The main results of this case-control study are that the T allele of the MTHFR 677C > T variant was associated with increased Hcy serum levels only PAOD patients but not in health controls, independently of extraneous variables but there are no associations between the genotypes and Fontaine or TASC classification. In this study, we demonstrated that patients with PAOD showed increased Hcy serum levels when compared with controls but the Hcy levels did not differ according to disease severity. To our knowledge, the present study is the first that evaluated the association between the MTHFR 677C > T variant, Hcy levels and the severity of PAOD in Brazilian patients.
Our data are in agreement with a previous study that found increased Hcy in PAOD patients compared with controls , and increased Hcy levels have been considered an important risk factor for cardiovascular diseases [33, 34]. Hcy may affect both atherogenesis and the resistance of the endothelium to thrombosis through different mechanisms including the direct toxic effect to endothelial cells, interference with the antithrombotic and vasodilator functions of nitric oxide , enhancement of leukocyte adhesion and extravasation, with the potency to contribute to vascular injury .
The MTHFR 677C > T (rs1801133) variant of MTHFR consists in a cytosine (C) to thymine (T) transition at the nucleotide 677 in the exon 4 and this nonsynonymous variant results in the alanine-to valine (Ala to Val) amino acid change leading to a thermolabile enzyme with reduced activity . The reduced MTHFR enzyme activity and the increased thermolability lead to a decrease in 5-methyltetrahydrofolate and an increase in the accumulation of the substrate 5,10- methylenetetrahydrofolate and, consequently HHcy .
Our data showed that the allelic and genotypes frequency of the MTHFR 677C > T variant did not differ between the PAOD patients and control group in different genetic models that were evaluated, suggesting that this genetic factor, by itself, may not contribute to the development of PAOD in our population. These results are in agreement with previous studies reported in worldwide population [23} as well as in Brazilian population . However, the observed frequency of the MTHRF 677C > T genotypes in the present study agrees with previous studies that showed the presence of the TT genotype among 5.0–15.0% of the general population and its role and impact on the Hcy levels may vary among different populations [37–40]. In addition, we demonstrated that patients with PAOD carrying the TT genotype (in recessive model) showed higher levels of Hcy than those carrying the CT + TT genotypes. Moreover, the patients with PAOD carrying the TT + CT genotypes (in dominant model) showed higher levels of Hcy than those carrying the CC genotype, suggesting that this variant may be related to HHcy in PAOD Brazilian patients. It is important to highlight that these associations were independently of age, sex, ethnicity, and BMI, folate, and vitamin B12.
Divergent results have been reported regarding the association between Hcy levels and MTHFR 677C > T genotypes. Khandanpour and coworkers  showed that PAOD was associated with elevated levels of Hcy and the TT genotype. However, other studies reported that the T allele could be a protector factor for PAOD , and that high levels of Hcy were associated with the T allele only in those individuals with decreased levels of folate, vitamin B12 or vitamin B6 . These results can be justified by the fact that folate and vitamins B12 and B6 are, respectively, substrate and cofactors in the Hcy metabolic pathway . Moreover, no significant association between the MTHFR 677C > T genotypes and Hcy levels was also reported [23, 43].
It is important to emphasize that our data showed that the presence of the T allele was associated with an increase in Hcy levels exclusively in the PAOD patients (but not in controls) with the CT + TT genotypes, regardless of the other risk factors. That association could be explained, in part, through the presence of other genetic and/or epigenetic factors associated with the disease that may modulate the metabolism of Hcy in these patients Several risk factors for PAOD could regulate gene expression through the epigenetic mechanisms. Studies reported the epigenetic effects of T2DM, SAH, Hcy levels, aging, smoking, and sedentary lifestyle in the DNA methylation and chromatin remodeling, histone acetylation, microRNA expression and regulation [44, 45].
Gene-by-environmental interactions are hypothesized to play an important role in the expression of a disease. Therefore, the effects of environmental factors in association with the risk factors for PAOD that were not evaluated in the present study may regulate the expression of genes involved in the Hcy metabolism and could explain why the high levels of Hcy were observed only in PAOD patients, independently of the risk factors controlled in this study. PAOD has complex phenotypes that depend on multiple other genetic and environmental factors, as well as epigenetic mechanisms  that could also explain the high levels of Hcy among the patients with PAOD carrying the T allele of MTHFR 677C > T variant. The reduced enzyme activity associated with the MTHFR 677C > T variant has been linked to decreased DNA methylation  and HHcy . In turn, decreased DNA methylation may involve aberrant expression of structural and matrix proteins or reduced DNA integrity resulting in premature aging of the vascular tissue .
This study has some potential limitations. First, it is a case-control design, which does not allow inferences on causal relationship between the MTHFR 677C > T variant and the PAOD, as well as the Hcy levels and the severity of the disease. Second, we evaluated one MTHFR variant, but this gene presents other single nucleotide variants, and it might be interesting to evaluate whether genetic haplotypes play a role in subjects with PAOD. However, this study has some strengths, such as controlling confounding effects by the statistical analysis.
In conclusion, the T allele of MTHFR 677C > T variant was associated with HHcy in PAOD patients, but not in controls. In addition, this variant was not associated with the clinical stage and the anatomoradiological categories of PAOD. Our data suggested a possible interaction between MTHFR 677C > T variant and a presence of other genetic, epigenetic and environment factors associated with PAOD on modulation of metabolism of Hcy.