atherosclerosis is a common disease primarily affecting large 
 arteries , which begins in childhood and progresses with age . it is the principal cause of heart attack , stroke , and gangrene of the extremities 
 . several pathophysiologic mechanisms , including the inflammatory response , immune response , cellular growth and proliferation , lipoprotein metabolism and coagulation , each of which are regulated by numerous gene products , can contribute to atherosclerosis either individually or in concert . one of the candidate genes for the development of atherosclerosis 
 regardless of localization is methylenetetrahydrofolate reductase 
 ( mthfr ; ec 1.5.1.20 ) . mthfr is a regulatory enzyme of the 
 homocysteine ( hcy ) metabolism , and is also necessary in the 
 metabolism of tetrahydrofolate , as well as in the synthesis of 
 purine , dna , and rna . elevated plasma total homocysteine ( thcy ) 
 concentration is now widely accepted as a major independent risk 
 factor for cerebrovascular , peripheral vascular disease and may 
 explain , at least in part , the occurrence of coronary artery 
 disease ( cad ) in patients who do not have dyslipidemia , 
 hypertension , and other conventional risk factors [ 3 , 4 ] . hcy is a sulphur - containing amino acid , formed by demethylation 
 of the essential amino acid methionine . it can be either degraded 
 by transsulfuration , involving the vitamin b6-dependant enzyme 
 cystathionine -synthase , or remethylated to methionine , 
 involving the cobalamine ( vitamin b12-dependant enzyme ) 
 methionine synthase . elevations of thcy might result from 
 nutritional deficiencies , including folate , pyridoxal phosphate 
 ( vitamin b6 ) , and methylcobalamin ( vitamin b12 ) , or from 
 genetically determined abnormalities of hcy metabolism ( e.g. , 
 mthfr ) , or a combination of these [ 5 , 6 ] . mthfr is an enzyme that reduces 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate , the main circulating 
 form of folate and the methyl donor for the remethylation of hcy 
 to methionine . a thermolabile form of mthfr has been identified 
 [ 7 , 8 ] and found to be caused by a missense mutation in its 
 encoding gene , with the cytidine residue at nucleotide position 
 677 being replaced by thymidine ( mthfr c677 t ) , resulting in the 
 substitution of valine for alanine in the enzyme . the homozygous 
 677tt genotype of this mutation has been found to specify a 
 variant enzyme with reduced activity and to be associated with 
 elevated thcy levels , particularly in the setting of low folate 
 levels , as compared to the wild - type ( 677cc ) and heterozygous 
 ( 677ct ) genotypes . also , data have shown that the frequency of the 
 mutation varies among different populations [ 9 , 10 ] . these 
 considerations have raised the possibility that the relatively 
 common c677 t mutation in mthfr might be an important genetic risk 
 factor for cardiovascular disease through its effects on 
 homocysteine metabolism [ 11 , 12 ] . the aim of this study was to investigate the frequency of mthfr 
 c677 t mutation and its association to cad and to fasting plasma 
 thcy concentrations in moroccan population . we also evaluated 
 interactions between mild elevated thcy levels , mthfr c677 t 
 polymorphism , and conventional risk factors of cad . the study sample consisted of individuals undergoing coronary 
 angiography because of either symptoms of suspected cad or 
 unrelated conditions requiring angiographic evaluation ( e.g. , 
 cardiomyopathy , valvular disease ) . patients were designated as 
 having cad if they had 50% stenosis of at least one 
 coronary artery , and having no cad if < 10% stenosis was 
 present in all major vessels . the protocol was ethically approved by the review board of the 
 national league of cardiology , rabat , morocco . the study consisted 
 of 400 unrelated individuals ( age mean = 50  10 ) in whom 
 coronary angiography was clinically indicated ( 218 men , 182 
 women ) . the group of cases was formed by 210 ( 52.5% ) patients 
 with angiographically confirmed cad . the group of controls 
 included 190 ( 47.5% ) subjects with normal coronary angiograms . patients with an acute illness , such as 
 myocardial infarction or coronary artery bypass graft surgery 
 occurring three months prior the study , or with a chronic disease , 
 such as chronic renal , hepatic or thyroid failure , were excluded 
 of the protocol . arterial hypertension was defined in the presence of active 
 treatment with antihypertensive agents or otherwise as systolic 
 blood pressure of 140 mmhg and/or diastolic blood pressure 
 of 90 mmhg on at least two separate occasions . patients were considered to have diabetes 
 mellitus if they were receiving active treatment with insulin or 
 oral hypoglycemic agents , or if fasting glucose in the serum was 
 126 mg / dl . dyslipidemia was defined as triglycerides ( tg ) 
 value of 1.50 mmol / l or ldl cholesterol ( ldlc ) value of 
 3.92 the weight and height were measured and the 
 body mass index ( bmi ) was obtained from the ratio of weight ( kg ) 
 to height squared ( m ) . subjects were considered 
 substantially obese when the bmi was > 30 kg / m . twelve hour fasting venous blood samples were obtained from all 
 subjects at the day of the coronary angiography . venous blood was 
 collected into heparinized tubes and immediately centrifuged at 
 1.550 g for 10 minutes . total serum cholesterol , hdl cholesterol , 
 and triglycerides were measured by enzymatic methods on a hitachi 
 analyzer using standard kits ( roche diagnostics ) . ldl cholesterol 
 was calculated with the friedewald formula in subjects with 
 triglycerides levels below 4.40 mmol / l . plasma total homocysteine was determined by immunoassay ( axis 
 biochemicals , oslo , norway ) . the assay method was based on an 
 enzymatic conversion of homocysteine to s - adenosyl - l - homocysteine , 
 followed by quantification of s - adenosyl - l - homocysteine by an 
 enzyme - linked immunoassay . the coefficients of variation 
 within and between days for the assays were 5% or less . cross 
 reactivity with glutathione , l - cysteine , adenosine , 
 l - cystathionine was below 0.1% . dna was extracted from white cells of fresh or frozen peripheral 
 blood by phenol - chloroforme - isoamylalcool method . the region 
 surrounding the 677 mthfr mutation site ( a 223 bp fragment ) 
 was polymerase chain reaction - amplified , using the following 
 primers ( mthfr 618a : 5-agctttgaggctgacctgaag-3 and mthfr 14 - 5b : 5-aggacggtgcggtgagagtg-3 ) ( oligo express ) 
 . pcr amplification reactions were performed with 300 ng of genomic dna in pcr buffer containing 1.5 mm 
 mgcl2 , 200 m dntps , 0.1 m of each primer , and 1u taq polymerase ( applied biosystems ) . the reaction mixture ( 20 l ) was denatured at 94c 
 for 1 minute , annealed at 58c for 15 seconds and extended 
 at 72c for 30 seconds for a total of 34 cycles . since the 
 polymorphism creates a hinfi recognition site , 5 l of 
 the pcr product was then digested with 5u hinfi , 2 l 10x 
 reaction ne buffer 2 ( new england , biolabs ) and 12.5 l of 
 water at 37c for 3 hours . the digestion product was 
 electrophoresed on a 2.5% agarose gel . the wild - type allele ( 677c ) 
 corresponds to the presence of one band of 223 bp , and the mutant 
 allele ( 677 t ) corresponds to the presence of two bands of 175 bp 
 and 48 bp . the heterozygous genotype ( ct ) corresponds to the 
 presence of three bands of 223 , 175 , and 48 bp . skewed variables were naturally 
 log - transformed to normalize the distribution before any 
 statistical analysis . the student 's t - test and 
 one - way anova procedures were used for mean comparison 
 between groups . hardy - weinberg equilibrium was assessed by 
 chi - square test , which was equally used for the 
 comparison of categorical variables between two groups . the 
 comparison between three groups was assured by the 
 kruskal - wallis test and the adequate post hoc 
 multiple comparisons . model selection log - linear analysis based on 
 backward elimination was used for highly correlated variables 
 identification , and the discriminant analysis with stepwise method 
 was computed for identifying variables that have significant 
 effects on cad and on hhcy . on the basis of the previous 
 statistical analysis , logistic regression model with backward 
 likelihood ratio method was used to compute odds ratio ( ors ) and 
 their 95% confidence interval ( 95% ci ) . all statistical 
 analysis was performed with spss for windows ( release 7.5.1 ; 
 standard version ) . all tests were two - tailed and p - value 
 of < .05 was deemed significant . the demographic characteristics of the study population and the 
 main risk factors are presented in table 1 . as 
 expected , the mean age , frequency of occurrence in 
 men , hypertension , smoking , diabetes , dyslipidemia , and obesity 
 were significantly higher in cases than in controls ( p < .05 ) . the risk of cad were significantly increased by the presence 
 of conventional risk factors ( all 95% ci not contain the value 
 of 0 ) , and varying from  1.5 fold for male compared to 
 female ( or = 1.60 , 95% ci : 1.072.38 , p < .05 ) to  3 fold for smoking habits compared to no smoking ( or = 
 2.97 , 95% ci : 1.844.78 , p < .001 ) . independently of cad , no significant difference was found between 
 men and women for mthfr c677 t genotype frequencies ( p = .880 ) . 
 in women , the genotype frequencies were 53.3% ( 21.4% in 
 cases and 31.9% in controls ) , 35.7% ( 17.0% in cases and 
 18.7% in controls ) , and 11.0% ( 7.7% in cases and 3.3% 
 in controls ) for 677cc , 677ct , and 677tt , respectively . in men , 
 the genotype frequencies were 53.7% ( 28.4% in case and 
 25.2% in controls ) , 33.9% ( 21.6% in cases and 12.4% in 
 controls ) , and 12.4% ( 7.8% in cases and 4.6% in controls ) 
 for 677cc , 677ct , and 677tt , respectively . t 
 polymorphism and cad were significantly independent in men ( p = 
 .302 ) ; but in women , the association was at the limit of the 
 significance ( p = .049 ) . for the overall population , the prevalence of 
 677cc , 677ct , and 677tt genotypes were 53.5% , 34.8% , and 
 11.8% , respectively . the mutant homozygous , 677tt , was recorded 
 in 8.4% of controls and 14.8% of cad patients . the wild - type 
 genotype , 677cc , was found in 59.5% of controls and 48.1% of 
 cad subjects . the difference 
 between controls and cad patients was statistically significant 
 for mthfr genotype and allele frequencies ( p < .05 ) . taking the 
 wild - type genotype as a reference , the mutant genotype increases 
 the risk of cad by  2 fold ( or = 2.17 , 95% 
 ci : 1.124.20 , p < .05 ) , while the cad risk increase associated to the mutant allele ( 677 t ) was  1.5 fold compared to the 
 wild - type allele 677c ( or = 1.54 , 95% ci : 1.111.84 , 
 p < .01 ) . subjects with cad had significantly ( p < .001 ) higher thcy levels ( mean  sd = 14.9  3.1 l as 
 the cut - off point to classify mild hyperhomocysteinaemia ( hhcy ) , 
 49.5% of cad patients ( 49.2% men , 50.0% women ) and 
 21.1% of controls ( 27.2% men , 15.3% women ) had hhcy 
 ( table 2 ) . mild hhcy was significantly associated to 
 cad ( p < .001 ) in men and women separately . in all subjects , cad was significantly associated to hhcy , with a risk increase of 
 about 4 fold compared to non - hhcy ( or = 3.68 , 95% ci : 
 2.375.72 , p < .001 ) . thcy levels were positively and 
 significantly associated to mthfr gene polymorphism ( spearman 's 
 rho = 0.38 , p < .001 ) . this relationship was observed in 
 case ( spearman 's rho = 0.44 , p < .001 ) and control ( spearman 's rho = 0.32 , p < .01 ) groups . based on the post hoc multiple comparison appropriate for equal variances not 
 assumed , there is a graded and significant ( p < .01 ) 
 increase in thcy levels from 677cc to 677tt genotypes of mthfr 
 gene in both cases and controls groups . the frequencies of the simultaneous presence of cad and hhcy were 
 16.4% , 30.2% , and 57.4% in 677cc , 677ct , and 677tt 
 genotypes , respectively ( see table 2 ) . discriminant analysis and model selection log - linear analysis were computed prior to the logistic regression   analysis based on the backward likelihood ratio method . the final model shows that hhcy is mainly and only significantly ( p < .01 ) affected by the mthfr polymorphism and smoking habits , and their combined effects were not significant ( see table 3 ) . the mutant genotype had the more adverse effect ( or = 11.72 , 95% ci : 4.9127.96 , p < .001 ) than smoking habits ( or = 6.92 , 95% ci : 3.5013.70 , p < .001 ) . as expected , traditional risk factor frequencies were higher in 
 cases than controls , with exception for dyslipidemia and obesity 
 in the 677tt genotypes . in cases subjects , all conventional risk 
 factor frequencies were lower in 677tt genotype than in the 
 remainders ( see table 4 ) . those differences lead to 
 think that conventional risk factors do not have the same effect 
 on cad according to mthfr genotypes . in fact , in subjects with the 
 wild - type genotype , the cad risk increase was significantly 
 associated to smoking habits , obesity , dyslipidemia , and 
 hypertension . in heterozygous individuals , the risk increase was 
 more likely associated to smoking habits and hypertension ; while 
 the hhcy was the sole factor that significantly increases the risk 
 of cad in subjects with the mutant genotype . in addition , it is 
 important to note that hhcy was associated to a significant and 
 graded cad risk increase from  2.5 fold in subjects with 
 677cc genotype ( or = 2.47 , 95% ci : 1.314.65 , 
 p < .01 ) to  9 fold in individuals with 677tt genotype 
 ( or = 8.68 , 95% ci : 2.0536.69 , p < .01 ) . in consequence , it appears that the 677 t allele multiplies by 2 the risk of cad 
 associated to hhcy ( see table 5 ) . on the basis of the results from model selection log - linear 
 analysis and discriminant analysis procedures , logistic regression 
 model based on backward likelihood method was used to compute the 
 or of cad associated to the studied parameters ( see table 6 ) . dyslipidemia , smoking habits , hypertension , and diabetes , as well as hhcy , were independent risk factors for cad . therefore , mthfr 
 polymorphism and hhcy interactive effect were found to be a strong 
 predictor of cad risk ( p < .01 ) . the negative interactive 
 effects between smoking habits and mutant genotype , as well as 
 between dyslipidemia and hhcy , should be more likely the 
 consequence of the small observed frequencies of smokers with 
 677tt genotype and the lowest frequency of hhcy among subjects 
 with dyslipidemia , rather than an eventual protective effect . in accordance with a previous study ( see ) , the present report confirms that cad is strongly and significantly associated 
 to hhcy in moroccan population . this is in agreement with the 
 meta - analysis by christen et al . , in which 43 studies were analyzed . in another meta - analysis , boushey et al .   evaluated 27 studies and showed that hcy is an independent and gradual risk 
 factor for cad . the ors for cad ranged from 1.6 for men to 1.8 for 
 women , for each thcy level elevation of 5 m . the increase 
 in coronary risk resulting from this increase is similar to an 
 increase of 20 mg / dl in total cholesterol levels . the authors considered that 10% of the 
 risk of the general population for the development of cad can be 
 attributed to hcy . the homocysteine hypothesis of atherosclerosis was largely based 
 on the clinical and pathological observations in patients with 
 homocystinuria , a metabolic disorder characterized by markedly 
 increased levels of homocysteine . biologic support of the theory was derived from experimental studies in which homocysteine 
 concentrations far exceeded the levels encountered , even under the 
 most severe pathological conditions , putting doubt on 
 pathophysiological relevance in the clinical setting . the strongest support for a possible causal link between homocysteine 
 levels and atherosclerosis came from retrospective , 
 cross - sectional , or case - control studies . some identified 
 hcy as an independent risk factor [ 18 , 19 ] , but others did not 
 [ 20 , 21 ] . recently , wald et al .   published a 
 comprehensive meta - analysis of 16 prospective studies . the or 
 for a 5 m increase hcy level was 1.23 ( 95% ci = 
 1.141.32 ) . published the results of 
 a 24-year follow - up evaluating hcy quintile , relative risk was 
 1.86 ( 95% ci = 1.063.26 ) for acute myocardial 
 infarction and 5.14 ( 95% ci = 2.2211.92 ) for death due to 
 acute myocardial infarction . our study was of interest because , 
 despite a high prevalence of coronary morbidity and mortality in 
 morocco , the total cholesterol concentrations are relatively low and are not the major cause for the development of 
 atherosclerosis . in the light of these findings , we thought that 
 plasma thcy concentrations might be an important risk factor for 
 cad in morocco . mol / l were a significant and independent risk 
 factor for cad [ 25 , 26 ] . our results confirmed this effect which 
 suggests that smoking increases hhcy , which in turn increases risk 
 of cad . smoking affects the vascular tree , 
 platelet activation , lipid peroxidation , enhanced tissue factor 
 activation , and reduced von willebrand factor , via several 
 different interactive mechanisms [ 28 , 29 ] . nicotine and carbon monoxide separately produce tachycardia , hypertension , and vasoconstriction and both produce direct endothelial damage . smoking also affects vaso - occlusive factors such as platelet aggregation , plasma viscosity , and fibrinogen levels 
 [ 31 , 32 ] . homocysteine may damage 
 endothelial cells and has been shown to be associated with 
 elevated levels of von - willebrand factor in vivo , and with enhanced nitric oxide production in cultured cells 
 , and thus would increase risk of atherosclerosis . also , woo et al . demonstrated impaired flow - mediated dilatation 
 of the brachial artery in 17 healthy individuals who had no other 
 risk factor except hhcy . furthermore , thcy levels and endothelial 
 function were corrected with folic acid supplementation . in 
 addition , some studies have demonstrated an association between 
 homocysteine and an increase risk of initial and recurrent venous 
 thrombosis , and although this has not been shown in all studies , it has been confirmed in a meta - analysis in patients with fasting hhcy . first , as in previous studies , we found a strong 
 link between the mthfr gene c677 t polymorphism and plasma thcy 
 levels . second , our study provides a very reliable phenotype 
 characterization of cad , by performing coronary angiography in all 
 patients and controls . third , our study is the first and the 
 largest one conducted in moroccan population , this facilitates the 
 examination of the thermolabile mthfr genotype as an independent 
 risk factor for cad and of the relations between genotypes , thcy , 
 and risk . in the present study , mthfr gene c677 t polymorphism was 
 significantly associated with cad ( p   < .05 ) . compared with 
 homozygous wild - type , the homozygous mutant of mthfr gene was 
 associated to a significant cad risk increase . in particular , morita 
 et al .   reported that the frequency of this mutation was 
 correlated with the severity of stenotic lesions and the number of 
 stenotic coronary arteries , suggesting that this mutation is 
 closely associated with the severity of cad . therefore , the 
 finding of an increased risk of cad associated with mthfr is not 
 surprising because mthfr is strongly associated with an increased 
 risk of hhcy . in the original description of thermolabile mthfr , selhub 
 and d'angelo et al .   reported that thermolabile mthfr , 
 which is correlated with the 677tt genotype , can be an inherited risk factor for cad . they documented a prevalence of 17% in patients with cad and of 
 5% in control subjects . , in a prospective study of patients with angiographically proved cad , 
 found no relation between mthfr gene c677 t polymorphism and the 
 risk of cad . in a health report of us physicians , the frequency of 
 the mthfr genotypes was similar between patients and control 
 subjects . the discrepancy between the results from all those 
 studies is unclear and may be due to differences in nutritional 
 intake of cofactors required for the mthfr pathway , such as 
 vitamin b12 or folate , or other ethnic differences ( e.g. , weight , bmi ) . our study showed a positive association between the homozygous 
 mutant genotype and elevated plasma thcy levels in both cases and 
 controls . this association suggests that mthfr gene c677 t mutation may increase plasma thcy levels and could be an 
 independent predictor of plasma homocysteine levels , particularly 
 in the setting of low folate status . on this basis , but without 
 implying causality , it is reasonable to suggest that the effect of 
 the 677tt genotype on cad may be mediated through high plasma thcy 
 and low plasma folate concentration . therefore , it is important to note that when cad and sex 
 categories were considered together , mthfr c677 t polymorphism was 
 not associated to cad ( p > .05 ) in men ; but in women this 
 relation was at the limit of the significance ( p = .049 ) . our 
 data do not allow us to conclude clearly about mthfr c677 t 
 mutation and cad association according to sex differences . when we assess the distribution of conventional cardiovascular 
 risk factors according to mthfr c677 t polymorphism , we found that 
 homozygous 677tt with cad had significantly lower frequencies of 
 these risk factors than those having 677ct and 677cc . in 677cc and 
 677ct subjects , the cad risk is mediated by traditional risk 
 factors and hhcy , while in 677tt subjects , the cad risk is 
 conferred only by isolated hhcy . in the studied moroccan population , elevated thcy level is a 
 strong risk factor for cad independently of the traditional risk 
 factors , and this cad risk increase is strongly influenced by 
 mthfr c677 t polymorphism . traditional risk factors were 
 associated to a significant increased risk of cad in subjects with 
 677ct and 677cc genotypes , while hhcy were the main and sole risk 
 factor of cad in subjects with 677tt . for an eventual strategy of 
 cardiovascular disease prevention in morocco , it is obvious that 
 conventional cardiovascular risk factors should be viewed in the 
 context of mthfr genotype and , of course , of other genetic 
 determinants .
increased plasma total homocysteine ( thcy ) levels have been shown to be a risk factor for coronary artery disease ( cad ) . 
 the common methylenetetrahydrofolate reductase c677 t ( mthfr c677 t ) polymorphism has been reported to be a strong predictor of mild hyperhomocysteinaemia ( hhcy ) . 
 we assessed whether this mutation was associated with increased risk of cad and plasma levels of thcy . 
 we also evaluated interactions between this polymorphism , mild elevated thcy levels and conventional risk factors of cad . 
 method . using pcr - rflp analysis 
 , we studied the frequency of the c677 t genotypes and its effect on cad and on thcy concentrations in 400 subjects without and with cad angiographically confirmed . 
 there were 210 subjects with cad and 190 subjects without cad . 
 results . 
 the frequencies of the c677 t genotypes were 53% ( 59.5% in controls versus 48.1% in cases ) , 34.8% ( 32.1 in controls versus 37.1 in cases ) , and 11.8% ( 8.4% in controls versus 14.8% in cases ) , respectively , for 677cc , 677ct , and 677tt . 
 the genotype frequencies were significantly different between case and control groups ( p < .05 ) . 
 the 677 t allele enhances the risk of cad associated to hhcy ( p < .01 ) . in multivariate analysis models , 
 mthfr c677 t polymorphism effect on cad was masked by other risk factors . 
 hhcy was only and independently influenced by mthfr polymorphism and smoking habits , and it is a strong predictor of cad independently of conventional risk factors . conclusion . 
 our data suggest that hhcy is strongly and independently associated to cad risk increase ; and mthfr c677 t polymorphism and smoking habits were the main predictors of thcy levels . 
 the cad risk increase is mainly associated with mild hhcy in 677tt , whereas in 677ct and 677cc it is mainly associated with the conventional risk factors .