Apolipoprotein E polymorphism and the risk of aneurysmal subarachnoid hemorrhage in a South Indian population

The rupture of a brain aneurysm causes bleeding in the subarachnoid space. This is known as aneurysmal subarachnoid hemorrhage (aSAH). We evaluated the association of apolipoprotein E (APOE) polymorphism and the risk of aSAH in a South Indian population. The study was performed on 200 subjects with aSAH and 253 healthy control subjects. Blood samples (5 ml) were used to isolate DNA and genotyping was performed for rs7412 and rs429358 using a Taqman allelic discrimination assay. Statistical software R.3.0.11 was used to statistically analyze the data and a p value < 0.05 was considered as statistically significant. We found a significant association with the risk of aSAH in ε3/ ε4 genetic model (OR = 1.91, 95% CI = 1.16–3.14, p = 0.01). However, in the other genetic models and allele frequency, there was no significant association with the risk of aSAH. In subtyping, we found a significant association of ε2 allele frequency with posterior communicating artery (PCOM) aneurysm (OR = 3.59, 95% CI = 1.11–11.64, p = 0.03). Our results suggest that APOE polymorphism has an influence on the risk of aSAH in this South Indian population, specifically in the PCOM subtype.

Apolipoprotein E (APOE) is a 299-amino acid protein mainly synthesized by the liver and macrophages [7]. In human beings, it is located in the long arm of chromosome 19 and the protein is encoded by exon 4 [8]. In the brain, it is synthesized by astrocytes and helps in the transport of cholesterol to neurons [9]. APOE also helps in neuronal repair [10], cerebral glucose metabolism [11], and differentiation and migration of neurons [12]. The APOE gene is polymorphic with three different alleles (ε2, ε3, ε4) and six different genotypes [13]. The combination of polymorphisms of rs7412 and rs429358 will determine the six possible genotypes [14].
There is no study that has addressed the relationship between APOE polymorphism and the risk of developing aSAH in the Indian population. The aim of this study is to investigate the association of APOE polymorphism and aSAH in a South Indian population.

Study population
The subjects were 200 patients with aneurysmal subarachnoid hemorrhage recruited from the Department of Neurosurgery, NIMHNAS, Bangalore, India, and 253 ethnicity-, ageand sex-matched healthy controls were selected randomly from the general population in 2015-2017. The healthy controls were unrelated to the patients. The patients and control groups were all from the Dravidian Kannada-speaking population of South India.
The inclusion criteria for patients with aSAH were diagnosis of aneurysmal SAH with the presence of symptoms suggestive of aSAH combined with subarachnoid blood found with the CT scan and a proven aneurysm found via conventional angiography. The exclusion criteria for selecting patients were: the presence of neuropsychiatric conditions like dementia, Parkinson's disease, epilepsy and psychoses; or if SAH resulted from a mycotic aneurysm, arterio-venous malformation or trauma. Demographic and clinical details were collected directly from the patients using structured questionnaires and data from the medical records section.
The inclusion criteria for healthy controls were: the absence of aneurysm, checked with digital subtraction angiography; similar demographic characteristics to the patient group (age, sex, ethnicity and dietary habits); no medical history of hemorrhage and no family history of aSAH in first-degree relatives.
The study protocol was approved by the Institute of ethics committee for human studies, NIMHANS, Bangalore. Written informed consent was obtained from all the participants.

DNA extraction and genotyping
Blood samples (5 ml) were collected from all the participants. Genomic DNA was isolated from the blood using the commercially available Machery-Nagel (MN) kit according to the manufacturer's protocol. The purity and quantity of DNA was analyzed using a Nanodrop ND2000c spectrophotometer. DNA with a purity of 1.75-1.85 was used for genotyping analyses.
Genotyping of rs7412 and rs429358 was performed using a Taqman allelic discrimination assay (Applied Biosystems) with a commercially available primer probe set (assay ID C_904973_10, C_3084793_20). Genotyping was performed in duplicates using an Applied Biosystem7500 Fast Real-Time Cycler.

Statistical analysis
R.3.0.11 statistical software was used to statistically analyze the data. The continuous variables were expressed as means ± SD and categorical variables as absolute values and percentages. The demographic characteristics of the patients and controls were compared using the χ2 test for all categorical variables. Differences in genotype and allele frequencies between groups were analyzed using the χ2 test. Association between APOE genotypes or alleles and aSAH risk were expressed as the odds ratio (OR) with 95% confidence intervals (CI), adjusted for the confounding effects of smoking, hypertension, drinking and diabetes mellitus using the logistic regression model. The Hardy-Weinberg equilibrium calculation was performed using an online tool at http://www.oege.org/software/hwe-mr-calc.shtml. p < 0.05 was considered statistically significant.

Study population characteristics
The characteristics of patients with aSAH and healthy controls were shown in Table 1. Clinical data were available for all patients and healthy control subjects and there was no statistical significance in gender difference. The mean age was slightly higher in aSAH patients than in the controls with a p value of 0.170. Smoking and alcohol consumption were slightly more common in patients. The frequencies of hypertension and diabetes mellitus were also slightly higher in patients. The majority of the patients had aSAH with an ACOM (anterior communicating artery) aneurysm (43%) with a size of less than 15 mm (79.5%) and a WFNS grade of I (46%).
A comparison of different genotype models of aSAH with healthy controls showed no significant difference in the data. The results of logistic regression analyses are shown in Table 3. A statistically significant association was found in the genotype model ε3/ε3 vs. When the aneurysms were classified according to location, size and WFNS grade and compared with different APOE genotype models and allele frequencies, only the PCOM aneurysm was statistically significant with ε2 vs. ε3 allele frequency (OR = 3.59, 95% CI = 1.11-11.64, p = 0.03). Classification of aneurysms according to APOE genotype frequency is shown in Table 4. Similarly, we performed comparisons between male vs. female, hypertensive vs. non-hypertensive, and diabetic vs. non-diabetic patients with different APOE genotype model and allele frequencies. None of the comparisons showed statistical significance with the APOE allele or genotype model.

Discussion
Subarachnoid hemorrhage caused by the rupture of an aneurysm is a condition with a low chance of recovery and a strong chance of life-long locomotor disability [23]. Guiding physicians in predicting the occurrence and rupture of an aneurysm will help them to save patients' lives and their capability to be productive. Various genetic polymorphisms are associated with the risk of developing aSAH. Apolipoprotein E polymorphism has emerged as one of the major genetic factors associated with the risk and prognosis of many neurological disorders and of hemorrhagic stroke in various populations. Japan has the highest incidence rate of aSAH in the Asian population. One explanation for this higher incidence is the relatively high life expectancy in Japan [24]. Statistical reports state that the Japanese population has the highest median age (43 years old) [25].The reported incidence rate of aneurysm in India ranges from 0.75-10.3%, and the North West Indian population, (lowest median age-28 years old) only has an incidence rate of 1% [26]. Therefore, age can be considered a risk factor for aSAH.
Another independent risk factor for aSAH is being female [23]. In our study, incidence of aSAH was 1.6 times higher in the female population than in the male population. The mean age of the female and male population was 55and 40. The reason for the higher incidence rates in women are not still clear. Previously, it was observed that incident rate of aSAH was higher after menopause [27]. After menopause, a drop in sex hormones occurs [28], especially in estrogen. It has also been reported that estrogen has a protective role for SAH [29]. Estrogen has been reported to improve the lipid profile, reducing the risk of atherosclerosis [30,31], which has been considered an important pathogenic mechanism for the formation of an aneurysm [32].
De Rooij et al. reported that at a younger age, the incidence of aSAH was higher in men than women [1]. But in our study, male and female subjects of less than 50 years of age were equally affected. There were 62.5% females who had multiple aneurysms in   our study, which concurs with previous findings that women are more likely to have multiple aneurysms than men [33]. APOE polymorphism has been associated with the risk of developing many central nervous system disorders, like Alzheimer's disease [34][35][36], vascular dementia [37], multiple sclerosis [38], cerebral infraction [39] and Parkinson's disease [40]. Many investigations have reported a positive association of APOE polymorphism with a risk of aSAH in different populations. Liu et al. showed that ε2/ε2 and ε2/ε3 genotype frequencies were higher in patients with intracranial aneurysm in the Chinese population [41]. Tang et al. noted that ε4 allele carriers have unfavorable outcomes after aSAH in the Chinese population [42].
In the Japanese population, Kokubo et al. found that ε4 allele carriers have a 2.5-fold higher risk of aSAH [43]. Mineharu et al. suggested that the three alleles of APOE did not have any association with aSAH in Western Japan [44].
In the Caucasian population, McCarron et al. reported that the ε2 allele was significantly associated with the risk of different intracranial hemorrhage [13]. Kaushal et al. and Fontanella et al. found that ε2 and ε4 allele were not significantly associated with the risk of aSAH in United States and Italian population [45,46].
A meta-analysis of nine case control studies found that in the Asian population, ε2/ε2 vs. ε3/ε3, ε2/ε3 vs. ε3/ε3, ε2 vs. ε3 and ε2 allele frequency were associated with the risk of aSAH. However, the study concluded that only the ε2/ε2 vs. ε3/ε3 genetic model was associated with the risk of aSAH in the Caucasian population [21].
In terms of lipoprotein metabolism, the main difference between APOε3 and APOε4 isoforms was that APOε4 has greater affinity for very low-density lipoprotein (VLDL) receptor [47]. Therefore, APOε4 impairs lipolytic processing and leads to the accumulation of VLDL in plasma [48]. The reason for pro-atherogenic lipoprotein-cholesterol distribution in the plasma of the APOε4 isoform was because of the Cys112Arg substitution [49]. The presence of Arg112 in the APOε4 isoform led to an altered interaction between the LDL receptor-binding domain and lipid-binding domain region, which was the reason for the higher binding affinity of this isoform to VLDL [50]. In ischemic stroke patients, the ε3/ε4 genotype was associated with elevated levels of very low-density lipoprotein and triglycerides [22].
In our study, the ε3/ε4 genotype showed significant association with the risk of aSAH. The frequency for the ε3/ε4 genotype was higher in our patient group (23%) than the controls (14%). We also found that ε4 allele frequency was higher in the patient population, but we did not find any statistical significance.
One of the proposed mechanisms for aneurysm formation and progression is atherosclerosis [51]. It was reported that elevated levels of triglycerides and very low-density lipoprotein promote atherosclerosis [52]. ε4 carriers (ε3/ε4, ε4/ε4, ε2/ε4 genotype) have been associated significantly with risk for atherosclerosis and elevated levels of very low-density lipoprotein [53,54]. From all these findings, it can be summarized that the ε3/ε4 genotype is one of the risk factor for aSAH, since it can promote atherosclerosis.
In this study, of the aSAH subtypes, the ε2 allele has a significant association with PCOM aneurysm. The PCOM aneurysm is the third most common Circle of Willis aneurysm. PCOM arteries are present at the base of the brain and form a part of the Circle of Willis [55]. It was reported that the ε2 allele is associated with an elevated concentration of plasma triglycerides [56] and that ε2/ε3 genotype carriers were less likely to survive a stroke [57]. Also, the presence of the APOε2 allele may indicate susceptibility to the development of fibrinoid necrosis and microaneurysm [58].
The main difference between APOε3 and APOε2 isoform was that the latter rarely binds to low-density lipoprotein receptors [59]. APOε2 differs from the APOε3 isoform by Arg to Cys amino acid substitution at position 158, which is close to the LDL receptor-binding domain region [60]. Cys158 disrupts the natural salt bridge between Asp152 and Arg154 in the LDLR recognition site, which impairs the binding to the LDL receptor [61]. This makes the APOε2 isoform unable to promote the clearance of low density lipoprotein and triglycerides from the plasma [62]. Bolger et al. noted that one of the reasons for saccular aneurysmal disease is the elevated serum level of low-density lipoprotein [63]. This suggests that the ε2 allele has a role in elevating the levels of low-density lipoprotein and thereby can be a causal factor in aSAH.

Conclusion
APOE polymorphism can be associated with the risk of aSAH in the South Indian population. When compared with the site of aneurysm, the ε2 allele was found to be associated with PCOM aneurysm. Further comprehensive studies are required to confirm these findings.