• MTHFR;
  • homocysteine;
  • folate;
  • genetic polymorphisms


  1. Top of page
  2. Abstract
  7. References

Two genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene (C677T and A1298C) can influence the plasma homocysteine (Hcy) levels, especially in the presence of an inadequate folate status. The aim of this study was to evaluate the frequencies of C677T and of A1298C MTHFR polymorphisms and their correlation with Hcy and serum folate concentrations in a population of blood donors living in a region of middle-southern Italy (the Molise Region). One hundred ninety seven blood donors were studied for total plasma Hcy, serum folate and C677T and A1298C MTHFR genotypes. The frequency of C677T genotypes was 20.8% (CC), 49.8% (CT) and 29.4% (TT); for the A1298C genotypes: 48.7% (AA), 43.7% (AC) and 7.6% (CC). Hcy and serum folate concentrations were significantly different among genotypes of the C677T polymorphism (CC versus CT versus TT: <0.0001 both for Hcy and folate), with Hcy values increasing, and serum folate decreasing, from CC to TT subjects. Regarding to A1298C polymorphism, the difference among genotypes (AA versus AC versus CC; p: 0.026 for Hcy and 0.014 for serum folate), showed an opposite trend for both parameters, with Hcy higher in the wild-type and lower in the homozygotes and serum folate higher in CC than in AA subjects. In conclusion, we found a high frequency of MTHFR allele associated with high level of Hcy and low levels of folate in an Italian southern population. Copyright © 2013 John Wiley & Sons, Ltd.


  1. Top of page
  2. Abstract
  7. References

Homocysteine (Hcy) has been widely considered as an independent risk factor for cardiovascular diseases (CVD); however, some recent clinical trials have partially failed to confirm that Hcy-lowering is really effective in reducing CVD, with the exception of stroke.[1]

Homocysteine has been largely studied also in relation to cognitive disorders,[2] cancer,[3] pregnancy outcome[4] and congenital disorders such as neural tube defects[5] and as functional marker of folate status because of the closest relationship that both Hcy and folate have in the one-carbon metabolism.

Plasma Hcy concentrations depend on nutritional, environmental and genetic factors; these last regard three major enzymes of the Hcy pathway: methylenetetrahydrofolate reductase (MTHFR), methionine synthase and cystathionine beta synthase. A thermolabile variant of MTHFR, associated with a significant reduced activity of the enzyme, has been described[6] as a consequence of the homozygous substitution of the C677T polymorphism; this homozygous variant significantly influences Hcy levels especially when a lower folate status is present. A second polymorphism in the MTHFR gene, the A1298C, has been investigated;[7, 8] although also this polymorphic variant induces a decrease in MTHFR activity, it does not result in a thermolabile protein. The C677T polymorphism occurs in exon 4 and is characterized by an alanine-to-valine substitution at codon 222, whereas the A1298C polymorphism occurs in exon 7 and is characterized by a glutamate-to-alanine substitution at codon 429.

The aim of this study was to evaluate the prevalence of the C677T and A1298C MTHFR genotypes and to correlate them with the Hcy concentration and folate status in a healthy adult population, living in middle-southern Italy (Molise region), where a voluntary fortification or supplementation with folic acid is not yet common.


  1. Top of page
  2. Abstract
  7. References

Blood samples were obtained from 197 overnight fasting blood donors, attending the blood bank of the General Hospital ‘A. Cardarelli’ in Campobasso (Molise Region, Italy). Exclusion criteria were the current use of B-vitamin supplements for possible interference with serum folate assay. The study was approved by the local ethical committee (N° 95, 10 June 2008), and a written consent was obtained by all the subjects. Samples were immediately taken to the nearby central laboratory of the Catholic University hospital, where they were processed within 2 h from collection. Plasma obtained from the tri-potassium ethylendiaminetetraacetic acid treated blood samples (Vacuette, Greiner Bio-One GmbH, Kremsmünster, Austria) were used for total Hcy assay, according to the high-performance liquid chromatography method of Araki and Sako[9] with slight modifications, as reported elsewhere.[10]

Serum samples were used for total folate assay and analysed within 2 h from collection by using a chemiluminescent microparticle immunoassay technology on the Architect i2000 analyzer (Abbott Laboratories, Abbott Park, IL, USA), according to the instructions of the manufacturer. Internal quality control samples were daily processed with samples analysis (Architect Folate Controls; Abbott Laboratories, Abbott Park, IL, USA).

The adequate status of serum folate and Hcy was defined according to Dhonukshe-Rutten et al.[11] for CVD, as follows: serum folate > 15 nmol l−1, fasting plasma Hcy < 10 µmol l−1.

MTHFR C677T and a 1298C genotype analysis

DNA was isolated from peripheral blood cells. MTHFR C677T and A1298C single nucleotide polymorphisms were analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with HinfI and Mbo II restriction endonucleases, respectively, as reported elsewhere.[12]

Statistical analysis

The distribution of genotypes for each polymorphism was assessed for deviation from Hardy–Weinberg equilibrium with χ2-test. Data on levels of serum folates and Hcy related to selected genotype were compared using the ANOVA. We considered a two tailed p value < 0.05 as significant.

The data analysis was generated using sas/stat software, version 9.1.3 of the sas system for Windows©2009 (Cary, NC, USA).


  1. Top of page
  2. Abstract
  7. References

The frequency of C and T alleles of MTHFR C677T polymorphism was 0.457 (95%CI: 0.408–0.506) and 0.543 (95%CI: 0.494–0.592), respectively. The frequency of TT subjects was 29.4%, in agreement with data from other regions of southern Italy[13, 14] and with our previous study.[12]

The frequency of A and C alleles of MTHFR A1298C polymorphism was 0.706 (95%CI: 0.661–0.751) and 0.294 (95%CI: 0.249–0.339), respectively, in agreement with data from other Italian studies.[12]

Genotype frequencies of both polymorphism agreed with those predicted by the Hardy–Weinberg equilibrium for both polymorphisms (p = 0.97 and 0.48, respectively).

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms were in almost complete negative linkage disequilibrium (D’ = −0.92, p < 0.0001). The genotype combination TT C677T /AA A1298C was present in 27.92% of the population, while 3.25% had CC C677T /AA A1298C combination, 7.11% had CC C677T /CC A1298C combination (Table 1). There was only one double homozygous 677TT/1298CC, whereas 64 subjects (32.5%) were double heterozygotes, a frequency markedly higher than in other countries.[8, 15, 16]

Table 1. Characteristics of the population
  • MTHFR, methylenetetrahydrofolate reductase.

  • *

    Values are expressed as mean ± SD or number (%).

Age (yrs)*40.6 ± 10.6
Gender (n, %) 
Men131 (66.5)
Women66 (33.5)
Smoking habits (n, %) 
No139 (70.6)
Yes58 (29.4)
Fruit and vegetable consumption (n, %) 
<3 portion/day139 (70.6)
≥3 portion/day58 (29.4)
Coffee consumption (n, %) 
No18 (9.1)
Yes179 (90.9)
Plasma homocysteine (µmol/l)15.4 ± 7.4
Serum folate (ng/ml)5.33 ± 2.9
MTHFR C677T genotypes (n, %) 
CC41 (20.8)
CT98 (49.8)
TT58 (29.4)
MTHFR A1298C genotypes (n, %) 
AA96 (48.7)
AC86 (43.7)
CC15 (7.6)
Genotype combination (677/1298; n, %) 
CC/AA7 (3.6)
CC/AC20 (10.2)
CC/CC14 (7.1)
CT/AA34 (17.3)
CT/AC64 (34.5)
CT/CC0 (0)
TT/AA55 (27.9)
TT/AC2 (1.01)
TT/CC1 (0.51)

Methylenetetrahydrofolate reductase plays a relevant role in Hcy metabolism, and the T allele of the C677T polymorphism is known to induce an impaired MTHFR in vitro activity, ranging from 30% to 70%, due to the increased enzyme thermolability;[6] as a consequence, a moderate hyperhomocysteinemia can be found in TT and CT genotypes, especially in the presence of a lower plasma folate concentration. Considering the C677T polymorphism (Table 2), Hcy and serum folate concentrations were significantly different among genotypes (CC versus CT versus TT: <0.0001 both for Hcy and folate) with Hcy values usually increasing, and serum folate decreasing, from CC to TT subjects. In the A1298C polymorphism, the differences among the genotypes (AA versus AC versus CC), although significant (p: 0.026 for Hcy and 0.014 for serum folate), show an opposite trend for both parameters, being Hcy higher in the wild-type and lower in the homozygotes and serum folate higher in CC than in AA subjects (Table 2). Contrasting opinions exist about the effects of A1298C polymorphism, on enzyme activity and Hcy levels; although some authors[7, 8] have described a reduced enzyme specific activity in the presence of A1298C mutation, albeit to a lower percentage than that due to the C677T polymorphism, they did not always find an association with Hcy levels.[7, 8] In our study, we found an increased plasma total Hcy associated with the A1298C polymorphism being Hcy levels higher in AA homozygotes as compared with AC or CC genotypes. A similar trend was found by Friedman et al.[17] MTHFR C677T variant allele was found associated with circulating folate levels by us and others,[18-20] while we and others reported conflicting results on the association of A1298C polymorphism with the circulating folate levels.[17, 21-23]

Table 2. Homocysteine and folate levels according to methylenetetrahydrofolate reductase gene polymorphisms
GenotypesHomocysteine (µmol/l)P for trend univariableP for trend multivariable*Folic acid (ng/ml)P for trend univariableP for trend multivariable*
  • MTHFR, methylenetetrahydrofolate reductase.

  • *

    Adjusted for age, sex, smoking habits, vegetable and fruit consumption and coffee consumption.

MTHFR C677T 0.00010.0001 0.00010.0001
CC12.8 ± 2.8  7.00 ± 4.1  
CT13.5 ± 3.9  5.3 ± 2.5  
TT20.3 ± 11.0  4.1 ± 2.1  
MTHFR A1298C 0.0260.0002 0.0140.0031
AA17.1 ± 9.2  4.7 ± 2.4  
AC13.8 ± 4.7  5.7 ± 3.2  
CC13.1 ± 3.2  6.7 ± 4.3  
Genotype combination C677T/A1298C      
  0.00030.0001 0.0050.010
CC/AA12.5 ± 3.6  5.7 ± 2.3  
CT/AA13.0 ± 3.0  5.5 ± 2.7  
TT/AA20.3 ± 11.0  4.1 ± 2.1  
  0.0710.083 0.0110.0003
CC/(AC or CC)12.8 ± 2.9  7.3 ± 4.3  
CT/(AC or CC)13.8 ± 4.3  5.2 ± 2.5  
TT/(AC or CC)20.8 ± 14.2  4.2 ± 0.8  

As already reported,[24, 25] Hcy and folate were associated with gender (p < 0.01) and smoking habits (0.035) and age (p < 0.001); however, their addition to a multivariate model did not change the association between Hcy, folate and genotype.

Several authors[7, 17, 26-28] report an increased total Hcy concentration associated with combined heterozygosity 677 T and 1298C. There were significant differences in the plasma Hcy and folate concentrations between various combined C677T or A1298C genotypes also in our study (Table 2). In particular, 677TT/1298AA and 677TT/1298 AC or CC showed higher Hcy levels than all the other genotype combinations. Because the T allele of 677 polymorphism is associated with high Hcy levels in combination of either A or C allele of 1298 polymorphism, therefore, the effect on Hcy level seems likely to be attributed the C677T polymorphism.

Low study power and limited sample size may not only explain the inability to analyse the double homozygosity for these two polymorphisms, but it might as well explain the conflicting results obtained by different studies.[7, 17, 26-28]

We compared the results of this study, performed in an adult healthy population, with those previously published in a newborn population, obtained in the same geographical area of Italy (Molise region); the C677T polymorphism shows similar genotype frequencies, whereas, for the A1298C polymorphism, some difference can be observed; in the newborn group, in fact, the frequency of CC subjects is 12.5% and in the adult population is 7.6%, closer to the frequency found in most of the studies performed in Italy.[14, 28, 29] Although this difference could be interesting being obtained in two well-defined subject groups, it could be invalidated by the small number of CC subjects (n = 13 for newborns and n = 15 for adults).

In conclusion, we found a high frequency of MTHFR allele associated with high level of Hcy and low levels of folate in an Italian southern population.


  1. Top of page
  2. Abstract
  7. References

The authors have declared that there is no conflict of interest.


  1. Top of page
  2. Abstract
  7. References
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