ORIGINAL ARTICLE

Association of methylenetetrahydrofolate reductase C677Tand A1298C polymorphisms with myocardial infarctionin Tunisian young patients

Dhouha Berredjeb Ben Slama & Najiba Fekih Mrissa &

Abdeddayem Haggui & Brahim Nsiri & Habib Haouala &

Nasreddine Gritli

Received: 21 December 2012 /Accepted: 30 October 2013# Springer-Verlag London 2013

Abstract Myocardial infarction (MI) is an important clin-ical problem because of its large contribution to mortality.The objective of this study is to assess whether two meth-ylenetetrahydrofolate reductase (MTHFR) polymorphisms,C677T and A1298C, were associated with MI amongTunisian patients. One hundred young patients (<47 yearsold) with MI were recruited and compared with 200 controlsubjects with no history of MI. The most common MI riskfactors were investigated. Fasting plasma homocysteinelevels were measured. Genotypes of the MTHFR C677Tand A1298 polymorphisms were studied by polymerasechain reaction. The mean plasma homocysteine level inthe study group was raised when compared with the controlgroup. Homozygous MTHFR C677T mutation was ob-served in 2 (2 %) patients and in 17 (8.5 %) control subjects,whereas heterozygous MTHFR C677T mutation was de-tected in 82 (82 %) patients versus only 79 (39.5 %) incontrol subjects. The mean total homocysteine concentra-tions were significantly higher in individuals with the677TT and CT genotypes. Our results indicate that C677Tand A1298C MTHFR mutations and hyperhomocysteine-mia contributed to the risk factors for MI.

Keywords Myocardial infarction .MTHFRpolymorphisms .

Elevated homocysteine levels

Introduction

The pathogenic mechanism of myocardial infarction (MI) iscomplex and involves the interaction of multiple environmen-tal and genetic factors. The main causal and treatable riskfactors for MI include diabetes and smoking. In addition tothese risk factors, studies have shown the importance ofgenetic factors and interactions between multiple genes andenvironmental factors in this condition (Collins 1999).Although various association studies have attempted to iden-tify genetic variants that contribute to MI, the genetic compo-nents of these conditions have not been determined definitive-ly (Weiss et al. 1996; Helgadottir et al. 2006). Among thesegenetic factors, the methylenetetrahydrofolate reductase(MTHFR) gene has received special attention since it has beenimplicated in hyperhomocysteinemia that, in turn, has beenassociated with an increased risk of MI (Kölling et al. 2004).Plasma homocysteine (Hcy) levels are modulated by bothnutritional and genetic factors. One such genetic factor ischaracterized by a point mutation at position 677 of theMTHFR gene resulting in the substitution of valine for alanineand is responsible for reduced enzymatic activity (Frosst et al.1995). A second common polymorphism (A1298C), in theMTHFR gene, results in a glutamate to alanine substitutionwithin a presumed regulatory domain of MTHFR. The 1298Callele has been reported to lead to decreased enzyme activity,although not to the same extent as the 677Tallele (van der Putet al. 1998). Therefore, the purpose of this study was to

D. B. B. Slama :N. Fekih Mrissa (*) :B. Nsiri :N. GritliLaboratory of Molecular Biology, Department of Hematology,Military Hospital of Tunisia, 1008 Mont Fleury, Tunis, Tunisiae-mail: fnajiba@yahoo.fr

A. Haggui :H. HaoualaDepartment of Cardiology, Military Hospital of Tunisia,1008 Mont Fleury, Tunis, Tunisia

Comp Clin PatholDOI 10.1007/s00580-013-1836-z

examine the possible association of the two MTHFR poly-morphisms, C677T and A1298C, in Tunisian young patientsunder 47 years of age with MI.

Material and methods

Selection of case patients and control subjects

This study population comprised 100 young patients whosuffered an MI (mean age 46.92±7.627). They had beenadmitted to the Cardiology Department of the MilitaryHospital of Tunisia between January 2009 and June 2010.Control subjects comprised 200 individuals who were healthyindividuals with no history of MI or thromboembolic disease(mean age 31±9.6). Two conventional risk factors for MI,smoking and diabetes, were present in both patients andcontrols. All patients participated in the study after they hadgiven their full informed consent, and the study protocol wasapproved by the ethics committee of the hospital.

Blood collection and DNA analysis

Peripheral blood from all individuals in the study was collectedin EDTA-containing tubes. DNAwas extracted from the whiteblood cells according to a salting-out method, and the DNAwas stored at 4 °C until amplification (Miller et al. 1988). Amultiplex amplification with biotinylated primers was used toamplify the genomic DNA. The PCR products then underwenta reverse hybridization by the use of a commercial kit(ThromboType® plus, Hain Lifescience, Nehren, Germany).This test was based on the DNA•STRIP® technology andpermits the combined molecular genetic characterization ofthe C677TandA1298Cmutations of the humanMTHFR gene.

Biochemical analysis

Blood was drawn from fasting patients and collected in tubescontaining EDTA. Plasma Hcy specimens were placed on iceand transported to the laboratory within 30 min of collection.The blood was centrifuged within 1 to 2 h after venipuncture.Plasma was frozen at −20 °C until analysis. The automaticanalyzer (DPC Immulite, Siemens, Tarrytown, NY, USA)based on a competitive immunoassay was used to measuretotal homocysteine (tHcy) concentration in plasma accordingto the manufacturer’s instructions; tHcy levels exceeding15 μmol/l were considered elevated.

Statistical analysis

All analyses related to the case–control study were performedusing the Statistical Package for the Social Sciences v.16(SPSS Inc., Chicago, IL, USA). Differences between cases

and controls were evaluated by using the chi-square test forqualitative variables and the Student’s t test for quantitativevariables. In addition, the odds ratio (OR) and 95 % confi-dence intervals (CI) were calculated. Cohen’s d was providedas a measure of effect size for the difference between thequantitative data of pair-wise groups. Probability valuesP <0.05 were considered statistically significant.

Results

The demographic characteristics and prevalence of traditionalrisk factors in the 100 patients of MI and 200 control subjectsare shown in Table 1. The two groups were sex-matched; thevast majority of patients and control subjects were men with 84male patients (84 %) and 129 male controls (64.5 %). Therewas a significantly higher prevalence of smoking in patients(73 %) than in controls (39.5 %). There were no femalesmokers in this study, but male smokers were four times morelikely than non-smoking males to be MI patients (OR=4.2,CI=[2.03–8.68]). Diabetes was also far more common in pa-tients than in control subjects (41 % versus 7.5 %). Malediabetics were more than six times more likely to suffer anMI than non-diabetic males (OR=6.6, CI=[3.09–14.1]), andfemale diabetics had more than three times the risk of malediabetics to suffer anMI (OR=21.5, CI=[5.25–88.4]). It shouldbe noted that the total number of female diabetics in this studywas small, and therefore, the results should be confirmed bystudies with larger populations. The mean plasma Hcy level inthe study group was raised when compared with the controlgroup (Table 2). Women overall had higher homocysteinelevels compared with men, and within each gender, the homo-cysteine levels among patients were statistically significantlyhigher as compared with their respective control group.Cohen’s d for both groups indicated a medium-to-high effect(d(males)=.593, d(females)=.669). Genotype frequencies ofthe MTHFR C677T and A1298C mutations are shown inTable 3. There were 82 (82 %) patients who suffered an MIcompared with 79 (39.5 %) controls who were heterozygousfor the C677T mutation and 2 (2 %) patients who were homo-zygous for the same mutation versus 17 (8.5 %) controls.Although there were no subjects who were homozygous forthe A1298C mutation, 17 (17 %) patients were heterozygousfor this mutation versus 14 (7 %) controls. Both males andfemales had similar risks associated with the C677T mutation.There were no female 677CC patients; however, the risk ratio(provided in Table 3) for females was 1.48 compared to 1.67 formales. The A1298C mutation was statistically significant formales but not for females. The female population of 1298ACcarriers was small, and although the P value did not meet apriori standards of significance, it should be noted that the effectsize is slightly larger for females than for males (OR(males=2.38), OR(females)=3.87). A larger study of female patients

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will be necessary to understand more clearly the role of thismutation in MI. The interaction between tHcy level andMTHFR genotypes was studied in patients and controls. Themean tHcy concentrations were significantly higher in individ-uals with the 677TTand CT genotypes than in individuals withthe 677CC (P <10−3) genotype. However, no association wasobserved between 1298AC genotypes or the combined geno-types and tHcy concentrations. An analysis by gender showedthat only the C677T mutation was a statistically significant riskfactor for elevated homocysteine levels in males alone(P <10−3, d =.508). Although other genotypes did not meetthe threshold for statistical significance, female 1298AA car-riers indicated a small-to-medium effect on elevating homocys-teine levels compared to 1298AC carriers (P=0.3, d =.375).Double wild-type females also indicated a medium-to-largeeffect on elevating homocysteine levels for females (P=0.2,d =.522) (see Table 4).

Discussion

Myocardial infarction is a leading cause of mortality world-wide. Moreover, the prevalence among young people(<45 years old) has increased during the last decade andcurrently represents almost 10% of all myocardial infarctions.Several factors and probably several genes are involved in thepathogenesis of MI (Isordia-Salas et al. 2010). Previous re-ports from several populations have identified an associationof homozygous MTHFR 677T genotypes with an increase inboth plasma Hcy concentrations and risk of MI, particularly ifindividuals have a low folate status (Kölling et al. 2004;Ghazouani et al. 2009; Gülec et al. 2001). Two of the principalresults of our study of Tunisian subjects were the confirmationof increased concentrations of tHcy in patients with MI com-pared with control subjects and the significant associationbetween the C677T polymorphism of the 5,10-MTHFR geneand MI. The association between elevated Hcy levels and MIwas significant in males; however, no effect was found infemales possibly due to the small number of females in thestudy group. A meta-analysis study found only a weak asso-ciation between elevated Hcy and coronary artery disease(CAD) in males (Humphrey et al. 2008), but a recent studyinvolving a relatively high number of women found signifi-cantly increased risk of CAD in females with elevated Hcy(Page et al. 2010). However, another study found no signifi-cant differences according to gender (Balogh et al. 2012).

The association between the C677T polymorphism of the5,10-MTHFR gene and MI was found in both genders, al-though some studies found that the C677T MTHFR variantconferred a higher risk of MI only in males (Tomaiuolo et al.2012). The current study demonstrated a significant associa-tion but other studies have yielded conflicting results (Rallidiset al. 2008; Helfenstein et al. 2005). The MTHFR C677Tvariant has been implicated with higher serum homocysteine

Table 1 Ensemble and by gender characteristics of risk factors among MI patients and control subjects

Characteristic Patients (N=100) Control subjects (N =200) OR (95 %CI) P value

Sex (male/female) 84/16 (84 %/16 %) 129/71 (64.5 %/35.5 %) – –

Age (year) ± SD (all) 46.92±7.62 31±9.6 – –

Age (year) ± SD (males) 46.48±7.32 32.86±8.54 – –

Age (year) ± SD (females) 48.87±8.78 29.28±10.97 – –

Diabetes (all) 41 (41 %) 15 (7.5 %) 8.57 (4.24–17.54) <0.05

Diabetes (males) 32 (38.1 %) 11 (8.5 %) 6.60 (3.09–14.1) <10−3

Diabetes (females) 9 (56.3 %) 4 (5.6 %) 21.5 (5.25–88.4) <10−3

Smoker (all) 73 (73 %) 79 (39.5 %) 4.14 (2.38–7.25) <0.05

Smoker (males) 73 (86.9 %) 79 (61.2 %) 4.20 (2.03–8.68) <10−3

Smoker (females) 0 (0 %) 0 (0 %) – –

Significant P values are in italics

SD standard deviation, P value probability value, CI confidence interval, OR odds ratio

Table 2 Ensemble and by gender homocysteine levels in the study andcontrol groups

Subjects Plasma homocysteinelevels

Cohen’s d P value

(μmol/l, mean ± SD)

Patients (all) (n =100) 17.65±7.89 .574 <0.01Controls (all) (n=200) 13.90±5.77

Patients (males) (n =84) 17.37±8.23 .593 <10−3

Controls (males) (n =129) 13.62±4.62

Patients (females) (n =16) 19.15±5.77 .669 0.02Controls (females) (n =71) 14.41±7.43

Cohen’s d =(difference in means / pooled standard deviation). SignificantP values are in italics

SD standard deviation, P value probability value

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levels. In our study, when considered by gender, only menshowed a statistical significance in the association betweenhigher level of Hcy and carriers of the 677CT genotype. Otherresearch has also hinted that gender (as well as other concom-itant factors) may have a role in hyperhomocysteinemia. Astudy of healthy children showed that 677TT males, whoreported lower dietary folate, exhibited tHcy that was signif-icantly higher than that of C-allele carriers (Papoutsakis et al.2006). Another study of healthy adults found higher serum

homocysteine levels in women carriers of the C variant of theA1298C polymorphism (Clifford et al. 2012).

Several epidemiological studies have identified moderatelyelevated concentrations of tHcy as a potentially modifiablerisk factor for coronary artery disease which may contribute tothe development of atherosclerosis (Boushey et al. 1995; deRuijter et al. 2008; Hanratty et al. 2001). The results of thisstudy dissent with a previously published study by Rallidiset al. (2008) who investigated a very young population

Table 3 Ensemble and by gender distribution of MTHFR C677T and A1298C in MI patients and controls

MTHFR Genotypes Patients Control subjects OR (95 % CI) P value(N=100) (N =200)n (%) n (%)

C677T (all) 677CC 16 (16) 104 (52) 5.69 (3.00–10.9) <0.05677(CT + TT) 82 (82)+2 (2) 79 (39.5)+17 (8.5)

C677T (males) 677CC 16 66 4.45 (2.33–8.48) <0.05677(CT + TT) 66 (66)+2 (2) 52 (26)+11 (5.5) (RR=1.67)

C677T (females) 677CC 0 (0) 38 (19) – <10−3

677(CT + TT) 16 (16)+0 27 (13.5)+6 (3) (RR=1.48)

A1298C (all) 1298AA 83 (83) 186 (93) 2.72 (1.28–5.78) <0.051298AC 17 (17) 14 (7)

A1298C (males) 1298AA 70 (70) 119 (59.5) 2.38 (1.00–5.60) 0.0491298AC 14 (7) 10 (5)

A1298C (females) 1298AA 13 (13) 67 (33.5) 3.87 (0.770–19.3) 0.111298AC 3 (3) 4 (2)

P calculated for carriers of the polymorphism (heterozygotes and homozygotes) versus wild type. SignificantP values are in italics. Note: There were nocarriers of the 1298CC genotype

CI confidence interval, OR odds ratio, P value probability value, RR risk ratio

Table 4 Ensemble and by genderinfluence of MTHFR C677T andA1298C polymorphisms on plas-ma homocysteine concentration

P calculated for carriers of thepolymorphism (heterozygotesand homozygotes) versus wildtype. Cohen’s d =(difference inmeans / pooled standard devia-tion). Significant P values are initalics

P value probability value,SD standard deviation

MTHFR Genotypes Plasmahomocysteinelevels (μmol/l,mean ± SD)

Cohen’s d P value

C677T (all) CC (n =120) 13.30±6.30 .465 <10−3

CT (n =161) + TT (n=19) 16.36±6.80

A1298C (all) AA (n =269) 15.28±7.00 .216 0.2AC (n =31) + CC (n =0) 13.82±4.20

C677T/A1298C (all) CC/AA (n =120) 16.03±6.84 .259 0.1(CT or TT) and (AC or CC) (n =31) 14.41±4.40

C677T (males) CC (n =82) 13.12±2.52 .508 <10−3

CT (n =118) + TT (n =13) 16.34±7.86

A1298C (males) AA (n =189) 15.10±6.56 .002 0.8AC (n =24) + CC (n =0) 15.11±6.51

C677T/A1298C (males) CC/AA (n =82) 15.40±4.40 .216 0.4(CT or TT) and (AC or CC) (n =24) 14.48±3.90

C677T (females) CC (n =38) 15.02±7.66 .064 0.8CT (n =43) + TT (n=6) 15.49±7.20

A1298C (females) AA (n =80) 15.50±7.50 .375 0.3AC (n =7) + CC (n =0) 12.77±5.40

C677T/A1298C (females) CC/AA (n =38) 17.38±6.30 .522 0.2(CT or TT) and (AC or CC) (n =7) 14.17±6.23

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(<35 years of age) with premature MI. Those authors did notfind an independent association between the carriers with theMTHFR 677TT genotype and the risk of premature MI. Infact, some studies have actually showed a negative associationbetween C677TandMI (Verhoef et al. 1997; Christensen et al.1997). Those discrepancies observed may be due to severalfactors: the definition of patients and controls, the size and ageof the sample, the ethnic background, and the influence ofenvironmental factors (Franco et al. 1998).

Our results, as in other studies (e.g., Chen et al. (2012)),indicate that the MTHFR A1298C polymorphism is associat-ed with an increased risk of MI. It appears, however, in thisstudy that the A1298C polymorphism contributes significant-ly neither to hyperhomocysteinemia alone nor in combinationwith the C677T polymorphism. Friso et al. (2002) have re-ported similar results. The relationship between the MTHFRC677T and A1298C gene polymorphisms and CAD risk hasnot been clearly established.

Conclusion

In the present study, the C677T and A1298C polymorphismsin the 5,10-MTHFR gene were associated with the develop-ment of MI in the Tunisian population. The most importantlimitation of this study was the small number of patients. Also,the disparate findings when gender was considered may pos-sibly be due to the study group containing more men thanwomen. Modifiable risk factors such as smoking and diabetesrepresented independent risk factors for MI. Because MI is amultifactorial disease involving strong interactions betweengenes and modifiable factors, more studies are needed todetermine the specific role of the C677T and A1298C poly-morphisms and homocysteine level variations in our youngpopulation.

Acknowledgments We would like to thank Dr. Christian Winchell forhis precious help in correcting this manuscript.

Conflict of interest None declared.

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