Background

Subarachnoid hemorrhage (SAH) is a neurological emergency and a severe subtype of stroke with high morbidity and mortality, which occurs at younger ages than ischemia stroke or intracerebral hemorrhage. It is reported that about 85% of SAH is due to the rupture of an intracranial aneurysm, leading to aneurysmal subarachnoid hemorrhage (aSAH) [1]. As the incidence of aSAH is urgent and dangerous, the epidemiological data have shown an estimated 10% of aSAH patients die before getting medical treatment [2]. Meanwhile, it is reported that with the 3-month mortality rate reaching as high as 47% to 49%, and half of all survivors are left severely disabled [3,4,5]. Therefore, until now the treatment of aSAH in clinical remains unpredictable and the rate of mortality and disability is still high.

Accumulating evidence have shown that aSAH was a multifactorial disorder associated with genetic and environmental factors [6]. Many studies have evaluated modifiable risk factors for aSAH, including age, female sex, hypertension, and tobacco use [7, 8]. Meanwhile, a lot of evidence have suggested that genetic predisposition is also involved in aSAH [9]. So, the discovery of the effects of gene polymorphisms in the course of aSAH, and development of personalized diagnosis and therapy might show attractive prospect in the management of aSAH.

Recently, a number of evidences have suggested that apolipoprotein E (APOE for gene, ApoE for protein) is a candidate gene that is associated with aSAH [10,Statistical analysis

Statistical analysis was performed with SPSS 18.0 software (Chicago, USA). The data were expressed as the mean ± standard deviation (SD) for numerical variables, or as the number (%) for categorical variables. The Hardy-Weinberg equilibrium was estimated in healthy controls. The independent t-test or the Chi-square test was used to compare outcomes. A multivariate logistic regression analysis adjusted for age and gender was carried out using aSAH as dependent variable, the data was showed as the odds ratios (ORs) and 95% confidence intervals (CIs). All tests of significance were two-tailed, and P < 0.05 was considered statistically significant.

Results

Clinical and laboratory characteristics of studying subjects

In our present study, 159 patients with aSAH within 48 h of onset were enrolled, 19 patients were excluded because their clinical data were not gotten completely. Nine patients who did not verified the responsible vascular lesion were also not included. The remaining 131 patients were recruited as aSAH group finally.

The general characteristics and serum lipids in this study are shown in Table 1. As to general characteristics, there were 268 subjects which included 131 patients with aSAH (55 males and 76 females) as aSAH group and 137 age- and gender-matched healthy adults (57 males and 80 females) as control group. The average age of the aSAH group was 54.65 years, with a standard deviation of 13.53, and those of healthy controls was 55.23 years, with a standard deviation of 10.44. There were no significant difference of age, sex, smoking, drinking, hypertension and BMI between the two groups (All P>0.05).

Table 1 Clinical and laboratory characteristics of patients with aSAH and healthy controls
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As to the levels of serum lipids (including TC, TG, HDL-C, LDL-C, ApoA1, and ApoB), there were no significant differences in the levels of TG, LDL-C and ApoB between the aSAH groups and control groups (all P > 0.05). However, the concentration of TC (aSAH groups: 4.52 ± 1.38 mmol/L; control groups: 5.11 ± 0.86 mmol/L), HDL-C (aSAH groups: 1.23 ± 0.46 mmol/L; control groups: 1.44 ± 0.32 mmol/L), and ApoA1 (aSAH group: 1.20 ± 0.32 g/L; control groups: 1.38 ± 0.25 g/L) were significant differences between two groups (all P < 0.001). As shown in Table 1, the serum TC,HDL-C and ApoA1 levels in aSAH were significantly lower than those in the controls.

APOE polymorphism distribution in aSAH and controls

The genotype and allele frequencies between patients with aSAH and healthy controls are shown in Table 2. The ApoE allelic frequency in controls was consistent with Hardy-Weinberg equilibrium (P > 0.05). Six common genotypes of human APOE were identified in our study. Among them, the genotype ε3/ε3 had the largest proportion both in aSAH groups and control groups, and then ε2/ε3 and ε3/ε4, while ε2/ε2, ε4/ε4 were the least, which is comparable to the values found in other studies performed in Asian populations [27, 28]. We further observed the differences in the distribution of the 6 common genotypes in the two groups, we found that the distribution of ε2/ε3 genotype was higher in the aSAH group than the healthy control group (aSAH group: 19.08%; control groups: 9.49%, P < 0.05). Although the ratio of ε3/ε4 in aSAH was lower, there was no statistically significant difference compared with controls (P > 0.05).

Table 2 Genotypes and allelic frequency of APOE in aSAH group and control group
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Then, we also observed the allele frequencies between the two groups, the ε3 allele was greatest (82.82%), while those of ε2 and ε4 were 11.07 and 6.11%, respectively in aSAH patients. Meanwhile, ε3 allele was 84.67%, and ε2 and ε4 were 5.84 and 9.49% in controls. We found that the frequency of ε2 in aSAH group was higher than that in control group (aSAH group: 11.07%; control groups: 5.84%, P < 0.05), indicated that ApoE ε2 may be play some roles in aSAH.

The association of APOE polymorphism with the incidence of aSAH

Using the multivariate logistic regression analysis adjusted for age and gender, we further explore the correlation between APOE Polymorphism and the incidence of aSAH.

As shown in Table 3, after adjusted for the age and gender, there was no significant difference in the incidence of aSAH between the allele ε4 and ε3 (OR = 0.650, 95% CI = 0.308-1.371, and P = 0.258), while, allele ε2 was significantly increased compared to allele ε3 in aSAH (OR = 2.083, 95% CI = 1.045-4.153, and P = 0.037), suggesting that ε2 is a risk factor for aSAH compared with ε3.

Table 3 Odds ratios and 95% confidence interval for aSAH under three major genetic models
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The association of plasma levels of ApoE with aSAH

As shown in Table 4, the differences of serum ApoE between the two groups were analyzed by 2-sample independent t-test. There was a statistically significant difference between the two groups (P < 0.05), the levels of ApoE in aSAH (53.03 ± 24.64 mg/L) was higher than those in controls (45.06 ± 12.84 mg/L).

Table 4 the plasma levels of ApoE in aSAH group and control group
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Meanwhile, as shown in Table 5, in order to explore the effects of genotype on serum ApoE, we further analyzed the plasma levels of ApoE in the three common genotypes (ε2/ε3, ε3/ε3 and ε3/ε4) in aSAH patients (The proportion of other three genotypes was too low, not yet compared). The data showed the plasma levels of ApoE in the three common genotypes were as follow: ε2/ε3 > ε3/ε3 > ε3/ε4, but there was not yet significant difference (all P > 0.05).

Table 5 the plasma levels of ApoE in aSAH group under three genetic models
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Discussion

As we known, aSAH is a critical clinical problem with less chance for patient recovery and survival even after surgical management and medication. So, the discovery of genetic predisposition of aSAH show attractive prospects for improve its diagnosis and treatment, and making medical decisions in clinical practice by personalized medicine.

It has been suggested that APOE polymorphism play a critical role in the incidence and prognosis of aSAH, which has been shown in various studies among different population [10,25].

As to the different results, there are some key factors should be mentioned, such as: (1) geographical area difference, (2) the bias in patients selection, (3) the population size and statistical processing, (4) the differences of objective case: some studies used the overall SAH as the objective case, including the traumatic or spontaneous SAH and so on.

For our current study, we focused on the Chinese Fujian Han population, which was located on the southeast of China. Moreover, every patient of aSAH was confirmed by DSA, CTA or surgery. Meanwhile, the ApoE genotype was determined by commercial ApoE genoty** chip (Sinochips, China), which had get the Certification of CFDA (China Food and Drug Administration) and widely used in many hospital in China, and further confirmed by Sanger sequencing.

Limitations

However, there were also some limitations in our current study. Firstly, the effects of diet on lipid profile in this study should be considered. A lot of studies have reported that the nutraceuticals and functional substances contained in food, such as proanthocyanidins, resveratrol, red wine, and fish oil, maybe have protective effect on vascular system and reduced the overall cardiovascular risk induced by dyslipidemia [42]. Secondly, the patients of aSAH in our study were all come from one hospital which may be a selection bias. Thirdly, those who died or give up during emergency treatment were not included, which could cause Neyman bias. Furthermore, the size of the cases admitted in this study was relatively small. These limitations make us be careful on drawing the conclusions.

Conclusions

In conclusion, our study have suggested that there was a significant correlation between serum lipids and aSAH, and APOE polymorphism might be associated with the incidence of aSAH in Chinese Fujian Han population and the ApoE ε2 allele was a risk factor for the incidence of aSAH. The serum ApoE was significant higher in aSAH compared with the healthy controls. The mechanisms of ApoE ε2 in the incidence of aSAH may be related with the impacts of ApoE genotypes for the serum lipids. Additional studies of larger population size are needed to confirm this finding.