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The Correlation of Endothelial Nitric Oxide Synthase (eNOS) Polymorphism and Other Risk Factors with Aneurysmal Subarachnoid Hemorrhage: A Case-Control Study
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.266231
Keywords: Aneurysm, endothelial nitric oxide synthase gene, outcome, subarachnoid hemorrhage
Aneurysm formation, rupture, and subsequent outcomes are due to multifactorial processes various modifiable and nonmodifiable risk factors are involved. Despite extensive attempts to identify the risk factors and the pathophysiology related to aneurysm formation, rupture, there has been limited success. Several genes have been analyzed in relation with aneurysm pathobiology.[1],[2],[3],[4] A study in the North American population analyzed the role of three eNOS gene polymorphisms as potential risk factors that can predict susceptibility to aneurysmal subarachnoid hemorrhage (aSAH) and cerebral vasospasm.[5] The exact role of eNOS gene and its polymorphism as a predictive factor has not been reported. However, evidence showed that the polymorphism of T-786C SNP variant reduced the eNOS gene promoter activation. Even the polymorphism of 27VNTR and T-786C variant produced decreased eNOS protein expression and enzymatic activity.[6] The purpose of this study was to evaluate the role of eNOS gene polymorphism in the clinical course of aSAH and investigate the predictive factors determining the outcome of aSAH.
This was a prospective, case-control study conducted at the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore. The study protocol was submitted for review by the Institute Human Ethics Committee and approval obtained prior to starting the study. Informed consent was taken from cases and controls before taking blood samples. Case group The study was carried out from December 2008 to July 2012 in NIMHANS, Bangalore. 100 patients diagnosed with SAH due to a cerebral aneurysm were included in the study. Patients with other vascular malformations (AVM, venous angioma, and congenital venous anomaly, dural arteriovenous fistula, cavernous angioma), recent onset myocardial infarction, intracranial mass lesion, and coagulation disorder were excluded from the study. Control group Gender-matched healthy subjects of similar age without a previous history suggestive of SAH were recruited by the Institute staff. Volunteers were recruited as controls. Demographic details along with past medical and family history were obtained. The subjects were examined in detail to rule out neurological deficits.
Sample collection and DNA isolation Six milliliters of blood were collected from the subjects in tubes containing EDTA. Genomic DNA was isolated from EDTA blood according to the conventional phenol–chloroform method. DNA was quantified using Nano Drop 1000 (Thermo Fisher Scientific, DE 19810, USA). Genotyping eNOS 4a/4b polymorphism screening Genomic DNA was amplified using PCR (primers 5'-AGG CCC TAT GGT AGT GCC TTT-3' (sense) and 5'-TCT CTT AGT GCT GTG GTC AC-3' (antisense) with Taq DNA Polymerase (Solis BioDyne, Estonia) and supplied buffer under the following conditions: 95°C for 5 min, followed by 35 cycles at 94°C for 30 s, 59°C for 45 s, and 72°C for 45 s, followed by a 10-min 72°C final extension. PCR products were electrophoretically separated on a 2% agarose gel. The 4/4 homozygous 4a allele (containing 4 of 27 bp repeats) gave rise to PCR product of 393 bp, the 5/5 homozygous 4b allele (containing 5 of 27 bp repeats) gave rise to PCR product of 420 bp whereas the 4/5 heterozygous 4ab allele gave rise to 420 and 393 bp bands [Figure 1].
eNOS–786T>C polymorphism screening Genomic DNA was amplified using PCR (primers 5'-GTG TAC CCC ACC TGC ATT CT-3' (sense) and 5'-CCC AGC AAG GAT GTA GTG AC-3' (antisense) with Taq DNA Polymerase (Solis BioDyne, Estonia) and supplied buffer under the following conditions: 95°C for 5 min, followed by 35 cycles at 94°C for 30 s, 63°C for 45 seconds, and 72°C for 45 seconds, followed by a 10-min 72°C final extension. PCR products of 308 bp were then digested with the restriction enzyme NaeI (New England Biolabs, Ipswich, Massachusetts, United States) at 37°C for 16 h and were electrophoretically separated on a 2% agarose gel. The -786 C allele is characterized by diagnostic NaeI restriction fragments of 227 and 81 bp. Thus, the gel pattern for individuals with homozygous mutant genotype (-786CC) showed 227 and 81 bp bands whereas -786 TT homozygous wild type did not undergo digestion by NaeI and showed PCR band of 308 bp. Individuals with -786 TC heterozygous genotype showed both intact PCR band of 308 bp and two cleaved bands of 227 and 81 bp [Figure 2].
eNOS 894G>T polymorphism screening Extracted DNA was amplified using PCR (sense primer 5'-AAG GCA GGA GAC AGT GGA TGG A-3'; anti-sense primer 5'-CCC AGT CAA TCC CTT TGG TGC TCA-3') with Taq DNA Polymerase (Solis BioDyne, Estonia) and supplied buffer under the following conditions: 95°C for 5 min, followed by 35 cycles at 94°C for 30 seconds, 67.5°C for 45 s, and 72°C for 45 seconds, followed by a 10-min 72°C final extension. PCR products of 248 bp were then digested with restriction enzyme Dpn II (New England Biolabs, Ipswich, Massachusetts, United States) at 37°C for 16 h and were electrophoretically separated on a 2% agarose gel. The 894T allele is characterized by diagnostic Dpn II restriction fragments of 163 and 85 bp. Thus, the gel pattern for individuals with homozygous mutant genotype (894 TT) showed 163 and 85 bp bands whereas 894 GG homozygous wild type did not undergo digestion by Dpn II and showed PCR band of 248 bp. Individuals with 894 GT heterozygous genotype showed both intact PCR band of 248 bp and two cleaved bands of 163 and 85 bp [Figure 3].
Outcome scale Glasgow outcome scale (GOS) was used to quantify the functional outcome. The primary goal was to assess the functional outcome at 6 months follow-up. The detail of GOS (5 = good recovery—able to return to work; 4 = moderate disability—able to live independently; unable to return to work; 3 = severe disability—able to follow commands/unable to live independently; 2 = vegetative state; 1 = dead). Statistical analysis Demographic data (patient related) and aneurysm-related data were categorized to determine predictive factors controlling the outcome. Chi-square test, univariate analysis were applied for analysis of individual factors. Later, regression analysis was done to finalize the predictive factors related to unfavorable outcome. Analyses were performed utilizing the SPSS software (v21, IBM Inc., Armonk, NY). P < 0.05 was considered statistically significant.
A total of 100 patients were included in the test group and 100 volunteers were included in the control group. Age of the aSAH patients ranged from 18 to 75 years, with a mean age of 51.61 years. Only one patient was 18 years and the polymorphism status was positive for 4BB, TT (T-786-C), and GT (G894T). The controls were in the range of 24–71 years, with the mean age being 45.81 years. Among the cases, 42 were males and 58 were females. Among the controls, 48 were males and 52 were females. Four cases had a family history of SAH though further details are not known. Among them, three cases were 4BB positive and one case was 4AB positive, all four were positive for TT (T-786-C) and three cases were positive for GG (G894T). Three different eNOS polymorphisms were analyzed from each blood sample. The most frequent 24-VNTR polymorphism types were 4BB (65%) followed by 4AB (30%) and 4AA (5%). The frequency was almost similar in the control group. TT (71%) was the frequent T-786-C polymorphism followed by TC (23%) and CC (6%). Among the G894T polymorphism, GG (71%) most frequent followed by GT (23%) and TT (6%). No significant differences in the distribution of allele and genotype frequencies in the patients and control groups were observed. Allele frequency distributions in controls and patient populations were found to be in Hardy–Weinberg equilibrium [Table 1].
The clinical presentations of aSAH cases are shown [Table 2]. The incidences of comorbidities were as follows: 32% patients with hypertension, 37% with a history of smoking, and 9% with diabetes. The usual presentation was sudden onset headache (96%) followed by loss of consciousness (64%). 17% of patients patients presented with seizures at the onset.
53% of patients were patients were in WFNS grade 1, 23% grade 2, 11% grade 3, and 10% were in grade 4. According to Fisher's grading, most of the patients were in grade 3 (57%) followed by grade 4 (28%). The most common location was anterior circulation (97%) followed by the posterior circulation (3%). Among 100 aneurysms, 83% aneurysms were clipped and 10% underwent coiling. Most of the aneurysms were in the middle group (6–9 mm) followed by bigger group (>10 mm) (37%) and only 6% aneurysms were in a small aneurysm (<6 mm) group. 33% of patient patients had evidence of vasospasm (angiographic) and those who developed clinical symptoms received treatment for vasospasm. TT of G894T polymorphisms had the highest occurrence (60%) of vasospasm among all polymorphism and T786C-TT (29.3%) had the lowest number of vasospasm though all individual polymorphism were not statistically significant with the outcome [Figure 4]. Seven cases had multiple aneurysms and most common polymorphisms were 4BB (71%) (24-VNTR), TT (71%) (T-786-C), and GG (57%) (G894T).
Follow-up was available for 95 patients and 5 patients expired (2 in hospital and 3 during follow-up). The median follow-up was 8 months (6-50 months). The mean GOS at discharge and follow-up (6 months) were 3.86,4.24. Association of eNOS gene polymorphism and outcome Results of the univariate analysis of patient related factors [Table 3] showed smoking was significantly associated with unfavorable outcome (OR: 3.19, CI: 1.19–8.84, P = 0.01). However, age, sex, preoperative hypertension, and diabetes were not associated with an unfavorable outcome. AA (OR: 12.15, CI: 1.13–624.9, P = 0.03) variety of 24-VNTR polymorphism and CC (OR: 15.39, CI: 1.60–762.8, P = 0.01) variety of T786C polymorphism were influencing the 6 month poor functional outcome in univariate analysis.
Among the aneurysm-related factors [Table 4], Fisher grade 4 (OR: 3.43, CI: 1.24–9.68, P = 0.01), WFNS grade (poor vs. good) (OR: 3.42, CI: 1.17–10.12, P = 0.02), Vasospasm (OR: 3.84, CI: 1.42–10.75, P = 0.006), and intraoperative rupture (OR: 4.77, CI: 1.55–15.27, P = 0.004) were strong predictors of an unfavorable functional outcome. The regression analysis revealed [Table 5] smoking (CI: 0.06–0.69, P = 0.01), Fisher grade 4 (CI: 0.09–1.00, P = 0.05) and intaroperative rupture (CI: 0.05–0.89, P = 0.03) were related with with a six month unfavorable outcome. Individual polymorphism incidence in relation to WFNS grade, Fisher grade, and type of circulation, and an unfavorable outcome was mentioned in table [Table 6].
Endothelial nitric oxide synthases (eNOS) expressed in endothelial cells play a major role in shear-stress response related endothelial dysfunction, which has been recognized as the underlying pathological processes of intracranial aneurysms.[7] The eNOS polymorphism is correlated with the size of an aneurysm,[1],[8] the risk for rupture,[9] and the risk for aneurysm formation.[5],[10],[11] However, literature is sparse regarding the correlation between eNOS and functional outcome. The present cohort attempts to analyze the correlation of the genetic (eNOS gene polymorphism) and as morphological risk factors associated with an unfavorable outcome after aSAH. eNOS polymorphism and SAH The eNOS T-786C SNP [12] is one of the most frequently investigated mutations associated with a cerebral aneurysm because it is located in the eNOS promoter region, which potentially affects eNOS expression. Mutations in other areas like intron-4 27-bp VNTR,[13] the exon-7 G894T SNPs [10] also affect the expression of eNOS gene and increase risk for a cerebral aneurysm. Khurana et al.[5] had reported that the eNOS 27 VNTR (in particular, the “4a” allele) is significantly associated (P = 0.007) with the occurrence of aneurysmal SAH and that the eNOS T-786C SNP (in particular, the “C” allele) is significantly associated with the occurrence of post-SAH vasospasm. Alexander et al.[14] showed that a combination of variant alleles of all three SNP of eNOS was associated with cerebral vasospasm. Our study showed that the homozygous variety, 4BB genotype (65%), TT (71%) of T-786C, and GG (71%) of the G894T polymorphism was most common haplotype frequency in the aneurysmal group. The allelic frequency in controls and patients population was following the Hardy–Weinberg equilibrium. As there was no association between the eNOS gene variants and aSAH in our population, the distribution of the genotypes did not differ much between cases and controls. The various environmental factors and ethnic background, played a major role in aneurysm formation but in our population, the association is lacking. Risk factors and outcome The outcome of aSAH correlates with patient associated factors as well as the severity of SAH. A recent meta-analysis [15] of epidemiological studies suggested that smoking (OR, 3.1; 95% CI, 2.7–3.5), hypertension (OR, 2.6; 95% CI, 2.0–3.1), and excessive alcohol intake (OR, 1.5; 95% CI, 1.3–1.8) are major risk factors of aSAH whereas diabetes was associated with a reduced risk of aSAH (OR, 0.7; 95% CI, 0.5–0.8). In the SAHIT study,[16] hypertension contribute to an inferior outcome at 3 months follow-up after SAH. Female gender was associated with poor outcome.[17],[18] However, other studies reported no influence of female gender with outcome.[19] McGirt et al.[20] found older age and hypertension was associated with a poor outcome after SAH in univariate as well as multivariate analysis. WFNS grades 4, 5, and shorter hospital stay was closely related with poor outcome.[19] Rosengart et al.,[21] reported in their study that various factors like age, poor neurological grade, high fisher grade, hypertension, intraventricular hemorrhage, intracerebral hemorrhage were associated with unfavorable outcome. Koshy et al.[22] did a study in a similar population and found that hypertension and smoking were related to aSAH but unrelated to the eNOS polymorphism. The International cooperative study [17] also reported the similar predictive factors for poor outcome. In another study,[23] only age (>60 years), and hydrocephalus were the predictors of poor functional outcome. However, Fisher grade, aneurysm size, intraoperative rupture, vasospasm were not related to poor outcome. The location of aneurysms, size, intraoperative rupture, and vasospasm are closely correlated with patient outcome after aSAH. Hamdan et al.[19] found that internal carotid artery aneurysm (P = 0.01) and posterior circulation (P = 0.006) location were related with poor outcome regression analysis. Posterior circulation aneurysm (P = 0.0001) and larger aneurysm size (P = 0.001) were associated with an unfavorable outcome.[21] In our cohort, hypertension, smoking, Fisher grade 4, intraoperative rupture, and vasospasm were closely related with a poor outcome in univariate as well as regression analysis after treatment of aSAH. Genetic polymorphism and functional outcome after subarachnoid hemorrhage Various genes [5],[24],[25],[26],[27],[28],[29] (eNOS, Endothelin, RAS, Apo e, Epoxide hydrolase, BDNF) were analyzed related to functional outcome after aSAH. Alexander et al.[14] found that a single copy of the variant allelles of SNP (rs1799983) of the eNOS gene (P < 0.001), was associated with a poorer outcome at 6 months GOS follow-up. Song et al.[30] found a relationship between T-786 C polymorphism and unfavorable outcome (CI: 1.131–16.108, P = 0.032). The polymorphism associated with high degrees of vasospasm is indirectly related with poor outcome at follow-up. Stark et al.,[31] reported T allele of T-786-C is strongly related to severe vasospasm. Hendrix et al.,[32] found C allele of T-786-C is related to delayed cerebral ischemia in univariate analysis. But in regression analysis, the same factor is not related with the final functional outcome. Our cohort showed that the AA of 24-VNTR polymorphism and CC of T-786C polymorphism was associated with an unfavorable outcome at the six month follow-up in univariate analysis. However, this was not significant in regression analysis. Few other genes are correlated with functional outcome after aSAH. The gene polymorphism,[28] and the T allele of the END1 T/G SNP (rs1800541) were associated with aSAH but not correlated with the functional outcome. The Renin system polymorphism [27] (ACE I/D) was analyzed with aSAH and the recessive effect of allele I (insertion) (OR: 2.76, 95% CI: 1.17–6.50; P = 0.0206) was correlated with an unfavorable outcome. The K genotype group of soluble epoxide hydrolase [26] was correlated with a poor functional outcome. Apolipoprotein E ε2 or ε4–genotypes of APO E gene polymorphism were not related to inferior outcome.[24] The allele of brain-derived neurotropic factor (BDNF) was associated with a poor outcome.[25] In the near future, the genetic parameter also should be included as a predictive marker for prognostication. Limitations Our study sample demonstrates the trend toward the outcome. A larger sample may perhaps show conclusively. The other factors that may have an association are the duration of hypertension, the amount, and duration of smoking as well as the time of collection of the blood sample for genetic analysis. The present study included the generic parameters, non-modifiable factors, and aneurysmal factors for analysis of functional outcome after treatment.
In this case-control study, we have explored the hypothesis of whether polymorphisms in the eNOS gene are associated with aSAH in Indian population. In our study, smoking, clinical grade (WFNS), Fisher grade 4, intraoperative rupture of an aneurysm, 4AA of 24-VNTR, CC of T-786C polymorphism, and vasospasm had an impact on the six month outcom. A larger multicenter study may perhaps give a more definite answer whether the above predictive factors are useful in the prognostication of aSAH. Disclosure Indian Council of Medical Research (ICMR) Grants. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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