The Methylenetetrahydrofolate Reductase C 677 T Polymorphism Influences Risk of Esophageal Cancer in Chinese

Esophageal cancer (EC) is a global health problem, and an estimated 482,300 new esophageal cancer cases and 406,800 deaths occurred in 2008 worldwide (Jemal et al., 2011). Its incidence rates vary internationally, and the highest rates found in Southern and Eastern Africa and Eastern Asia were nearly 16-fold, compared with lowest rates observed in Western and Middle Africa and Central America in both males and females (Jemal et al., 2011). In China, there are about 250,000 cases diagnosed yearly, contributing to about half of the world’s cases (Yang et al., 2003). Many studies demonstrated that the risk factors of EC included poor nutritional status, low intake of fruits and vegetables, cigarette smoking, alcohol drinking, and drinking beverages at high temperatures (Gao et al., 1994; Islami et al., 2009; Wu et al., 2009; Wu et al., 2009). Folate deficiency caused by low consumption of vegetables and fruits increase the risk of several cancers, including esophageal cancer (Jaskiewicz et al., 1988; Prasad et al., 1992; Giovannucci et al., 1995; Tseng et al., 1996). Providing methyl groups required for intracellular methylation reactions and de novo deoxynucleoside triphosphate synthesis is an important biological function of folate; therefore, folate deficiency is thought to be carcinogenic through disruption of DNA methylation and synthesis or impaired DNA repair (Choi


Introduction
Esophageal cancer (EC) is a global health problem, and an estimated 482,300 new esophageal cancer cases and 406,800 deaths occurred in 2008 worldwide (Jemal et al., 2011).Its incidence rates vary internationally, and the highest rates found in Southern and Eastern Africa and Eastern Asia were nearly 16-fold, compared with lowest rates observed in Western and Middle Africa and Central America in both males and females (Jemal et al., 2011).In China, there are about 250,000 cases diagnosed yearly, contributing to about half of the world's cases (Yang et al., 2003).
Many studies demonstrated that the risk factors of EC included poor nutritional status, low intake of fruits and vegetables, cigarette smoking, alcohol drinking, and drinking beverages at high temperatures (Gao et al., 1994;Islami et al., 2009;Wu et al., 2009;Wu et al., 2009).Folate deficiency caused by low consumption of vegetables and fruits increase the risk of several cancers, including esophageal cancer (Jaskiewicz et al., 1988;Prasad et al., 1992;Giovannucci et al., 1995;Tseng et al., 1996).Providing methyl groups required for intracellular methylation reactions and de novo deoxynucleoside triphosphate synthesis is an important biological function of folate; therefore, folate deficiency is thought to be carcinogenic through disruption of DNA methylation and synthesis or impaired DNA repair (Choi
Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, catalyzes the reduction of 5,10-methylene-tetrahydrofolate to 5-methyltetrahydrofolate (Bailey et al., 1999), the dominant circulating form of folate, which provides the methyl group with the ability to convert homocysteine to methionine (Banerjee et al., 1990).Two common functional polymorphisms of MTHFR gene, C677T and A1298C, have been identified, and the variant genotypes are associated with a significant reduction of enzyme activity (Frosst et al., 1995;Weisberg et al., 1998).Individuals with the MTHFR 677TT genotype have significantly lower plasma folate levels and higher plasma homocysteine levels than those with 677CC genotype (Larsson et al., 2006;Larsson et al., 2006).It also has been indicated that genomic DNA methylation is diminished in subjects with the MTHFR 677TT genotype, especially when folate status is low (Stern et al., 2000;Friso et al., 2002).
Currently, there are some researches concerning of the polymorphisms of MTHFR and the risk of esophageal cancer.However, the conclusions of these researches were inconsistent due to either small sample size or populations with mixed genetic backgrounds.Therefore, the present study was carried out to further investigate the association of MTHFR C677T and A1298C polymorphisms with esophageal cancer susceptibility in Henan Province, the highest incidence area of EC in China.

Study population
This study population included 381 EC patients and 432 healthy controls.All subjects were unrelated Chinese with Han ethnicity in Henan Province.Patients with newly diagnosed and histologically confirmed primary EC before undergoing any treatment were recruited at the First Affiliated Hospital of Zhengzhou University between March 2008 and January 2010.Healthy controls were recruited from a census of digestion diseases carried out in Xinxiang County and Xin'an County of Henan Province.The controls matched to cases on gender and age were required to be free of any digestion diseases, having no cancer history and related clinical signs.
Uniform trained interviewers used a special questionnaire to collect data from the study subjects on age, gender, tobacco smoking, alcohol drinking, family history of cancer, etc.The venous blood (5 ml) obtained from each study subject was collected in EDTA and stored at -70°C for extraction of genomic DNA.Smokers were defined as former or current individuals smoking at least one cigarette per day for at least one year.Alcohol intake was defined as drinking at least once a week with more than 100 gram every time for at least six months.The study was approved by the Ethics Committee of Zhengzhou University and the written informed consent was obtained from all study subjects.

Genotyping analysis
Genomic DNA was extracted from buffy coats using the DNA Blood Mini kit (TIANGEN BIOTECH (BEIJING) CO. , LTD) according to the manufacture's instructions.
The genotypes of MTHFR A1298C were analyzed by PCR-confronting two-pair primers (PCR-CTPP) as described in our previous report (Wang et al., 2011).
For quality control, genotyping was performed with blinding to case/control status, and a 10% random sample of subjects (n=82) was genotyped again by different persons, and the consistency was 100%.Each type of PCR products was randomly sequenced to ascertain the genotype.

Statistical analysis
χ 2 test was used to compare the frequency distributions of demographic variables, smoking status, drinking status, family history of cancer and MTHFR genotypes in the study groups.Unconditional logistic regression models were applied to calculate odds ratios (OR) and 95% confidence intervals (95%CI), which were adjusted by age, gender, smoking, drinking and family history of cancer.The Hardy-Weinberg equilibrium of the genotype frequency was assessed by χ 2 test in the control group.D' linkage disequilibrium (LD) value was calculated using Haploview4.0 software.Haplotypes were inferred by using the SNPHAP2.0 software.Gene-environment  interactions were evaluated by using Multifactor dimensionality reduction (MDR, V2.0 Beta 2).A detailed explanation about MDR has been provided elsewhere (Ritchie et al., 2001;Hahn et al., 2003).All analyses were performed using SPSS12.0software.All tests were two sided and P values<0.05were considered as statistically significant.

Subjects characteristics
The demographical characteristics of the study population were showed in Table 1.There were no significant differences in the distribution of gender, age, smoking and alcohol drinking between case group and control group.However, the family history of cancer (P=0.00) had significant difference between case and control groups.

Haplotype analysis
The linkage disequilibrium and haplotype of MTHFR C677T and A1298C polymorphisms were examined, given their close relation in a chromosome location.The analysis indicated strong linkage disequilibrium between the two loci (D'=0.88).Table 3 showed totally four possible haplotypes (CA, CC, TA, TC).Comparing to haplotype CA with both wild allele, haplotypes TA and TC which contain the variant T allele of polymorphism C677T could reduce the susceptibility to esophageal cancer.The adjusted OR (95% CI) were 0.61(0.47-0.79)and 0.06(0.01-0.43),respectively.

Combined genotypes analysis of MTHFR C677T and A1298C
There were totally nine combined genotypes of MTHFR C677T and A1298C, which were shown in Table 4.The combined wild genotype CC/AA was taken as the reference, there were significant differences in the distribution of frequencies for CT/AA, CT/AC and TT/ AA, which contain minor allele T of C677T, between case and control groups, and the adjusted OR (95% CI) were 0.48 (0.24-0.98), 0.46 (0.22-0.98) and 0.31 (0.15-0.62), suggesting that these combined genotypes could reduce the risk of esophageal cancer.

Gene-environment interaction analysis
MDR acts by reducing multiple variables, attributes into single, binary attribute with ''low risk'' and ''high risk'' categories.The specifics of the algorithm used have been elsewhere (Ritchie et al., 2001;Hahn et al., 2003).Analysis was performed with MTHFR polymorphisms (C677T and A1298C), smoking, drinking and family history of cancer.The results of MDR analysis were shown in Table 5.The best model of four factors (family history of cancer, smoking, C677T and A1298C) with testing balance accuracy (TBA): 0.6104 and cross validation consistency (CVC): 10/10, which could increase the EC risk in the "high risk group" with 4.18fold (OR: 4.18, P<0.0001, 95% CI: 3.08-5.69),compared to the "low risk group".

Discussion
There have been several studies showing that the MTHFR 677T allele could increase the esophageal cancer risk (Song et al., 2001;Zhang et al., 2004;Wang, 2005;Wang, 2007), while no risk change has been observed among the other studies (Wu et al., 2002;Wu et al., 2002;Stolzenberg-Solomon et al., 2003).However, the present study was the first report suggesting the MTHFR 677T allele could decrease the risk of EC, which was consistent with the result of Nelofar Kureshi et al. (Kureshi et al., 2004;Kureshi et al., 2004), who have shown that C allele was associated with susceptibility to head and neck cancer, although their results did not reach statistical significance.Yang et al. (2005) found high folate consumption and the MTHFR 677TT genotype to be associated with a non-significant decrease in the risk of esophageal cancer.The association of MTHFR 677T allele with malignancy was best studied in colon cancer.Huang et al. (2007) performed a meta-analysis, which has suggested the 677T allele showed a small but significant protective effect against colorectal cancer compared to the 677C allele for a worldwide population.There may be a possible explanation, the cancer risk associated with MTHFR polymorphisms depending on the level of folate intake, which has been suggested in the studies on colorectal cancer (Chen et al., 1996;Ma et al., 1997).Therefore, based on this hypothesis, when folate intake is sufficient, individuals with the MTHFR CT or TT genotypes may have a reduced risk of cancer, since decrease in MTHFR activity due to 677T allele might lead to an elevation in 5,10-methylene-tetrahydrofolate, which would facilitate DNA synthesis and diminish DNA damage, while adequate provision of methyl donors could still be ensured.However, under the condition of low folate intake, both impaired DNA methylation and DNA synthesis/repair may become the primary mechanisms of carcinogenesis.Although, there were not data for folate intake in the present study, the result might be consistent with this hypothesis.
For the MTHFR A1298C polymorphism, the present study was consisted with Stolzenberg et al. (2003), who did not find it associated with susceptibility to esophageal cancer, while Song et al. (2001) reported a significant 4.43-fold increased EC risk with the MTHFR 1298CC genotype.There was a small number of subjects with the CC genotype in each study, which limited the conclusions of any results.The variant MTHFR A1298C genotypes clearly reduce MTHFR activity, albeit to a lesser extent than the C677T variant genotypes.Consequently its effect on homocysteine levels is also attenuated and, in fact, may only be significant when an individual carries both variants and/or has poor nutrient status (Weisberg et al., 1998).
The analysis of haplotype and combined genotypes supplied a greater amount of information than a single SNP.The association between the haplotype of MTHFR C677T and A1298C polymorphisms and the susceptibility to EC was not reported previously.The present study found the TA and TC haplotypes decreased the risk of esophageal cancer, with adjusted OR (95%CI) of 0.61 (0.47-0.79) and 0.06 (0.01-0.43), respectively.Song et al. (Song et al., 2001) reported a joint effect between MTHFR 677CT and 1298CC variant genotypes on rising risk of ESCC.However, in this study, the combined genotypes CT/AA, CT/AC, and TT/AA diminished the risk of EC as compared with MTHFR 677CC/1298AA genotype, which was in line with Mastuo et al. (2004), who determined that possible  hypomethylation inducible alleles (MTHFR 677T, 1298C) were protective for malignant lymphoma.Although MTHFR C677T polymorphism was associated with significant reduced risk and the A1298C polymorphism showed no effect, when they were separately analyzed.For this inconsistency, there was a possible that phenotypic effect of A1298C may be limited compared with the C677T polymorphism.Yamada et al. (2001) previously verified that the recombinant human MTHFR activity in vitro to be more strongly influenced C677T than A1298C.
Esophageal cancer is a complex disease likely resulting from multiple interacting genetic polymorphisms and gene-environment interactions.Some earlier studies have shown an interaction of MTHFR C677T polymorphism with environmental risk factors such as smoking and alcohol (Weinstein et al., 2002;Yang et al., 2005;Boccia et al., 2007).However, this study performed the analysis of gene-environment interaction by MDR method, and found a best model including familial history of cancer, smoking and MTHFR polymorphisms (C677T and A1298C).Highand low-risk groups were generated by MDR and the OR value was 4.18 (95% CI: 3.08-5.69).
The limitation of this study was nonavailability of folate levels in study subjects.Because in the presence of low folate intake, both impaired DNA methylation and DNA synthesis/repair may become the primary mechanism of carcinogenesis in those who had the variant MTHFR genotypes.However, it has been suggested that in those with the variant MTHFR 677T allele, decreased risk of colorectal neoplasia was observed among subjects with adequate folate levels (Chen et al., 1996;Ma et al., 1997).Therefor the result of the present study was reliable.
In conclusion, the current study suggested that MTHFR C677T polymorphism could reduce the risk of EC, and MTHFR A1298C was not associated with the risk of EC.However, the haplotype and combined genotypes of MTHFR C677T and A1298C could reduce the risk of esophageal cancer.In addition, this study showed an interaction in MTHFR polymorphisms, smoking and familial history of cancer.

Table 3 . Haplotype Analysis of MTHFR C677T and A1298C
*Adjusted for age, gender, smoking, drinking and family history of cancer