Role of ACE Polymorphism in Acute Ischemic Stroke
Keywords: ACE, angiotensin converting enzyme, genetic polymorphism, ischemic stroke
Stroke is a big health problem and requires special attention. Around the world, 15 million people every year have a stroke and of these five million eventually succumb to the disease. Stroke burden is projected to rise from 38 million DALY's in 1990 to nearly 61 million DALY's in 2020. Even in India, stroke is a leading cause of death and disability with the an estimated prevalence rate of 84–262/100,000 in rural and 334–424/100,000 in urban areas. Stroke is also on the rise in India as a result of increasing longevity of population and lifestyle changes due to urbanization. The traditional stroke risk factors like increasing age, hypertension, diabetes mellitus, smoking, dyslipidemia, ischemic heart disease, alcohol consumption are not found in all cases of ischemic stroke and the mechanism of stroke remains unexplained in about 30% of cases. Therefore, newer genetic markers such as Interleukin-6, Methyl tetrahydrofolate reductase, haptoglobin, angiotensin-converting enzyme (ACE), Apolipoprotein E Angiotensinogen, endothelial nitric oxide synthase, ICAM-1 (Intercellular cell adhesion molecule-1), MIF (Macrophage migratory inhibitory factor), MCP-1 (Monocyte chemoattractant protein-1), gene encoding Factor V, gene encoding prothrombin, PDE4D (Phosphodiesterase 4D), C-reactive protein, homocysteine, and Lipoprotein(a),, are being extensively looked for in order to detect stroke early and institute primary as well as secondary prevention strategies. In developing countries, individuals develop stroke around a decade and a half earlier than that in developed countries. Newer genetic markers might therefore be important in the setting of young stroke. In order to establish the role of these genes as stroke risk factors various types of studies such as candidate gene association, linkage and genome wide association studies can be conducted. Out of these, candidate gene association studies (using a case control design) are the ones which are conducted most frequently. Multiple candidate gene association studies have been conducted in the Indian population but have failed to show a conclusive role of genetic polymorphisms in ischemic stroke.
Lately the focus has been on ACE, whose polymorphism might have a role in acute ischemic stroke (AIS). ACE plays a pivotal role in the pathogenesis of hypertension, atherosclerosis, and cardio and neurovascular disease. According to literature, the presence of either a deletion (D)/insertion (I) polymorphism of a 287-bp fragment in intron 16 of the ACE gene could be a plausible genetic risk factor for ischemic stroke. Certain studies have found that the homozygous expression of the D allele is associated with greater enzyme activity and the DD genotype may cause a greater occurrence of stroke, via its effect on essential hypertension., A study conducted in India showed lack of significant association between ACE insertion/deletion (I/D) polymorphism and ischemic stroke. In this study, the DD genotype led to an increase in small vessel disease-related strokes, but with borderline significance. Another Indian study found that the presence of DD genotype and ADD1 GW genotype significantly increased the risk of IS compared to controls. Thus, multiple studies have yielded conflicting results regarding the role of ACE polymorphisms in ischemic stroke., Since scant literature is available in India regarding ACE polymorphism, the present study sought to find an association between ACE polymorphism and AIS in the North-Indian population.
The study included 260 subjects which comprised of 130 AIS cases and 130 controls. The study was approved by Institutional ethics board before starting recruitment. Prior to enrolment, all subjects or their legally authorized representatives gave a written and informed consent.
One hundred thirty consecutive ischemic stroke patients of age >18 years reporting within 1 week of onset of symptoms and who had clinical signs and symptoms and/or radiological evidence of AIS on CT/MRI brain were recruited as cases. In the study, a patient was considered to have hypertension if the systolic or diastolic blood pressure recordings were above 140 or 90 mmHg, respectively, on at least two different occasions. Subjects with a fasting blood glucose >126 mg% or with a prior diagnosis of diabetes were considered to have diabetes in the study.
Patients with history of traumatic brain injury, any preexisting neurodegenerative disorder, or transient ischemic attacks were excluded from the study. One hundred and thirty age and sex matched healthy controls with no history of prior stroke or any vascular illnesses or thromboembolic complications were recruited as controls in the study.
All the patients were evaluated by means of a proforma, which included detailed history, examination, investigations and evaluation of risk factors for stroke like smoking, hypertension, alcoholism, dyslipidemia, and carotid atherosclerosis. 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 of blood was used for routine hematological and biochemical analysis and the remaining 4 mL of sample was stored in refrigerator at 4°C for extraction of DNA within 24 h of collection. DNA extracted from the blood was subjected to PCR amplification in the peltier thermo cycler by using following primers:
Forward Primer: 5' CCC CTC CAT CCC ACC CAG TCA AC 3'
Reverse Primer: 3' GTC GTG CAG CTT CGC TGG CAA AGG A 5'
The PCR product was checked on 2% agarose gel. Restricted digestion was done and the genotypes were visualized by running the PCR product on 3% agarose gel at 100 V and the result documented in gel Documentation system. This process yielded three genotypes: II, ID, and DD genotypes, which were identified by the presence of corresponding bands: II-490, ID-490, 190, and DD-190 bp.
Descriptive data was presented as mean ± SD and percentages where applicable. Microsoft Excel spreadsheets were used to analyze the data wherever suitable. Measures of strength of association were calculated by Chi-square test taking P value of <0.05 to be statistically significant. Statistical analysis was done using Microsoft Office Excel and SPSS software version 20.
Demographic characteristics of the subjects included in the study
The cases had a mean age of 55.02 years, while the age was 53.44 years among the controls. Twenty-one cases were young strokes, having age less than 40 years. Of the 130 stroke cases, 67 (51.5%) were females and 63 (48.5%) were males, whereas among the 130 controls, 61 (46.9%) were females and 69 (53.1%) were males. Among the cases, 17.6% were smokers, while 6.1% consumed alcohol regularly. Around 17% of cases had hypertension, whereas 17.6% of the cases had evidence of diabetes. Coexisting coronary artery disease was seen in 8.4% and a history of prior stroke was found in 3.8% cases. Out of the 130 cases, nine presented with sensory abnormalities and 88 presented with motor abnormalities; out of which, 26 had right-sided hemiparesis, 44 had left-sided hemiparesis, seven had right monoparesis, five had left monoparesis, and six had cerebellar signs. We found that 4.6% cases had the presence of carotid atherosclerosis and lipid profile was deranged in 33% of the cases [Table 1].
Frequency distribution of ACE genotypes among cases and controls
Among the 260 subjects of the study population, the ID genotype was present in 185 subjects out of 260 accounting for 71.2% of genotypes. The genotype frequency distribution among Cases was: DD-13.8, ID-73.1, and II-13.1%. The frequency distribution among Controls was: DD-0.8, ID-69.2, and II-21.5%. Harboring the DD genotype showed an increased risk of stroke occurrence which was highly significant with a P value of 0.000. All allelic frequencies among cases and controls followed the Hardy Weinberg equilibrium [Table 2].
Dominant and recessive genetic models and OR's calculated for various genotypes
To measure the degree of association of the DD allele with AIS, odds ratio was calculated between cases having DD alleles with those having II and ID alleles (recessive model of inheritance) and was found out to be significant (OR = 20.732; 95% CI = 2.7241–157.7864; P value = 0.003). To study the association of even a single D allele with AIS, odds ratio were calculated comparing cases with ID genotype versus II genotype (Homozygous common v/s heterozygous model) and was found out to be significant (OR = 2.421; 95% CI = 1.2789–4.5853; P value-0.007). The DD genotype significantly increased the odds of developing ischemic stroke by the dominant model as well (OR = 2.848; 95% CI = 1.5127–5.3649; P value = 0.001) [Table 3].
We performed a multivariate analysis with stroke as a dependent variable and traditional risk factors along with DD genotype as independent variables. The analysis revealed that DD genotype, hypertension, diabetes, and smoking were independent risk factors for developing AIS [Table 4].
Ischemic stroke is known to have a heterogeneous risk factor profile comprising of modifiable as well as genetics risk factors. In our study, it was found that harboring the DD genotype greatly increased the odds of having a stroke, which was independent of the traditional stroke risk factors. Our results are thus similar to other studies which too have shown a positive association between DD genotype and ischemic stroke.,,, However, in a case control study involving 224 cases and 224 controls, there was a lack of significant association between ACE I/D polymorphism and ischemic stroke. Even in this study, the frequency of the DD genotype was higher among small vessel disease stroke subtypes; however, it failed to reach statistical significance. A meta-analysis comprising of three Indian studies showed that DD genotype increased the odds of having an ischemic stroke. However, in this meta-analysis, the overall heterogeneity was 85%, which shows a lack of combinability of these three studies. Zhang et al. synthesized a meta-analysis consisting of 50 studies (comprising 10,070 cases and 22,103 controls), which indicated that the D allele of ACE I/D conferred a higher risk of ischemic stroke albeit with a low penetrability. In the previous studies conducted in India, the frequency of DD genotypes reported have ranged from 15 to 37·8% among cases, whereas the frequency varied from 8 to 31.8% among the controls.,,, We recorded a lower DD genotype frequency compared to prior studies with the frequencies being 13.8% among cases and 0.8% among controls. One of the reasons for this could be the variable genetic pool which is available in our country. Some of the prior studies have been conducted from the southern part of India, which has a different genetic pool compared to our study which included primarily the north Indian population. Another reason for the smaller frequency could be due to small sample size of our study.
The frequency distribution of the ACE genotypes in cases who were males was: DD: 25.4%(16/63), ID-54% (34/63), and II-20.6% (13/63), and in cases who were females was: DD: 3%(2/67), ID-91% (61/67), and II-6% (4/67). Thus, ACE polymorphism was found to be significantly associated with male AIS cases than with their female counterparts. An increased risk of stroke with the II genotypes has been reported in south Indian males by Vijayan et al.; however, other studies have not reported such findings. Hence, larger population-based studies would be required before any drawing any conclusions.
Stroke in the young is not a rare event, especially in countries of oriental origin, such as China, Korea, and India.,, Indian studies have shown that 10–15% of strokes in the Indian population occur below the age of 40 years. 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. Most studies on ACE polymorphism have targeted the elderly population and paid less attention to young onset strokes. In our study, 21 out of 130 cases (comprising 16.2%) were below the age of 40 and there was no increased risk portended by the DD genotype among these cases. Further studies with adequate number of young strokes are needed to draw more accurate conclusions regarding the association of ACE polymorphism with young stroke.
Our study showed that harboring the D allele as such itself carried a greater risk of stroke occurrence. Having even a single D allele increased the odds of developing a stroke by 2.4 times in our study. The risk of stroke attributed by a single D allele has been supported by prior studies as well as by large meta-analysis conducted around the world. However, inconclusive results among other studies might be due to the low penetrance of this allele explaining the negative association in certain other studies.
Both the dominant and recessive genetic models showed that the DD genotype confers a higher stroke risk, which is in line with prior studies and meta-analysis., [Table 5] summarizes the results of some prior studies investigating the role of ACE polymorphism as a stroke risk factor.
Since ACE plays a key role in the development of essential hypertension, which itself is a major stroke risk factor; we carried out multivariate analysis in order to establish the DD genotype as an independent stroke risk factor. We found that the DD genotype was significantly associated with the risk of developing AIS independent of other traditional risk factors like hypertension, diabetes, and smoking. Thus, the DD genotype leads to an increased risk of developing acute ischemic which was independent of hypertension and other confounding stroke risk factors.
Our study had limitations which include selection, referral filter, and ascertainment biases. Another limitation was that the study had 260 patients including 130 stroke cases, which is small as far as genetic studies are concerned. We did not study ACE enzymatic activity in stroke cases and its corresponding correlation with genotypes. This may be one future research area which can give better information regarding genotypic, enzymatic activity correlation, and stroke risk. Young strokes accounted roughly 16% of cases and further studies with a younger target population could help in finding association between ACE polymorphism and young strokes.
In conclusion, our study showed that the DD polymorphism in the ACE gene is strongly associated with the risk of AIS. This might be an important consideration in planning strategies for primary stroke prevention.
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Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]