Genetics of ischemic stroke: Indian perspective
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.103195
Source of Support: None, Conflict of Interest: None
A stroke is still a major cause of long-term disability and the third largest killer in the world after heart attack and cancer. Inherited genetic variation has been shown to play a role in its pathogenesis and therefore, there is a need to identify the culprit genetic variants. They may provide novel targets for preventive therapeutics. The most intensively investigated candidate gene is PDE4D. There are several positive replication studies of PDE4D gene with stroke. The genetic contribution to ischemic stroke risk in India has not been explored adequately. Reports on few candidate genes are available but we are still lagging behind in this aspect. Most of the reports are from Andhra Pradesh, a province in south India and a few parts of north India. PDE4D has been identified as a predisposition gene for ischemic stroke in Southern as well as the Northern population of India.
Keywords: India, ischemic stroke, multifactorial stroke, single gene disorders
A stroke, also known as 'Brain Attack' is defined as a sudden loss of neurological function resulting from focal disturbance of cerebral blood flow, due to ischemia or hemorrhage. It is a major cause of mortality and morbidity around the world, especially in the elderly population. Approximately 85% of all strokes are ischemic, mainly caused by acute thromboembolic occlusion of intracranial arteries. Over 300 risk factors have been associated with ischemic stroke and associated disorders. Established risk factors for stroke include old age, sex, cardiovascular disease, hypertension, diabetes, hypercholesterolemia, alcohol consumption, and smoking and other definite risk factors.  Stroke seems to run in some families. A three fold increased incidence has been reported in the risk of stroke in men whose mothers had died of stroke in comparison with men without a maternal history of stroke. Stroke may be the outcome of single gene disorders or more commonly, a polygenic multifactorial disease. Many studies have been carried out to investigate potential risk genes for multifactorial stroke. Mutations in some candidate genes are likely to predispose or give protection against stroke. There are obvious racial differences in the prevalence of these mutations and therefore, genetic variants associated with stroke vary among different ethnic groups. However, most robust findings have been on single gene disorders in stroke. In India, stroke genetics is still in infancy. Most of the studies have been carried out by our group, focussing on genes involved in coagulation and hemostasis, endothelial function and nitric oxide release, the rennin angiotensin aldosterone system and inflammation. There are few association studies from north India as well. The purpose of this article is to review the genetic determinants evaluated in association with stroke in India till date.
Mendelian conditions are important causes of stroke in young patients without known risk factors. These disorders include cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL, a rare form of small vessel disease), Fabry's disease, sickle cell disease, the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and moyamoya disease, Ehler Mayer's syndrome and more. According to their genetic-inheritance model, these diseases can be inherited in a classical Mendelian pattern as X-linked (e.g. Fabry's disease), autosomal recessive (e.g. Maeda syndrome, sickle cell disease and homocystinuria), or autosomal dominant (e.g. CADASIL, polycystic kidney disease, Ehler-Danlos syndrome Type IV and Marfan syndrome) disorders. In addition, they can be also inherited as maternal disorders when they are associated with the mitochondrial DNA, such as the MELAS syndrome.
CADASIL may start with attacks of migraine with aura or sub cortical ischemic attacks or stroke or mood disorders between 35-55 years of age. CADASIL is the only form of the isolated stroke displaying heritable patterns of inheritance which is caused by mutations in the Notch3 gene.  The underlying genetic mutations were identified using a linkage strategy. Although two cases of CADASIL have been reported from India, one each from north west India and Mumbai, genetic analysis has been carried out only in the latter case. , Genetic studies were done by sequencing exons 3 and 4 of the Notch3 gene. A substitution of a nucleotide (C>T) at position 499 was reported in exon 4. 
Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) directly affects the cerebral small blood vessels, that is caused by mutations in the gene encoding HtrA serine peptidase/protease 1 (HTRA1).  To the best of our knowledge there are no reports of CARASIL from India. Marfan syndrome is an inherited connective tissue disorder transmitted as an autosomal dominant trait characterized by abnormalities of the musculoskeletal system, cardiovascular system and the eye.  Marfan syndrome is caused by defects in a gene called fibrillin-1 located onchromosome 15, which encodes for the glycoprotein fibrillin. Precise statistics for the incidence of Marfan syndrome in India are not readily available. No genetic studies have been carried out so far in India. In case of Fabry's disease, most patients carry missense or nonsense mutations in the coding region of α-galactosidase A (GLA) gene. Stroke in patients with Fabry disease occurs typically by the third or fourth decade of life and manifests as both large artery disease and small vessel disease, with a preference for the posterior circulation. So far, one case of Fabrys disease has been reported from India.  The mutation analysis revealed a mutation in the GLA gene. The nonsense mutation, W236X confirmed Fabry disease in this case. Sickle-cell disease is an autosomal recessive disorder caused by a mutation in hemoglobin resulting in hemoglobin S (HbS). Although sickle cell disease is quite prevalent in India, no data on the incidence of stroke in sickle cell disease is available. The syndrome of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disorder characterized by stroke like episodes often involving the cortex, typically in the occipitoparietal region, migraine headaches, nausea and vomiting. About 80% of MELAS patients exhibit a heteroplasmic mutation in the dihydrouridine loop of the tRNA Leu (UUR) gene at nucleotide position 3243.  No genetic studies have been carried out on MELAS in India till date.
The genetic contribution to common multifactorial stroke is polygenic. However, identifying the underlying genetic variants has been a major challenge. Until recently, the candidate gene association study was the main methodology used. Most human genetic studies have focused on polymorphic variants that promote stroke predisposing phenotypes or mediators, including targets in inflammatory pathways (interleukin-10 (IL-10),  Tumor necrosis factor-α (TNF-α)1),  cyclo oxigenase-1and 2 (COX-1 and COX-2) genes, modulators of vascular tone and endothelial functions [(endothelial nitric oxide synthase (eNOS)], lipid and homocysteine metabolisms [(lipoprotein lipase (LPL),  estrogen receptor 1 (ESR-1),  aldosterone synthase (CYP11B2),  cytochrome P450 4F2 (CYP4F2),  CYP2C9 genes)], methylene tetrahdrofolate reductase (MTHFR), components of haemostatic, fibrynolytic and coagulation system: Prothrombin (PT) gene,  factorV Leiden (FVL),  tissue plasminogen activator (tPA) or PLAT, , plasminogen activator inhibitor-1 (PAI-1) genes,  and extracellular matrix degradation matrix metalloproteinases (MMP's) [Table 1].  Some studies have shown a significant association of these genes with ischemic stroke while, others have failed to do so.
The coagulation system, together with endothelial cells and platelets, is responsible for maintaining blood in a fluid state, but, when activated, rapidly results in the development of a fibrin clot by conversion of the plasma protein, fibrinogen, to the polymer fibrin by the key enzyme, thrombin. In addition to production of fibrin, thrombin has wide-reaching functions that range from platelet activation to stimulation of endothelial cells, vascular smooth muscle cells (VSMC), monocytes,
Tlymphocytes, and fibroblasts, so the production and inhibition of thrombin is tightly regulated.
As far as the genetic variants affecting coagulation and fibrinolytic cascade in association with stroke are concerned in India, we have evaluated G20210A polymorphism of PT gene, 1691G>A polymorphism of FVL gene, I/D and-7351C/T polymorphism of tPA as well as 4G/5G polymorphism of PAI-1 gene. The results of our study revealed that all these genetic variants except for tPA I/D polymorphism, no other variants could be associated significantly with stroke in our study population from Andhra Pradesh. , We also found a significant association of I/D polymorphism of tPA gene with intracranial large artery atherosclerosis and stroke of undetermined etiology. Various studies have indicated that PT 20210A is uncommon in the indigenous population from north and western India. , A case-control study carried out by Rees et al. detected one heterozygous individual in the control group; on studying 150 DNA samples from India.  Two recent studies carried out in the states of Karnataka and Tamil Nadu, south India, have also shown the absence of factor II G20210A polymorphism. ,
Previous studies have evaluated the plasma lipid profile in different ischemic stroke subtypes, and notably in lacunar infarctions and cardioembolic strokes.  Genes encoding proteins involved in plasma lipoprotein metabolism are interesting candidates, when looking for genetic risk factors of ischemic stroke. The genes mainly involved in stroke are lipoprotein lipase (LPL), apolipoprotein E (ApoE), ATP-binding cassette, subfamily A, member 1 (ABCA1), lipoprotein, Lp(a) or apolipoprotein (a) (LPA), apolipoprotein A5 (ApoA5), epoxide hydrolase 2 (EPHX2), paraoxonases 2 and 3 (PON1,2 and 3), estrogen receptor ESR-1 etc.
We evaluated the Hind III polymorphism of LPL gene in association with ischemic stroke risk and found that this polymorphism is significantly associated with overall stroke, especially intracranial large artery atherosclerosis in south Indian populations.  Another case-control study was carried out in our laboratory on PvuII and XbaI polymorphism of ESR1 gene, with stroke risk in our population. There was a significant association of this polymorphism with ischemic stroke, especially in postmenopausal women. Further, we evaluated the association of this polymorphism with stroke subtypes and found significant association of PvuII polymorphism with extracranial atherosclerosis, lacunar and cardioembolic stroke classified according to the TOAST classification. 
The renin-angiotensin-aldosterone system (RAAS) is important for cerebrovascular research because it influences blood pressure, vasoconstriction, thrombosis, and vessel wall damage. Increased RAAS activity has been linked with an increased risk of stroke and other cardiovascular diseases. Gene polymorphisms of renin-angiontensin aldosterone system (RAAS) have been suggested to be risk factors for hypertension, cardiovascular diseases and stroke.  A study from our laboratory revealed that angiotensin-converting enzyme (ACE)gene I/D polymorphism contributes to the risk of developing stroke in the south Indian population, especially with intracranial large artery atherosclerosis, which is the most common subtype in our population.  To the best of our knowledge, there are no other reports on the association of this gene polymorphismwith stroke in India.
CYP11B2 gene is a natural candidate for sodium homeostasis and blood pressure. The association of CYP11B2 − 344C/T gene polymorphism with hypertension and stroke was carried out by our group. The results of the study suggest that −344T allele of CYP11B2 gene is an important risk factor for hypertension and ischemic stroke in the south Indian population from Andhra Pradesh. Further evaluation of the association of this polymorphism with stroke subtypes revealed a significant association with intracranial large artery atherosclerosis, lacunar stroke and cardioembolic stroke. 
A case-control study was also carried out by our research team to evaluate the association of 1347 G/A polymorphism in the 11 th exon region of CYP4F2 gene with hypertension and ischemic stroke. Our findings suggested that AA genotype and A allele significantly associate with hypertension and stroke, especially the cardioembolic stroke subtype.  No other studies from India are available.
It has been found that genetic factors may play a role in ischemic stroke through endothelial dysfunction.  Nitric oxide (NO) synthesized from Larginine by endothelial nitric oxide synthase (eNOS) is a key mediator of endothelial function, which plays a central role in the maintenance of vascular homeostasis, including regulation of cerebral circulation.
We investigated whether the variable number of tandem repeats (VNTR) polymorphism in intron 4 of the endothelial nitric oxide synthase (eNOS) gene is associated with ischemic stroke in a South Indian population from Andhra Pradesh. We found that this polymorphism is significantly associated with ischemic stroke in the south Indian population from Andhra Pradesh. However, we did not find a significant association of this polymorphism with any specific stroke subtype. 
Matrix metalloproteinases (MMPs) are a family of calcium-requiring and zinc dependent proteases involved in the degradation of extracellular matrix proteins such as collagen, elastin, gelatins, casein, and others.  Accumulating evidence suggests that several genetic variations of MMPs are predisposed to the development of atherosclerotic pathology, among which MMP-3 was associated with plaque progression (PP) and leads to ischemic stroke.  We evaluated the MMP-3 5A/6A polymorphism in the South Indian population but we did not find a significant association. 
Accumulated evidence suggests important roles for inflammation in the process of atherosclerosis and specifically in stroke. Clinical and experimental evidence implicates inflammation in multiple phases of stroke etiology and pathology. , Examples of functional candidates, which have been tested for their impact in stroke pathogenesis include pro-inflammatory genes (TNF-α, IL-6, COX and CRP) and anti inflammatory genes (IL-10). 
Recently, we analyzed +488 G/A polymorphism in TNF-α gene and found a significant association of this polymorphism with ischemic stroke risk in the south Indian population from Andhra Pradesh, especially intracranial large artery atherosclerosis, extracranial large artery, atherosclerosis, cardio embolism, and stroke of undetermined etiology.  However, a study carried out by Banerjee et al. in the north Indian population on 308 G/A polymorphism of TNF-α could not establish an association with ischemic stroke. 
A recent case-control study carried out by Chakraborty et al. on IL-6 -174 G/C polymorphism, in association with severity and outcome in acute ischemic stroke patients from North India reported that the patients with the GC genotype had more severe strokes with poorer short and long-term outcomes and increased mortality but GG genotype appears to be protective against stroke severity, outcome, and mortality. 
We also carried out a study on-1082G/A polymorphism of IL-10 gene with ischemic stroke in the south Indian population from Andhra Pradesh.The results of our study indicated that the AA genotype and A allele of IL-10 gene is strongly associated with ischemic stroke in the study population.We found significant association of this polymorphism with stroke of undetermined etiology. 
A linkage and association study in Icelandic families led to the identification of the first candidate gene, PDE4D.  It is the first putative gene associated with common polygenic stroke. Replication studies in non-Icelanders have yielded variable results. However, the identification of this gene has set the stage for research that would eventually lead to better understanding and treatment of the stroke.
We found a significant association of SNPs 56, 83 and 41of Phosphodiesterase 4D (PDE4D) gene with ischemic stroke in the south Indian population from Andhra Pradesh. However SNPs 37 and 87 were found to be monomorphic in our population. , Another study by Banerjee et al. from India have also confirmed the association of SNP83 with ischemic stroke. A study from North India also found an association of SNP83 and SNP87 with ischemic stroke and extracranial large artery atherosclerosis respectively.  Therefore, PDE4D gene plays a key part in the pathogenesis of stroke.
Among biological factors, plasma homocysteine levels are considered a major risk factor for vascular diseases, including stroke.  Several studies suggest a positive and dose-dependent association between the serum concentration of total homocysteine and the risk of stroke, which is independent of other vascular risk factors.  Mutations in genes of the homocysteine metabolic pathway may confer an increased risk for ischemic stroke because of elevated plasma homocysteine levels. A study carried out by Alluri et al. in a South Indian population found that C677T MTHFR mutation is strongly associated with arterial stroke, especially in young adults and concluded that MTHFR allele evaluation will help in preventing/reducing morbidity caused by stroke.  All the gene variants evaluated in India have been summed up in [Table 2].
Conventional risk factors for stroke have been recognized and include high blood pressure, dyslipidemia, diabetes and defects in hemostasis. Although these conventional risk factors account for a significant proportion of stroke risk, but much risk still remains unexplained. It is not understood why some individuals with risk factors develop stroke while others with similar risk factors remain stroke free. Some of this unexplained risk might be on account of genetic factors and it is hoped that the discovery of these might allow better prediction of the risk as well as the identification of novel stroke mechanisms, which could be targeted with new therapeutic strategies.
With the advent of new high throughput techniques like Genome Wide Association Studies (GWAS) and also important advances in understanding the monogenic stroke, major advances in stroke genetics are taking place. Since most of the strokes have multifactorial pathogenesis, identifying the genetic basis is usually impossible and clinically irrelevant. On the contrary the rare monogenic disorders which cause stroke, identifying the underlying genetic abnormality is not only realistic, but may also have important implications for clinical management as well as genetic testing of other family members.
As far as the genetics of stroke in India is concerned, it is till in its infancy and research into the genetics of stroke has lagged behind that of many other countries. It is worth noting that no GWAS has been carried out in India so far. In future, it is likely that the focus will be on genotyping studies to look for less common variant alleles of more significant effect and in studying copy number variants as additional genetic factors associated with ischemic stroke in the Indian population. However, we have a long way to go before we can accurately pinpoint the genes responsible for multifactorial stroke. These gene variants need to be investigated thoroughly, not only in relation to susceptibility but also with regard to responsiveness to treatment and gene environment interactions in the development of stroke.
[Table 1], [Table 2]