The results from our study suggest that the T allele is more likely to have a protective effect in CRC development in the Macedonian population. The prevalence of the MTHFR T allele was lower in patients with advanced CRC (Duke’s stages C and D) and with microsatellite instable tumors (MSI+), indicating an inverse association with the CRC aggressiveness and MSI status. This effect seemed to be independent of gender, age of onset and localization since there was no difference after the stratification regarding these variables. Methylenetetrahydrofolate reductase regenerates the methionine from homocysteine, to maintain the supply of SAM for diverse methylation reactions of DNA, RNA, proteins and lipids. Total plasma homocysteine (tHcy) is a functional indicator of MTHFR activity and is correlated with the possession of the thermolabile form of the enzyme [32] and is inversely associated with DNA methylation [33]. DNA hypomethylation, particularly within promoter regions, could potentially reverse methyl-mediated silencing of oncogenes. Because of all of these, it could be expected that MTHFR 677TT is likely to increase the risk of CRC. Although some studies support this hypothesis [25-27], the majority of the studies, as well as two recent meta-analyses, have found a reduced risk of CRC associated with the MTHFR C677T homozygous variant (TT) genotype [14,18-24,34,35] that agree with our results (Table 4). An alternative to the DNA hypomethylation hypothesis to account for the reduced risk associated with the C677T polymorphism has been proposed [18]. The higher activity of the wild-type enzyme may reduce the availability of 5,10-methyleneTHF for synthesis of dTMP from dUMP. Low erythrocyte folate levels (<140 ng/mL) were associated with increased frequency of DNA double-strand breaks as a consequence of the mis-incorporation of uracil.[36] The TT genotype would slow down the conversion of 5,10-methyleneTHF to 5-methylTHF and close this avenue for DNA damage [31]. Therefore, the MTHFR C677T polymorphism would modify the CRC risk and CRC advancement by influencing the availability of 5,10-methyleneTHF for DNA synthesis and repair rather than through DNA methylation. The conflicting results from different studies (Table 4), may result from dissimilarity in lifestyle and dietary habits in different populations (e.g., diet rich or low in folate, alcohol consumption), and from polymorphisms in genes encoding for other enzymes (Dihydrofolate reductase; Methionine synthase; Thymidylate synthase or others), interconnected in folate metabolism.

      The inverse association between the MTHFR 677T allele and the MSI+ that we observed, can be explained by the DNA hypomethylation hypothesis. The lower enzyme activity in homozygotes for the variant allele (677TT) could lead to reduced epigenetic silencing of the mismatch repair (MMR) genes, this being the main reason for MSI in sporadic CRC.         

      In conclusion, our results suggest that the MTHFR 677T allele is more likely to have a protective effect in CRC development and progression in the Macedonian population, and support previous findings of an inverse association of the MTHFR 677T allele with CRC. Knowledge of the interaction between folate intake and the MTHFR polymorphisms could be useful in elucidating the role of the MTHFR polymorphisms in colorectal carcinogenesis.