Clinical Relevance of MTHFR, eNOS, ACE, and ApoE Gene Polymorphisms and Serum Vitamin Profile among Malay Patients with Ischemic Stroke

Clinical Relevance of MTHFR, eNOS, ACE, and ApoE Gene Polymorphisms and Serum Vitamin Profile among Malay Patients with Ischemic Stroke Loo Keat Wei, PhD,*†‡x Anthony Au, MSc,x Saras Menon, PhD,‡ Siew Hua Gan, PhD,x and Lyn R. Griffiths, PhD‡

Background: The purpose of this study was threefold. First, it was to determine the relationship between serum vitamin profiles and ischemic stroke. The second purpose was to investigate the association of methylenetetrahydrofolate reductase (MTHFR), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE), and apolipoprotein-E (ApoE) gene polymorphisms with ischemic stroke and further correlate with serum vitamin profiles among ischemic stroke patients. The third purpose of the study was to highlight the interaction of MTHFR and eNOS haplotypes with serum vitamin profiles and ischemic stroke risks. Methods: Polymorphisms of these genes were analyzed in age-, sex-, and ethnicity-matched case–controls (n 5 594); serum vitamin profiles were determined using immunoassays. Results: The MTHFR 677C.T, 1298A.C, eNOS intron 4a/b, and ApoE polymorphisms were significantly associated with the increased risk of ischemic stroke. Elevated serum homocysteine and vitamin B12 levels were associated with MTHFR 677C.T and eNOS intron 4a/b polymorphisms. The ApoE and eNOS 2786T.C polymorphisms were associated with increased serum vitamin B12 levels. However, none of the polymorphisms influenced serum folate levels except for the MTHFR 1298A.C. Different patterns of MTHFR and eNOS haplotypes tend to affect serum vitamin profiles to different degrees, which contribute to either different susceptibility risk or protective effect on ischemic stroke. Overall, increased levels of serum homocysteine and vitamin B12 levels were associated with higher risk of ischemic stroke in the investigated population. Conclusions: The present study suggests that the genotypes and haplotypes of MTHFR 677C.T and eNOS intron 4a/b polymorphisms are potential serum biomarkers in the pathophysiological processes of ischemic stroke, by modulating homocysteine and vitamin B12 levels. Key Words: Gene polymorphisms—folate—homocysteine—vitamin B12— stroke. Ó 2015 by National Stroke Association

Stroke is the leading cause of death worldwide, with an average of one stroke mortality in every four minutes.1 In Malaysia, stroke is one of the top five leading causes of death and is also one of the top 10 causes for hospitaliza-

tion.2 Several factors have been associated with ischemic stroke risk, such as hyperhomocysteinemia, hypercholesterolemia, atherosclerosis, diabetes and hypertension. For example, hyperhomocysteinemia is detected in 40% of

From the *Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, Perak, Malaysia; †Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Perak, Malaysia; ‡Genomics Research Centre, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia; and xHuman Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia. Received March 6, 2015; accepted April 9, 2015.

The authors declare that they have no conflicts of interest. Address correspondence to Loo Keat Wei, PhD, Centre for Biodiversity Research, UTAR, 31900 Kampar, Perak, Malaysia. E-mail: [email protected]; [email protected]. 1052-3057/$ - see front matter Ó 2015 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2015.04.011

Journal of Stroke and Cerebrovascular Diseases, Vol. -, No. - (---), 2015: pp 1-9

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L.K. WEI ET AL.

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cerebral, coronary, and peripheral artery diseases as well as 15% of healthy individuals. It is reported to be actively involved in the oxidative stress event.3 In addition, high levels of low-density lipoprotein cholesterol that resulting in hypercholesterolemia tends to accelerate the rate of macrovascular complications.4 All in all, these risk factors underlie the pathophysiology of ischemic stroke.3-6 Several lines of experimental and clinical evidences indicate that methylenetetrahydrofolate reductase (MTHFR), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE), and apolipoprotein-E (ApoE) genetic polymorphisms also contribute to hyperhomocysteinemia, hypercholesterolemia, diabetes, and hypertension.7,8 MTHFR is an important enzyme that converts 5,10-methylenetetrahydrofolate to 5methyltetrahydrofolate which catalyzes the conversion of homocysteine to methionine.6 MTHFR 677C.T and 1298A.C are functional polymorphisms for MTHFR enzyme, which may affect its role in the metabolism of folate, thus contributing to hyperhomocysteinemia.7 eNOS involves in cerebral autoregulation, cerebral vasodilatation, and maintaining basal cerebral flow. Polymorphisms in eNOS gene, including the 2786T.C, intron 4a/b and 1894G.T, are reported to be associated with lower nitric oxide levels and adverse oxidative stress events.8 The ACE gene, which encodes for angiotensin converting enzyme, is a potent regulator in renin– angiotensin system where the ACE I/D accounts for approximately half of the phenotypic variance in serum ACE levels and contributes to hypertension.4,9 There has been mounting evidence which suggests that the E2, E3 and E4 polymorphisms of ApoE play a key role in lipid metabolism and hypercholesterolemia.4 The purpose of this study was threefold. First, it was to determine the relationship between serum vitamin profiles and ischemic stroke. The second purpose was to investigate the association of MTHFR, eNOS, ACE, and ApoE gene polymorphisms with ischemic stroke and further correlate with serum vitamin profiles among ischemic stroke patients. The third purpose of the study was to highlight the interaction of MTHFR and eNOS haplotypes with serum vitamin profiles and ischemic stroke risks.

Materials and Methods Sampling of Subjects The present study was approved by the Institutional Ethics Review Committee of Universiti Sains Malaysia (reference no: USMKK/PPP/JEPeM [231.1.(08)]), which complied with the Declaration of Helsinki. Ischemic stroke patients (n 5 297) referred from the Neurology Clinics and Wards at Hospital Universiti Sains Malaysia were recruited. After full explanation of the study protocol, the study benefits, and the risks of participation, written informed consents were obtained from the

subjects or their proxy. Ischemic stroke was defined based on clinical manifestations and/or evidence of occlusion(s), stenosis and/or lesion(s) in computerized tomography scan or magnetic resonance imaging. Patients who were ethnic Malays, aged between 18 and 70 years, and have been previously investigated for ischemic stroke severity based on the Modified Rankin Scale were included. Exclusion criteria included patients who (1) participated in any ongoing trial, (2) have taken any form of vitamin B supplementation 6 months before the study, (3) have a brain tumor or any form of cancers, or (4) have cerebral hemorrhage. Age-, sex-, ethnicity, and geographic location-matched individuals who exhibited normal findings on medical history and physical examinations and who had no family history of stroke were included in the control population (n 5 297).

Genotyping of the MTHFR, eNOS, ACE, and ApoE Polymorphisms Venous blood samples (2 mL) were collected from both case and control groups. Genomic DNA was extracted using the QIAampÒ Blood Midi Kit with a Spin Protocol (Qiagen, Hilden, Germany) according to the manufacturer’s instruction with some slight modifications. Genetic polymorphisms of MTHFR, eNOS, ACE, and ApoE were selected based on previous findings that have indicated that the polymorphisms have biological and clinical relevance.10-14 For the MTHFR 677C.T, eNOS 1894G.T, and 2784T.C polymorphisms, genotyping was performed based on the method described by Loo et al.15 The MTHFR 1298A.C, eNOS intron 4a/b, ACE I/D, and ApoE polymorphisms were genotyped using methods described by Shi et al,16 Xin et al,17 Ramachandran, et al18 and Pantelidis et al,19 respectively, with slight modifications.

Determination of Serum Vitamin Profiles Among the patients’ group, fasting serum samples were collected to determine serum vitamin profiles (homocysteine, folate and vitamin B12) within 48 hours of ischemic stroke onset. Serum vitamin profiles of controls who fasted more than 12 hours were determined. Blood (6 mL) was collected in clot activator gel tube (Becton Dickinson) for the determination of serum homocysteine levels using fluorescence polarization immunoassay (Architect ci8200; Abbott Diagnostics, Abbott Park, IL) whereas serum folate and vitamin B12 levels were determined using competitive immunoassay of direct chemiluminescence technology (ADVIA Centaur XP immunoassay system, Siemens Healthcare).

Statistical and Haplotype Analysis Statistical analysis was performed using PLINK version 1.07.20 The demographic characteristics of the

CLINICAL RELEVANCE OF MTHFR, eNOS, ACE, And ApoE GENE POLYMORPHISMS

Table 1. Demographic characteristics of study subjects

Parameters

Controls (n 5 297)

Cases (n 5 297)

Age (y) Gender (male/female) WSR Hypertension (%) Diabetes (%) Hypercholesterolemia (%)

51.8 6 8.7 2:3 .5 6 0.1 6.1 33.3 6.1

52.6 6 8.8 1:8 .5 6 1.4* 76.8** 54.9** 71.4**

Abbreviation: WSR, waist-to-stature ratio. Values are mean 6 standard deviation from unadjusted Student t test for continuous variables and chi-square test for categorical variables. *P , .05 versus controls. **P , .001 versus controls.

study subjects were analyzed using Student t test for continuous variables whereas Pearson’s chi-squared test was used for categorical variables. The odds ratios (OR) and 95% confidence intervals (CI) per 1 mmol/L, 1 nmol/L, or 1 pg/mL increases in log serum homocysteine, folate or vitamin B12 levels were calculated using logistic regression. Age, sex, waist-to-stature ratio (WSR), serum vitamin profiles, diabetes, hypertension, and hypercholesterolemia were controlled in multivariate analysis. As a quality control procedure, genotypic distribution for each polymorphism was tested among the controls for any possible deviations from the Hardy–Weinberg equilibrium. Differences in the genotype frequencies between cases and controls were determined using the likelihood ratio test. Logistic regression models were used to estimate OR and 95% CI for determining possible high-risk genotypes of each polymorphism for ischemic stroke. Potential confounders such as age, sex, WSR, cigarette smoking, hypertension and diabetes were adjusted. The haplotypes for MTHFR and eNOS polymorphisms were inferred by using PHASE software version 2.121 which estimates the haplotype frequencies for both cases and controls and deduces the possible haplotype combinations for each individual. Only haplotypes with observed frequencies larger than 1% were considered. The association between the inferred haplotypes and

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susceptibility to ischemic stroke was investigated using PLINK version 1.07, and the likelihood ratio test was calculated for haplotype–phenotype associations. The distributions of serum vitamin profiles were investigated for normality using Kolmogorov–Smirnov test. Positively skewed serum vitamin profiles were logarithmic-transformed for analysis before data presentation. Analysis of variance was used to compare the mean levels of serum vitamin profiles among different genotypes, whereas the influences of respective haplotypes on serum vitamin profiles were determined using linear regression models. The data were adjusted for 10000 permutations.

Results Demographic characteristics of study subjects are shown in Table 1. We observed that 1 mmol/L increase in log serum homocysteine and vitamin B12 levels were associated with 2.32 (95% CI, 1.25-4.30; P 5 .008) and 4.50 (95% CI, 1.43-14.12; P 5 .010) folds increased risks of ischemic stroke, respectively (Table 2). Although 1 mmol/L increase in log serum folate was associated with a .71 (95% CI, .51-.98; P 5 .038) fold decreased risk of ischemic stroke, the susceptibility risk became nonsignificant following multivariate adjustment (Table 2). Significant differences in the distribution of genotype frequencies were observed between cases and controls (Table 3). MTHFR 677CT (OR, 1.86; CI, 1.28-2.70; P 5 .001), 677TT (OR, 5.67; CI, 2.27-14.19; P , .001), MTHFR 1298CC (OR, 2.50; CI, 1.02-6.14; P , .05), eNOS intron 4 aa (OR, 16.77; CI, 8.71-32.29; P , .001), and ApoE E2E3 (OR, 7.05; CI, 3.64-13.67; P , .001) were significantly associated with increased risk of ischemic stroke (Table 3). The frequency of ApoE E4E4 genotype was higher among cases as compared to controls (22.90% versus 4.04%; OR, 3.83; CI, .78-18.83), but statistically insignificant (Table 3). The genotype distribution for each polymorphism among the control subjects is in agreement with Hardy–Weinberg equilibrium (Table 3), suggesting that genotyping error is almost unlikely. The influences of MTHFR, eNOS, ACE, and ApoE gene polymorphisms on the levels of serum vitamin profiles were determined. The MTHFR 677C.T and eNOS intron 4a/b polymorphisms were observed to be positively

Table 2. Serum vitamin profiles and the risk of ischemic stroke when compared to controls

Cases (n 5 297) Univariate OR (95% CI), P value Multivariate OR (95% CI), P value*

Homocysteine (mmol/L)

Folate (nmol/L)

Vitamin B12 (pg/mL)

2.01 (1.40-2.89), ,.001 2.32 (1.25-4.30), .008

.71 (.51-.98), .038 .78 (.45-1.36), NS

5.36 (2.33-12.29), ,.001 4.50 (1.43-14.12), .010

Abbreviations: CI, confidence interval; NS, not significant; OR, odds ratio; WSR, waist-to-stature ratio. Odds ratio and 95% confidence intervals of 1 mmol/L increases in log serum vitamin profiles. *Multivariate analysis adjusted for age, sex, WSR, serum vitamin profiles, diabetes, hypertension, and hypercholesterolemia.

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Table 3. Allelic and genotypic distributions of MTHFR, eNOS, ACE, and ApoE

Variants MTHFR 677C.T

MTHFR 1298A.C

eNOS 2786T.C

eNOS intron 4a/b

eNOS 1894G.T

ACE I/D

ApoE

Controls Frequency HWE Cases Frequency Genotypes (n 5 297) (%) P value* (n 5 297) (%) P valuey CC CT TT C T AA AC CC A C TT CT CC T C bb ab aa b a GG GT TT G T II ID DD I D E3E3 E3E4 E2E3 E2E4 E2E2 E4E4 E3 E2 E4

226 65 6 517 77 186 104 7 476 118 171 102 24 444 150 216 70 11 502 92 32 124 141 188 406 90 154 53 334 260 163 89 12 27 4 2 427 47 120

76.10 21.88 2.02 87.04 12.96 62.63 35.02 2.35 80.13 19.87 57.58 34.34 8.08 74.75 25.25 72.73 23.57 3.70 84.51 15.49 10.77 41.75 47.47 31.65 68.35 30.30 51.85 17.85 56.23 43.77 54.88 29.97 4.04 9.09 1.35 .67 71.89 7.91 20.20

.603

.085

.120

.086

.546

.358

.973

177 95 25 449 145 184 95 18 463 131 230 36 31 496 98 134 48 115 316 278 44 90 163 89 208 125 140 32 390 204 137 54 68 23 8 7 396 107 91

59.60 31.98 8.42 75.59 24.41 61.95 31.99 6.06 77.95 22.05 77.44 12.12 10.44 83.50 16.50 45.12 16.16 38.72 53.20 46.80 14.82 30.30 54.88 29.97 70.03 42.09 47.14 10.77 65.66 34.34 46.12 18.18 22.90 7.74 2.69 2.36 66.67 18.01 15.32

,.001 ,.001 NS

NS ,.001

.003 ,.001 ,.001 .012

NS .003

.001 ,.001

,.001

OR (95% CI) 1.00 1.86 (1.28-2.70) 5.67 (2.27-14.19) 1.00 2.03 (1.51-2.74) 1.00 .92 (.65-1.31) 2.50 (1.02-6.14) 1.00 1.14 (.86-1.52) 1.00 .26 (.17-.43) .96 (.54-1.70) 1.00 .66 (.51-.85), 1.00 1.12 (.73-1.72) 16.77 (8.71-32.29) 1.00 2.90 (2.29-3.67) 1.00 .52 (.31-.90) .84 (.50-1.40) 1.00 .93 (.74-1.18) 1.00 .66 (.46-.94) .44 (.26-.75) 1.00 .66 (.52-.84) 1.00 .70 (.46-1.06) 7.05 (3.64-13.67) 1.02 (.56-1.88) 2.46 (.72-8.38) 3.83 (.78-18.83) 1.00 .82 (.60-1.11) 2.45 (1.69-3.55)

P valuez

.001 ,.001 ,.001 NS .037 NS ,.001 NS .001 NS ,.001 ,.001 .018 NS NS .019 .002 ,.001 NS ,.001 NS NS NS ,.001 NS

Abbreviations: ACE, angiotensin converting enzyme; ApoE, apolipoprotein-E; CI, confidence interval; eNOS, endothelial nitric oxide synthase; HWE, Hardy–Weinberg equilibrium; MTHFR, methylenetetrahydrofolate reductase; NS, not significant; OR, odds ratio. *HWE P value is a measure of deviation from HWE calculated from the observed and predicted heterozygosity in the controls. yP value for the association between polymorphism in regard to cases and controls. zP value for ischemic stroke susceptibility.

associated with higher concentration of serum homocysteine (Table 4). Of all the investigated polymorphisms, only the MTHFR 1298A.C was inversely associated with serum folate levels. Additionally, it was also observed that the presence of the mutant allele in MTHFR 677C.T, ApoE, eNOS 2786T.C and intron 4a/b were significantly related to serum vitamin B12 levels. The constructed haplotypes for MTHFR 677C.T and 1298A.C as well as the eNOS 2786T.C, intron 4a/b,

and 1894G.T demonstrated a significant distribution of MTHFR and eNOS haplotypes between cases and controls except for the MTHFR C677C1298 and eNOS C-786aG1894 haplotypes (Table 5). Haplotype MTHFR T677A1298 and T677C1298 increase the risk toward ischemic stroke significantly (OR, 1.98; CI, 1.41-2.78; P , .001 and OR, 3.67; CI, 1.60-8.43; P , .001, respectively). For eNOS polymorphism, haplotype patterns of C-786aT1894 (OR, 2.13; CI, 1.01-4.52; P , .05), T-786aG1894 (OR, 3.75; CI, 2.07-6.79; P , .001), and T-786aT1894 (OR, 3.51;

CLINICAL RELEVANCE OF MTHFR, eNOS, ACE, And ApoE GENE POLYMORPHISMS

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Table 4. The influence of MTHFR, eNOS, ACE, and ApoE polymorphisms on serum vitamin profiles Genotypes MTHFR 677C.T CC CT TT P value MTHFR 1298A.C AA AC CC P value eNOS 2786T.C TT CT CC P value eNOS intron 4a/b bb ba aa P value eNOS 1894G.T GG GT TT P value ACE I/D II ID DD P value ApoE E3E3 E3E4 E2E3 E2E4 E2E2 E4E4 P value

Homocysteine (mmol/L)

Folate (nmol/L)

Vitamin B12 (pg/mL)

12.15 6 1.44 (403) 12.70 6 1.43 (160) 15.09 6 1.48 (31) .004

11.93 6 1.85 (403) 12.14 6 1.96 (160) 11.13 6 1.75 (31) NS

509.12 6 1.58 (403) 552.37 6 1.55 (160) 625.03 6 1.49 (31) .014

12.53 6 1.43 (370) 12.04 6 1.45 (199) 14.24 6 1.54 (25) NS

12.15 6 1.88 (370) 12.09 6 1.87 (199) 8.32 6 1.71 (25) .013

529.70 6 1.52 (370) 520.49 6 1.67 (199) 516.25 6 1.55 (25) NS

12.65 6 1.45 (401) 11.83 6 1.41 (138) 12.42 6 1.43 (55) NS

11.74 6 1.93 (401) 12.07 6 1.67 (138) 13.20 6 2.00 (55) NS

540.67 6 1.55 (401) 472.63 6 1.56 (138) 563.22 6 1.72 (55) .005

11.85 6 1.43 (350) 12.58 6 1.42 (118) 14.05 6 1.46 (126) ,.001

11.95 6 1.86 (350) 12.05 6 1.77 (118) 11.82 6 2.03 (126) NS

494.20 6 1.61 (350) 557.82 6 1.41 (118) 592.14 6 1.57 (126) ,.001

11.98 6 1.45 (76) 12.23 6 1.41 (214) 12.69 6 1.46 (304) NS

12.83 6 1.86 (76) 11.75 6 1.79 (214) 11.86 6 1.93 (304) NS

523.89 6 1.71 (76) 515.50 6 1.53 (214) 534.11 6 1.57 (304) NS

12.25 6 1.52 (215) 12.59 6 1.42 (294) 12.35 6 1.30 (85) NS

12.07 6 1.89 (215) 12.02 6 1.89 (294) 11.37 6 1.79 (85) NS

514.47 6 1.58 (215) 527.83 6 1.58 (294) 549.88 6 1.52 (85) NS

12.72 6 1.41 (300) 11.71 6 1.43 (143) 12.40 6 1.46 (80) 12.93 6 1.49 (50) 12.37 6 1.59 (12) 12.38 6 1.80 (9) NS

11.13 6 1.84 (300) 13.03 6 1.73 (143) 11.65 6 2.00 (80) 13.78 6 2.33 (50) 14.09 6 1.58 (12) 14.17 6 1.74 (9) NS

550.35 6 1.50 (300) 475.72 6 1.62 (143) 507.64 6 1.76 (80) 550.21 6 1.54 (50) 600.56 6 1.36 (12) 515.86 6 1.63 (9) .034

Abbreviations: ACE, angiotensin converting enzyme; ApoE, apolipoprotein-E; eNOS, endothelial nitric oxide synthase; MTHFR, methylenetetrahydrofolate reductase; NS, not significant. The number in bold represents statistically significant (P, .05).

CI, 2.51-4.89; P , .001) were significantly higher among patients with ischemic stroke (Table 5). Of the four possible MTHFR and eight eNOS haplotypes, two of the MTHFR and eNOS haplotypes showed significant association with serum homocysteine and vitamin B12 levels (Table 6). Whereas none of the MTHFR and eNOS haplotypes were significantly associated with serum folate levels (Table 6).

Discussion The role of hyperhomocysteinemia as an independent risk factor for ischemic stroke has been reiterated in our

study. Serum homocysteine, folate, and vitamin B12 levels were significantly elevated among cases when compared to controls, indicating a positive risk of ischemic stroke.22,23 It has been reported that vitamin B12 deficiency is positively correlated with hyperhomocysteinemia, which is partly attributed to low levels of folate.24 Therefore, inadequate folate or vitamin B12 can affect homocysteine excretions via urinations and thus lead to hyperhomocysteinemia.24 This event will promote thrombosis and atherosclerosis, which eventually leads to ischemic stroke by decreasing nitric oxide levels.25 Based on the genetic variation data, MTHFR 677CT, 677TT, and 1298CC variant genotypes were significantly

L.K. WEI ET AL.

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Table 5. Distributions and susceptibility risks of MTHFR and eNOS haplotypes among cases and controls Haplotypes* MTHFR CA CC TA TC eNOS CbG CbT CaG CaT TbG TbT TaG TaT

Controls

Cases

P valuey

OR (95% CI)

P valuez

.69 .18 .11 .02

.59 .17 .19 .05

,.001 NS ,.001 .005

.62 (.49-.80) .93 (.68-1.28) 1.98 (1.41-2.78) 3.67 (1.60-8.43)

,.001 NS ,.001 ,.001

.06 .16 .01 .02 .21 .41 .03 .09

.03 .08 .02 .04 .15 .28 .10 .30

.002 ,.001 NS .049 .005 ,.001 ,.001 ,.001

.31 (.15-.64) .47 (.32-.68) 2.86 (.84-9.79) 2.13 (1.01-4.52) .67 (.50-.90) .61 (.48-.76) 3.75 (2.07-6.79) 3.51 (2.51-4.89)

.001 ,.001 NS .041 .007 ,.001 ,.001 ,.001

Abbreviations: eNOS, endothelial nitric oxide synthase; MTHFR, methylenetetrahydrofolate reductase; CI, confidence interval; OR, odds ratio; NS, not significant. *Loci MTHFR 677C.T (rs1801133) and 1298A.C (rs1801131); eNOS 2786T.C (rs2070744), intron 4a/b and 1894G.T (rs1799983). yP value was obtained from chi-square test. zP value adjusted by 10000 permutation test.

associated with the risk of ischemic stroke, which is supported by previous meta-analyses.26-28 A meta-analysis on MTHFR 677TT genotype carriers from South Asians showed a lower magnitude of susceptibility risk of ischemic stroke when compared with carriers of the European descent,28 suggesting that different study populations may exhibit different extent of susceptibility risk. MTHFR 1298CC genotype was significantly associated with increased ischemic stroke risk, which is consistent with previous studies.29 Ulvik et al30 has suggested that the MTHFR 1298A.C polymorphism has less impact on

enzyme activity when compared to the MTHFR 677C.T polymorphism. Furthermore, the influence of MTHFR 1298A.C polymorphism on serum folate level is still questionable and to be reconfirmed in larger samples. For eNOS polymorphisms, the intron 4 variant aa genotype tends to demonstrate higher odds toward ischemic stroke, whereas the 2786T.C and 1894G.T showed a lack of significant association against ischemic stroke. These findings are in agreement with several case–control studies.10-12,31,32 Akhter et al.33 reported that lower levels of nitric oxide is associated with ischemic stroke,

Table 6. The influence of MTHFR and eNOS haplotypes on serum vitamin profiles Homocysteine (mmol/L) Haplotypes MTHFR CA CC TA TC eNOS CbG CbT CaG CaT TbG TbT TaG TaT

b

t test

2.02 .00 .04 .02

6.32 .01 9.72 .46

2.04 2.02 .03 .03 2.02 2.02 .03 .04

2.63 2.34 .38 1.50 4.53 2.96 1.96 19.00

Folate (nmol/L)

Vitamin B12 (pg/mL)

b

t test

P value*

b

t test

.013 NS .002 NS

.02 2.04 .00 2.02

2.24 3.16 .00 .10

NS NS NS NS

2.01 2.02 .04 .09

1.85 2.26 4.73 6.18

NS NS .031 .013

NS NS NS NS .033 NS NS ,.001

.01 .03 .01 .04 .02 2.02 2.02 .00

.03 1.31 .01 .72 1.38 2.50 .41 .02

NS NS NS NS NS NS NS NS

2.02 2.02 .06 .00 2.03 2.02 .03 .05

.41 1.44 1.12 .09 3.30 4.53 2.52 18.00

NS NS NS NS NS .035 NS ,.001

P value*

Abbreviations: eNOS, endothelial nitric oxide synthase; MTHFR, methylenetetrahydrofolate reductase; NS, not significant. *P values derived from t test were adjusted for 10000 permutation test.

P value*

CLINICAL RELEVANCE OF MTHFR, eNOS, ACE, And ApoE GENE POLYMORPHISMS

indicating that eNOS has a neuroprotective effect and play the role of endothelial dysfunction in ischemic stroke pathogenesis. Recently, meta-analyses have reported the significant association between eNOS 4a/b polymorphism and increased risk of ischemic stroke, particularly in Asian population rather than Caucasian population.34-37 Lower nitric oxide plasma level was detected among 4a allele carriers than in the 4b wild-type allele.31 A similar trend found in genotype–phenotype studies on NOS protein concentration and enzyme activity, indicates that protein expression and enzyme activity were lower in 4a4b genotype as compared to 4b4b genotype.38 Moreover, the mutant 4a allele of eNOS may predispose a harmful effect on atherosclerosis, which is an independent risk factor for ischemic stroke.39 Our findings demonstrate that the ApoE E2E3 genotype frequency was significantly higher among ischemic stroke patients compared with the control group. This result is consistent with a recent meta-analysis, whereby, individuals with E2E3 genotype are 1.49 times more likely to develop ischemic stroke than individuals with the E3E3 genotype.40 Therefore, it is reasonable to hypothesize that the E2E3 genotype of ApoE is a risk factor for the development of ischemic stroke. Nevertheless, the distribution of ACE ID and DD genotype frequencies were significantly higher in the case group, which indicated that the heterozygous ID and variant DD genotypes are protective factors for the development of ischemic stroke. Our result is in agreement with Hong et al.13 and Abboud et al,14 whereas other studies demonstrated lack of significant association of this polymorphism with ischemic stroke risk.41-44 Identification of the susceptibility haplotypes demonstrated that different haplotypic patterns are associated with either protective or increased risk against ischemic stroke. Haplotype analysis revealed that MTHFR T677A1298, T677C1298, eNOS C-786aT1894, T-786aG1894, and T-786aT1894 haplotype combinations were significantly higher in the cases group with 1.98- to 3.75-fold increased risk of ischemic stroke. It can be clearly seen that MTHFR 677T and eNOS intron 4a alleles may act as independent biomarkers for predicting the risk of ischemic stroke. Although there is some evidence that MTHFR, eNOS, and ApoE polymorphisms are associated with ischemic stroke, little is known about its effect on the serum vitamin profiles.45 Genotypic and haplotypic findings confirmed that the MTHFR 677C.T and eNOS intron 4a/b polymorphisms are important mediators for serum homocysteine and vitamin B12 levels which may predispose individuals to ischemic stroke. The present study is in concordance with the previous studies on the positive association between MTHFR 677C.T and eNOS intron 4a/b polymorphisms with serum homocysteine and vitamin B12 levels.7,45 In contrast, elevated levels of serum homocysteine and vitamin B12 were observed among the MTHFR 677TT and eNOS intron 4a/b aa

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genotype carriers. Functionally, MTHFR enzyme activity is reduced 35% with the heterozygous CT genotype and 70% with the variant TT genotype.29 A recent study reported the association between MTHFR 677C.T gene polymorphisms and increased serum homocysteine levels and thus influences the risk of ischemic stroke, where the total homocysteine concentrations are typically higher among TT carriers as compared to CC carriers.28 Additionally, the eNOS 2786T.C and ApoE polymorphisms are reported to mediate serum vitamin B12 but not homocysteine levels as indicated by Fatini et al.45 Thus, this evidence further suggests that serum vitamin status has a major influence on serum homocysteine levels because there was no significant influence of eNOS 2786T.C and ApoE polymorphisms on serum folate levels. In addition, Arai et al46 reported that the ApoE E4 polymorphism contributed to a decrease levels of serum vitamin B12 and white matter integrity, which can affect the blood brain barrier and initiate the ischemic cascade leading to stroke. Haplotype pattern of MTHFR T677A1298 significantly influenced the homocysteine levels in the present study, which is in consistent with that of Han et al.47 Furthermore, eNOS T-786aT1894 haplotype demonstrated an increased level of serum homocysteine that resulting in an increased risk of ischemic stroke among Malays population.

Conclusions In conclusion, the MTHFR (677C.T and 1298A.C), eNOS intron 4a/b, and ApoE (E2/E3/E4) polymorphisms have been significantly associated with the increased risk of ischemic stroke, varying between 1.86- and 16.77-fold. The genotype and haplotype patterns of MTHFR 677C.T and eNOS intron 4a/b polymorphisms may be the predominant genetic determinants that predispose individuals to ischemic stroke by increasing serum homocysteine and vitamin B12 levels, which are likely to initiate the respective pathophysiology observed in ischemic stroke. Future retrospective cohort studies with larger sample size and meta-analyses are warranted to better understand the association of these polymorphisms with ischemic stroke, before translating it into clinical practice. Acknowledgment: We thank medical team members that involved in this research. The researchers wish to thank the Griffith Health Institute and to acknowledge financial support for this work from Griffith University. First author was the recipient of Endeavour Research Fellowship and MyBrain 15 (MyPhD) scholarship.

References 1. Go AS, Mozaffarian D, Roger VL, et al. Heart disease and stroke statistics–2014 update: a report from the American Heart Association. Circulation 2014;129:e28-e292.

8 2. Loo KW, Gan SH. Burden of stroke in Malaysia. Int J Stroke 2012;7:165-167. 3. Pezzini A, Del Zotto E, Padovani A. Homocysteine and cerebral ischemia: pathogenic and therapeutical implications. Curr Med Chem 2007;14:249-263. 4. Dominiczak MH, Caslake MJ. Apolipoproteins: metabolic role and clinical biochemistry applications. Ann Clin Biochem 2011;48(Pt 6):498-515. 5. McCully KS. Chemical pathology of homocysteine. IV. Excitotoxicity, oxidative stress, endothelial dysfunction, and inflammation. Ann Clin Lab Sci 2009;39:219-232. 6. Sen U, Tyagi SC. Homocysteine and hypertension in diabetes: does PPARgamma have a regulatory role? PPAR Res 2010;2010:806538. 7. Sazci A, Ergul E, Tuncer N, et al. Methylenetetrahydrofolate reductase gene polymorphisms are associated with ischemic and hemorrhagic stroke: dual effect of MTHFR polymorphisms C677T and A1298C. Brain Res Bull 2006; 71:45-50. 8. Hassan A, Gormley K, O’Sullivan M, et al. Endothelial nitric oxide gene haplotypes and risk of cerebral smallvessel disease. Stroke 2004;35:654-659. 9. Wei LW, Menon S, Griffiths LR, et al. Signaling pathway genes for blood pressure, folate and cholesterol levels among hypertensives: an epistasis analysis. J Hum Hypertens 2015;29:99-104. 10. Moe KT, Woon FP, De Silva DA, et al. Association of acute ischemic stroke with the MTHFR C677T polymorphism but not with NOS3 gene polymorphisms in a Singapore population. Eur J Neurol 2008;15:1309-1314. 11. Cheng J, Liu J, Li X, et al. Effect of polymorphisms of endothelial nitric oxide synthase on ischemic stroke: a case-control study in a Chinese population. Clin Chim Acta 2008;392:46-51. 12. Munshi A, Rajeshwar K, Kaul S, et al. VNTR polymorphism in intron 4 of the eNOS gene and the risk of ischemic stroke in a South Indian population. Brain Res Bull 2010;82:247-250. 13. Hong SH, Park HM, Ahn JY, et al. ACE I/D polymorphism in Korean patients with ischemic stroke and silent brain infarction. Acta Neurol Scand 2008;117:244-249. 14. Abboud S, Viiri LE, L€ utjohann D, et al. Associations of apolipoprotein E gene with ischemic stroke and intracranial atherosclerosis. Eur J Hum Genet 2008;16:955-960. 15. Loo KW, Griffiths LR, Gan SH. A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C . T, eNOS 1894 G . T and - eNOS -786 T . C variants among Malaysian Malays. BMC Med Genet 2012;13:34. 16. Shi M, Caprau D, Romitti P, et al. Genotype frequencies and linkage disequilibrium in the CEPH human diversity panel for variants in folate pathway genes MTHFR, MTHFD, MTRR, RFC1, and GCP2. Birth Defects Res A Clin Mol Teratol 2003;67:545-549. 17. Xin Y, Song X, Xue H, et al. A common variant of the eNOS gene (E298D) is an independent risk factor for left ventricular hypertrophy in human essential hypertension. Clin Sci (Lond) 2009;117:67-73. 18. Ramachandran V, Ismail P, Stanslas J, et al. Association of insertion/deletion polymorphism of angiotensinconverting enzyme gene with essential hypertension and type 2 diabetes mellitus in Malaysian subjects. J Renin Angiotensin Aldosterone Syst 2008;9:208-214. 19. Pantelidis P, Lambert-Hammill M, Wierzbicki AS. Simple sequence-specific-primer-PCR method to identify the three main apolipoprotein E haplotypes. Clin Chem 2003;49:1945-1948.

L.K. WEI ET AL. 20. Purcell S, Neale B, Todd-Brown K, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. The American Journal of Human Genetics 2007;81:559-575. 21. Stephens M, Donnelly P. A comparison of bayesian methods for haplotype reconstruction from population genotype data. The American Journal of Human Genetics 2003;73:1162-1169. 22. Khan U, Crossley C, Kalra L, et al. Homocysteine and its relationship to stroke subtypes in a UK black population: the south London ethnicity and stroke study. Stroke 2008; 39:2943-2949. 23. Ma Y, Zhao X, Zhang W, et al. Homocysteine and ischemic stroke subtype: a relationship study in Chinese patients. Neurol Res 2010;32:636-641. 24. Liew SC, Gupta ED. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: epidemiology, metabolism and the associated diseases. Eur J Med Genet 2015;58:1-10. 25. Sung ML, Wu CC, Chang HI, et al. Shear stress inhibits homocysteine-induced stromal cell-derived factor-1 expression in endothelial cells. Circ Res 2009;105:755-763. 26. Bentley P, Peck G, Smeeth L, et al. Causal relationship of susceptibility genes to ischemic stroke: comparison to ischemic heart disease and biochemical determinants. PLoS One 2010;5:e9136. 27. Wang XM, Wu HY, Qiu XJ. Methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism and risk of preeclampsia: an updated meta-analysis based on 51 studies. Arch Med Res 2013;44:159-168. 28. Yadav S, Hasan N, Marjot T, et al. Detailed analysis of gene polymorphisms associated with ischemic stroke in South Asians. PLoS One 2013;8:e57305. 29. Zhou BS, Bu GY, Li M, et al. Tagging SNPs in the MTHFR gene and risk of ischemic stroke in a Chinese population. Int J Mol Sci 2014;15:8931-8940. 30. Ulvik A, Ueland PM, Fredriksen A, et al. Functional inference of the methylenetetrahydrofolate reductase 677C . T and 1298A . C polymorphisms from a large-scale epidemiological study. Hum Genet 2007; 121:57-64. 31. Kim OJ, Kim UK, Oh SH, et al. Association of endothelial nitric oxide synthase polymorphisms and haplotypes with ischemic stroke in Korean individuals with or without diabetes mellitus. Mol Med Rep 2010;3:509-513. 32. Hou L, Osei-Hyiaman D, Yu H, et al. Association of a 27-bp repeat polymorphism in ecNOS gene with ischemic stroke in Chinese patients. Neurology 2001; 56:490-496. 33. Akhter MS, Biswas A, Rashid H, et al. Screening of the NOS3 gene identifies the variants 894G/T, 1998C/G and 2479G/A to be associated with acute onset ischemic stroke in young Asian Indians. J Neurol Sci 2014;344:69-75. 34. Guo X. Endothelial nitric oxide (eNOS) gene G894T and VNTR polymorphisms are closely associated with the risk of ischemic stroke development for Asians: metaanalysis of epidemiological studies. Mol Biol Rep 2014; 41:2571-2583. 35. Yao YS, Chang WW, Jin YL, et al. An updated metaanalysis of endothelial nitric oxide synthase gene: three well-characterized polymorphisms with ischemic stroke. Gene 2013;528:84-92. 36. Wang M, Jiang X, Wu W, et al. Endothelial NO synthase gene polymorphisms and risk of ischemic stroke in Asian population: a meta-analysis. PLoS One 2013;8:e60472. 37. Niu PP, Yang G, Zheng BK, et al. Relationship between endothelial nitric oxide synthase gene polymorphisms

CLINICAL RELEVANCE OF MTHFR, eNOS, ACE, And ApoE GENE POLYMORPHISMS

38.

39.

40.

41.

42.

and ischemic stroke: a meta-analysis. Acta Neurol Scand 2013;128:202-212. Song J, Yoon Y, Park KU, et al. Genotype-specific influence on nitric oxide synthase gene expression, protein concentrations, and enzyme activity in cultured human endothelial cells. Clin Chem 2003;49(6 Pt 1):847-852. Sivri N, Unlu A, Palabiyik O, et al. Endothelial nitric oxide synthase intron 4a/b polymorphism in coronary artery disease in Thrace region of Turkey. Biotechnol Biotec Eq 2014;28:1115-1120. Gu L, Su L, Chen Q, et al. Association between the apolipoprotein E gene polymorphism and ischemic stroke in Chinese populations: new data and meta-analysis. Exp Ther Med 2013;5:853-859. Pera J, Slowik A, Dziedzic T, et al. ACE I/D polymorphism in different etiologies of ischemic stroke. Acta Neurol Scand 2006;114:320-322. Prabhakar P, De T, Nagaraja D, et al. Angiotensinconverting enzyme gene insertion/deletion polymorphism and small vessel cerebral stroke in Indian population. Int J Vasc Med 2014;2014:305309.

9

43. Kumar A, Vivekanandhan S, Srivastava A, et al. Association between angiotensin converting enzyme gene insertion/deletion polymorphism and ischemic stroke in north Indian population: a case-control study and metaanalysis. Neurol Res 2014;36:786-794. 44. Tuncer N, Tuglular S, Kilic G, et al. Evaluation of the angiotensin-converting enzyme insertion/deletion polymorphism and the risk of ischaemic stroke. J Clin Neurosci 2006;13:224-227. 45. Fatini C, Sofi F, Gori AM, et al. Endothelial nitric oxide synthase -786T.C, but not 894G.T and 4a4b, polymorphism influences plasma homocysteine concentrations in persons with normal vitamin status. Clin Chem 2005; 51:1159-1164. 46. Arai K, Lok J, Guo S, et al. Cellular mechanisms of neurovascular damage and repair after stroke. J Child Neurol 2011;26:1193-1198. 47. Han IB, Kim OJ, Ahn JY, et al. Association of methylenetetrahydrofolate reductase (MTHFR 677C.T and 1298A.C) polymorphisms and haplotypes with silent brain infarction and homocysteine levels in a Korean population. Yonsei Med J 2010;51:253-260.