Lin Huang, Dan-Xin Wang, Li-Min Zhou, Tao Wang, Hai-Ying Zhang, Yun-Xia Zhang, Yin-Dong Zhang

1Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China

2Department of Nursery, The Affiliated Hospital of Hainan Medical College, Haikou 571199, China

3Public Research Laboratory of Hainan Medical University, Haikou 571199, China

1. Introduction

Cholesteryl ester transfer protein (CETP) is a single-chain,highly hydrophobic glycoprotein with a molecular weight of 74 kDa[1]. CETP is a lipid carrier between lipoproteins, playing an important role in cholesterol retrograde transport system and thus promoting the exchange of neutral fat and phospholipids in plasma lipoproteins[2-4]. Studies have shown that genetic polymorphisms can affect CETP activity, thereby affecting lipid metabolism and leading to the formation of atherosclerosis.

Atherosclerosis is an important cause of cerebral infarction. As one of the susceptible genes[5], CETP gene plays a decisive role in lipid transport activity. At present, more and more polymorphic sites have been found[6]. In this study, two polymorphic loci of rs1532624 and rs289741 in CETP gene were selected to analyze the association between gene polymorphism and atherosclerotic cerebral infarction(ACI).

2. Materials and methods

2.1. Research objects

Cerebral infarction group: 95 cases of cerebral infarction were selected from Cadre Sanatorium of Hainan Province, including 51 males and 44 females, with average age (67.8±11.2). All patients met the criteria of International Classification of Diseases 10thEdition(ICD10). Patients with persistent, sudden onset of focal neurological deficits above 24 h were diagnosed by head CT and/or head MRI and vascular imaging.

Healthy control group: 177 healthy subjects with the same physical examination center in Cadre sanatorium of Hainan Province,including 116 males and 61 females, with an average age of(68.9±10.4) years. All healthy people had no coronary heart disease or past cerebral infarction and no family history of cerebrovascular disease. All subjects were from Hainan Province and had no blood relationship with each other. This study was approved by the Hospital Ethics Committee, and all participants signed an informed consent form.

2.2. Blood collection and DNA extraction

Fasting venous blood samples were drawn into 2 mL EDTA anticoagulation tube. DNA extraction was done by Genomic DNA extraction kit, and the products was stored at -20 ℃.

2.3. Genetic polymorphism detection

DNA sequencing and primer designed were accomplished by Shenzhen Huada Gene Technology Services Ltd., using ADS2.0 software design primers (Pre-PCR primer and Extension primer,Table 1) and MassARRAY mass spectrometry single nucleotide polymorphism (SNP) typing method.

Pre-PCR reaction system: total system is 5.0 μL, containing template DNA 1.0 μL, 10× PCR Buffer 0.5 μL, 25 mmol/L MgCl20.4 μL, 25 mmol/L dNTP mix 0.1 μL, Primer mix 1.0 μL, 0.5 U HotstarTaq 0.1 μL, and H2O 1.9 μL. PCR reaction program:denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 20 s,annealing at 56 ℃ for 30 s, extension at 72 ℃ for 60 s, 45 cycles and extension at 72 ℃ for 5 min.

SAP digestion reaction system and procedures: SAP buffer 0.17 μL,SAP enzyme 0.30 μL, H2O 1.53 μL, 37 ℃ water bath for 40 min,85 ℃ inactivated 5 min.

EP reaction system: H2O 0.62 μL, iPLEX buffer 0.20 μL, iPLEX Termination mix 0.20 μL, iPLEX Extend Primer mix 0.94 μL, and iPLEX enzyme 0.04 μL. EP reaction program: 94 cycles 2 min,40 cycles × 52 ℃ 5 s, 80 ℃ 5 s, 72 ℃ 3 min. Then the extension product was purified by resin, spotted on the machine, and genotyped by mass spectrometer.

2.4. Statistical analysis

SPSS16.0 software was used for statistical analysis. Deference of genotypes and allele frequencies between groups was tested by χ2test with a=0.05.

3. Results

3.1. Analysis of rs1532624 and rs289741 polymorphism

The results of MassARRAY mass spectrometry showed that the genotypes of rs1532624 in 95 patients with cerebral infarction and 177 healthy controls were CC, CA, and AA. CC and AA showed a single peak at Mass 4 824.2 and Mass 4 864.1, respectively.However, the CA genotypes had bimodal peaks at Mass 4 824.2 and Mass 4 864.1 (Figure 1).

The genotypes of rs289741 were AA, AG, and GG. AA and GG showed a single peak at Mass 4 809.2 and Mass 4 825.2,respectively, but the AG genotype showed double peaks at Mass 4 809.2 and Mass 4 825.2 (Figure 2).

Figure 1. Detection for genotypes of rs1532624.

Figure 2. Detection for genotypes of rs289741.

3.2. Correlation analysis of rs1532624 and rs289741 locus polymorphism and ACI

There was no significant difference in allele frequency distribution between rs1532624 (χ2=1.723, P=0.189) and rs289741 (χ2=2.466,P=0.116) in the cerebral infarction group and healthy control group.The frequency distribution of rs1532624 genotype between the cerebral infarction group and healthy control group was statistically significant (χ2=7.096, P=0.029), while rs289741 genotype frequency distribution between the two groups was not statistically significant(χ2=2.906, P=0.234) (Table 2).

4. Discussion

Atherosclerosis refers to the chronic inflammatory process of lipid deposition, extracellular matrix proliferation, and cell infiltration in cardiovascular and cerebrovascular diseases. It can cause stenosis and plaque loss in the arterial lumen, leading to ischemia and hypoxia necrosis of brain tissue[7]. ACI is a complex disease that interacts with many environmental and genetic factors. Atherosclerosis is the most basic pathological feature. With the development of molecular biology and genetics, the study of the correlation between gene polymorphism and disease has become a hot spot. In this study,CETP gene, which is closely related to lipid transport activity,was selected to investigate the association between rs1532624 and rs289741 polymorphisms and ACI in Hainan population.

The human CETP gene is near the lecithin cholesterol acyltransferase gene and consists of 16 exons and 15 introns at 16q21, about 25 kbp in length. Mature CETP protein consists of 476 amino acid residues, of which 45% hydrophobic amino acids, is a hydrophobic glycoprotein[8,9]. The process of reverse cholesterol transport refers to the process by which plasma high density lipoprotein (HDL) transports the excess cholesterol in the body to the liver for further metabolism. Cholesterol reverse transport process can reduce plasma cholesterol levels and thus is considered to have anti-atherosclerotic function, and CETP plays a key role in the reverse cholesterol transport process. CETP can also change the level of HDL-cholesterol and HDL particle size by mediating cholesterol from HDL to apolipoprotein-containing very low (VLDL) and low density lipoprotein (LDL) conversion[10-14]. CETP gene mutations can affect its activity, thereby changing the concentration of plasmaHDL and particle size and promoting or resisting the occurrence and development of atherosclerosis[13].

Table 1 Pre-PCR prime and extension primer.

Table 2 Genotype and allele frequency distribution comparison of rs1532624 and rs289741 loci [n(%)].

For rs1532624 polymorphic locus, the results showed that there was no significant difference between the C allele frequency of cerebral infarction group and the healthy control group (68.4% vs.73.7%, χ2=1.723, P=0.189). The frequency of AA genotype in the cerebral infarction group was 10.5% higher than AA genotype in healthy control group (2.8%), with statistical significance (χ2=7.096,P=0.029). This suggests that there may be a correlation between CETP rs1532624 polymorphism and ACI, and AA genotypes may be risk factors of ACI and promote the occurrence and development of ACI.

However, the rs289741 locus had no significant difference in genotype frequency and allele frequency between the cerebral infarction group and healthy control group (χ2=2.906, P=0.234;χ2=2.466, P=0.116 respectively). Therefore, we cannot draw the conclusion that the rs289741 polymorphism was associated with ACI and it may be caused by small sample bias.

In summary, the study obtained some results, but the sample size collected in this study was small. A single factor analysis is not enough to represent all patients. Thus, in order to explore the exact relationship between CETP gene polymorphism and ACI, we need to further expand the sample size and systematically study the larger genetic background population.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgments

The study was supported by grants from Natural Science Foundation of China (31501018, 31760310, and 81660224), and the Social Development Project of Hainan Province (SF201401).

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