Prevalence and Role of Methylenetetrahydrofolate Reductase 677 C→T and 1298 A→C Polymorphisms in Coronary Artery Disease in Arabs Open Access

Elevated plasma homocysteine has been recognized as a major risk factor for coronary artery disease (CAD) in several populations.1 A number of enzymes and cofactors are known to play a role in the metabolism of homocysteine. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate into 5-methyl-tetrahydrofolate, which is the major circulating form of folate. Folate is, in turn, used in many biochemical pathways, including the methylation of homocysteine to methionine. Mutations in the MTHFR gene could reduce the enzyme activity and cause hyperhomocysteinemia as well as vascular complications, including CAD.1,2 

Increased plasma homocysteine levels in humans have been associated with the presence of a common 677 C→T mutation (alanine-to-valine substitution) in the MTHFR gene, which results in thermolability and reduced MTHFR activity.3 Recently, a second less common polymorphism in the MTHFR gene was reported, which results in a glutamate-to-alanine (A→C) change at position 1298 leading to mildly decreased MTHFR activity.4 Both of these polymorphisms have been suggested as risk factors for CAD.5–7 Whether these 2 MTHFR polymorphisms contribute independently to cardiovascular diseases is debatable. Studies attempting to establish the relationship between the presence of these MTHFR variants and CAD have yielded conflicting results. While some studies have shown an association,1,5,7–12 others have failed to establish any such link.2,13–20 The association of MTHFR polymorphisms with CAD was recently reviewed.21,22 

The prevalence of the 677 C→T polymorphism in the European population was 22%, 32%, 37%, and 44% in studies from Norway, Ireland, Britain, and Italy, respectively.23 Among whites outside Europe, the prevalence was similar to that of Britain and Ireland.23 The prevalence ranged from 34% to 37% among whites from Australia, Brazil, Canada, and the United States. The prevalence of this polymorphism in other ethnic groups was 78%, 45.2%, 25.6%, and 44.4% in Mexicans,24 Hungarians,25 Thais,26 and Greek-Cypriots,27 respectively. The majority of studies of Asians have been performed on the Japanese population, and the prevalence among this ethnic group is 34%.23 To date, few reports are available on the prevalence of the 1298 A→C polymorphism among different ethnic groups. The frequency is 9% in 2 studies from Canada4 and the Netherlands,28 while it is 13.8%, 17%, and 41.1% for studies conducted on populations from Germany,13 China,29 and Brazil,30 respectively. In addition, the compound heterozygosity C677T/A1298C was reported to be associated with increased homocysteine and lowered plasma folate levels.23 The frequency of this compound heterozygosity is 15%, 20%, and 17% in studies from Canada,4 the Netherlands,28 and the United States,31 respectively. To our knowledge, no reports are available on the prevalence of the 677 C→T and/or 1298 A→C MTHFR variants in Arab populations. The first aim of this study was to determine the prevalence of these MTHFR variants among the Middle-Eastern Arab population using healthy blood donors (BDs). The second aim was to determine the potential relevance of these 2 MTHFR variants, either individually or combined, and to assess whether these 2 MTHFR variants can act as an independent genetic risk factor for CAD in the Arab population.

The study group was composed of 545 individuals (514 men and 31 women) of Middle-Eastern Arabic descent with angiographically documented CAD (CAD group) and a mean age of 44 ± 0.3 years. Additionally, 625 healthy Middle-Eastern Arab BDs (600 men and 25 women) with a mean age of 37 ± 0.4 years visiting the BD clinic at the King Faisal Specialist Hospital and Research Centre (Riyadh, Saudi Arabia) were recruited as a control group to determine the carrier distribution of the MTHFR 677 C→T and 1298 A→C variants in the general population.

Five milliliters of peripheral blood was collected from all individuals who participated in this study after obtaining their consent. Peripheral blood was anticoagulated by collection in EDTA blood tubes. DNA extraction was performed using the PURGENE DNA isolation kit from Gentra Systems (Minneapolis, Minn) and stored in aliquots at −20°C until required.

Determination of the 677 C→T MTHFR polymorphism was carried out by polymerase chain reaction (PCR) amplification, followed by HinfI restriction enzyme digestion and detection of the fragments on 4% MetaPhor agarose gel. For DNA amplification, we used the forward primer 5′-CCT TGA ACA GGT GGA GGC CAG-3′ and the reverse primer 5′-GCG GTG AGA GTG GGG TGG AG-3′, designed to amplify a 294-base pair (bp) fragment of the MTHFR gene. Each 25-μL PCR reaction contained 2.5 μL of 10;ts reaction buffer with MgCl₂ (Amersham Pharmacia Biotech, Piscataway, NJ), 10 ρmol of each primer, 100 ρmol/μL each of the deoxynucleoside triphosphates (deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate) in Tris HCl buffer (Perkin-Elmer Corporation, Foster City, Calif), 1 U of Taq DNA polymerase (Amersham), and 100 ng of genomic DNA template. The mixture was denatured at 95°C for 5 minutes, and the PCR reaction was carried out for 35 cycles in a GeneAmp 9600 PCR system (Perkin-Elmer) under the following conditions: denaturation at 95°C for 1 minute, annealing at 65°C for 30 seconds, and extension at 72°C for 1 minute. The final extension cycle of 72°C was for 7 minutes. The PCR products were electrophoresed on a 1% agarose gel and detected with 0.5 μg/mL of ethidium bromide to confirm the correct amplicon size. Restriction enzyme digestion was performed using the HinfI restriction enzyme (Stratagene, La Jolla, Calif). After digestion, all fragments were resolved on a 4% MetaPhor agarose gel (FMC Bio-products, Rockland, Me) made up with Tris-buffered ethylenediaminetetraacetic acid buffer containing 0.5 μg/mL of ethidium bromide, and the sizes of the digested amplicons were determined using the 100-bp ladder (Amersham). The 677 C→T MTHFR genotypes were determined according to the method described previously.32 The homozygous 677 C/C genotype resulted in a single fragment of 294 bp. The heterozygous 677 C/T genotype produced 3 fragments of 294, 168, and 126 bp, and the homozygous 677 T/T resulted in 2 fragments of 168 and 126 bp.

Determination of the 1298 A→C MTHFR polymorphism was carried out by PCR, followed by MboII restriction enzyme digestion and detection of the fragments on 4% MetaPhor agarose gel. For DNA amplification, we used the forward primer 5′-CTT TGG GGA GCT GAA GGA CTA CTA C-3′ and the reverse primer 5′-CAC TTT GTG ACC ATT CCG GTT TG-3′, designed to amplify a 163-bp fragment of the MTHFR gene.32 The PCR conditions were as described for the 677 C→T variant, except for the annealing temperature, which was performed at 62°C for 1 minute. Restriction enzyme digestion was performed using the MboII restriction enzyme (Stratagene). After digestion, all fragments were resolved on a 4% MetaPhor agarose gel as described for the 677 C→T variant. The 1298 A→C MTHFR genotypes were determined according to the method described previously.32 Heterozygous 1298 A/C produced 6 fragments of 84, 56, 31, 30, 28, and 18 bp, and homozygous 1298 C/C produced 4 fragments of 84, 31, 30, and 18 bp. Fragments of 31, 30, 28, and 18 bp cannot be seen on the gel; however, the identification of the 1298 A/C variants relied only on the 84- and/or 56-bp fragments, which was sufficient to determine the different 1298 A/C genotypes.

The chi-square test for homogeneity of proportions was used to test the equality of the prevalence of different genotypes in the BD and CAD groups. The test was calculated using S-Plus2000 statistical software (Insightful Corporation, Seattle, Wash). A P value less than .05 was considered significant.

The 677 MTHFR genotypes were determined by PCR and digestion using the HinfI restriction enzyme as described in “Materials and Methods.” Among the healthy BD group (n = 625), the homozygous 677 C/C genotype was found in 451 individuals (72.2%), the heterozygous 677 C/T genotype was found in 161 individuals (25.8%), and the homozygous 677 T/T genotype was found in 13 individuals (2%). Within the CAD group (n = 545), the homozygous 677 C/C genotype was found in 350 individuals (64.2%), the heterozygous 677 C/T genotype was found in 175 individuals (32.1%), and the homozygous 677 T/T genotype was found in 20 individuals (3.7%). The frequency of the T allele was similar in patients with CAD compared with the BD group (0.20 vs 0.15). The frequency of the C allele was almost the same in patients with CAD compared with the BD group (0.80 vs 0.85). Chi-square analysis was used to evaluate significant differences in the distribution of genotypes between both groups. P values of .19 and .86 for genotypes C/T and T/T, respectively, were obtained, which indicates no significant difference in the distribution of genotypes between the BD and CAD groups.

The 1298 MTHFR genotypes were determined by PCR and digestion using the MboII restriction enzyme as detailed in “Materials and Methods.” Among the healthy BD group (n = 625) tested for this variant, the homozygous 1298 A/A genotype was found in 246 individuals (39.4%), the heterozygous 1298 A/C was found in 322 individuals (51.5%), and the homozygous 1298 C/C was found in 57 individuals (9.1%). Within the CAD group (n = 540) tested for this variant, the homozygous 1298 A/A genotype was found in 247 individuals (45.7%), the heterozygous 1298 A/C was found in 253 individuals (46.9%), and the homozygous 1298 C/C was found in 40 individuals (7.4%). The frequency of the A allele was similar in patients with CAD compared with the BD group (0.69 vs 0.65). The frequency of the C allele was almost the same in patients with CAD compared with the BD group (0.31 vs 0.35). Chi-square analysis was used to evaluate significant differences in the distribution of genotypes between the study groups. P values of .75 and .21 for A/C and C/C genotypes were obtained, which indicates no significant difference in the distribution of the genotypes between the BD and CAD groups. The results are summarized in the Table.

The prevalence of the 677 C→T and 1298 A→C compound heterozygous variants was 9.6% (60 of 625) for the healthy BD group and 12.3% (67 of 545) for CAD group. Among the 161 individuals heterozygous for 677 C/T within the healthy BD group, 60 (37.3%) were also heterozygous for the 1298 MTHFR variant. Among the 175 individuals heterozygous for 677 C/T within the CAD group, 67 (38.3%) were also heterozygous for the 1298 A/C MTHFR variant. A P value of .94 was obtained, which indicates no difference in the distribution of compound heterozygosity between the BD and CAD groups.

To our knowledge, no previous investigations concerning the prevalence of the 677 C→T and 1298 A→C MTHFR variants have been performed in Arab populations. Hence, the first aim of this study was to determine the prevalence of these 2 MTHFR variants among our population of Middle-Eastern Arabs. Among the healthy BD group tested for the 677 C→T variant, the heterozygous 677 C/T prevalence of 25.8% was comparable to the prevalence rates observed in other ethnic groups. A prevalence rate of 51.5% was observed for the heterozygous 1298 A/C variant in this population, which was slightly higher than rates observed in other ethnic groups.4,13,28–30 The prevalence for compound heterozygosity among the BD group was 9.6%, which is lower than the prevalence of 15%, 20%, and 17% available so far from limited studies conducted in Canada,4 the Netherlands,28 and the United States,31 respectively.

In this study, we did not attempt to associate these 2 MTHFR variant polymorphisms with altered plasma homocysteine because previous studies have failed to establish a link between the two.33–35 In addition, other major risk factors such as smoking, poststroke, postmyocardial infraction, increasing age, and insufficient dietary intake of vitamin B6 and folic acid play a major role in elevating the plasma homocysteine levels.33,34 On the other hand, to date, studies attempting to establish an association between the presence of the 677 C→T and 1298 A→C MTHFR polymorphisms and CAD have yielded conflicting results. Furthermore, little is known about the association of these 2 MTHFR variants with CAD in Arab patients. However, at least one study concluded that hyperhomocysteinemia, which may result from mutations in the MTHFR gene, is independently associated with CAD in Arab men.36 Therefore, our second aim was to evaluate the possible association of these 2 MTHFR polymorphisms with CAD in Arab patients and to assess whether these MTHFR variants can act as a genetic risk factor for CAD in this population, totally independent from homocysteine levels and other CAD risk factors.

Results show that, among the angiographically confirmed CAD group tested in this study, the prevalence observed was 32.1% and 46.9% for the heterozygous 677 C/T and 1298 A/C variants, respectively. These results are comparable to the prevalence of 25.8% and 51.5% observed in the healthy BD group for the heterozygous 677 C/T and 1298 A/C variants, respectively. P values of .19 and .75 were obtained for the 677 C/T and 1298 A/C variants in the BD and CAD groups, respectively, suggesting that the differences in the prevalence between the 2 study groups were not statistically significant.

The same applies to the compound heterozygosity of both variants in which the prevalence of 9.6% and 12.3% was observed in the healthy BD and CAD groups, respectively (P = .94). The frequencies of the T and C alleles in the 677 C →T MTHFR variant were almost similar in patients with CAD compared to the BD control group. The same is true for the A and C alleles in the 1298 A→C MTHFR variant. The distribution and frequency of alleles of these 2 MTHFR variants followed the Hardy-Weinberg equilibrium and were similar in the CAD and BD study groups. This indicates that the presence of these 2 MTHFR variants, either individually or combined, does not predispose individuals to CAD, at least in our population. Some studies have reached a similar conclusion.2,13–20 However, other studies, conducted on individuals from different ethnic groups, have concluded that there is a strong association between the presence of these 2 MTHFR variants and the occurrence of CAD.1,5,7–12 Therefore, it seems that the association is influenced by the ethnic origins of the examined subjects.

It is noteworthy that, with an average age of 44 years for the CAD study group, the population being studied is fairly young compared to those studied elsewhere, particularly in developed countries. This may be because, in the past 3 decades, there have been major changes in the lifestyle of the Saudi population. High-fat diets, obesity, diabetes, and smoking, all of which are considered CAD risk factors, have become more prevalent, and people are leading a more sedentary lifestyle. Hence, the disease manifests itself much earlier in life than in developed countries, where the level of awareness to risk factors might be higher. Two independent studies developed an incremental scale for predicting the development of CAD in the Saudi population because of sharp increases in CAD risk factors such as obesity, hypercholesterolemia, diabetes, hypertriglyceridemia, and high blood pressure.37,38 Early detection of individuals who are genetically susceptible to CAD can lead to early intervention. However, data are lacking regarding the efficacy of this approach in preventing clinical symptoms. Despite this lack of evidence, knowledge of genetic CAD susceptibility has value in providing risk information and guiding decision making. Further research that investigates outcomes regarding genetic risk assessment for CAD is necessary. In conclusion, neither the 677 C→T variant nor the 1298 A→C MTHFR variant, either individually or combined, can be used as an independent genetic risk factor for CAD in Middle-Eastern Arab populations. Other CAD risk factors such as obesity, diabetes, and hypertension should be considered, and we note also that the origin of CAD is basically an interaction between environmental influences in conjunction with other genetic predisposition factors, as has been recently reported.39