Genetics of ischemic stroke: An Indian scenario
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.173645
Source of Support: None, Conflict of Interest: None
Stroke, a heterogeneous multifactorial disorder, is known to be a major cause of death and adult disability within both the developed and developing countries. Approximately 85% of stroke cases are ischemic, whereas the remaining 15% are hemorrhagic. It is caused by multiple genetic factors, environmental factors, and interactions among these factors. Several candidate genes have been found to be associated with ischemic stroke. The most extensively studied genes include those involved in hemostasis, inflammation, nitric oxide production, homocysteine and lipid metabolism, and rennin–angiotensin–aldosterone system. Combined linkage/association studies have demonstrated that genes encoding phosphodiesterase 4D (PDE4D) and arachidonate 5-lipoxygenase-activating protein (ALOX5AP) confer risk for stroke. Even though there is substantial evidence for the genetic basis of stroke as provided by the epidemiological data from twin- and family-based studies, the contribution of genetic factors identified till now is either not enough or very less to explain the entire spectrum of encountered phenomena associated with ischemic stroke. Till date, no genome-wide association studies (GWAS) have been carried out in India. We aim to extensively review the studies on candidate genes that may have potential applications in the early diagnosis, prevention, and treatment of ischemic stroke in the Indian population. This article further emphasizes the role of GWAS in ischemic stroke and the need for an extensive GWAS in the Indian population.
Keywords: Candidate gene; genetics; genome-wide association studies; multifactorial stroke; single-gene disorders; stroke
Stroke is among the leading causes of death in the world and a common cause of disability. Stroke is a multifactorial, polygenic and complex disease resulting from the combination of vascular, environmental, and genetic factors. The incidence of stroke in the developing countries is continuously increasing. A 42% decrease in the incidence of stroke in high-income countries and a >100% increase in its incidence in low- to middle-income countries have been estimated in the past four decades., Lifestyle changes, migration from rural to urban areas, and an increased exposure to the risk factors responsible for stroke may aggravate the stroke burden in the future.
A considerable risk of developing ischemic stroke (IS) is due to non-modifiable risk factors (age, race, and male sex) and acquired risk factors (hypertension, cigarette smoking, diabetes, atrial fibrillation, and obesity)., Genetics may contribute up to 50% of an individual's risk of developing a stroke in the future (International Stroke Genetics Consortium 2013; www.strokegenetics.org). Nevertheless, it is difficult to predict the risk of developing a stroke using these factors. A large body of evidence has suggested a genetic component in the development of stroke. Animal model studies as well as twin- and family-based association studies have proposed that a substantial genetic predisposition to the development of stroke exists. There is a several fold increase in the prevalence of stroke among the monozygotic twin pairs compared with the dizygotic ones, suggesting the substantial contribution of genetics to the risk of developing stroke.
Several types of studies may be conducted for the identification of genetic risk factors for stroke. They include the following: (i) Candidate gene association study: This includes a hypothesis-driven approach in which genes that may be involved in the pathogenesis of stroke are tested for association; these studies are focused on the selection of genes that have been previously related to the disease in some way or the other. This is, therefore, conducted with prior knowledge about the gene function. This approach uses a case–control study design and is the most common approach for identifying genes for polygenic genetic disorders; (ii) Linkage study: This study maps a possible disease locus by studying how genetic markers have segregated in large multigenerational pedigrees with many affected family members, in relation to the affection status of the pedigree members; (iii) Genome-wide association study (GWAS): It is the study of genetic variation across the (entire) human genome; and, (iv) Exome sequencing study: This includes efficient strategies to selectively sequence the coding regions of the genome.
Candidate gene association studies in stroke
The major approach used to find stroke genes has been the candidate gene approach using case–control methodologies. In this approach, the genes that may be associated with the pathogenesis of stroke are tested for association to determine the frequency distribution of the allele and allelic variants compared with the controls. Candidate gene contribution to stroke is mild to moderate, and to detect this small difference, a large sample size is required. Important candidate gene association studies in realtion to stroke are summarized below:
Several case–control and prospective studies have demonstrated that a moderate elevation of the plasma homocysteine (Hcy) level is a potential risk factor for the development of cardiovascular diseases and venous and arterial thrombosis, that may precipitate a stroke. Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in the metabolism of Hcy. A recent meta-analysis of 38 case–control studies comprising 6,310 patients with ischemic stroke (IS) and 8,297 control subjects conducted by Kumar et al., in 2015 showed a significant association between MTHFR C677T genetic polymorphism and the risk of IS in both the dominant (odds ratio [OR] =1.09, 95% confidence interval [CI] =1.06–1.12, P < 0.001) and recessive (OR = 1.31, 95% CI = 1.19 to 1.44, P < 0.001) models of inheritance.
Apolipoprotein E (Apo E) has a major role in the lipid transport and metabolism and is also significantly expressed in the brain. It is one of the commonly studied genes in vascular and neurodegenerative diseases. Its protein component comprises of glycoproteins, with three common isoforms: E2, E3, and E4, encoded by the respective alleles ɛ2, ɛ3, and ɛ4, giving rise to six genotypes. One study reported from North India showed a significant association between Apo E polymorphism and IS. The Apo E polymorphism can modify the risk of other modifiable risk factors; for example, the effect of cigarette smoking on IS may be higher in young adults who carry the variant Apo €4 allele.
Endothelial nitric oxide synthase
The endothelial nitric oxide synthase (eNOS) gene is located on chromosome 7 (7q35–q36) and consists of 26 exons. It codes for an enzyme that generates nitric oxide (NO) in the vascular endothelium. eNOS polymorphism may lead to a reduced activity of vascular endothelial NO synthase, due to which partly impaired endothelium-dependent vasodilatation occurs (which is a common feature of atherosclerotic vessels), thereby leading to the development of stroke. Thus, genetic variations may contribute to the risk of developing a stroke. A recent meta-analysis of 31 studies including 8,547 patients and 9,117 control subjects performed by Guo et al., (2014) suggested the positive association between eNOS gene 4b/a, T-786C and G894T polymorphisms and IS. For eNOS G894T polymorphism, TT genotype was significantly associated with an increased risk of IS when compared with the G allele (OR = 1.25 and 95% CI = 1.09–1.42 for TT vs. GT + GG, P < 0.001).
Factor V Leiden
Factor V Leiden (FVL) mutation is the most prevalent prothrombotic genetic mutation and causes activated protein C resistance. It is associated with venous thromboembolism in IS patients. A meta-analysis published in 2010 that included 18 case–control studies conducted by Hamedani et al., involving 2,045 cases and 5,307 controls showed a significant association of FVL G894T gene polymorphism with the susceptibility to develop a stroke (OR = 2.00, 95% CI = 1.59–2.51).
β-Fibrinogen gene polymorphism (C148→T)
Fibrinogen concentration is controlled by genetic and environmental factors; for example, smoking, obesity, use of contraceptives, trauma, and lack of exercise and have been reported to elevate fibrinogen levels. It also increases with age and in the presence of diabetes mellitus, hypertension, or lipid abnormalities. Several polymorphisms have been identified within the region encoding for fibrinogen, of which C148→T polymorphism is located close to an interleukin-6–responsive element and may affect fibrinogen gene expression, mainly in response to the acute-phase reaction. A meta-analysis of 18 independent case–control studies that included 2,159 IS patients and 3,222 control subjects done by Zhang et al., in 2014 showed that − 148C>T polymorphism in the FBG gene was associated with an increased risk of IS ([TT + CT] vs. CC: OR = 1.40, 95% CI = 1.20–1.45, P < 0.0001; T vs. C: OR = 1.35, 95% CI = 1.18–1.56, P < 0.0001).
Phosphodiesterase 4D (PDE4D) gene located on 5q12 has 24 exons. The gene expresses nine different functional protein isoforms through alternative splicing or the use of differential promoters. PDE4D is the family of enzymes which breaks the phosphodiester bond of cyclic adenosine monophosphate (cAMP), degrades them and maintains the appropriate level and duration of action of cAMP within the cell. The cAMP is a secondary signaling molecule that involves provoking genes to produce inflammatory mediators by including several types of inflammatory cells and atherosclerosis. In 2002, the deCODE group published the results of a genome-wide screening for stroke susceptibility genes in Iceland. A recent meta-analysis of 25 studies comprising 8,878 cases and 12,306 controls conducted by Yan et al., in2014 showed a significant association between SNP 83 of PDE4D gene and the risk of developing an IS, especially in the Asian and Chinese populations, but not in the Caucasian subjects (OR = 0.87, 95% CI = 0.69–1.11).
Arachidonate 5-lipoxygenase–activating protein
Arachidonate 5-lipoxygenase–activating protein (ALOX5AP) acts as a critical mediator in the biosynthesis of leukotrienes, involved in the progress and pathogenesis of atherosclerosis. Its gene is located on chromosome 13p12–13. A recent study published by Wang et al., (2014) showed a significant association between TT/TA genotype of ALOX5AP SG13S114 A/T and an increased risk for the development of an acute cerebral infarct (ACI) in the northern Han Chinese population (OR = 1.82, 95% CI = 1.14–2.92, P = 0.012). A recent meta-analysis of 11 case–control studies comprising 5,361 cases and 5,676 controls conducted by Ye et al., (2014) showed that the AA genotype of ALOX5AP SG13S114 A/T is significantly associated with an increased risk of developing an IS compared with the TT genotype in the Chinese population (OR = 1.47, 95% CI = 1.13–1.91, P = 0.005).
The linkage studies aim to map a possible disease locus by studying how genetic markers have segregated in large multigene rational pedigree with many affected family members. For several reasons, the linkage approach is difficult to apply to locate genes that are contributing to the occurrence of stroke. Stroke is a late-onset disease due to which collection of information from other family members becomes difficult. The polygenic nature of stroke and shared environmental exposures contribute to the difficulties with the linkage approach as these studies cannot detect genes with minimal or modest effect on stroke risk., Genome-wide linkage studies on Icelandic individuals with stroke have indicated the presence of a gene on chromosome 5, which may have a significant role in the development of the stroke phenotype. The same group have reported that the likely candidate gene was phosphodiesterase 4D (PDE4D) gene, especially in large-vessel disease stroke and cardioembolic stroke, but not in small-vessel disease stroke.
Genome-wide association studies in stroke
There have been enormous advances in the sequencing and genotyping technologies in the past decade. The field of complex genetics has been revolutionized by the Genome-Wide Association Studies ( GWAS) approach, which uses microarray technology to genotype up to 1 million or more single-nucleotide polymorphisms (SNPs) spanning the whole genome in an individual subject. This technology provides the opportunity for typing thousands of SNPs together in GWAS to find genetic variations associated with a specific disease. Such studies are particularly useful in finding genetic variations that contribute to common complex diseases. The first GWAS on IS, published by Matarin et al., in 2008, included 249 white patients with IS and 268 white control subjects, in which they identified more than 400,000 unique SNPs and observed that no single locus conferred a large effect on IS risk; however, there was a moderate, but statistically significant, association between CDKN2A (Chr 9p21) and IS. Another GWAS reported by Kubo et al., in the same year included 188 Japanese individuals with IS and 188 matched control subjects in the first phase, using 52,608 gene-based tagging SNPs. In the second phase, they genotyped 924 IS patients and 924 matched control subjects for the 1,098 SNPs identified as associated in the first phase with P values less than 0.01. In this study, they observed an association between the SNP 15 in PRKCH and lacunar infarction. Sequencing of all the exons in this gene in 48 cases and 48 controls showed SNP 1425G/A to be associated with lacunar infarction by direct sequencing. This SNP causes an amino acid substitution at position 374 from valine to isoleucine. This position is within the ATP-binding site of the protein, a member of the protein kinase C (PKC) family, thus enhancing the PKC activity. The SNP 1425G/A, however, is in almost complete linkage disequilibrium with the SNP 15, which has very minor allele frequencies in European and African subjects, suggesting that the obtained results are likely to be specific to Asian populations. The meta-analysis published by Anderson et al., in 2010 showed that variants on chromosome 9p21.3 are significantly associated with IS, and analysis limited to large-artery stroke increases the effect size towards that observed in prior association studies of coronary artery disease and myocardial infarction. Recently, the Wellcome Trust Case Control Consortium on IS GWAS identified a novel association at 7p21; the most likely underlying gene is HDAC9, encoding histone deacetylase. Its association was observed with large-vessel artery stroke. HDAC9 is a member of a large family of genes that encode for proteins responsible for deacetylation of histone, and, therefore, regulate chromatin structure and gene transcription. Its exact role in the mechanism of stroke is not clear; however, it is believed that abnormal functioning of this gene leads to an increase in the atherogenic process. Very recently, a European Union–funded multicenter study (EuroCLOT) conducted an analysis of genetic variants in a three-stage study design to identify the common variants influencing coagulation and fibrin structure/function, and observed that ABO gene variants (rs505922) are associated with large-vessel and cardioembolic stroke, but not with small-vessel stroke. The important GWAS conducted in stroke are listed in [Table 1].
Several candidate gene association studies have been reported across the globe. Three studies investigated the role of beta-adrenergic receptor polymorphism in stroke. One study published in 2007 showed that the Glu27 allelic variant of the beta-2 adrenergic receptor gene may be a determinant of IS, with a significantly increased risk of stroke when assuming a recessive mode of inheritance (OR = 1.68; 95% CI = 1.17–2.41, P = 0.005). Another study published in 2003 observed no association of the Glu27 homozygote and carrier of Glu27 allele with stroke. A study published in 2009 observed no association regarding IS in white women. Several studies have been reported on angiotensin converting enzyme insertion/deletion (ACE I/D) polymorphism and stroke. A recent meta-analysis evaluating 10,070 subjects versus 22,103 controls from 50 studies concluded that the ACE genotypes conferred a significant effect, with an OR of 1.37 (95% CI = 1.22–1.53) assuming the recessive mode of inheritance. Subgroup analysis in Asian population also showed a significant association of DD genotype with stroke in the recessive mode of inheritance (OR = 1.87, 95% CI = 1.5–2.3). Several candidate gene studies supported the view that ACE insertion/deletion polymorphism is an important candidate gene for the IS because rennin–angiotensin system plays a central role in atherosclerosis and hypertension.
Till date, not a single GWAS on IS has been published in the Indian setting. A number of candidate gene studies published in India have shown inconclusive findings for the association of genetic polymorphisms with IS [Table 2]. A study published by Banerjee et al., (2008) showed that the CC genotype of PDE4D (SNP 83T/C) was associated with the risk of IS. Munshi et al., (2010) further showed an association of PDE4D (SNP83 T/C) with two stroke subtypes, namely the intracranial large-artery atherosclerosis and the small-artery occlusion. In another study, Munshi et al., (2012) also confirmed the association of PDE4D (SNP 41 and SNP 56) with the risk of developing an IS. Studies published by Alluri et al., (2005), Panigrahi et al., (2006), and Narayan et al., (2013) on the South Indian population showed a strong association of MTHFR C677T polymorphism with IS, whereas Somarajan et al., (2011) did not show any significant association of MTHFR C677T gene polymorphism with either of the two stroke subtypes. A study published by Biswas et al., (2009) showed an association of FVL G1691A polymorphism with stroke. Studies published by Munshi et al., (2010) and Sultana et al., (2011) showed a significant association of IL-10-1082 G/A with stroke. Munshi et al., (2010) and Majumdar et al., (2010) did not observe a significant association of the eNOS 4a/b polymorphism with any stroke subtype. Another study by Sultana et al., (2011) on the IFNγ +874A/T polymorphism showed a significant association of the TT genotype with IS in the South Indian population. Kumar et al., (2014, 2015) found that the ADRB2 Gln27Glu and ADRB1 Ser49Gly polymorphisms were significantly associated with the risk of IS in the North Indian population., An exome genotyping study on stroke has been initiated at the All India Institute of Medical Sciences under the leadership of Dr. Kameshwar Prasad to identify the exonic variants which may be linked with the risk of stroke in the Indian setting. Genetic studies reported from the Indian population have been depicted in [Figure 1].
Stroke genetics has emerged as a pivotal field that may alter/modify the management strategies for prevention of stroke through the development of novel approaches. PDE4D and ALOX5AP are the newly identified genes that encode for enzymes involved in specific pathways which may be targeted in stroke. Specifically regulating the cAMP levels by altering the cyclase activity or the cAMP effector proteins could provide an additional target for rationally designed stroke therapy. It has been established that the metabolic rates of several cytochrome P (CYP) 450 enzymes vary because of the genetically determined polymorphisms. Identifying a gene polymorphism profile that can predict the atherosclerotic plaque formation and its activation susceptibility can provide a new vista for exploring the gene-directed therapy.
Studies have shown a significant influence of gene profiling in response to medications such as statins. Gene polymorphism in the toll-like receptor-4 gene has shown an association with an increased beneficial effect in risk reduction of cardiovascular events in patients treated with pravastatin. This data along with other studies support the concept that a comprehensive genetic profiling is needed to use the currently available as well as future medications more efficiently in patients with atherosclerotic disease. It further illustrates that genetic profiling combined with serotyping of specific organisms depicts the interrelation between genetic and immune activity responsible for the increased risk of atherothrombotic stroke. Stroke pharmacogenomics can most commonly be used in the outpatient clinic to facilitate primary and secondary stroke prevention. The interaction between antihypertensive diuretic therapy and the adducing gene variant was found to be associated with a lower risk of stroke.
In future, stroke genetic studies would also be able to identify variants that predict the lack of efficacy for specific treatments. This can lead to an overall improvement in cost-effectiveness and can limit the potential side effects of drugs received by patients suffering from stroke.
Genetics has the potential to transform neurological research. It may lead to the identification of stroke-causing genes and other new genes that in future could be directly used for developing a genetic testing kit for an early, accurate, and presymptomatic diagnosis of stroke. This method of genetic testing could be used to identify several individuals with a high risk of developing stroke. Early preventive measures could, therefore, be instituted in these subjects that may lead to an early prevention of stroke; or, these measures may delay the onset of the disease by an aggressive treatment of the known risk factors. Eventually, genes responsible for the development of stroke may be used as potential targets for newer pharmaceutical advancements, which may lead to both prevention and treatment of stroke as well as to a paradigm shift from modern medicine to personalized medicine. This would pave way for the concept of 'appropriate medicine/therapy for the appropriate patient.'
We would like to thank the Department of Biotechnology, Government of India for providing resources through the Programme support in stroke-phase II projects for successfully completing this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2]