Endothelial Nitric Oxide Synthase (Glu298Asp) Polymorphism is Associated Significantly with Ischemic Stroke Presenting with Seizures and Altered Sensorium
Keywords: Endothelial nitric oxide synthase (eNOS), gene polymorphism, ischemic stroke, genotypeKey Message: The role of eNOS as a risk factor for ischemic stroke is still questionable. The TT genotype (Glu298Asp) of eNOS is associated with an increased risk of acute ischemic stroke in the Indian population.
Stroke being a big public health problem,,, deserves special consideration. Stroke incidence is rising in developing countries like India with the annual average stroke incidence estimated to be 145 per 100,000 persons per year., Since stroke has a huge socioe-conomic impact, genetic markers are being extensively looked for in order to institute primary and secondary stroke prevention in the community. Indian studies have shown that 10–15% of strokes in the Indian population occur below the age of 40 years., Newer genetic markers are therefore being sought for, in young stroke cases. Some of these novel genetic polymorphisms are: IL-6, MTHFR, ACE, endothelial nitric oxide synthase (eNOS), hyper-homocysteinemia, factor V and prothrombin mutations, PDE4D, and Lp(a).
Nitric oxide (NO) may have an antithrombotic effect by reducing both platelet adhesion and aggregation. NO is synthesized by the three nitric oxide synthase (NOS) isoenzymes which are: Endothelial (eNOS), neuronal (nNOS), and cytokine-inducible NOS. Studies suggest that the eNOS isoenzyme is the most likely for maintaining resting cerebral blood flow. eNOS exon 7 polymorphism (Glu298Asp, G894T) has been considered to influence the risk of coronary artery disease and carotid atherosclerotic plaque formation. However, the role of eNOS polymorphism in ischemic stroke is still unclear and requires further evaluation.,
Multiple investigators have studied different populations in order to find an association between eNOS and acute ischemic stroke (AIS). In 2013, Wang et al. reported that the eNOSG894T polymorphism is associated with a high risk of AIS but another detailed meta-analysis of genetic polymorphisms associated with ischemic stroke in the south Asian population failed to find a conclusive association.,
From India, scant literature is available on the role of eNOS in AIS. Two prior case control studies from India have reported opposite results. The study from the southern part of India did not report any difference in the distribution of genotypic and allelic frequency of G894T gene polymorphism between patients and controls. However, the North Indian study showed a significant association between eNOS G894T (rs1799983) polymorphism and risk of ischemic stroke. Since only a limited number of studies with conflicting results have been carried out in the Indian subcontinent, this study sought to find out any relationship between eNOS polymorphism and AIS in the Indian population.
A total of 307 subjects were recruited in the present study which included 153 cases of AIS and 154 age and sex matched controls.The study was approved by Institutional ethics board before starting recruitment. Prior to enrollment, all subjects or their legally authorized representatives gave a written and informed consent. Patients aged >18 years presenting within 48 h to the emergency department, with clinical signs and symptoms of AIS as per the WHO definition and/or radiological evidence of stroke on CT/MRI brain were included as cases in the study. One hundred fifty-four age and sex matched healthy controls without any vascular disease were also included. Patients with history of traumatic brain injury, intracranial bleed, prior neurodegenerative disorders, transient ischemic attacks, and individuals refusing consent were excluded from the study.
All the subjects underwent a stepwise evaluation which included: a detailed history, general physical and neurological examination, routine biochemistry, haematological investigations and electrocardiogram. A systolic or diastolic blood pressure of more than 140 or 90 mmHg, respectively, on two different occasions, was considered as evidence of hypertension. Subjects who had a fasting blood glucose >126 mg% or with a prior diagnosis of diabetes were considered to have diabetes in the study.
Ten milliliters of fasting venous blood sample was taken from the antecubital vein of the patients under all aseptic precautions within 24 h of presentation. Six milliliters was used for routine hematological and biochemical analysis, which were done by Sysmex KX 21 and the Backman CX 4 clinical chemistry autoanalyzers using standard kits.The remaining 4mLwas divided into two parts (2.5 mLin EDTA vial and 1.5 mLin plain vial) and transported within 48 h of collection to the Genetic Lab in ice, for DNA extraction. DNA was extracted from EDTA blood, using the protocol established by Miller et al.
PCR amplification was done in a peltier thermocycler using the following primers: Forward Primer: 5' CCC CTC CAT CCC ACC CAG TCA AC 3'and Reverse Primer: 3' GTCGTGCAGC TTCGC TGG CAA AGG A 5'
The eNOS Glu298Asp polymorphism was identified by PCR amplification followed by restriction digestion and genotyping on 3% agarose gel as per the protocol given by Norma C, et al. The PCR product was checked on 2% agarose gel following which restriction digestion was done and the genotypes were visualized by running the PCR product on 3% agarose gel at 100V, and the result documented in a gel documentation system. Three bands corresponding to the three genotypes appeared as GG-152, GT-152,103,49, and TT-103,49 bp.
Continuous variables were depicted as mean ± SD and percentages wherever applicable. The student's t-test was used for continuous variables. We used the Chi-square test for analyzing categorical data and a P-value <0.05 was taken to be statistically significant. The odds of developing a stroke in the presence of the T allele was expressed in terms of corresponding Odd's ratio (OR's) and 95% confidence intervals. The association between TT genotype and stroke risk was done using dominant and recessive genetic models. In order to evaluate the independent association between the eNOS TT genotype and clinical features among ischemic stroke cases, we used a multivariate logistic regression analysis model. All data analysis was done using SPSS version 20.0.
Demography and baseline characteristics of the study population
The mean age in the study was 59.44 years among cases and 51.76 years among the controls. The sex distribution among cases and controls was comparable, with 152 males (49.5%) and 155 females (50.5%) making up the study population[79 (51.6%) males and 74 (48.4%) females among the cases, while there were 73 (47.4%) males and 81 (52.6%) females among the controls]. Among the cases, 44 (28.8%) were smokers with an average of 25.23 pack years and a minimum of 5 pack years, whereas the control group had 34 (22.1%) subjects who were smokers.Twenty-one cases (13.7%) were alcohol consumers. Out of 153 cases, 53 (34.6%) had hypertension, whereas 36 (23.5%) cases had diabetes. Dyslipidemia was present in 37 (24.2%) cases on analyzing fasting lipid profiles. Among the cases, 41 (26.8%) had loss of consciousness at presentation, 15 (9.8%) cases had a seizure history, 54 (35.3%) had slurring of speech, and 26 (17%) had bladder/bowel involvement. Cranial nerve involvement was found in 57 (37.3%) cases; motor deficits were present in a total of 119 (77.8%) cases, whereas only 16 (10.5%) patients had sensory deficits at presentation. Age, hypertension, and dyslipidemia were found to be independently associated with ischemic stroke. Systolic and diastolic blood pressure levels were independently associated with a greater risk of AIS (Mean systolic BP among cases versus controls being 136.56 v/s 125.00 mmHg, respectively; P-value = 0.000. Mean diastolic blood pressure among cases versus controls being 82.50 v/s 78.84 mm Hg, respectively; P-value = 0.032) [Table 1].
Genotype and allele distribution of the eNOS variants in cases and controls
The genotype distributions among the cases were GG: 44.4%, GT: 43.8%, and TT: 11.8%. Among the controls, the distribution was GG: 39.6%, GT: 55.2%, and TT: 5.2% [Figure 1]. A significant association between the eNOS genotypic polymorphism Glu298Asp G894T (TT) and the development of AIS was noted.
The allele frequencies followed the Hardy Weinberg equilibrium principle and the frequency of T allele was 33.7% among cases and 32.8% among controls [Table 2].
The association of eNOS genotype distribution with the clinical features among stroke cases was investigated, and the results are shown in [Table 3]. The TT genotype was found to be significantly associated with the risk of AIS with OR: 2.43;95% CI: 1.025–5.779, and P-value = 0.0387 using the recessive inheritance model but not in the other models [Table 4]. On using multivariate regression analysis, we found that cases harboring the TT genotype had higher odds of developing seizures and loss of consciousness at presentation (OR: 7.98;95% CI: 1.99–32.09, P-value: 0.007 for seizures and OR: 5.27;95% CI: 1.59–17.04, P-value-0.003 for loss of consciousness at presentation), which was independent of other traditional risk factors.
Ischemic stroke is known to have a heterogeneous risk factor profile comprising of modifiable as well as genetic risk factors. Sometimes the mechanism of stroke remains uncertain despite exhaustive evaluation and genetic risk factors may be important in these cases. In this study, we found that the TT genotype portended a greater risk of developing AIS. Stroke cases harboring the TT genotype were more likely to have seizures or loss of consciousness at presentation. These results may be interpreted in the light of deficiency of NO in the cerebral vasculature predisposing to increased platelet and leukocyte adhesion, impaired vasodilation, and increased risk of atherosclerosis., This makes eNOS a biologically plausible candidate for study as a susceptibility gene for ischemic stroke.
Similar to results of Wang et al. and Saidi et al., this study too found an association between eNOS Polymorphism G894T (Glu298Asp) and AIS. In a hospital-based case control study conducted in North India comprising of 221 subjects, G894T eNOS polymorphism was found to be significantly associated with the risk of AIS, but the eNOS 4a polymorphism was not. A recent case control study conducted in north India by Kumar et al. had shown an association between G894T polymorphism and the risk of ischemic stroke (particularly large vessel disease) using a dominant inheritance model. The study by Kumar et al. however, did not investigate for an association between eNOS polymorphism and the clinical features or risk factors among ischemic stroke patients. Our study focused on finding an association between eNOS polymorphism with both stroke and risk factors. It also sought to find any difference in clinical findings among stroke cases harboring the TT genotype versus the GG/GT genotype, which had not been studied previously. We found that patients with the TT genotype had a significantly higher frequency of seizures and altered consciousness at presentation. This would be clinically meaningful in terms of finding out whether genetic polymorphisms lead to severe clinical presentations among stroke patients.
In the study conducted by Brass et al., it was found that the concordance rate for stroke was much higher among monozygotic twins compared to dizygotic twins (17.7% v/s 3.6%), thus suggesting a genetic component to this disease. Many genetic association studies have been carried out but have yielded inconsistent results., A population with less environmental risk exposure would be ideal in studying the role of genetic factors. Hence, targeting a younger population would be better in testing the genetic associations with ischemic stroke. In our study, there were a total of 14 cases below 40 years, constituting 9.15% of the total cases and is similar to the results of the Kolkata study. Our study did not find a significant association between eNOS polymorphism and the risk of developing stoke in young (P-value = 0.475). This is similar to the results obtained by Majumdar et al., who did not find a significant association between eNOS G894T (Glu298Asp) polymorphism and strokes in the 15–45 year age group.
The T allele frequency observed in our study was 32.8% among the controls, which is much higher than that reported from previous studies in the north Indian population., This might be due to the limited overall sampling of the genetic pool.
Among studies from India, there is conflict regarding the role of eNOS polymorphism in AIS. The study conducted by Majumdar et al. failed to find a significant association between G894T polymorphism and AIS. They however reported that intron 4a/4b polymorphism might portend an increased risk of stroke among young South Indian females. These findings might be due to ethnic differences and the different genetic pool among South Indians compared to North Indians. Meta-analysis has found G894T and intron 4a eNOS polymorphisms to be significantly associated with stroke in Asians but not in Caucasians. Thus, ethnic and genetic differences might decide which eNOS polymorphism (G894T, intron 4a, T-786C) leads to an increased stroke risk in a particular population. Our study, however, is in line with other North Indian population studies, which have reported an association between G894T polymorphism and occurrence of AIS.,
Our results show that G894T eNOS polymorphism portends a greater risk of developing ischemic stroke via the recessive genetic model. Saidi et al. too showed that eNOS polymorphism increased the risk of developing acute stroke via a recessive as well as a dominant model of inheritance. In 2017, a meta-analysis supported the role of eNOS polymorphism in increasing the risk of AIS via the recessive model of inheritance, whereas borderline significance was seen with the dominant model. Prior meta-analysis has reported an increased risk via only a dominant model of inheritance in the Chinese population.,, The study by Kaur et al. found an association between G894T polymorphism with ischemic stroke via the dominant and codominant model but not by the recessive model. Thus, both dominant and recessive models of inheritance have been found to explain the increased risk attributed by the eNOS G894T polymorphism across multiple studies in different parts of the world. These conflicting findings across studies could be due to ethnic/racial differences and genetic heterogeneity in the sampled subjects even from the same ethnicity. This heterogeneity could influence the frequency distribution of T allele within populations, thus resulting in disease causation via different inheritance models.
Seizures after ischemic stroke have been estimated to occur in 9% cases. Seizures signify a greater severity of the initial stroke episode and a larger infarct size. The TT genotype independently increased the odds of developing seizures at initial presentation among stroke cases. However, this finding has to be viewed with caution as the total number of patients presenting with seizures was small (15 out of 153 cases, only 9.8% of the total cases). Thus, further large prospective cohort studies would be required before any definite conclusions can be drawn.
In this study, 41 out of 153 stroke cases (26.8%) had altered consciousness at presentation. The TT genotype increased the odds of developing altered consciousness at stroke onset in our study. An altered level consciousness at presentation in an ischemic stroke is suggestive of a large stroke with raised intracranial tension or due to brainstem compression. This genetic marker may, thus, be useful in identifying patient at risk of having severe strokes associated with altered level of consciousness and seizures at presentation. However, larger prospective studies would be required before any recommendations can be made.
Limitations of the study
Our study had 307 patients including 153 stroke cases, which is small as far as genetic studies are concerned. Sampling a younger population would be preferable for independently assessing the risk attributed by eNOS polymorphism. The role of eNOS polymorphism as a risk factor for young strokes still needs to be determined as too few cases were included in our study. Thus, in light of these limitations, further studies are required before establishing eNOS polymorphism as a risk factor for developing AIS.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
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Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2], [Table 3], [Table 4]