000, P.R. China Zhejiang Provincial Key Laboratory of Pathophysiology, School of Medicine, Ningbo University, Ningbo, Zhejiang 315000, P.R. China Correspondence to: Dr Jianqing Zhou, Ningbo Medical Center, Lihuili Hospital, Ningbo University, 57 Xingning Road, Ningbo, Zhejiang 315000, P.R. China E-mail: hjmpin@163.com Professor Shiwei Duan, Zhejiang Provincial Key Laboratory of Pathophysiology, School of Medicine, Ningbo University, Ningbo, 818 Fenghua Road, Zhejiang 315000, P.R. China E-mail: duanshiwei@nbu.edu.cn

Contributed equally

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Coronary artery disease (CAD) is mainly caused by atherosclerosis, which is closely associated with the C-reactive protein (CRP), a systemic inflammatory mediator. The aim of the present study was to examine whether the CRP rs2794520 polymorphism played a role in the risk of CAD. A total of 459 CAD patients and 432 non-CAD controls were recruited in the case-control study. Genotyping was performed on the SEQUENOM® Mass-ARRAY iPLEX® platform according to the manufacturer's instructions. The results showed that CRP rs2794520 was not associated with CAD. A further breakdown analysis by age or gender also indicated a lack of association between rs2794520 and CAD. In addition, the CRP rs2794520 polymorphism was not associated with the severity of CAD, which was represented by the number of coronary arteries with stenosis. In conclusion, there was no contribution of the CRP rs2794520 polymorphism to the risk of CAD.

 C-reactive protein coronary artery disease rs2794520 no association Introduction

Coronary artery disease (CAD) is characterized by the narrowed or blocked coronary arteries that can cause a lack of myocardial oxygen supply. CAD is the main cause of human fatalities worldwide (1). CAD is a complex disorder contributed by environmental and genetic factors (2). C-reactive protein (CRP) is a widely used inflammatory factor (3) in the detection of systemic inflammation diseases, such as atherosclerosis (4), which is an inflammation process closely linked to CAD (5,6). The circulating CRP level was found to be significantly associated with the risk of CAD (7–10). Aggregated C reactive protein was found to bind to low-density lipoproteins and very low-density lipoprotein (11) in the atherosclerotic plaques (12).

Recent genetic and genome-wide association studies have identified a number of genetic loci that are associated with CRP levels (9,13–16). Approximately 40% of CRP level variation was determined by genetic factors (10), including the CRP rs2794520 polymorphism, which was one of most significant markers in the meta-analysis of the genome-wide association studies among >80,000 European subjects (9).

In association with the previous studies, we hypothesized that the CRP rs2794520 polymorphism may influence the risk of CAD. Thus, a case control study was performed to assess the contribution of the CRP rs2794520 polymorphism to CAD in the Han Chinese population.

 Materials and methods <sec> <title>Study population

A total of 891 unrelated individuals were carefully selected, which included 459 CAD patients (males, 69.9%; age, 59.7±9.48 years; diabetes mellitus, 22.1%; hypertension, 60.3%; body mass, 23.4±3.4 kg/m2; and smokers, 44.5%) and 432 non-CAD patients (males, 54.4%; age, 59.7±9.48 years; diabetes mellitus, 11.5%; hypertension, 49.3%; body mass, 23.3±3.4 kg/m2; and smokers, 31.7%). All were Han Chinese residents of Ningbo city in Eastern China. The standards required were that the involved CAD patients should have one or more major coronary arteries with a diameter stenosis ≥50%, or had a history of a coronary artery bypass surgery or prior angioplasty. The non-CAD patients were diagnosed with a diameter stenosis <50% of the major coronary arteries and without any atherosclerotic vascular disease. The diagnosis was made by at least two cardiologists (JZ and JL). All the samples were recruited between May 2008 and April 2014 from Ningbo Lihuili Hospital (Ningbo, China). Blood samples were collected in 3.2% citrate sodium-treated tubes and subsequently stored at −80°C. The protocol was approved by the Ethical Committee of Ningbo Lihuili Hospital and all the involved individuals provided signed informed consent.

 Single-nucleotide polymorphism (SNP) genotyping

The genomic DNA was isolated from peripheral blood lymphocytes using a conventional phenol/chloroform method, and subsequently DNA was quantified using the PicoGreen® double strand (dsDNA) DNA Quantification kit (Molecular Probes, Inc., Eugene, OR, USA). Amplification of the genomic DNA was performed on the ABI Gene Amp® PCR System 9700 Dual 384-Well Sample Block Module (Applied Biosystems, Foster City, CA, USA) for the quantitative polymerase chain reaction (qPCR) analysis. The surrounding DNA sequence of the tested polymorphism was downloaded from the NCBI dbSNP. Online program (http://www6.appliedbiosystems.com/support/techtools/calc/) was used to design the primers. The sequences of the primers are as follows: Forward, 5′-GCGGGCAGGGCGGCCTGTGTGTATGAAGGGCAT AGGAC-3′; and 5′-GATTACCGCTGTGTGTATGAAGGG CATAGGAT-3′; and reverse, 5′-CAGGCCTCATTCAGTGTG GACC-3′. PCR amplification procedures consisted of an initial denaturation at 95°C for 30 sec to activate the enzyme activity, a 40-cycle denaturation (95°C for 30 sec, annealing stage at 59°C for 30 sec, and an extension at 72°C for another 30 sec), and a final extension for 5 min at 72°C. The amplified DNA was held at 4°C. DNA amplification for genotyping was performed on the SEQUENOM® Mass-ARRAY iPLEX® platform according to the manufacturer's instructions (17).

 Data analysis

Arlequin program (version 3.5) was used to estimate the Hardy-Weinberg equilibrium (HWE) (18). Clump 16 software with 10,000 Monte Carlo simulations was used to compare the frequency of the genotype and allele between cases and controls (19). Odd ratio (OR) with 95% confidential interval (CI) was calculated using an online program (http://faculty.vassar.edu/lowry/odds2x2.html). The power of the study was assessed by Power and Sample Size Calculation software (v3.0.43) (20). χ2 analysis was used to compare the severity of CAD and the rs2794520 polymorphism (21). A two-sided P-value <0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Association between the SNP and CAD

In the present study, no significant association was found between rs2794520 and CAD (P=0.12; OR, 1.16; 95% CI, 0.96–1.41; Table I). As gender and age are two important factors in the risk of CAD, a subgroup analyses was further performed by age or gender; however, no positive results were revealed in all the tests (P>0.05; Tables I and II). Additional subgroup analysis by age and gender showed a negative result in rs2794520 with CAD (P>0.05; Table III). Under the dominant model, a significant result occurred between male CAD patients and controls (P=0.04; OR, 1.64; 95% CI, 1.02–2.63; Table IV), although the significance was not retained following the correction by the number of statistical tests. No significant association was found for the other genotype analyses (Table IV). The power of each analysis in the study was also calculated (Tables I–IV). These results showed that a moderate power existed in the current association test, indicating that the negative association in the study may be due to a lack of power (power=0.474; Table I). Future study or meta-analysis are required to establish the link of this polymorphism to CAD.

In addition, the association between the severity of CAD and the rs2794520 polymorphism was also examined. All the cases were divided into three groups based on the number of major coronary artery with stenosis ≥50%. The correlation test revealed no significant association between the severity of CAD and rs2794520 (P=0.61; Table IV).

 Discussion

CAD has become the leading cause of fatalities in developed and developing countries. The plasma cardiac troponin I (cTnI) level was regarded as a gold standard for the diagnosis of acute myocardial infarction (AMI) worldwide (22). However, the rise of the plasma cTnI level comes after 4–6 h when the clinical events occurred, and this may delay the diagnosis (23). CRP levels had higher sensitivity to cardiovascular events compared to cTnI (24). Increased concentrations of plasma CRP have been found in patients with unstable angina (25). The elevation of CRP was found at the time of hospital admission in patients with myocardial infarction (MI) and history of unstable angina (24). The level of CRP was able to predict cardiovascular events in the healthy population and CAD patients (26,27). A study with 911 typical exertional angina patients indicated that increased CRP levels had a positive correlation with the CAD risk (28). Additionally, a number of studies identified CRP polymorphisms as reliable biomarkers of CAD (9). A case-control study indicated that there is a significant association between the CRP 1059 G/C polymorphism (rs1800947) and AMI in Italian population (29). In the present study, a case-control study was performed that involved 459 CAD patients and 432 non-CAD patients to assess the association between CRP rs2794520 and CAD risks.

There was no association between rs2794520 and CAD (P=0.12). As gender and age are two well-known factors that contribute to the CAD risk (30), subgroup analyses were performed by stratifying the samples into gender and age groups. However, there was no association between the age and gender groups (P>0.05). Additionally, the subgroup analyses were performed by different genetic models that comprised of dominant and recessive models. A close significant result was found in the dominant model between male CAD patients and controls (P=0.04); however, possible multiple testing may exist in the analysis, and therefore, this result may not be considered significant. Other significant associations based on the genetic models in all the stratifications did not occur (P>0.05).

There were certain limitations in the present study. Firstly, the samples of the study were relatively small, and the power was 0.474 in the association between rs2794520 and CAD, which may influence the results of the study. Therefore, studies with larger-scale samples and stronger power are required in future research to confirm the current findings. Secondly, CAD is a complex disease that environmental and genetic status may alter the results of the study, although the cases and controls in the present study were carefully selected with the help of multiple professional doctors, any hidden factors that may influence the results of the current study could not be excluded. More carefully and precisely designed studies are required to strengthen the results of the study. Thirdly, there are 2,587 polymorphisms in the CRP based on the information in the dbSNP in PubMed. The present study only focused on one polymorphism that could not represent the whole contribution of CRP to CAD. Other CRP polymorphisms, such as CRP 1059 G/C (rs1800947), were previously found to be associated with the risk of CAD (29). Examining more CRP polymorphisms is required to assess their contribution to CAD.

In conclusion, the present study showed that the CRP rs2794520 polymorphism had no association with the risk of CAD in the Han Chinese population. Studies with different populations and stronger power are required to further confirm the findings of the study.

 Acknowledgements

The present study was supported by the grants from the National Natural Science Foundation of China (nos. 31100919, 30772155 and 81371469), Natural Science Foundation of Zhejiang Province (no. LR13H020003), K. C. Wong Magna Fund in Ningbo University, Zhejiang provincial Program for the Cultivation of High 1 level Innovative Health Talents, Natural Science Foundation of Zhejiang Province (no. Y206608), the Scientific Innovation Team Project of Ningbo (no. 2011B82014), and the Youth and Doctor Foundation of Ningbo (no. 2005A610016).

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Comparison of the genotype and allele frequencies between the cases and controls by gender.

Allele, counts
rs2794520 Genotype, counts P-value
P-value
Subjects group (n) TT/TC/CC χ2 (df=2) HWE T C χ2 (df=1) OR (95%CI) Power
All CAD case (459) 146/230/83 0.70 522 396
Non-CAD controls (432) 153/217/62 2.77 0.25 0.31 523 341 2.47 0.12 1.16 (0.96–1.41) 0.474
Male CAD case (321) 100/159/62 1.00 359 283
Non-CAD controls (235) 81/124/30 4.25 0.12 0.13 286 184 2.71 0.10 1.23 (0.96–1.56) 0.314
Female CAD case (138) 46/71/21 0.60 163 113
Non-CAD controls (197) 72/93/32 0.59 0.74 0.88 237 157 0.08 0.78 1.05 (0.76–1.43) 0.209

df, degrees of freedom; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; CI, confidence interval; CAD, coronary artery disease.

Comparison of the genotype and allele frequencies between the cases and controls by age.

Genotype, counts Allele, counts
rs2794520 P-value P-value
Age, years group, n TT/TC/CC χ2 (df=2) HWE T/C χ2 (df=1) OR (95%CI) Power
55≤ CAD case (115) 40/61/14 0.24 141/89
Non-CAD controls (159) 57/81/21 0.13 0.94 0.41 195/123 0.000015 1.00 1.00 (0.71–1.42) 0.179
55–65 CAD case (162) 49/83/30 0.75 181/143
Non-CAD controls (166) 59/86/21 2.52 0.28 0.25 204/128 2.11 0.15 1.26 (0.92–1.72) 0.209
≥65 CAD case (182) 57/86/39 0.55 200/164
Non-CAD controls (107) 37/50/20 0.47 0.79 0.69 124/90 0.49 0.48 1.13 (0.80–1.59) 0.182

df, degrees of freedom; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; CI, confidence interval; CAD, coronary artery disease.

Comparison of the genotype and allele frequencies between the cases and controls by age and gender.

rs2794520 Genotype, counts P-value Allele, counts P-value
Age, years Gender group (n) TT/TC/CC χ2 (df=2) HWE T/C χ2 (df=1) OR (95%CI) Power
≤55 Male CAD case (85) 30/44/11 0.50 104/66
Non-CAD controls (99) 35/55/9 0.75 0.69 0.08 125/73 0.15 0.70 1.09 (0.71–1.66) 0.136
Female CAD case (30) 10/17/3 0.44 37/23
Non-CAD controls (60) 22/26/12 2.01 0.37 0.43 70/50 0.18 0.67 0.87 (0.46–1.64) 0.089
55–65 Male CAD case (118) 35/62/21 0.58 132/104
Non-CAD controls (78) 27/43/8 2.23 0.33 0.16 97/59 1.51 0.22 1.30 (0.86–1.96) 0.137
Female CAD case (44) 14/21/9 1.00 49/39
Non-CAD controls (88) 32/43/13 0.75 0.69 1.00 107/69 0.63 0.43 1.23 (0.74–2.07) 0.107
≥65 Male CAD case (118) 35/53/30 0.27 123/113
Non-CAD controls (58) 19/26/13 0.27 0.88 0.59 64/52 0.29 0.59 1.13 (0.72–1.77) 0.125
Female CAD case (64) 22/33/9 0.61 77/51
Non-CAD controls (49) 18/24/7 0.08 0.96 1.00 60/38 0.03 0.87 1.05 (0.61–1.79) 0.101

df, degrees of freedom; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; CI, confidence interval; CAD, coronary artery disease.

Comparison of the genotype distribution between the cases and controls in the recessive and dominant models.

Recessive Dominant
rs2794520
P-value
Subjects group (n) TT+CT CC χ2 (df=1) OR (95%CI) Power TT CC+CT χ2 (df=1) OR (95%CI) Power
All CAD case (459) 376 83 146 313
Non-CAD controls (432) 370 62 2.27 0.13 1.32 (0.92–1.89) 0.284 153 279 1.30 0.25 1.18 (0.89–1.55) 0.442
Male CAD case (321) 259 62 100 221
Non-CAD controls (235) 205 30 4.21 0.04 1.64 (1.02–2.63) 0.175 81 154 0.68 0.41 1.16 (0.81–1.66) 0.292
Female CAD case (138) 117 21 46 92
Non-CAD controls (197) 165 32 0.06 0.80 0.93 (0.51–1.69) 0.146 72 125 0.37 0.54 1.15 (0.73–1.82) 0.195

? df, degrees of freedom; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; CI, confidence interval; CAD, coronary artery disease.

Comparison of the genotype and allele frequencies between rs2794520 and the severity of CAD patients.

rs2794520 Genotype, counts P-value Allele, counts P-value
Group case, n TT/TC/CC χ2 (df=4) T/C χ2 (df=2)
Single-vessel disease 240 76/123/41 275/205
Double-vessel disease 110 36/57/17 129/91
Triple-vessel disease 109 34/50/25 2.53 0.64 118/100 0.98 0.61

df, degrees of freedom; CAD, coronary artery disease.