Cryptogenic Stroke in the Young: Role of Candidate Gene Polymorphisms in Indian Patients with Ischemic Etiology
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.333441
Source of Support: None, Conflict of Interest: None
Keywords: Cryptogenic young ischemic stroke, PCR-RFLP, single nucleotide polymorphisms
Stroke, both ischemic and hemorrhagic, is a major cause of death and disability in the industrialized countries. Although young adults are at a lower risk of stroke compared to older people, its incidence ranges from 60 to 200 new cases per year per million inhabitants., Most of the strokes in the young age are of ischemic etiology, and they are important in Indian context too.
Compared with older adults, young patients (18-45 years) with ischemic stroke (IS) have a lower prevalence of the classical risk factors for atherosclerosis such as dyslipidemia, hypertension, diabetes mellitus, atrial fibrillation, and are also less likely to have an associated coronary or peripheral artery disease.,,, This may suggest an underlying etiology only targeting the cerebrovascular system. Cryptogenic strokes are strokes that remain without a definite cause even after extensive workup and normally 30–40% of ISs in the young are cryptogenic.,,
Data suggests that some of these cryptogenic strokes in the young can have a genetic basis and environmental factors can make them more susceptible. 32% of stroke deaths and 17% of stroke hospitalizations or stroke deaths in twins have been attributed to heritability., Having a positive family history of stroke was associated with a ≈30–76% increase in stroke risk. It is also reported that younger stroke cases are more likely to have a positive family history of stroke than older cases or that age at stroke was inversely related to a family history of stroke.,,,,
There are some well-established genetic causes for strokes. Among the more well-known disorders are Fabry disease, cerebral autosomal dominant [or recessive] arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL [or CARASIL]), mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), sickle-cell disease, homocystinuria, and Marfan syndrome.,,, Even collectively, these disorders are rare and account for only a small percentage of young stroke cases.
The molecular basis for the genetic risk of IS is likely to be multigenic and influenced by environmental factors. It is possible that there may be other high-penetrance mutations segregating in some families also contribute to the burden of early-onset stroke. Candidate genes and pathways for IS generally fall into one of the following categories: (a) genes involved in coagulation and fibrinolytic system or genes encoding platelet glycoproteins,,,, (b) genes in homocysteine metabolism,,,, (c) genes involved with lipid metabolism,, and (d) inflammation genes. As with many candidate gene studies, the associations of these genes with stroke have generally been inconclusive because of insufficient sample size, heterogeneity in the study design, diversity of study populations, inconsistent phenotype classifications, and diverse inclusion/exclusion criteria for each study. Furthermore, relatively few of these studies have focused on younger populations, a group in which rare high-penetrance variants could theoretically account for disproportionately more of the disease burden.
This controversial situation led us to evaluate the association between cryptogenic young IS with some candidate gene polymorphisms along with the established Factor V Leiden, MTHFR C677T, and Prothrombin G20210A mutations. [Table 1] shows the SNPs screened for and the probable outcomes of these SNPs which could contribute to the manifestation of stroke.
A case-control study with young (age 15 to 45 years) IS patients, comprising of both South and North Indians, and age-, sex-, and ethnicity-matched control subjects was carried out. Patients were consecutively recruited during their acute hospitalization or when attending as outpatients for follow up treatment in the Department of Neurological Sciences. Control subjects were healthy blood donors without any risk factors for stroke, randomly selected from the respective populations, matched for age and ethnicity. The study was approved by the Institutional review board.
An extensive workup of all the cases, apart from a detailed history and clinical examination, was carried to rule out an etiology. MRI with MRA was done to establish the diagnosis of the IS and to look for any radiological clues toward an etiology. In patients suspected to have vasculitis (primary or secondary vasculitis of the CNS), additional digital subtraction angiogram (DSA) was done. CSF analysis was carried out in cases when a CNS infection was suspected as the cause of the stroke. In patients suspected to have embolic stroke of undetermined source (ESUS), a complete work up ruled out any source for emboli. Transthoracic ECHO was done in all patients and in suspected cases of paradoxical emboli, a Transcranial Doppler Bubble study was carried out. HOLTER was done in patients suspected to have an underlying cardiac arrhythmia.
Apart from the routine blood tests which included lipid profile; all patients were screened for antinuclear antibody (ANA), C & P antineutrophil cytoplasmic antibodies (ANCA) and C-reactive protein (CRP), and in suspected cases of Sjogren's syndrome screening of anti-SSA and SSB, and salivary gland biopsy was also carried out. All the patients had tested negative for HIV infection. A complete thrombotic workup was done which included assays of: Protein C (functional estimation was done using a chromogenic assay and if low, antigenic assay was done using ELISA), activated protein C resistance (APCR—estimated by clot-based method), Protein S (bound & Free—by ELISA), antithrombin III (using chromogenic assay), Factor VIII level (APTT-based single staged factor assay), Lupus anticoagulant (Dilute Russell Viper Venom Test), and anticardiolipin antibodies. Other tests such as serum homocysteine levels, paroxysmal nocturnal hemoglobinuria (Ham's and sucrose lysis test), and Sickle cell preparation were also carried out. Markers for thrombosis, which could be influenced by the acute thrombotic event (protein assays), were repeated after six months if they were found to be abnormal, to avoid a false positives result. For patients on oral anticoagulants, this was done after stopping oral anticoagulants for a minimum period of 6 weeks.
Genomic DNA was isolated using QiaAmp Blood mini kit (Qiagen) from 2 ml peripheral blood sample collected in EDTA tubes from the participants. Genotyping was carried out by PCR-RFLP method. The primer sequences used for PCR and the restriction enzymes used for RFLP are given in [Table 1].
Statistical analysis was carried out using Vassar Stats. The prevalence of the variants was compared between patients and controls using Chi-square tests with Yates correction. Hardy–Weinberg equilibrium (HWE) for each genotype polymorphism was determined for control subjects with the Chi-square test. Univariate odds ratios (ORs) and 95% confidence intervals (CIs) were estimated whenever necessary. P values of <0.05 were considered to be statistically significant.
A total of 105 young cryptogenic IS patients and 215 control subjects were recruited for this study. The mean age of the patients at the onset of stroke was 32.7 (± 7.7) years and the mean age of the controls was 31.8 (± 8.4) years. 77% of the cases (n = 79) were males. Among the study samples, 26 cases and 44 controls were from the North India. [Table 2] shows the prevalence of the various genotypes of the screened polymorphisms in the study population. All the polymorphisms were found to be well within the limits of Hardy–Weinberg equilibrium.
MTHFR rs1801133 and KNG1 rs710446 showed significant statistical association with cryptogenic young IS (P = 0.0261 and 0.0157, respectively) in Indian population. The T allele of MTHFR rs1801133 was found to be statistically associated with cryptogenic young IS in Indian population (OR 1.8725; 95% CI: 1.1858–2.9570; P = 0.0071). While in KNG1 rs710446, the significance was found to be in having CC genotype (OR 3.0412; 95% CI: 1.3818–6.6934; P = 0.0057). We also found a trend of association (P = 0.0743) in Factor XII (FXII) rs1801020 polymorphism with cryptogenic young IS but the allelic frequency analysis showed a borderline protective effect (OR 0.7265; 95% CI: 0.5211-1.0128; P = 0.0595).
When the results were analysed based on their ethnicity, i.e., South and North Indians, KNG1 rs710446 showed significant association with cryptogenic young stroke only in South Indians (P = 0.0032) but not in North Indians [Table 2] and having CC genotype increased the risk around four times (OR 4.3885; 95% CI: 1.7576–10.9573; P = 0.0015). Though the genotype of FXII rs1801020 also showed an association (P = 0.0307) with cryptogenic young stroke in South Indians, the allelic frequencies revealed that the 'T' allele showed a protective effect in these patients (OR 0.6951; 95% CI: 0.4753–1.0166; P = 0.0608). Likewise, the CYP4V2 rs13146272 polymorphism was found to be associated to cryptogenic stroke significantly in North Indians (P = 0.03) and having AA genotype was found to increase the risk three times (OR 3.3333; 95% CI: 1.1508–9.6554; P = 0.0265). The FXIII V34L and MTHFR rs1801133 polymorphisms showed only a trend of association (P = 0.084 and 0.094, respectively) in South Indians, the allelic frequencies showed significant association and having T allele increased the risk of cryptogenic IS almost two times (OR 2.1372; 95% CI: 1.0286–4.4409; P = 0.0418 for FXIII V34L and OR 1.8079; 95% CI: 1.0513–3.1088; P = 0.0323 for MTHFR rs1801133).
When the results were analysed further based on their gender, we found a significant association of KNG1 rs710446 with cryptogenic young IS only in males (P = 0.0224) but it was more prominent in South Indian males (P = 0.0036) in which having C allele increased the risk approximately two fold (OR 1.9310; 95% CI: 1.1885–3.1373; P = 0.0079). Likewise, though FXII rs1801020 polymorphism was also found to be associated with stroke in males (P = 0.0305), the association was mainly because of South Indian males (P = 0.0376); however, the presence of the minor allele had a protective effect (OR 0.6043; 95% CI: 0.3763–0.9705; P = 0.0372). SERPINC1 rs2227589 showed a significant association in female cryptogenic young stroke patients (P = 0.0426), mainly because of the significant association in South Indian female patients (P = 0.0374) in whom having A allele increased the risk significantly (OR 2.7182; 95% CI: 1.2276–6.0185; P = 0.0137). On the other hand, CYP4V2 rs13146272 polymorphism was associated with stroke in North Indian males (P = 0.0293) and having A allele increased the risk of stroke almost two fold (OR = 1.963; 95% CI: 0.9732–3.9595; P = 0.0595) but the North Indian females could not be analyzed as very few of them were included in the study. MTHFR rs1801133 showed a trend of association only in males (P = 0.0699) and the T allele was found to be statistically associated with cryptogenic young IS in males (OR 1.9; 95% CI: 1.1063-3.266; P = 0.02). On the contrary, FXIII V34L showed a trend of association with the cryptogenic young IS in South Indians females (P = 0.061), with T allele showing significant association (OR 2.8134; 95% CI: 1.0579–7.4819; P = 0.0382). Except CYP4V2 rs13146272 polymorphism, we could not analyze the gender differences in other polymorphisms in North Indian population as the numbers were inadequate.
Genetic predisposition resulting from polymorphisms in the coagulation factor genes is linked to an increased risk of thrombosis and Factor V Leiden, Prothrombin G20210A mutation, and MTHFR C677T are some of the inherited polymorphisms reported to be involved in genetic risk of thrombosis., Neither Factor V Leiden mutation nor Prothrombin G20210A showed any association with cryptogenic young IS in our study population.
Several studies have shown an association between MTHFR rs1801133 polymorphism, hyperhomocysteinemia, and IS.,,, In the present study, we have observed that the T allele of MTHFR rs1801133 polymorphism is associated with IS in males, more prominently in South Indian population but not in females. It is also worthy to note that 53% of those with this allele in our study had hyperhomocysteinemia. The contribution of this polymorphism in North Indian population can only be ascertained in a study with more number of samples as we faced limitations of recruiting young cryptogenic IS of North Indian origin.
The T>C substitution at the KNG1 T1742C polymorphic site which results in Ile581Thr substitution, has been reported to be associated with decreased aPTT values. Shortening of activated partial thromboplastin time (aPTT) is associated with risk of thrombosis and IS.,, Though this polymorphism showed significant statistical association with young IS, when we separately analyzed the data based on the two major ethnic groups (South and North Indian), the significance of this association was confined to South Indian male patients.
The risk allele A of rs2227589 (786 G>A), on intron 1 of SERPINC1 gene which codes for antithrombin has been reported to be associated with lower anticoagulant activity, decreased plasma anti-factor Xa, and decreased antithrombin levels. Various studies have shown an association of IS with antithrombin deficiency., In our study also, a trend of association between the A allele of SERPINC1 rs2227589 and IS in young was noted. More careful analysis of the data has revealed a significant association of this polymorphism in only South Indian females. Its significance in North Indian patients could not be ascertained due to lack of enough cases and controls.
It has been reported that FXII plasma levels exhibit a 67% of heritability and that these levels are strongly determined by FXII gene variants. Although low FXII plasma levels have been reported to affect the development of cerebrovascular diseases, it has been recently proposed that rather than the cause of thrombosis, these low levels might actually be the result of it., The FXII rs1801020 polymorphism which leads to 46C>T transition affects the translation efficiency, leading to reduced FXII plasma levels. Previous genetic association studies between 46 C>T and cerebrovascular risk showed that genotype TT increases significantly the risk for venous thrombosis, IS, and acute coronary artery disease.,, In our study, though FXII rs1801020 showed only a trend of association over all, it was found to be negatively associated with stroke in males especially in South Indian males i.e., the T allele showed a protective effect rather than a risk of stroke. Conversely, there are also studies that either do not detect genetic association between this genotype and thrombosis or alternatively report association between the CC genotype and cerebrovascular risk., Further studies in larger populations are needed to clarify the role this polymorphism.
FXIII Val134Leu, a common genetic single nucleotide polymorphism (SNP), is critically located near the thrombin activation site (R37-G38) of FXIII-A and has been demonstrated to affect the rate of FXIII activation peptide cleavage by thrombin, resulting in faster FXIII activation and more rapid fibrin clot formation thus increasing the risk of thrombosis. In the present study, FXIII V34L showed a trend of association with the cryptogenic young IS in the South Indian females.
CYP4V2 rs13146272 is located close to the gene encoding coagulation factor XI and factor XI levels have been reported to be associated with IS. This SNP is a missense variant (K259Q) of CYP4V2, and this SNP was most strongly associated with deep vein thrombosis (DVT) in many SNP association studies.,, Ironically, the CYP4V2 gene encodes for a protein which is not known to be related to thrombosis, but its genetic effect on DVT susceptibility may be indirectly mediated through FXI protein with which level this marker is correlated. The rs13146272 lies 67 kb upstream of the F11 gene, and this indirect effect may arise from a long-range transcriptional regulatory element of F11 encompassing the CYP4V2 SNP or its proxies. Alternatively, the CYP4V2 SNP may be in high LD with another variant in or more proximal to the F11 gene that is capable of directly regulating F11 expression. We found that this polymorphism significantly increased the risk of stroke only in North Indian population, more evidently in men, but not in South Indian population.
To conclude, our study indicates that in the Indian population MTHFR rs1801133, KNG rs710446, FXII rs1801020, SERPINC1 rs2227589, CYP4V2 rs13146272, and FXIII V34L might be significant risk factors for cryptogenic IS in the young, and also ethnicity and gender play a significant role. However, the limitations of the study include the small population size, especially the North Indian population, and lack of sufficient study on the functional effects of these polymorphisms. Therefore, further validation of this study with the functional effects in a larger population size is required to completely establish these polymorphisms as risk factors for cryptogenic IS in the young Indian population.
The authors would like to thank the Christian Medical College and Hospital, Vellore, India, for funding this work under the fluid research grant scheme. We also acknowledge the help received from Dr. Joy J Mammen, Professor and Head, Department of Transfusion medicine and Immunohaematology, Christian Medical College, Vellore for the help in recruiting age-, sex-, and ethnicity-matched control subjects from among the blood donors.
Financial support and sponsorship
Fluid research grant (IRB Min. No. 9753 [Observe] dated 18-11-2015) from Christian Medical College and Hospital, Vellore, India.
Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2]