Association between renin–angiotensin–aldosterone system genotypes andhaplotypes and risk of ischemic strokeof atherosclerotic etiology

Introduction

Ischemic stroke is a complex heterogeneous diseaseof multiple etiologies and major clinical manifes-tations (1, 2). While traditional risk factors (hyper-tension, hyperlipidemia, smoking and diabetesmellitus) were viewed as more significant thaninherited factors, a number of recent case–controlstudies and meta-analysis demonstrated strongcontribution of genetic factors and genetic back-ground in influencing stroke susceptibility (2–4).This was evidenced by the clustering of stroke infamilies (5, 6) and by the association of specificgene variants with the risk of stroke (2, 3, 7, 8),which in turn act directly in impacting strokedevelopment or indirectly through synergism with

classical risk factors in enhancing the overallrisk of stroke (7, 9, 10). These include geneticpolymorphisms of the paraoxonase gene whichregulates plasma lipid profile (11, 12) and therenin–angiotensin–aldosterone system (RAAS), anendocrine system responsible for systemic bloodpressure regulation, salt and water homeostasisand for maintaining vascular tone (13–15). Poly-morphisms in many components of RAAS wereshown to cooperate in modifying angiotensin II(ATII) effects (16), including vasoconstriction andextracellular collagen matrix synthesis, and werelinked with stroke development (8, 17–20).In addition to hypertension, a well-established

risk factor for ischemic stroke (14, 21), and giventhe key role RAAS plays in blood pressure

Acta Neurol Scand 2009: 119: 356–363 DOI: 10.1111/j.1600-0404.2008.01105.x � 2009 John Wiley & Sons A/S

ACTA NEUROLOGICASCANDINAVICA

Saidi S,Mallat SG,AlmawiWY,Mahjoub T.Association between renin–angiotensin–aldosterone system genotypes and haplotypes and risk ofischemic stroke of atherosclerotic etiology.Acta Neurol Scand 2009: 119: 356–363.� 2009 John Wiley & Sons A/S.

Objective – The association of renin C-4063T and angiotensinogen(AGT) T174M, AGT M235T and AGT A-6G polymorphisms withischemic stroke of atherosclerotic etiology was investigated in 329Tunisian patients with stroke and 444 controls. Materials andmethods – Genotyping was performed using PCR-RFLP and thecontributions of polymorphisms to the risk of stroke were analyzedusing haplotype and multivariate regression analysis. Results – AGT235T and AGT-6G allele and AGT 235T ⁄T, AGT-6A ⁄G andAGT-6G ⁄G genotype frequencies were higher in patients. Linkagedisequilibrium (LD) was noted for AGT174T with AGT235M andAGT(-6)A in patients, while AGT235M was in LD with AGT(-6)Ain controls and AGT235T was in LD with AGT(-6)G in bothgroups. The AGT 174T ⁄ 235T ⁄ -6A and AGT 174T ⁄ 235M ⁄ -6Ghaplotypes were positively and negatively associated with strokerespectively. Multivariate regression analysis identified AGT174T ⁄ 235M ⁄ -6A, AGT 174T ⁄ 235T ⁄ -6G, AGT 174T ⁄ 235T ⁄ -6A andAGT 174M ⁄ 235T ⁄ -6A haplotypes to be significantly associatedwith an increased risk of stroke. Conclusions – Renin–angiotensin–aldosterone system polymorphisms influence the risk ofatherosclerotic stroke in Tunisians.

S. Saidi1, S. G. Mallat2,W. Y. Almawi3, T. Mahjoub1

1Research Unit of Hematological and AutoimmuneDiseases, Faculty of Pharmacy, University of Monastir,Monastir, Tunisia; 2Universite� St Joseph and Hotel Dieudu France Hospital, Beirut, Lebanon; 3College ofMedicine and Medical Sciences, Arabian GulfUniversity, Manama, Bahrain

Key words: angiotensinogen; polymorphism; renin;stroke

Wassim Y. Almawi, PhD, Department of MedicalBiochemistry, College of Medicine & Medical Sciences,Arabian Gulf University, PO Box 22979, Manama,BahrainTel.: +973 39717118Fax: +973 17271090e-mail: wassim@agu.edu.bh

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homeostasis (13, 22), evidenced by the benefits ofRAAS inhibition with angiotensin-convertingenzyme (ACE) inhibitors and angiotensin II type1 receptor (AGTR1) blockers in stroke prevention(23), previous studies investigate whether RAASpolymorphisms constituted independent risk fac-tors for ischemic stroke and cerebral abnormalities(9, 18, 19). The ACE insertion–deletion (1, 9),AGTR1 A1166C (9), angiotensinogen (AGT)M235T (17, 24) and AGT G-6A promoter (16,24) polymorphisms were associated with anincreased risk of hypertension and stroke. Otherstudies yielded conflicting findings (25); however,this may be explained in part by ethnic variationsin the association of RAAS gene variants withstroke (26), or on the focus of many such studieson a single gene variant. Given the fact that thecontribution of a single gene is usually small, thismay have masked the contribution of related ornearby elements to the risk of stroke.The association between elevated blood pres-

sure, RAAS polymorphisms and ischemic strokeprompted us to investigate whether renin andAGT polymorphisms influence the risk of strokeamong Tunisian patients. Using haplotype anal-ysis, we examined the association of geneticvariation in renin and AGT genes to evaluatethe risk of stroke.

Subjects and methods

Study subjects

This was a retrospective case–control study involv-ing unrelated Tunisian patients with stroke,recruited from the service of Neurology in CHUSahloul in Sousse, Tunisia. The patient groupcomprised 329 consecutive hospitalized individualswith first ischemic stroke (mean age 61.8 �12.2 years), defined as rapid development of clin-ical signs of focal or global disturbance of cerebralfunction, with symptoms lasting 24 h or longer, orleading to death, with no apparent cause otherthan vascular origin. Stroke subtype assignmentwas as per the Trial of ORG 10172 in Acute StrokeTreatment (TOAST) criteria; test reliability wasoptimized using in-house computer system. Themajority of hospitalized patients with ischemicstroke in CHU Sahloul (study locale) were thosewith atherosclerotic origin (53%), followed bycardioembolic stroke (18%), lacunar stroke(small-artery occlusion; 21%) and stroke of othercauses (8%). We focused on ischemic stroke casesof atherosclerotic etiology; a total of 329 unrelatedpatients (180 men and 149 women) were includedin the study.

Exclusion criteria included non-atherosclero-tic causes, rheumatic heart disease, ventriculararrhythmias, uncompensated heart failure, strokesecondary to atrial fibrillation, hematoma, braintumors, accidental or iatrogenic stroke, arterialmalformation, recent acute myocardial infarction,and history of alcohol ⁄drug abuse, pregnancy,and thrombolytic therapy (including oral antico-agulants). Assessment of patients included brainCT, MRI, cerebral angiography, 24-h ECG, echo-cardiography and carotid ultrasound, togetherwith functional evaluations (speech, occupationaltherapy, etc.).Controls comprised 444 unrelated healthy indi-

viduals (245 men, 199 women; mean age60.3 � 12.8 years); all study subjects (patientsand controls) were Tunisian Arabs. Completeclinical history, including risk factors for stroke,was recorded for all participants. The study wascarried out in accordance with the guidelines of theHelsinki Declaration of 1975, and had theapproval of the University of Monastir EthicsCommittee; written informed consent was obtainedfrom all participants.

Data collection

Information on conventional cardiovascular riskfactors, including hypertension, diabetes mellitus,cigarette smoking (designated as current, former ornever) and medication (anti-coagulants and anti-platelet) was obtained from study participants atbaseline. Cardiovascular disease was positive ifsubjects reported a history of heart disease orstroke. Diabetes mellitus was assessed according tofasting blood glucose and ⁄or use of glucose-low-ering drugs (including insulin). Obesity was definedas a body mass index (BMI, kg ⁄m2) of 30 orhigher. Blood pressure (BP) was measured twicewith participants in the sitting position following a5-min rest, and hypertension was defined asBP >160 ⁄95 mm Hg on two separate occasionsand ⁄or the use of antihypertensive therapy. Fast-ing blood samples were obtained from all studyparticipants for the determination of CPK, glucoseand serum lipid levels.

RAAS genotyping

AGT T174M polymorphism was assessed usingPCR-RFLP analysis of the 674-bp PCR product(Nco I digestion) using the following primers:(sense) 5¢-CAATTCAGGCC AAGACATCC-3¢and (antisense): 5¢-GCCAGAGCCAGCAGA-GAG-3¢; the C allele being visualized as 502- and172-bp bands, while the T allele was visualized as

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256-, 246- and 172-bp fragments. AGT M235Tpolymorphism was assessed by BstUI digestion ofthe 302-bp PCR product using the followingprimers: (sense) 5¢-GATGCGCACAAGGTC-CTG-3¢ and (antisense): 5¢-CAGGGT GCTGT-CCACACTGGCTCGC-3¢; the T (302 bp) and C(278 and 24 bp) alleles were identified on 2.5%agarose gel. AGT A-6G polymorphism wasassessed by Bst NI digestion of the 308-bp PCRproduct using the following primers: (sense)5¢-CCCTCAGCTATA AATAGAGCATC-3¢ and(antisense): 5¢-GCAGGAAGACCTGACCATCT-3¢; the G (245 and 63 bp) and A (209, 63 and36 bp) alleles were identified on 2.0% agarose gel.Renin C-4063T polymorphism was determined byTaq I digestion of the 235-bp PCR product usingthe following primers: (sense) 5¢-AAACTAGAATGGGCTACCAGA-3¢ and (antisense): 5¢-GCT-GTGACTTGTCTCTTCCTG A-3¢; the C (163and 68 bp), and T (235 bp) alleles were identifiedon 2.5% agarose gel. A number of patients andcontrol samples were independently and blindlygenotyped twice with concordant results.

Statistical analysis

Statistical analysis was performed on SPSS v. 13.0software (SPSS Inc., Chicago, IL, USA). Data wereexpressed as mean � SD (continuous variables) oras percentages of total (categorical variables).Pearson chi-squared or Fisher�s exact test wereused to assess inter-group significance and Stu-dent�s t-test was used to determine differences inmean values. Gene polymorphisms were analyzedfor Hardy–Weinberg equilibrium using chi-squared goodness-of-fit test and for allelic fre-quency with the gene-counting method, usingHPLUS 2.5 software (available at http://qge.fhcrc.org/hplus). Linkage analysis, the non-randomassociation between loci on the same chromosomearm, as defined by the delta (D¢) coefficient, wascalculated using the HLA STAT-2000 software(courtesy of Dr M. Busson, Hopital St Louis,Paris, France).Haplotype estimation was performed using the

expectation maximization method (HPLUS 2.5),where the sum of probability estimates for allpossible haplotypes equals 1.0. Where haplotypeassignment was uncertain (heterozygous carriers),the haplotype assignment probability estimate wasused to determine the individual�s contribution tothat haplotype. Univariate and later multivariateregression analyses were performed to estimateodds ratios (OR) and 95% confidence intervals(CI) using HPLUS 2.5 and HAPSTAT haplotypeanalysis software (available at http://www.bios.

unc.edu/�lin/hapstat), taking healthy subjects asthe reference group. The structure of the modelused was checked using the confidence interval forthe parameters tested, and the presence of 0.0 inthe CI meant removal of that parameter from themodel. The confounding variables included in thefinal model were age, gender, hypertension andtotal cholesterol concentrations. Statistical signifi-cance was set at P < 0.05.

Results

Clinical characteristics of study subjects

The characteristics of patients with stroke andhealthy control subjects are shown in Table 1. Thetwo groups were matched for age (P = 0.104),gender (P = 0.942), BMI (P = 0.583) and to theprevalence of smoking (P = 0.881). Significantlyhigher percentage of hypertensive individuals(P < 0.001) was in the patient group than in thecontrol group. Elevated serum glucose(P < 0.001), total cholesterol (P = 0.006), LDL(P = 0.005), HDL (P = 0.029) and triglycerides(P = 0.006) were seen in the patient group.

Genotype analysis

Genotype frequency distributions of the fourpolymorphisms did not deviate from Hardy–Wein-berg equilibrium among participants (v2 = 0.031,P = 0.861 for AGT T174M; v2 = 0.481,P = 0.488 for AGT M235T; v2 = 1.128,

Table 1 Clinical characteristic of patients and controls

Characteristic Controls1 Patients1 P 2

Age (years; mean � SD) 60.3 � 12.8 61.8 � 12.2 0.104Age distribution [<60 years; n (%)] 202 (45.5) 152 (46.2) 0.903Male:female 245:199 180:149 0.942Body mass index (kg ⁄ m2; mean � SD) 26.3 � 2.8 26.4 � 1.9 0.583Systolic BP (mmHg; mean � SD) 122.7 � 15.6 160.1 � 30.0 <0.001Diastolic BP (mmHg; mean � SD) 79.1 � 11.1 88.7 � 14.7 <0.001Hypertensives, n (%)3 103 (23.2) 208 (63.2) <0.001Smokers, n (%)4 172 (38.7) 130 (39.5) 0.881Serum chemistry

Total cholesterol5 4.8 � 1.1 5.2 � 1.1 0.002LDL cholesterol5 2.9 � 0.5 3.6 � 1.0 0.008HDL cholesterol5 1.0 � 0.2 1.2 � 0.7 0.038Triglycerides5 1.2 � 0.6 1.5 � 0.7 <0.001Glucose5 5.9 � 2.0 7.6 � 3.3 <0.001Creatinine (lmol ⁄ l) 79.3 � 26.7 102.3 � 60.5 0.007CPK (U ⁄ l) 64.9 � 19.9 59.9 � 31.5 0.028

1Study subjects comprised 444 controls and 329 patients with stroke.2Student�s t-test (two-tailed) for continuous variables, Pearson�s chi-squared testfor categorical variables.3Blood pressure of ‡140 ⁄ 90 mmHg and ⁄ or use of antihypertensive medications.4Includes both current and ex-smokers for the past 10 years.5Concentrations in mmol ⁄ l; values indicate mean � SD.

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P = 0.288 for AGT A-6G and v2 = 0.591,P = 0.442 for renin C-4063T). The frequencies of(mutant) AGT 235T (P < 0.001) and AGT-6G(P < 0.001) alleles were higher in patients withstroke than in control subjects (Table 2). Thefrequency of homozygous AGT 235T ⁄T (P <0.001) in addition to AGT-6A ⁄G (P = 0.047) andAGT-6G ⁄G (P < 0.001) was higher in the patientgroup than in the control group. The allele andgenotype distribution of the AGT T174M andrenin C-4063T SNPs was comparable betweenpatients with stroke and control subjects (Table 2).

Renin and angiotensinogen genotypes with hypertension

The renin and AGT genotype distribution innormotensive and hypertensive patients and con-trols are shown in Table 3. AGT M238T wasassociated with hypertension in both controls(P = 0.002) and in patients with stroke(P < 0.001). By contrast, AGT A-6G(P = 0.002), and to a lesser extent AGT T174M(P = 0.057), were associated with hypertensiononly in patients. Significantly higher systolic BPwas associated with AGT A-6G (P = 0.031), andto a limited extent AGT T174M (P = 0.065), inpatients but not in control subjects (data notshown).

Linkage disequilibrium analysis

Linkage disequilibrium (LD) analysis [defined bythe delta (D¢) coefficient] was determined for reninC-4063T (1q32) and the AGT T174M, AGTM235T and AGT A-6G (1q42-q43) SNPs. No

LD was noted for renin C-4063T with any of theAGT SNPs. AGT 174M was in LD with AGT235T and with AGT-6G among controls more thanamong patients (Table 4). By contrast, strong LDwas noted for AGT 174T with AGT 235M andwith AGT-6A among patients only. Furthermore,AGT 235M was in LD with AGT-6A in controls,while AGT 235T was in LD with AGT-6G in bothpatients and controls (Table 4).

Haplotype distribution

The three-locus AGT haplotype analysis, stratifiedby study subjects, is shown in Table 5. Of the eightmajor AGT haplotypes identified, select haplo-types were positively or negatively associated withstroke. These comprised the AGT 174T ⁄235T ⁄ -6A(P = 0.001; OR 1.70; 95% CI 1.24–2.31) haplo-type which was higher and AGT 174T ⁄235M ⁄ -6G(P < 0.001; OR 0.52; 95% CI 0.43–0.64) haplo-type which was lower among patients comparedwith among control subjects, thus conferringdisease susceptibility and protective nature tothese haplotypes respectively. These differencesremained significant after applying the Bonferronicorrection for multiple testing.

Regression analysis

The association of AGT polymorphic variants withstroke was examined first at the univariate andthen at the multivariate levels. Taking the AGT174T ⁄235M ⁄ -6G haplotype as the reference (OR1.00), univariate analysis identified AGT 174T ⁄235M ⁄ -6A (P = 0.001), AGT 174T ⁄235T ⁄ -6G(P = 0.001), AGT 174T ⁄235T ⁄ -6A (P < 0.001)and the triple-mutant AGT 174M ⁄235T ⁄ -6A(P = 0.003) haplotype to be positively associatedwith stroke (Table 6). Multivariate analysis con-firmed the association of all four haplotypes withstroke, after adjustment for the covariates: age,gender, systolic and diastolic blood pressure, totalcholesterol, LDL and triglyceride levels (Table 6).

Discussion

Although RAAS gene polymorphism werereported to be involved in blood pressure regula-tion and salt balance (13, 15, 22), their contributionto the risk of stroke remains controversial. Previ-ous studies documented association of severalgenetic variants with an increased risk of stroke.While positive correlation between AGT genevariants, plasma AGT concentrations, and withrisk of hypertension (22, 24, 26), inconsistentfindings were reported regarding their association

Table 2 Allele and genotype frequencies

Allele ⁄ genotype Controls1 Patients1 P-value2 OR (95% CI)

Renin )4063T 161 (0.18)3 135 (0.21) 0.265 1.17 (0.90–1.50))4063CC 300 (0.68) 208 (0.63) 0.237 0.83 (0.61–1.11))4063CT 127 (0.29) 107 (0.33) 0.274 1.20 (0.88–1.64))4063TT 17 (0.04) 14 (0.04) 0.910 1.12 (0.55–2.29)

AGT T174M 174M 81 (0.09) 76 (0.12) 0.139 1.30 (0.94–1.81)174TT 367 (0.83) 261 (0.79) 0.281 0.81 (0.56–1.16)

174TM 73 (0.16) 60 (0.18) 0.577 1.13 (0.78–1.65)174MM 4 (0.01) 8 (0.02) 0.159 2.74 (0.82–8.19)

AGT M235T 235T 246 (0.28) 265 (0.40) <0.001 1.76 (1.42–2.18)235MM 235 (0.53) 122 (0.37) <0.001 0.52 (0.39–0.70)235MT 172 (0.39) 149 (0.49) 0.080 1.31 (0.98–1.75)235TT 37 (0.08) 58 (0.18) <0.001 2.35 (1.51–3.63)

AGT A(-6)G )6G 261 (0.29) 274 (0.42) <0.001 1.71 (1.39–2.12))6AA 226 (0.51) 115 (0.35) <0.001 0.52 (0.39–0.70))6AG 175 (0.39) 154 (0.47) 0.047 1.35 (1.01–1.80))6GG 43 (0.10) 60 (0.18) <0.001 2.08 (1.36–3.15)

1Study subjects comprised 444 controls and 329 patients with stroke.2Pearson�s chi-square test.3Number (frequency).

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with stroke. This was mainly due to the concen-tration of most studies on analyzing the contribu-tion of single variant to the risk of stroke, therebydisregarding the epistatic interaction between AGTgene variants and neighboring or distant loci, aswas suggested (3, 4). As stroke is multifactorial innature, it is very likely that multiple candidate gene

variants synergize in determining the overall risk(3, 4).In this study, we investigated on the Tunisian

Arab population the contributions of the renin-4063C ⁄T and three AGT (M235T, T174M andG-6A) polymorphisms to the risk of stroke at thesingle-locus and haplotype levels in 329 patientswith stroke and 444 control subjects, using multi-variate regression analysis, in which potentiallyconfounding variables (age, gender, systolic anddiastolic blood pressure, total cholesterol, LDLand triglyceride levels) were controlled for. At thesingle-locus level, AGT M235T and AGT G-6A,but not AGT T174M or renin-4063C ⁄T SNPs,were significantly associated with an increased riskof stroke. Haplotype analysis confirmed the asso-ciation of the AGT variants with stroke, evidencedby the negative (174T ⁄235M ⁄ -6G) and positive(174T ⁄235T ⁄ -6A) association of select AGT hapl-otypes with stroke, which remained significant evenafter applying the Bonferroni correction, thereby

Table 3 Association of renin and angiotensinogen genotypes with hypertension1

SNP Genotype

Controls Patients with stroke

Normotensive Hypertensive P 2 Normotensive Hypertensive P

Renin-4063C ⁄ T C ⁄ C 228 (66.9)3 72 (69.9) 0.505 70 (57.9) 138 (66.3) 0.305C ⁄ T 98 (28.7) 29 (28.2) 45 (37.2) 62 (29.8)T ⁄ T 15 (4.4) 2 (1.9) 6 (5.0) 8 (3.8)

AGT T174M T ⁄ T 287 (84.2) 80 (77.7) 0.304 104 (86.0) 157 (75.5) 0.057T ⁄ M 51 (15.0) 22 (21.4) 16 (13.2) 44 (21.2)M ⁄ M 3 (0.9) 1 (1.0) 1 (0.8) 7 (3.4)

AGT M235T M ⁄ M 193 (56.6) 42 (40.8) 0.002 62 (51.2) 60 (28.8) <0.001M ⁄ T 127 (37.2) 45 (43.7) 47 (38.8) 102 (49.0)T ⁄ T 21 (6.2) 16 (15.5) 12 (9.9) 46 (22.1)

AGT A-6G A ⁄ A 179 (52.5) 47 (45.6) 0.475 56 (46.3) 59 (28.4) 0.002A ⁄ G 130 (38.1) 45 (43.7) 51 (42.1) 103 (49.5)G ⁄ G 32 (9.4) 11 (10.7) 14 (11.6) 46 (22.1)

1Hypertension defined as BP > 160 ⁄ 95 mmHg on two separate occasions, and ⁄ or the use of antihypertensive therapy.2Fisher�s exact test.3Number of subjects (percent of total within subgroup).

Table 4 Linkage analysis1

Locus 1 Locus 2

Patients Controls

D ¢ v2 P 2 D ¢ v2 P 2

174M 235T 0.020 4.137 0.042 0.026 15.65 <0.001174T 235M 0.080 27.57 <0.001174M )6G 0.038 15.46 <0.001 0.031 20.81 <0.001174T )6A 0.068 17.72 <0.001235M )6A 0.075 23.88 <0.001235T )6G 0.056 11.81 <0.001 0.040 13.59 <0.001

1Analyzed using HLA Stat2000 software.2Determined using the Fisher�s exact test.

Table 5 Angiotensinogen haplotypes in ischemic stroke

Haplotype1 Controls Patients P 2 Pc3 OR (95% CI)

174T ⁄ 235M ⁄ -6G 0.545 � 0.026 0.384 � 0.044 <0.001 <0.001 0.52 (0.43–0.64)174T ⁄ 235M ⁄ -6A 0.137 � 0.012 0.176 � 0.017 0.043 0.296 1.34 (1.02–1.77)174T ⁄ 235T ⁄ -6G 0.131 � 0.010 0.174 � 0.016 0.024 0.177 1.39 (1.05–1.85)174T ⁄ 235T ⁄ -6A 0.095 � 0.014 0.150 � 0.032 0.001 0.008 1.70 (1.24–2.31)174M ⁄ 235T ⁄ -6A 0.033 � 0.014 0.061 � 0.022 0.012 0.092 1.92 (1.17–3.10)174M ⁄ 235M ⁄ -6A 0.029 � 0.010 0.029 � 0.028 0.915 1.000 0.99 (0.55–1.80)174M ⁄ 235T ⁄ -6G 0.018 � 0.011 0.017 � 0.035 1.000 1.000 0.93 (0.44–2.01)174M ⁄ 235M ⁄ -6G 0.012 � 0.062 0.009 � 0.171 0.717 1.000 0.73 (0.29–2.00)

1Haplotype (T174M ⁄ M235T ⁄ A-6G) frequency determined using the maximum likelihood method.2Fisher�s exact test.3Pc = corrected P, as per the Bonferroni method [Pc = 1 ) (1 ) P)n], where n is the number of comparisons.

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supporting associations of these AGT SNPs withstroke.Insofar as renin plays a key role in blood

pressure control, and as renin gene variants wereassociated with an increased risk of hypertension(27) and carotid artery stenosis (28), we testedthe contribution of the renin-4063C ⁄T promotervariant to the risk of stroke. In this study, therewas no separate association of renin-4063C ⁄Tpromoter SNP with stroke. This was in agree-ment with recent studies which demonstrated thelack of association of renin gene variants withthe risk of stroke development (29, 30). Bearingthis in mind, our study was limited in size, andthus a larger study which will be sufficientlypowered will be needed to confirm or rule outthe contribution of renin-4063C ⁄T polymor-phisms to stroke.Although several SNPs were identified in the

AGT gene (located on chromosome 1q41-qter), thefunctional attribute for most remains to be seen.This was exemplified by the association of the 235Tallele with increased AGT blood levels in some (31)but not all (32) studies. Our results demonstrated asignificant association of the M235T and A-6GAGT SNPs with the risk of stroke. This was inagreement with studies on Caucasian (15, 33) andKorean (10) patients linking the 235T allele withhigher stroke-associated mortality rates, but inapparent disagreement with Japanese (34) and US(30) studies demonstrating no link between AGTM235T and stroke. Ethnic differences may explainthe observed discrepancies in the stroke association,highlighted by the wide variability in the 235T alleledistribution among different ethnic groups, ranging20–21% in whites (35, 36) and 74–91% in Africans(36, 37).The 174M allele was shown to be associated with

vascular diseases, such as coronary atherosclerosis

and hypertension (18, 38). No significant associa-tion was found between the T174M allele andstroke in our population; in fact, there seems to bea trend for a protective effect of the 174M allele, inagreement with previous studies that reported onthe lack of association of the T174M allele withstroke (39), or the (marginal) protective effect ofthe 174M allele on brain infarction (33). It remainspossible that the underlying genetics of stroke maynot be due to single mutation with major effects,but on epistatic interactions between multiple lociwhich exert a more profound effect in influencingthe risk of disease, as was demonstrated in anearlier meta-analysis (39). To test this possibility,we examined the possible linkage between 174Mallele and other AGT alleles, and indeed found thatthe presence of the 174M allele either increases [OR2.38 (174M ⁄235T ⁄ -6A) vs OR 2.03 (174T ⁄235T ⁄-6A)] or reduces [OR 0.71 (174M ⁄235M ⁄ -6A) vsOR 1.78 (174T ⁄235M ⁄ -6A)] the overall riskdepending on the specific AGT haplotype ana-lyzed. Given the modest OR and wide confidenceintervals, one cannot exclude the possibility ofchance finding and spurious association betweenthe T174M SNP and the risk of stroke.In conclusion, we found that the AGT M235T

and G-6A, but not the renin-4063C ⁄T or AGTT174M mutations, were associated with anincreased risk of stroke. The mechanism underly-ing the observed positive association of individualAGT genotypes (AGT M235T and G-6A) orhaplotypes (174M ⁄235T ⁄ -6A) with the risk ofstroke remains to be seen. Enhanced RAASactivity, induced in part by specific gene variants,including AGT A-6G and M235T SNPs, is oftenincreased in patients with cerebrovascular andcardiovascular disease (13, 15, 17), and is linkedwith inappropriate sodium and water retention (27,31). This in turn results in increased peripheral

Table 6 Multivariate regression analysis

Haplotype1

Unadjusted Multivariate2

Z-score P aOR (95% CI) Z-score P aOR (95% CI)

174T ⁄ 235M ⁄ -6G 1.00 1.00174T ⁄ 235M ⁄ -6A 3.37 0.001 1.78 (1.27–2.49) 2.51 0.012 1.78 (1.13–2.79)174T ⁄ 235T ⁄ -6G 3.46 0.001 1.82 (1.30–2.56) 2.01 0.044 1.58 (1.01–2.47)174T ⁄ 235T ⁄ -6A 3.59 <0.001 2.26 (1.45–3.53) 2.83 0.005 2.03 (1.24–3.33)174M ⁄ 235T ⁄ -6A 2.98 0.003 2.22 (1.31–3.74) 2.35 0.019 2.38 (1.15–4.90)174M ⁄ 235M ⁄ -6A 0.70 0.482 1.37 (0.57–3.30) )0.70 0.484 0.71 (0.27–1.86)174M ⁄ 235T ⁄ -6G 0.66 0.511 1.43 (0.49–4.16) 1.28 0.202 1.99 (0.69–5.70)174M ⁄ 235M ⁄ -6G 0.13 0.894 1.10 (0.28–4.30) )0.46 0.646 0.62 (0.08–4.69)

aOR, adjusted odds ratio.1T174M ⁄ M235T ⁄ A-6G haplotype.2Adjusted for age, gender, hypertension and total cholesterol concentrations.

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vascular resistance, precipitation of adverse vascu-lar remodeling, vasoconstriction, thrombosis andvessel wall damage. Future prospective replicationstudies on different ethnic groups would be usefulin assessing population differences in the contri-bution of the RAAS gene polymorphisms to therisk of stroke. Studies addressing the interactionbetween AGT and neighboring positional candi-date gene variants would also be useful in rulingout, or alternatively confirming, spurious associa-tion of AGT as a potential stroke-associated locus.

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