In the present study, among CRC patients an increase in 677TT MTHFR polymorphisms has been indicated.
Previous studies on the association of C677T polymorphism and susceptibility to CRC revealed no constant results. Some studies suggested a protective effect of TT genotype for colon cancer because, a reduced risk of CRC progress was observed in TT individuals with a sufficient folate intake . Chen et al. performed the first study to investigate at the relation between the MTHFR C677T polymorphism and CRC. According to their results, the MTHFR C677T polymorphism affected enzyme activity and was involved in aberrant methylation and DNA synthesis, resulting in colorectal tumorigenesis . Similar findings were subsequently reported by Slattery et al , Ma et al  and Le Marchand et al . However, due to statistically non-significant findings, several other published studies failed to support an impact of MTHFR gene polymorphisms on CRC risk [24–26].
It has been shown that the effect of 677TT MTHFR polymorphism, the phenotype of valine amino acid, significantly depends on folate intake. An in-vitro study on HCT116 colon carcinoma cells reported the association of valine amino acid (TT genotype) with increased genomic DNA methylation in an adequate folate level and a significantly lower DNA genomic methylation in folate deficiency suggested folate as a genotype modifier. In fact, biochemical changes in the valine-containing enzyme is important, which shows the enzyme stabilization by the addition of 5-methyltetrahydrofolate (5-MTHF) to the culture medium. Therefore, folate might modify correlation between SNPs and the CRC risk . According to Kennedy et al., MTHFR 677TT genotype was linked to a lower incidence of CRC. Furthermore, the associated risk of CRC was decreased for both the MTHFR 677 CC and TT genotypes when total folate intake was high .
Migration and proliferation of cancer cells are two important events in cancer development. The main cause of death for cancer patients is metastasis, migration of cancerous cells from organ to other organs. It has been demonstrated that about 10 µM folic acid reduced the migration and proliferation of human cell lines (COLO-205, LoVo and HT-29) .
It has also been reported that MTHFR 677TT genotype is one strong reason to lower the risk of proximal colon cancer. The site-specific analysis indicated the role of different molecular alterations in carcinogenesis in proximal and distal of colon and rectum. The more frequent genetic alterations in distal site of colon are K-ras and P53 mutations but microsatellite instability (MSI) is more frequent in the proximal site in CRC [15, 30, 31]. Totally, decreased risk of distal colon cancer, rectal cancer and proximal colon cancer have been reported to be associated with 677TT genotype . However, in the present study, we found an increase of CRC risk in MTHFR TT and TT + CT genotypes compared to CC genotype (OR = 3.68 95%CI 2.35–5.75; OR = 1.42 95%CI 1.08–1.87, respectively). We also found that regardless of tumor site individuals with MTHFR 677TT genotype were associated with higher risk than C allele carriers (Colon: OR = 3.16 95%CI 1.9–5.26; Rectosigmoid: OR = 4.5 95%CI 2.73–7.41). Similarly, two studies on Iranian population on 406 (175 cases, 231 controls) and 491 (234 cases, 257 controls) subjects, respectively reported that CC genotype has a protective effect on CRC [5, 32]. Although a more recent combined case-control study and meta-analysis on 2421 subjects has shown no significant association between MTHFR C677T polymorphism and the risk of CRC in Iranian population suggesting the need for a bigger sample size for MTHFR association studies . There are also some reports indicating the increased risk of TT genotype in other populations [34, 35].
Some studies have reported a reduced risk of developing CRC with only TT genotype with a sufficient folate intake, suggesting a protective effect against CRC [21, 22]. In high folate intake cases, the risk of CRC risk is reduced for both MTHFR 677CC and 677TT genotypes . Similarly, others have also reported the association of high-methyl diets like high folate dietary intake and low alcohol consumption with the protective effect of MTHFR 677TT genotype [16, 20, 23, 37, 38], although not everyone were able to demonstrate the protective effect of the MTHFR 677TT genotype even in high folate dietary intake [39, 40]. . However, it seems to be an association between the MTHFR polymorphism and dietary methyl supply, although the relation remains inconsistent.
Even though we genotyped the MTHFR polymorphism in 1017 blood samples, there was still a limited sample size for folate and Vit B12 measurement that hindered us from incorporating this part of data into a comprehensive association study. Regard to the pilot study based on the limited number of samples for folate and Vit B12 measurement, further investigations is needed to support our findings. Furthermore, due to the heterogeneity of the Iranian population, there is an inevitable need for more multicentric studies in the Iranian population.