CD36 Gene Polymorphisms Are Associated with Intracerebral Hemorrhage Susceptibility in a Han Chinese Population

The CD36 gene encodes a membrane glycoprotein (type B scavenger receptor, SR-B2) that plays a crucial role in lipid sensing, innate immunity, atherogenesis, and glycolipid metabolism. In this study, we aimed to investigate the association between CD36 gene polymorphisms and intracerebral hemorrhage (ICH) in a Han Chinese population. We performed genotype and allele analyses for eleven single nucleotide polymorphisms (SNPs) of CD36 in a case-controlled study involving 292 ICH patients and 298 control participants. Eleven SNPs were genotyped by the Improved Multiple Ligase Detection Reaction (iMLDR) method. The results indicated that the SNP rs1194182 values were significantly different between ICH group and control group in a dominant model after adjusting for confounding factors. The subgroup analysis conducted for rs1194182 showed that the allele G frequencies were significantly different between ICH patients and controls in hypertension group via a dominant model. We then analyzed the rs1194182 genotype distributions among different groups of the serum lipid groups, including BMI, TC, TG, HDL, and LDL. However, no significant differences were found in the analysis of other subgroups. Taken together, these findings indicate that rs1194182 polymorphism in the CD36 gene was associated with ICH, and genotype GG could be an independent predictor.


Introduction
Intracerebral hemorrhage (ICH) is a crucial classification of stroke and has a high rate of mortality and morbidity [1,2]. The pathogenesis of ICH is complex and influenced by environmental factors and genetic factors. However, its pathogenesis is not yet clear. Hypertension is the most common contributing factor to primary intracerebral hemorrhage. Factors including smoking, drinking, diabetes, and male gender increase the risk further [3][4][5]. In addition, many studies have shown an inverse association between the level of serum cholesterol and ICH [6]. More importantly, the role of genetics in the pathogenesis of ICH has received wide attention [7,8]. Epidemiological studies have demonstrated that a history of a first-degree relative with ICH is an independent risk factor for lobar and nonlobar ICH [4]. It has also been reported that a family history of any stroke was a significant risk factor for patients with ICH who were <70 years compared with those who were >70 years [9]. Taken together, the observed differences based on family history indicate that genetic factors play an important role in the incidence of ICH.
CD36 is a type B scavenger receptor, now officially designated as SR-B2, and CD36 gene located in the 7q11.2 chromosome with 17 exons and 16 introns and is expressed on the surface of various cells: platelets, microvascular endothelial cells, monocytes/macrophages, dendritic cells, adipocytes, striated muscle cells, and hematopoietic cells [10,11]. As a result of its expression in multiple cell types, it can be involved in a variety of biological processes, including transport of oxidized LDL (oxLDL) and fatty acids by macrophages and monocytes, and it participates in processes of inflammation, phagocytosis, and endocytosis [12,13]. In addition, a wide variety of studies have investigated the important roles CD36 plays in many disorders, such as coronary heart disease, hypertension, Alzheimer's Disease, insulin resistance, and metabolic syndrome [14][15][16][17]. However, the relationship between CD36 gene polymorphisms and the risk of ICH has not yet been studied. Thus, to clarify the association of CD36  Table 2.
Venous blood were collected in EDTA-coated vials after at least a 12-hour overnight fasting period. And the ICH patients' venous blood were collected within 24 h after ICH occurred. Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, USA) following the manufacturer's instructions. Improved Multiple Ligase Detection Reaction (iMLDR) was applied for genotyping [18]. Data analysis was carried out using GeneMapper Software version 4.0. Genotyping was carried out blind to group status.

Statistical Analysis.
Statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). The presence of the Hardy-Weinberg equilibrium was determined using the chi-square test. The categorical variables were tested using the chi-squared ( 2 ) test. Categorical variables are expressed as proportions (%) and continuous variables as the mean ± standard deviation. For continuous variables, all comparisons between ICH group and the control group were made using independent -test and among genotype groups by one-way ANOVA followed by LSD test and multiple comparisons. The genotype and allele distributions in ICH patients and control subjects were determined using the chisquared ( 2 ) test. Multivariate logistic regression was used to analyze the genotype frequency of the subjects specified by different genetic models (additive, dominant and recessive comparison) and to calculate the value, odds ratios (ORs), and 95% confidence intervals (CIs) after adjustment for covariates. Statistically significance was set at < 0.05.

Results
A total of 292 ICH patients and 298 control subjects were included in this study. The clinical and laboratory parameters of all subjects in this study are presented in Table 1. The average age and sex distribution were similar between the ICH group and the control group ( > 0.05). As previous studies found, the prevalence of hypertension, drinking history, and family history in the ICH group were significantly higher than in the control group, and there were significant differences between the two groups ( < 0.05). BMI values and TC, TG, and LDL-C levels were also slightly raised in ICH patients. There were no significant differences between the two groups in gender, HDL, smoking history, and prevalence of coronary heart disease and diabetes mellitus. Basic information of the eleven selected SNPs is shown in Table 2. The genotype and allelic frequencies of the SNPs in the ICH group and control group are shown in Table 3. All of the genotypes of eleven SNPs were in agreement with the HWE for the control group ( > 0.05), which indicates that the data remain constant in the population (data not shown).
Logistic regression analysis was performed after adjusting for age, gender, body mass index, hypertension, coronary heart disease, diabetes mellitus, smoking and drinking habits, TC, TG, HDL, LDL, and family history of stroke. A significant association between CD36 and ICH was seen in rs1194182, showing that genotype GG was a risk factor for ICH compared with genotype GC-CC (dominant model, OR = 0.674, 95% CI 0.457-0.992, = 0.046). However, allelic frequencies of rs1194182 in the ICH group and control group have no significant difference. In addition, no associations between the other ten SNPs and the risk of ICH were observed in this study.
All the participants were divided into subgroups to examine whether there were associations between CD36 and ICH in gender or hypertension, ICH location, which are listed in Table 4. After logistic regression analysis for rs1194182, the genotype GG distribution (dominant model, OR = 0.578, 95% CI 0.341-0.98, = 0.042) and the allele G frequencies (OR = 1.533, 95% CI 1.095-2.145, = 0.012) were significantly different between ICH patients and controls in the hypertension group. And among nonlobar ICH group, we found a significant difference in the genotype of GG distribution compared to controls (dominant model, OR = 0.645, 95% CI 0.461-0.997, = 0.043). No significant association could be found in the normotensive group, nonlobar ICH group, the male group, and the female group. CD36 is involved in a variety of roles in lipid metabolism. We then analyzed the rs1194182 genotype distributions among different subgroups, including BMI, TC, TG, HDL, and LDL. However, no significant differences were found in different lipid groups (Table 5).

Discussion
In our study, we identified eleven SNPs of CD36 and found that SNP rs1194182 has significant differences in this casecontrolled study among a Chinese Han population after adjusting for age, gender, body mass index, hypertension, coronary heart disease, diabetes mellitus, smoking and drinking habits, TC, TG, HDL, LDL, and family history of stroke. Subgroup analysis conducted for the SNP rs1194182 in CD36 showed that GG genotype frequencies were significantly different between ICH patients and controls in hypertension group via a dominant model, especially in the hypertension group.
ICH is an important subtype of stroke and is etiologically diverse. The pathogenesis of ICH involves many factors. As found in our study, CD36 SNP rs1194182 may increase the risk of ICH. However, the mechanism by which CD36 increases the risk of ICH is unknown. It is known that hypertension plays an important role in the pathogenesis of ICH [19], and many studies have found that CD36 is closely related to the development of hypertension [14,20,21]. In a microarray analysis study of the differential gene expression between hypertensives and normotensives, 31 genes were upregulated and 18 genes were downregulated, including the CD36 gene with 4.8-fold changes and significant differences between hypertensive and normotensive groups [22]. CD36 deficient individuals were found to have increased blood pressure levels [23]. The +273A/G polymorphism in CD36 was associated with essential hypertension especially in males [14]. Thus, CD36 may contribute to the development of ICH by the following mechanisms. One possibility is through regulating the function of endothelin-1 and altering the properties of vascular smooth muscle, which lead to the development of hypertension and atherosclerosis [24]. Pravenec et al. found that deficiency in renal expression of CD36 could increase blood pressure [21,25]. In our study, the results showed that    [4]. Thus, we assume that CD36 gives rise to ICH through blood pressure pathways. In addition, SNP rs1194182 in the CD36 gene could be a molecular marker for ICH particularly in hypertensive patients. CD36 is involved in a variety of lipid metabolism pathways. CD36 has the ability to facilitate the uptake of long-chain fatty acids in muscle and adipose tissues, which contributes to the regulation of lipid metabolism and insulin resistance [26,27]. CD36 is also expressed on macrophages as a receptor for oxidized low-density lipoproteins and plays an important role in the development of atherosclerosis [28]. Previous Japanese studies have reported an association between rare CD36 variants and high blood levels of free fatty acid and triglycerides [29,30]. CD36 sequence variants were also associated with HDL-C levels [31]. A metaanalysis found that the CD36 gene was significantly linked to triglycerides and triglycerides/HDL-C ratio but not linked to LDL or total cholesterol [32]. In addition, many studies have demonstrated that the polymorphism of CD36 influences the serum lipid levels in the patients of atherosclerosis, coronary heart disease, and metabolic syndrome [33][34][35]. Importantly, as the Rotterdam Study found, low serum triglycerides levels were also associated with an increased risk of ICH [36]. To evaluate the influence of those factors on the genotype of CD36 in this study, we conduct subgroups analysis according to the BMI, TC, TG, HDL, and LDL. However, for rs1194182, there was no significant difference among lipid subgroups found in the analysis. These findings may indicate that the CD36 increased risk of ICH is not through dyslipidemia pathways.
To our knowledge, this study was the first to report an association between SNPs of CD36 and ICH in a Chinese Han population. This association may be stronger for those individuals labeled as ICH. The SNP rs1194182 in CD36 can be recognized as molecular markers of ICH even though the mechanism is not clear. However, the main limitations of this study are the small sample size and the mechanisms of the CD36 gene in ICH are still unknown. These results should be validated in a large population and in different ethnicities. In our future studies, we will work on the mechanisms of the CD36 gene in ICH with a larger sample size in more diverse areas. As the association between SNPs of CD36 and ICH has been confirmed in a larger sample size and more diverse areas, we would estimate individual risk of ICH just according the venous blood sample.