Journal of the Neurological Sciences 315 (2012) 72–76

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Journal of the Neurological Sciences

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Association of C3435T multi drug resistance gene-1 polymorphism with aspirinresistance in ischemic stroke and its subtypes

Vandana Sharma a,b, Subhash Kaul c, Amal Al-Hazzani d, T. Surya Prabha c,Polugari Prem Kumar Manohar Rao a, Sneha Dadheech a, A. Jyothy a, Anjana Munshi a,d,⁎a Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad-500016, Indiab Dr. NTR University of Health Sciences, Vijayawada, Andhra Pradesh, Indiac Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad-500082, Indiad Dept. of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia

⁎ Corresponding author at: Department of Molecularand Hospital for Genetic Diseases, Begumpet, Hyderaba27762776.

E-mail address: anjanadurani@yahoo.co.in (A. Muns

0022-510X/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.jns.2011.11.027

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 July 2011Received in revised form 9 November 2011Accepted 22 November 2011Available online 15 December 2011

Keywords:AspirinIschemic strokeMultiple drug resistance-1 (MDR-1) geneRespondersNon-responders

Aspirin is the most commonly used antiplatelet drug for treatment of a serious vascular event, most notablymyocardial infarction and stroke. Significant fraction of aspirin treated patients is resistant to the antiplateleteffects of the drugs. Previous studies have suggested that a genetic basis for aspirin resistance exists. There-fore the present study was taken up to investigate the role of C3435T polymorphism (rs 1045642) of multipledrug resistance-1 (MDR-1) gene with aspirin resistance in stroke patients. Five hundred and sixty ischemicstroke patients and 560 age and sex matched healthy controls were involved in the study. Baseline clinicaldata were collected and follow-up telephone interviews were conducted with patients at 3, 6 and 12 monthspost event to determine stroke outcome. Blood samples were collected and genotypes determined. Signifi-cant difference was observed in the genotype distribution and allele frequency between patients and con-trols. The results were confirmed by a step wise multiple logistic regression analysis controlling all otherconfounding risk factors [adjusted Odds ratio=3.132 (95% CI; 2.043–4.800; pb0.001)]. There was a signifi-cant difference in genotype distribution between drug responders and non-responders. The risk of aspirin re-sistance was significantly high in patients with TT genotype in comparison to those with CC genotype [(TT vs.CC, χ2=6.268; p=0.012, Odds ratio=1.85) (95% CI; 1.142–3.017) (adjusted Odds ratio=2.465; 95% CI;1.895–4.625 and pb0.001)]. As far as the stroke subtypes are concerned TT genotype associated significantlywith aspirin resistance in intracranial large artery atherosclerosis. Our results indicate that the risk of aspirinresistance is more in patients with 3435TT genotype than in those with CC genotype. However, this is a pre-liminary study and a large study of replication is needed to confirm our results.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Many patients are treated with antiplatelet drugs like aspirin andclopidogrel to prevent a serious vascular event, like myocardial in-farction and stroke. Despite the development of new antiplateletdrugs, aspirin is still considered as golden standard of antiplatelettherapy [1]. Aspirin is an O-acetyl derivative of salicylic acid and isobtained by acylating the hydroxyl group with acetic anhydrideusing sulfuric acid as catalyst [2]. Aspirin inhibits platelet cyclooxy-genase system involved in the formation of thromboxane A2 (TXA2).Thromboxane A2 triggers reactions that lead to platelet activationand aggregation. Aspirin acts as a potent antiplatelet agent by

Biology, Institute of Geneticsd-500016, India. Tel.: +91 40

hi).

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inhibiting generation of this mediator. However, despite the demon-strated benefit of aspirin, several patients develop vascular events,an observation that gave rise to the concept of aspirin resistance(AR) [3]. Studies have shown that aspirin resistance occurs in 5% to65% of people with ischemic stroke [4]. Aspirin resistance is a poorlydefined term. The term has been used to describe not only an absenceof the expected pharmacological effect of aspirin on platelets but alsopoor clinical outcome such as recurrent vascular events in patients onaspirin treatment. The mechanisms of aspirin resistance are multifac-torial. It might be on account of inadequate dose of aspirin, reducedabsorption, and/or increased metabolism of aspirin. Poor glucose con-trol and body weight have also been proposed to contribute to aspirinresistance [5]. Apart from all these factors a genetic etiology to aspirinresistance has also been proposed. Genetic variants like COX-1 andCOX-2 and several receptors on the surface of platelets have been ex-amined for the association with aspirin resistance [6,7].

Previous studies have shown that multi drug resistance gene-1(MDR-1/ABCB 1) polymorphism influences oral bioavailability of

Table 1Clinical characteristics of stroke patients and controls.

Patients(n=560)

Controls(n=560)

p Value

Age 49.3 (17.34) 49.01 (16.78)Male:Female 393:167 387:173Systolic BP (mm Hg) 142 (17.2) 128 (16.2) pb0.001Diastolic BP (mm Hg) 88.4 (20.2) 79 (15.3) pb0.001Total cholesterol (mg/dl) 197.45 (40.45) 195.36 (47.50) p>0.05Triglycerides (mg/dl) 178.5 (40.02) 138.68 (43.3) pb0.001Random Glucose (mg/dl) 128.08 (7.2) 118.73 (23.28) pb0.001HDL cholesterol (mg/dl) 53.25 (20.23) 59.56 (22.62) pb0.001Hypertension 57.2% 30.5% pb0.001Diabetes 45.1% 28.9% pb0.001Smokers 44.3% 32.5% p=0.001Alcohol use 32.6% 26.1% pb0.001Family history of stroke 25.5% 11.7% pb0.001

Age, systolic BP, diastolic BP, total cholesterol, high density lipoprotein (HDL)cholesterol, random glucose and triglycerides are given as mean (SD). Stroke patientshave significantly higher levels of systolic and diastolic blood pressure, higher serumtriglycerides and random glucose and a lower level of HDL(pb0.001). p Values werecalculated using students' paired t-test (SPSS 18).

Table 3Multiple logistic regression analysis including the genotypes and all other risk factors.

Variables Oddsratio

95% CI p

Lower Upper

Genotype (TT) 3.132 2.043 4.800 b0.001Diabetes 2.087 1.528 2.850 b0.001Hypertension 2.255 1.667 3.050 b0.001Smoking 1.489 1.060 2.093 0.022Alcoholism 1.801 1.259 2.576 0.001Total cholesterol 2.999 2.118 4.245 b0.001

Only the variables with significant values are given.

73V. Sharma et al. / Journal of the Neurological Sciences 315 (2012) 72–76

antiplatelet drugs like clopidogrel and influences the prognosis ofpatients with cardiovascular diseases [8,9]. MDR-1 gene codes forp-glycoprotein (P-gp), a large (170 kDa) transmembrane protein.P-gp plays an important role in the bioavailability of a variety ofdrugs including chemotherapeutic agents, cardiac drugs, antibiotics,steroids, immunosuppressants, HIV-protease inhibitors and warfarin[10–13]. Taubert et al. have reported that two variant alleles ofMDR-1 (TT) genotype had lower plasma concentration of clopido-grel and its active metabolite in comparison with carriers of CCand CT genotypes [9]. There are no studies evaluating the associa-tion of MDR-1 C3435T polymorphism with aspirin resistance in is-chemic stroke or a particular subclass of stroke. Therefore thepresent study was taken up with an aim to study the associationof C3435T polymorphism (rs 1045642) with aspirin resistance in is-chemic stroke and its sub types in a South Indian population fromAndhra Pradesh.

2. Materials and methods

2.1. Subjects

Five hundred and sixty ischemic stroke patients (Males:females=393:167) presenting with new stroke, evaluated in thestroke clinic of Nizam's Institute of Medical Sciences, Hyderabad(AP, India) between January 2007 and May 2010, were included inthe study. The study was approved by ethical committee of thestudy hospital and written informed consent was obtained from allthe subjects included in the study. All patients were examined by aqualified stroke neurologist to confirm diagnosis. Patients were con-firmed to have suffered an ischemic stroke by computed tomographyscans and magnetic resonance imaging. Patients with major renal, he-patic, cardiac diseases and cancers were excluded from the study.

Table 2Distribution of MDR 1 (C3435T) genotypes and allelic frequencies of the study population.

Study group MDR 1 genotypes (%)

CC CT TT

Patients n (%) 103 (18.3) 256 (45.7) 201 (35.8)Control n (%) 160 (28.5) 263 (46.9) 137 (24.4)

For TT vs. CC, χ2=24.35; pb0.001, Odds ratio=2.27 (95% CI; 1.64–3.168). For TT vs. CT+CCpb0.001, Odds ratio=1.54 (95% CI; 1.308–1.827).

Ischemic stroke was classified according to Trial of ORG 10172 inAcute Stroke Treatment (TOAST) classification [14].

Five hundred and sixty healthy individuals matched for sex andage formed the control group (Males:females=387:173). The con-trols were volunteers recruited simultaneously from the same demo-graphic area. They were blood donors from the study hospital andstaff members of the Institute of Genetics and Hospital for GeneticDiseases. They underwent a routine medical checkup in the outpa-tient clinic of the Institute of Genetics. The controls had no clinical ev-idence of any cerebrovascular disease. Information on demographiccharacteristics and risk factors were collected by using a structuredquestionnaire. Hypertension, alcohol use, diabetes and smokingwere defined as reported previously [15].

2.2. Follow up

All the patients were on antiplatelet therapy with aspirin. Thedose of aspirin ranged from 75 to 325 mg/day. The follow-up tele-phone interviews were conducted with patients at 3 months,6 months and 12 months after hospital discharge. Poor outcome wasdefined as a score of more than 2 on modified Rankin Scale Score (in-cluding death at 3 months) and b5 on an extended Glasgow OutcomeScale (GOS-E) from stroke onset. The aspirin resistance was definedas poor clinical outcome and recurrent stroke events and death in pa-tients on aspirin treatment.

2.3. DNA isolation and genotyping

5 ml of blood was collected in EDTA tubes. Genomic DNA wasextracted from blood samples using standard phenol-chloroformmethod. The MDR-1 C3435T polymorphism was detected by PCR-RFLP technique. PCR was performed using following primers: for-ward: 5′-GTTTTCAGCTGCTTGATGGC-3 and reverse: 5′CATTAGG-CAGTGACTCGATG3′. The amplified 216 bp PCR product was digestedwith Mbol restriction enzyme (Fermentas Fast digest) by incubatingat 37 °C for 5 min followed by separation of fragments on 3% agarosegel. −3435C allele was detected as fragments of 159 bp and 57 basepairs (bp) while as −3435T allele was detected as 216 bp productand CT genotype was detected as fragments of 216 bp, 159 bp and57 bp.

Allele frequency

Total C T Total

560 462 (0.41) 658 (0.58) 1120560 583 (0.52) 537 (0.47) 1120

, χ2=17.34; pb0.001, Odds ratio=1.72 (95% CI; 1.335–2.239). For T vs. C, χ2=26.25;

Table 4Genotypic frequencies of MDR-1 in response to aspirin in patients.

Total no. MDR-1 (C3435T) genotype n (%)

CC CT TT

Responders 338 54 (15.9) 149 (44) 135 (39.9)Non-responders 222 49 (22) 107 (48.1) 66 (29.7)

TT vs. CC, χ2=6.268; p=0.012, Odds ratio=1.85 (95% CI; 1.142–3.017).

74 V. Sharma et al. / Journal of the Neurological Sciences 315 (2012) 72–76

2.4. Statistical analysis

Hardy–Weinberg equilibrium was tested for MDR-1 gene poly-morphism. Association between genotypes and stroke was exam-ined by odds ratio with 95% confidence interval (CI) and Chi-square analysis using Open EPI6 software (Open Epi Version 2.3.1from Department of Epidemiology, Rollins School of Public Health,Emory University, Atlanta, GA 30322, USA). Allelic frequencieswere calculated according to the number of different alleles ob-served and the total number of alleles examined. Multiple logisticregression analysis with forward stepwise selection (Wald) wasperformed using SPSS15software. The independent variables weredecoded as the following dummy variables: genotype,0 for normalhomozygotes and 1 for mutant homozygotes or heterozygotes;sex,1 for male and 2 for female; smoking, 1 for smokers and 2 fornon-smokers; hypertension, 1 for hypertension and 2 for normo-tension; diabetes mellitus, 1 for presence and 2 for absence; andtotal cholesterol, 1 for high and 2 for low; triglycerides, 1 for highand 2 for low and HDL, 1 for low and 2 for high. The associationof responders and non-responders with genotype was examinedby Odds ratio with 95% confidence interval (CI) and Chi-squareanalysis using the above mentioned software. The results were con-firmed by stepwise multiple logistic regression analysis. Statisticalsignificance was defined as pb0.05.

3. Results

Five hundred and sixty ischemic stroke patients and 560 controlswere included in the study. All patients belonged to a South Indianpopulation from Andhra Pradesh. The clinical characteristics of thestroke patients and controls have been presented in Table 1. Meanage was 49.3 years in the former group and 49.01 years in the later.Risk factor profile of the patients revealed hypertension in 57.2%, di-abetes in 45.1%, smoking in 44.3%, alcohol use in 32.6% and family

Table 5Genotypic frequencies of responders and non responders classified according to TOAST clas

TOAST classification No. of patients Genotypes Responders

Large artery atherosclerosis 195A. Intracranial large artery 122 CC (18) 8

CT (59) 31TT (45) 32

B. Extracranial large artery 73 CC (16) 10CT (31) 20TT (26) 18

Small artery occlusions (Lacunar) 90 CC (16) 9CT (41) 25TT (33) 22

Cardioembolism 89 CC (20) 8CT (36) 23TT (33) 19

Other determined etiology 86 CC (15) 9CT (43) 24TT (28) 21

Undetermined etiology 100 CC (18) 10CT (46) 26TT (36) 23

history of stroke in 25.5% subjects. In the control group 30.5% had hy-pertension, 28.9% were diabetic, 32.5% smokers, 26.1% alcohol usersand 11.7% had family history of stroke. Stroke patients also had signif-icantly higher levels of systolic and diastolic blood pressure, higherserum triglycerides and random glucose and a lower level of HDL(pb0.001).

The genotypic distribution of MDR-1 C3435T polymorphism andallele frequency of C and T alleles in patients and controls has beengiven in Table 2. We found statistically significant difference in thegenotypic distribution between patients and controls [for TT vs. CC,χ2=24.35; pb0.001, Odds ratio=2.27 (95% CI; 1.64–3.168)](Table 2). Significant difference was observed in the frequency of Cand T alleles in patients and controls (T vs. C, χ2=26.25; pb0.001,Odds ratio=1.54) (95% CI; 1.308–1.827). We performed multiple lo-gistic regression analysis with forward stepwise selection using allvascular risk factors and TT genotype of MDR-1 gene. It revealedthat the most predictive independent risk factors for stroke werethe TT genotype of MDR-1 gene [adjusted Odds ratio=3.132 (95%CI; 2.043–4.800; pb0.001)], hypertension, diabetes, alcoholism andtotal cholesterol (Table 3).

Based on the follow up studies of 3 months, 6 months and12 months we classified the patients into two groups as respondersand non-responders. All the patients were on antiplatelet therapywith aspirin. The non-responders had a poor clinical outcome definedas a score of more than 2 on modified Rankin Scale Score (Includingdeath at 3 months) and b5 on an extended GOS-E, from strokeonset. The frequency of recurrent stroke events and death was morein non-responders. We further evaluated the association of drug re-sponders and non-responders with MDR-1 genotypes. There was asignificant difference in genotype distribution between drug re-sponders and non-responders (Table 4). Patients with good outcomehad a higher frequency of CC genotype when compared to patientswith bad outcome classified as non-responders. The risk of aspirin re-sistance was significantly high in patients with TT genotype in com-parison to carriers of the homozygous CC genotype [(TT vs. CC,χ2=6.268; p=0.012, Odds ratio=1.85) (95% CI; 1.142–3.017)(adjusted Odds ratio=2.465; 95% CI; 1.895–4.625; pb0.001)]. ForCT heterozygotes no significant differences in genotype distributionwere observed in relation to responders and non-responders.

Evaluating the association of MDR-1 genotypes in responders andnon-responders belonging to different stroke subtypes classifiedaccording to TOAST classification, there was a significant difference ingenotype distribution between drug responders and non-respondersin stroke patients with intracranial large artery atherosclerosis (For TT

sification.

Non responders χ2 Odds ratio (95% CI) p Value

10 3.882 3.07 0.992–9.537 0.04828136 0.197 1.35 0.364–5.007 0.657

1187 0.492 1.55 0.457–5.292 0.482

161112 1.51 2.03 0.657–6.301 0.21913146 1.018 2 0.523–7.647 0.315

1978 0.344 1.41 0.447–4.478 0.557

2013

Fig. 1. Possible mechanism of aspirin resistance: Aspirin inhibits platelet cyclooxygenase, a key enzyme in thromboxane A2 (TXA) generation. TXA2 triggers reactions leading toplatelet activation and aggregation. In CC bearing individuals there is no platelet activation and aggregation because cyclooxygenase activity is inhibited by aspirin. In TT bearingindividuals cellular efflux of aspirin takes place and therefore cyclooxygenase leads to thromboxane A2 generation and platelet activation and aggregation.

75V. Sharma et al. / Journal of the Neurological Sciences 315 (2012) 72–76

vs. CC, χ2=3.882; p=0.048, Odds ratio=3.07) (95% CI; 0.992–9.537)(Table 5).

4. Discussion

The concept of aspirin resistance is a well known phenomenon inclinical practice. Despite aspirin intake several patients develop ad-verse vascular events. Previous studies have shown that adequateantiplatelet affects of aspirin are not achieved in 5–45% patients sug-gesting that many patients are resistant to aspirin [16]. Since there isno single definition or validated method to define aspirin resistance,its reported prevalence varies greatly. A study investigating aspirinresistance in Indian patients with coronary artery disease reported41.66% patients showing inadequate response to aspirin [17]. In onestudy it has been reported to be as high as 60% [18]. In the presentstudy 57.8% of the patients were found to be aspirin resistant ormore appropriately aspirin treatment failure. Aspirin resistance ismultifactorial in origin. Genetic etiology to aspirin resistance hasalso been suggested. A largest and most comprehensive review byGoodman et al. analyzed 31 candidate genes of aspirin resistance.50 polymorphisms in 11 genes were identified [19]. However, thereare no studies evaluating the influence of MDR-1 C3435T polymor-phism on the outcome of stroke patients treated with aspirin. Strokeis a multifactorial genetic disorder influenced by genetic and environ-mental factors. Twin studies, family studies and case control associa-tion studies suggest that significant proportion of stroke risk isexplained by genetic factors. However, vascular risk factors like hy-pertension, alcoholism, diabetes, triglyceride levels, HDL levels arealso important [20]. This is the first study to evaluate the associationof C3435T polymorphism with aspirin resistance in stroke patients.A stepwise multiple logistic regression analysis was carried out to ad-just all the vascular risk factors. Based on the results of multiple logis-tic regression analysis we found that TT genotype and T allele of theMDR-1 C3435T polymorphism were significantly high in stroke pa-tients in comparison with controls. It has been shown that MDR-1gene polymorphism influences the oral bioavailability of anotherantiplatelet drug clopidogrel which influences the prognosis of

patients with myocardial infarction [8, 9]. P-glycoprotein encodedby MDR-1 gene has been found to be responsible for cellular effluxof many medications [21]. A study carried out in German populationhas indicated that TT genotype of MDR1 gene leads to diminishedconcentration of clopidogrel and its active metabolite [9]. Simon etal. have demonstrated the association of TT genotype with worse clin-ical outcome in myocardial infarction patients receiving clopidogreltreatment [8]. In the present study also we found a significant associ-ation of TT genotype with worse clinical outcome. In non-respondersthe recurrence of stroke and death rate was high in comparison withresponders. The TT genotype might be influencing the oral bioavail-ability of aspirin and therefore the bearers of TT genotype showmore aspirin resistance in comparison with the patients bearing CCgenotype (Fig. 1). However, this remains to be confirmed and furtherstudies are required to establish this. Evaluating the association ofC3435T polymorphism of MDR1 gene with aspirin resistance withstroke subtypes, we found a significant association of TT genotypein intracranial large artery atherosclerosis. However, the same geno-type of C3435T polymorphism has been associated with increased,decreased or non-influenced plasma levels of substrates of p-glycoprotein, depending on the analyzed substrate and study popula-tion [22–26].

The limitations of our study are that we were unable to analyzethe plasma levels of aspirin and its active metabolite. We performedan analysis of only one polymorphism, where as other known poly-morphisms of this gene might be of relevance. Further studies evalu-ating the influence of MDR-1 polymorphism on aspirin resistanceshould include these measurements. In conclusion our study showsthat the risk of aspirin resistance was higher in patients with3435TT genotype than in those with 3435CC genotype. The risk of as-pirin resistance was found to be more in patients belonging to strokesubtype intracranial large artery atherosclerosis bearing TT genotype.A large study of replication is required to confirm the results.

Conflict of Interest

None of the authors has any conflict of interest.

76 V. Sharma et al. / Journal of the Neurological Sciences 315 (2012) 72–76

Acknowledgment

Subhash Kaul and Anjana Munshi are thankful to ICMR, India forfinancial support.

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