Methylenetetrahydrofolate reductase tagging polymorphisms are associated with risk of esophagogastric junction adenocarcinoma: a case-control study involving 2,740 Chinese Han subjects

In this study, we aimed to determine the potential association of MTHFR tagging single nucleotide polymorphisms (SNPs) with risk of developing esophagogastric junction adenocarcinoma (EGJA). MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms were genotyped in 1,677 healthy individuals and 1,063 patients with EGJA. We found that MTHFR rs1801133 G>A polymorphism was significantly associated with the risk of developing EGJA (AA vs. GG: adjusted P = 0.001; GA/AA vs. GG: adjusted P = 0.007 and AA vs. GA/GG: adjusted P = 0.001). However, for MTHFR rs4845882 G>A polymorphism, the decreased risk of EGJA was found in two genetic models (AA vs. GG: adjusted P = 0.002 and AA vs. GA/GG: adjusted P = 0.005). In addition, for MTHFR rs3753584 T>C and rs9651118 T>C polymorphisms, a tendency to decreased risk of EGJA was noted. In a subgroup analysis, a significantly decreased risk of EGJA in <64 years subgroup was identified. We found that MTHFR Grs1801133Trs3753584Grs4845882Ars4846048Crs9651118, Grs1801133Crs3753584Ars4845882Ars4846048Trs9651118 and Grs1801133Trs3753584Ars4845882Grs4846048Trs9651118 haplotypes significantly decreased the risk of EGJA (P = 0.002, P < 0.001 and P = 0.038, respectively). In conclusion, our study demonstrates that MTHFR rs1801133 G>A may be associated with the increased risk of EGJA. Meanwhile, MTHFR rs3753584 T>C, rs4845882 G>A and rs9651118 T>C polymorphisms and haplotypes may decrease the risk of EGJA in Eastern Chinese Han population. Further studies with large sample size and detailed gene-environmental data are needed to validate our conclusion.


INTRODUCTION
The increasing incidence of esophagogastric junction adenocarcinoma (EGJA) was observed worldwide [1][2][3] and was considered to have different etiology and risk factor compared with distal gastric carcinoma (GC) [4]. EGJA remains poor prognosis [5] and is a common public health problem. The vital risk factors contributing to the development of EGJA are obesity, gastroesophageal reflux disease, smoking, foods preserved by salting and low intake of fruits and vegetables et al [6,7]. However, these observed risk factors could not interpret the overall susceptibility to EGJA. Recently, more and more epidemiologic studies suggested that individual's genetic factor might influence the pathogenesis of EGJA.
Accumulating evidences indicate that folate insufficiency may increase the susceptibility of multiple malignancies [8,9]. In humans, the majority of methyl groups may be presented by folic acid for endocellular methylation reactions and DNA de novo deoxynucleoside synthesis. During DNA synthesis, lack of folate can cause uracil misincorporation and then affect the stability of DNA [10]. In folate metabolism and DNA synthesis, methylenetetrahydrofolate reductase (MTHFR) is an important enzyme which catalyzes the revivification of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. And 5-methyltetrahydrofolate is a main circulating and existing form of folate and is the methyl donor for DNA methylation and remethylation procedure of homocysteine to methionine. Based on the important role of participation in both DNA synthesis and methylation, any variant of MTHFR gene may involve in the carcinogenesis.
Human MTHFR is composed of 656 amino acids. MTHFR gene is located on the short arm of Chromosome 1. The human MTHFR gene is very polymorphic (http:// www.ncbi.nlm.nih.gov/SNP) and a number of loci have been established, such as rs1537514, rs3753584, rs9651118, rs1537516, rs4845882, rs1801131, rs1801133, rs2066462, rs4846048 and rs3737967 polymorphisms, etc. Interestingly, many previous case-control studies demonstrated that MTHFR polymorphisms were correlated with the risk of multiple human malignancies [e.g., esophageal squamous cell carcinoma (ESCC) [11], gastric cardia adenocarcinoma (GCA) [12], cervicalcancer [13], breast cancer [14,15] and childhood acute lymphoblastic leukemia [16] et al]. Thus, the single nucleotide polymorphisms (SNPs) in MTHFR genes on EGJA risk attracted our interest. Exploring the potential association of MTHFR SNPs with EGJA susceptibility may be conducive to the prevention and personalized diagnosis. In this study, we selected MTHFR tagging SNPs (rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C) and performed a case-control study to evaluate the effect of MTHFR genotypes for EGJA risk.

Baseline characteristics
A total of 1,063 sporadic patients with EGJA and 1,677 normal controls were recruited. Of the EJGA patients, 759 were male and 304 were female, with a mean age (± standard deviation) of 64.19 ±8.63 years. The normal controls comprised of 1,194 males and 483 females with a mean age of 63.91 ±10.22 years. The demographics (age and sex) was well matched (P = 0.165 and P = 0.909, respectively; Table 1). Of the smoking and alcohol consumption, a significant difference was observed between EGJA patients and controls (P < 0.001, Table 1 Table 3). For MTHFR rs4845882 G>A polymorphism, the decreased risk of EGJA was found in two genetic models (AA vs. GG: crude OR = 0.47, 95% CI: 0.29-0.75, P = 0.002 and AA vs. GA/GG: crude OR = 0.50, 95% CI: 0.31-0.80, P = 0.004; Table 3). Adjustment for age, sex, smoking and drinking, the results were not materially changed (AA vs. GG: adjusted OR = 0.47, 95% CI: 0.29-0.76, P = 0.002 and AA vs. GA/GG: adjusted OR = 0.50, 95% CI: 0.31-0.81, P = 0.005; Table 3). In addition, these associations were still significant after a Bonferroni correction for multiple comparisons.     The results of association between MTHFR rs4845882 G>A polymorphism and EGJA risk in the stratified analyses are summarized in Table 6. We found that MTHFR rs4845882 G>A polymorphism decreased the risk of EGJA in several subgroups  (Table 6)]. Table 7 lists MTHFR rs4846048 A>G genotype frequencies in the stratified analysis. We found no significant difference in genotype distribution of MTHFR rs4846048 A>G polymorphism among EGJA cases and non-cancer controls.

Association of
The results of relationship between MTHFR rs9651118 T>C polymorphism and EGJA risk in the stratified analyses are summarized in Table 8. We found that MTHFR rs9651118 T>C polymorphism was associated with the decreased risk of EGJA in <64 years subgroup [TC vs. TT: adjusted OR = 0.78, 95% CI 0.61-0.99, P = 0.040 (Table 8)].

DISCUSSION
Incidence of EGJA has increased over the past two decades [18,19]. Many studies demonstrated that the morbidity of EGJA was increased in Asian countries, such as China, Korea and Japan [19][20][21]. However, the etiology of EGJA remains unknown. In this study, we explored the association between MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms and EGJA risk in Eastern Chinese Han population. We found that MTHFR rs1801133 G>A might be associated with the increased risk of EGJA. Meanwhile, MTHFR rs3753584 T>C, rs4845882 G>A and rs9651118 T>C polymorphisms decreased the risk of EGJA.
MTHFR gene lies in 1p36.3 and contains 11 exons with a length of about 1980 bp. In exon 4, a G to A variant at nucleotide 677 locus (rs1801133 G>A) directly leads to valine substitution for alanine, which is relevant to a reduction of MTHFR activity [22]. The individuals who carry heterozygous genetype (GA genetype) of MTHFR rs1801133 G>A polymorphism have 70% of normal enzyme activity, however, those who carry homozygous  [23]. A case-control study reported that rs1801133 AA genotype was associated an increased risk of GCA [24]. Another case-control study also found that MTHFR rs1801133 AA and GA genotypes were associated the increased risk of GCA [25]. These results were in accordance with our conclusions. In the future, more replicated study should be conducted to verify these primary findings. MTHFR rs3753584 T>C is situated in the intron region of MTHFR gene. There were only a few studies focusing on the association between MTHFR rs3753584 T>C and cancer risk. A previous study found that there was an increased lung cancer risk in carriers of MTHFR rs3753584 CC genotype compared with carriers of rs3753584 TT genotype [26]. However, no association was found between ESCC risk and MTHFR rs3753584 T>C polymorphism [11]. In addition, Wang et al. also reported that MTHFR rs3753584 T>C was not associated with GCA risk [12]. The present study concluded that rs3753584 TC and TC/CC genotypes were related to a decreased EGJA risk in <64 years subgroup. These apparent discrepancy findings may be due to the insufficient sample size. In the future, more studies with large sample size and detailed environmental factors are indispensable to explore the relationship between MTHFR rs3753584 T>C and the risk of different cancers.
MTHFR rs4845882 G>A polymorphism lies in a intron region and is almost complete linkage disequilibrium (LD) with MTHFR rs1801131 A>C locus. Shen et al. found there was no significant relationship between MTHFR rs4845882 G>A polymorphism and gastric cancer risk [27]. Additionally, the association between MTHFR rs4845882 G>A and GCA risk was not concluded in a recent sutdy [12]. However, our study saw a decreased EGJA risk in the individuals carrying  [28]. Several studies implicated that MTHFR rs9651118 C allele was associated with a reduced risk of lung cancer and prostate cancer [28,29]. In addition, MTHFR rs9651118 C allele was associated with a decreased risk of breast cancer [30]. Our results suggested that MTHFR rs9651118 TC genotype may reduce EGJA susceptibility in <64 years subgroup, which were very similar to the findings of previous studies. In the future, these potential should be confirmed by functional studies.
In this case-control study, we constructed five MTHFR haplotypes to assess the potential inherited patterns of haplotype. We found that MTHFR G rs1801133 T r s3753584 G rs4845882 A rs4846048 C rs9651118 , G rs1801133 C rs3753584 A rs4845882 A r s4846048 T rs9651118 and G rs1801133 T rs3753584 A rs4845882 G rs4846048 T rs9651118 haplotypes significantly decreased the risk of EGJA. To the best of our knowledge, we first explore the relationship of haplotypes in MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms with EGJA susceptibility. We also found that MTHFR rs1801133 G and rs4845882 A alleles might be protective factors for haplotype to EGJA.
However, several limitations in our study should be presented. First, for the controls were recruited from the local hospitals, the selection bias of the study population should not be ignored. Second, the data of plasma folate level were not available, which may affect the association between MTHFR SNPs and EGJA susceptibility. Thirdly, for lack of cancer stage, disease progression and overall survival data, we did not consider the influence of MTHFR  [11], and 'ever drinkers' were subjects who drank no less than three times a week for more than 6 months [11]. The corresponding data are listed in

Selection of SNPs
The MTHFR tagging SNPs (upstream and downstream of MTHFR gene extending 5 Kb, respectively) were selected from the database of CHB population using the HapMap Project (http://hapmap.ncbi.nlm.nih.gov/ index.html.en) and Haploview 4.2 software. The major criterion were: (a) MAF ≥ 0.05 and call rate ≥ 95 %, (b) a HWE P ≥ 0.05, (c) a pairwise linkage disequilibrium (LD) r 2 threshold of 0.8 between polymorphisms (r 2 > 0.8) [11,31,32]. Finally, five MTHFR tagging SNPs (rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C) were eligible and included in this case-control study to evaluate the effect of MTHFR polymorphisms with EGJA risk. The primary information of MTHFR tagging SNPs is presented in Table 2.

DNA extraction and genotyping
Each participant donated 2ml blood sample which was stored in an EDTA-anticoagulated tube. We use the Promega Genomic DNA Purification Kit (Promega, Madison, USA) to extract the genomic DNA. SNPscan TM genotyping assay (Genesky Biotechologies Inc., Shanghai, China) [33,34] was harnessed to determine the genotyping of MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms. Briefly, 150ng DNA sample was denatured at 98°C for 5min. The ligation reaction was done in an ABI 2720 thermal cycler. We used a 48-plex fluorescence PCR reaction for each ligation product amplification. In an ABI 3730XL sequencer, PCR products were analyzed by capillary electrophoresis. The obtained raw data were conducted by GeneMapper 4.1 software (Applied Biosystems, USA). One hundred and ten DNA samples (4%) were randomly selected to reanalyze the genotypes by different laboratory technicians and the reproducibility was 100%.

Statistical analysis
Age of EGJA patients and controls was expressed as mean ± standard deviation. And a Student's t-test was harnessed to assess the difference for age. The Chisquare test (χ 2 ) was used to compare age, sex, smoking, drinking and the genotypes distribution of MTHFR SNPs in patients and controls. We used multivariate logistic regression analysis to assess the risk of MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms and considered the confounders such as sex, age, smoking and drinking status. The crude/adjusted ORs and 95% CIs were calculated using the SAS software (Version 9.4; SAS Institute Inc., Cary, NC, USA). A P < 0.05 (two sided) was considered as statistical significance. In this study, multiple comparisons were conducted by Bonferroni correction [35]. We used a SHESIS software (http:// analysis.bio-x.cn/myAnalysis.php) [17] to construct MTHFR haplotypes.