Abstract
Background
Attempts for early detection of gastric cancer have recently focused on host's genetic susceptibility factors and gene-environment interactions. We have, herein, studied the association of MTHFR C677T single nucleotide polymorphism (SNP) and its interaction with Helicobacter pylori infection, smoking, age and gender on the risk of gastric cancer among an Iranian population.
Methods
Gastric cancer patients (n = 450) and cancer-free controls (n = 780) were studied for serum H. pylori-specific IgG antibodies by ELISA and MTHFR C677T polymorphism (SNP) by PCR-RFLP. Demographic and life style data were collected through patient interviews. Unconditional logistic regression model estimated odds ratio (OR) and the corresponding 95% confidence intervals (CI).
Results
The interactions of MTHFR genotype with H. pylori infection (P = 0.03), age (P = 0.049) and gender (P = 0.007) were statistically significant. Accordingly, MTHFR C677T carriers who were also positive for H. pylori infection exhibited 80% (OR = 1.8, 95% CI = 1.0-2.9) significant excess risk of non-cardia gastric cancer. Furthermore, subjects over the age of 50 or female subjects carrying MTHFR C677T SNP showed 40 (OR = 1.4, 95% CI = 1.0-2.0) and 100 (OR = 2.0, 95% CI = 1.2-3.2) percent increased risk of gastric cancer, respectively.
Conclusion
MTHFR C677T SNP seems to increase the risk of gastric cancer and the effect is significantly inflated by interactions with H. pylori infection, age and gender.
Key Words: Helicobacter pylori, Smoking, Gender identity, Age group, Methylenetetrahydrofolate reductase
Introduction
Gastric cancer is the fourth most frequent cancer [1] and the second cause of cancer-related death [2] worldwide. Helicobacter pylori infection is the main established risk factor for gastric cancer with variable strengths of associations [3]. Nevertheless, gastric cancer has a multifactorial etiology and is co-modulated by different factors including host factors, such as age, gender and genetic predisposition in addition to environmental factors such as smoking, socioeconomic status and consumption of fruits and vegetables [3]. The question regarding what combination of risk factors predisposes affected individuals to gastric cancer remains the topic of investigations worldwide [4].
Recently, the influence of genetic factors such as single nucleotide polymorphisms (SNP), one of the largest types of inherited genetic variations, in regulatory genes, which may affect the individual's susceptibility to cancer, has come to the center of attention [5, 6].
MTHFR gene located on chromosome 1p36.3 encodes for a key enzyme in folate metabolism [7]. MTHFR C677T and A1298C are the two common functional polymorphisms [8]. The MTHFR C677T SNP results in the substitution of the amino acid alanine for valine, which results in a less active form of the enzyme. Consequently, the heterozygote genotype (CT) and homozygote mutant genotype (TT) respectively retain 60% and 30% of the original enzymatic activity of the wild type (CC) form [8]. The role of MTHFR in the metabolism of folate is to catalyze the reduction of 5, 10-methylene-tetra-hydrofolate to 5-methyltetrahydrofolate, the dominant form of folate in the circulation, which donates the methyl group for remethylation of homocysteine to methionine [9] and finally to S-adenosyl-L-methionine. A reduced enzyme activity may result in lower levels of S-adenosyl-L-methionine and an increased risk of cancers as a consequence of gene hypomethylation [10]. It may also potentiate cancer development by increasing the ratio of deoxyuridylate monophosphate to deoxythymidylate monophosphate, thereby increasing the incorporation of uracil into DNA instead of thymine, leading to point mutations and DNA/chromosome damage [11]. Association of MTHFRC677T polymorphism with various types of cancers, including leukemia [12], colorectal [13], breast [14], esophageal [15] and gastric [16] cancers have been reported.
Gastric cancer is the most common cancer in Iran with an incidence rate of about 20 per 100,000 [17]. However, the interactions between gene and environmental factors on the risk of gastric cancer have not been sufficiently investigated. In the current study, we have evaluated the impact of MTHFR C677T polymorphism on the risk of gastric cancer as well as its potential interaction with other established risk factors such as H. pylori infection in an Iranian case-control study.
MATERIALS AND METHODS
Cases and controls. Cases included patients with histologically confirmed gastric cancer (n = 405), who had undergone gastrectomy at Cancer Institute of Iran between 2002 and 2011. These patients were further subdivided based on anatomical subsites (cardia and non-cardia), and histopathological subtype (intestinal and diffuse) of their tumors according to the updated Lauren’s classification system [18]. Patients with tumors involving the entire stomach, of mixed histological subtype or undocumented subsite or subtype were excluded from the stratified analyses. Controls included 780 subjects over the age of 35 and comprised of 381 unselected healthy individuals having referred for routine laboratory check-ups and 399 ulcer/cancer-free subjects gastroscopied at Amiralam Hospital (Tehran, Iran) during the same time period. Due to similar distribution of the demographic data and established risk factors, including age, sex, and smoking among the two control groups, they were pooled together and used as a single control group for all statistical analyses. Each participant provided a written informed consent prior to the interview and collection of the exposure information and biological samples. This study was approved by the Iranian National Ethical Committee for Medical Research.
H. pylori infection. Sera were isolated from fasting blood samples and kept at -70°C for further studies. H. pylori-specific IgG antibodies were detected through an ELISA assay developed at Pasteur Institute of Iran according to the previously described protocol [19]. Sera with borderline titers (n = 50) were excluded from the statistical analyses.
Blood sampling and DNA extraction. Genomic DNA was extracted from white blood cells according to sodium salting out extraction method [20]. The DNA purity and concentrations were determined by spectrophotometric measurement of absorbance at 260/280 nm.
MTHFR genotyping. The C677T SNP (rs#1801133) of MTHFR gene was studied by PCR-RFLP method as previously described [21]. Briefly, we used the forward (5'-CGA AGC AGG GAG CTT TGA GGC TG-3') and reverse (5'-AGG ACG GTG CGG TGA GAG TG-3') PCR primers to amplify a 233-bp product, which was digested by HinfI at 37C overnight. Visualization of PCR products on 2% agarose gel revealed a 233-bp fragment for the wild-type (CC), 233, 176 and 57 bp fragments for heterozygotes (CT), and 176 and 57 bp fragments for homozygotes (TT). Due to the limited power and low prevalence of TT genotype (7.2%) among our study population, we compared the T carrier (including CT and TT) genotype with the wild type (CC) as the reference group.
Statistical analysis. Research subjects were classified into ever/never smokers, H. pylori positive/negative, younger/older than the age of 50 in all statistical analyses. Unconditional logistic regression model was used to estimate the crude and adjusted odds ratios (OR) and the corresponding 95% confidence intervals (CI) measuring the association between MTHFR C677T SNP and the risk of gastric cancer. We further evaluated the interactions of MTHFR C677T SNP with age, gender, H. pylori and smoking status on the risk of gastric cancer. P values of the interaction term in the logistic regression model were used for the homogeneity test. Every model was adjusted for age and gender unless stated as crude. The statistical package STATA version 10 was used to perform the statistical analyses.
Results
Demographic data. The average age was higher in the cases (62.6 ± 11.8) than in the control group (47.61 ± 14.1) (Table 1) and the male to female ratio was 3.0 and 0.7, respectively. The majority of cases and controls were H. pylori positive with a similar distribution (70.1% vs. 71.5%). Smoking habit was nearly twice as prevalent in the cases as compared to the controls (38.6% vs.18.2%). The most frequent subsite and subtype of gastric tumor in the cases were non-cardia and intestinal, respectively.
Table 1.
Distribution of age, gender, H. pylori and smoking status among cases and controls.
Variables |
Controls
(n = 780) |
All GC
(n = 405) |
|
Subtype
|
|
Subsite
|
|||
---|---|---|---|---|---|---|---|---|---|
Intestinal
(n = 142) |
Diffuse
(n = 80) |
Cardia
(n = 152) |
Non-cardia
(n = 210) |
||||||
Average age (SD) | 47.61 (14.1) | 62.6 (11.8) | 65.6 (10.3) | 58.4 (11.2) | 64.3 (11.9) | 61.0 (12.3) | |||
Male/female ratio | 0.70 | 3.0 | 3.6 | 2.6 | 3.0 | 2.9 | |||
Positive H. pylori | 71.50% | 70.1% | 74.1% | 79.7% | 74.0% | 65.8% | |||
Ever smoker | 18.20% | 38.6% | 40.9% | 46.8% | 39.9% | 35.6% | |||
MTHFR variants no. (%) | |||||||||
CC | 422 (54.1%) | 198 (48.9%) | 69 (48.6%) | 39 (48.8%) | 77 (50.7%) | 99 (47.1%) | |||
CT | 308 (39.5%) | 172 (42.5%) | 59 (41.5%) | 34 (42.5%) | 60 (39.5%) | 98 (46.7%) | |||
TT | 50 (6.4%) | 35 (8.6%) | 14 (9.9%) | 7 (8.8%) | 15 (9.9%) | 13 (6.2%) | |||
T Carriers (CT or TT) | 358 (45.9%) | 207 (51.1%) | 73 (51.4%) | 41 (51.3) | 75 (49.3%) | 111 (52.9%) |
MTHFR C677T polymorphism and the risk of gastric cancer. The T allele frequency amongst the control group was 26% and the genotype distribution confirmed no deviation from the Hardy-Weinberg Equilibrium (P>0.05). The associations between the MTHFR 677 genotypes and risk of gastric cancer are shown in Table 2. We found 20% increased risk of gastric cancer (OR = 1.2, 95% CI = 1.0-1.6) and 30 percent increased risk of tumors of the non-cardia subsite (OR = 1.3, 95% CI = 1.0-1.8) among MTHFR C677T carriers compared to the wild type. However, after adjustment for age and gender, the excess risk did not remain statistically significant (Table 2). We found no differences in the risk of diffuse (OR = 1.2, 95% CI = 0.9-1.8) and intestinal (OR = 1.2, 95% CI = 0.8-2.0) subtypes of gastric cancer in MTHFR C677T carriers.
Table 2.
Association of MTHFR C677T polymorphism and gastric cancer stratified by the tumor anatomic subsite and histological subtype.
MTHFR variants | Case/ Control |
Crude OR
(95%CI) |
Adjusted OR
(95%CI) |
|
---|---|---|---|---|
All GC | CC T carriers |
198/422 208/358 |
Ref. 1.2 (1.0-1.6) |
Ref. 1.1 (0.9-1.5) |
Carida GC | CC T carriers |
77/422 75/358 |
Ref. 1.1 (0.8-1.6) |
Ref. 1.1 (0.7-1.6) |
Non-cardia GC | CC T carriers |
99/422 112/358 |
Ref. 1.3 (1.0-1.8) |
Ref. 1.2 (0.8-1.7) |
Diffuse GC | CC T carriers |
39/422 41/358 |
Ref. 1.2 (0.9-1.8) |
Ref. 1.1 (0.7-1.7) |
Intestinal GC | CC T carriers |
69/422 73/358 |
Ref. 1.2 (0.8-2.0) |
Ref. 1.2 (0.7-1.9) |
Interaction of MTHFR C677T polymorphism with age, gender, H. pylori infection and smoking. The interactions of MTHFR genotypes with age (P = 0.01) and gender (P = 0.007) were statistically significant (Table 3). Subjects over the age of 50 showed 40% higher risk of gastric cancer (OR = 1.4, 95% CI = 1.0-2.0). In addition, female carriers of MTHFR C677T SNP demonstrated 2 folds increased risk of gastric cancer compared to the wild type (OR = 2.0, 95% CI = 1.2-3.2). The prevalence of H. pylori infection was similar and approximately 70% in both cases and controls. The interaction term between H. pylori infection and MTHFR677 genotype distribution in the regression model was statistically significant (P = 0.03, Table 3). Accordingly, among the H. pylori-positive stratum, the risk of gastric cancer was 1.5 folds higher in the MTHFR C677T carrier group compared to the wild type (OR = 1.5, 95% CI = 1.0-2.2) and the odds inflated to 1.8 for the non-cardia gastric cancer in this group (OR = 1.8, 95% CI = 1.0-2.9, Table 3). We found no interaction between the MTHFR 677 genotypes and smoking habits on the risk of gastric cancer (P = 0.9) or its subcategories. Stratification for the histological subtype did not change the relative risk for any of the above interaction analyses (data not shown).
Table 3.
Association of MTHFR C677T polymorphism with the risk of gastric cancer and its subsite categories among different age, gender, H. pylori and smoking strata.
≤50 y
|
>50 y
|
P values for interaction | ||||
---|---|---|---|---|---|---|
Case/ Control | Adjusted OR * (95%CI) | Case/ Control | Adjusted OR * (95%CI) | |||
All GC | 58/454 | 0.6 (0.3-1.0) | 341/307 | 1.4 (1.0-2.0) | 0.01 | |
Cardia GC | 18/454 | 0.5 (0.2-1.5) | 133/307 | 1.2 (0.8-1.8) | 0.2 | |
Non-cardia GC | 32/454 | 0.6 (0.3-1.2) | 174/307 | 1.5 (1.0-2.2) | 0.049 | |
Female
|
Male
|
|||||
All GC | 100/454 | 2.0 (1.2-3.2) | 303/322 | 0.8 (0.6-1.2) | 0.007 | |
Cardia GC | 38/454 | 2.0 (1.0-4.0) | 114/322 | 0.8 (0.5-1.3) | 0.03 | |
Non-cardia GC | 53/454 | 1.6 (1.0-3.0) | 156/322 | 1.0 (0.7-1.6) | 0.3 | |
H. pylori
-negative
|
H. pylori
-positive
|
|||||
All GC | 96/152 | 0.9 (0.5-1.5) | 274/453 | 1.5 (1.0-2.0) | 0.08 | |
Cardia GC | 30/152 | 1.1 (0.5-2.5) | 108/453 | 1.1 (0.7-1.7) | 0.9 | |
Non-cardia GC | 56/152 | 0.8 (0.4-1.6) | 114/453 | 1.8 (1.0-2.9) | 0.03 | |
Never smoker
|
Ever smoker
|
|||||
All GC | 225/404 | 1.1 (0.7-1.6) | 150/95 | 1.2 (0.7-2.1) | 0.9 | |
Cardia GC | 84/404 | 1.1 (0.6-1.8) | 59/95 | 1.2 (0.6-2.6) | 0.8 | |
Non-cardia GC | 123/404 | 1.1 (0.7-1.8) | 72/95 | 1.2 (0.6-2.4) | 0.9 |
*odds ratios were adjusted for age and gender
Discussion
A recent meta-analysis has investigated the controversies regarding the impact of MTHFRC677T polymorphism and gastric cancer by analyzing 22 qualified case-control studies and concluded that MTHFR C677T polymorphism increases the risk of gastric cancer by about 17% (OR = 1.17, 95% CI = 1.05-1.27) [16].
In our case control study, we have demonstrated that although the overall effect of MTHFR C677T polymorphism on the risk of gastric cancer was modest with a borderline significance, the risk was inflated by interaction with H. pylori infection, age and gender.
Our overall observation of 20% excess risk of gastric cancer among MTHFR C677T carriers did not retain statistical significance after adjustment of the analysis for age and gender. It was, however, in accordance with the summary OR reported by this meta-analysis [16]. Our study suggests that age and gender may interact with MTHFR C677T polymorphism in creating predisposition to gastric cancer. Consequently, stratification of our study population according to age and gender revealed 40 and 100 percent increased risk of gastric cancer for MTHFR C677T SNP carriers aged over 50 years and of the female gender, respectively. Similarly, De Re et al. [22] found an association between the 677TT genotype and gastric cancer in the female as well as older aged (>60 y) Italian population. The impact of age may be partly due to the increased methylation of CpG islands as a consequence of age [23]; older MTHFR 677T carriers may contain higher numbers of methylated CpG islands in comparison with the wild (CC) genotype [24]. Keeping in mind that our stratification for age has slightly dropped the case:control ratio below 1:1 in the stratum of less than 50 years of age, which reduces our strength of interpretation.
The higher susceptibility of female SNP carriers, with potentially defective folate metabolism, to gastric cancer development may be contributed by the low circulating folic acid during their reproductive age particularly in developing countries [25].
Infection with H. pylori is a strongly confirmed etiological factor for gastric cancer, which colonizes the gastric lumen of over 50% of the adult population worldwide [26]. H. pylori is a persistent mucosal pathogen that promotes gastric carcinogenesis through numerous mechanisms including induction of chronic inflammation, which is considered as a critical component in developing site-specific disease and tumor progression [27]. In addition, H. pylori infection eventually results in a decrease in folate absorption as a consequence of elevation of pH and/or reduction of vitamin C concentration in the gastric juice [28]. Hence, due to these and other outcomes, H. pylori infection may accentuate the existing deficiency in MTHFR enzyme activity. Accordingly, we have found a significant interaction between MTHFR C677T polymorphism and H. pylori in development of gastric cancer. In other words, MTHFR C677T polymorphism in H. pylori-positive subjects increased the risk of gastric cancer by 50%, which was inflated up to 80% for tumors of the non-cardia region. In addition, it has been demonstrated that H. pylori infection and chronic inflammation are factors which induce gene methylation in the stomach, which are mostly associated with non-cardia gastric cancer [29]. In addition, Tahara et al. [24] provided evidence that MTHFR C677T may affect DNA methylation of CpG island of p16 gene in H. pylori-infected gastric mucosa. Moreover, Neves Filho et al. [29] reported an association between infection with virulent strains of H. pylori and the methylation of CDKN2A in the distal stomach (non-cardia region) in patients with MTHFR 677TT genotype. Hence, it seems that H. pylori-infected mucosa with malfunctioning MTHFR enzyme due to gene polymorphism may influence gene methylation and create grounds for other carcinogenic activities leading to gastric cancer development particularly in the non-cardia location, where the H. pylori mostly resides.
In stratification analysis according to the histological subtype of the tumor, we found no differences in the risk of diffuse and intestinal gastric cancer in MTHFR C677T carriers. This finding is in agreement with data reported by hospital-based studies from Poland [30] and Germany [31]. These studies reported no significant added risk for GC histological subtypes for those carrying MTHFR 677T allele.
Previous studies have shown that smoking affects folate concentrations at plasma and tissue levels even after correction for folate intake [32, 33], which may cause disorders in DNA synthesis/repair and methylation. On the other hand, MTHFR has been shown to act as an essential component of the same pathway [34]. Contrary to our prior hypothesis, we observed no significant interaction between MTHFR C677T polymorphism and smoking for the risk of gastric cancer. There are, however, similar reports which have also failed to detect such an interaction [35].
In conclusion, although several studies from Iran have reported on the role of MTHFR SNP in various cancers, including breast [36], thyroid [37] and prostate cancers [38], to our knowledge, this is the first study reporting its impact on predisposition to gastric cancer. We, thereby, conclude that MTHFRC677T poly-morphism is associated with the risk of gastric cancer of the non-cardia region, specifically in the H. pylori positive Iranian population. This finding may be an informative clue on the etiology of this deadly cancer in a high prevalence area for H. pylori infection.
Acknowledgment
This study was supported by a generous technical assistance grant (IRN-072) co-funded by the Islamic Development Bank, Saudi Arabia, and Pasteur Institute of Iran.
References
- 1.Leon DA, Davey SmithG. Infant mortality, stomach cancer, stroke, and coronary heart disease: ecological analysis. BMJ. 2000 Jun;320(7251):1705–6. doi: 10.1136/bmj.320.7251.1705. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Lao-Sirieix P, Caldas C, Fitzgerald RC. Genetic predisposition to gastro-oesophageal cancer. Curr Opin Genet. 2010 Dec;20(3):210–7. doi: 10.1016/j.gde.2010.03.002. [DOI] [PubMed] [Google Scholar]
- 3.Stoicov C, Saffari R, Cai X, Hasyagar C, Houghton J. Molecular biology of gastric cancer: Helicobacter infection and gastric adenocarcinoma: bacterial and host factors responsible for altered growth signaling. Gene. 2004 Oct;341:1–17. doi: 10.1016/j.gene.2004.07.023. [DOI] [PubMed] [Google Scholar]
- 4.Bornschein J, Malfertheiner P. Gastric carcinogenesis. Langenbecks Arch Surg. 2011 Aug;396(6):729–42. doi: 10.1007/s00423-011-0810-y. [DOI] [PubMed] [Google Scholar]
- 5.El-Omar EM, Carrington M, Chow WH, McColl BreamJH, Young HA, et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature. 2000 Mar;404(6776):398–402. doi: 10.1038/35006081. [DOI] [PubMed] [Google Scholar]
- 6.Zabaleta J. Multifactorial etiology of gastric cancer. Methods Mol Biol. 2012;863:411–35. doi: 10.1007/978-1-61779-612-8_26. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Goyette P, Sumner JS, Milos R, Duncan AM, Rosenblatt DS, Matthews RG, et al. Human methylenetetrahydrofolatereductase: isolation of cDNA, mapping and mutation identification. Nat Genet. 1994 Jun;7(2):195–200. doi: 10.1038/ng0694-195. [DOI] [PubMed] [Google Scholar]
- 8.Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in Methyl-enetetrahydrofolate reductase. Nat Genet. 1995 May;10(1):111–3. doi: 10.1038/ng0595-111. [DOI] [PubMed] [Google Scholar]
- 9.Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in Methylenetetr hydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab. 1998;64(3):169–72. doi: 10.1006/mgme.1998.2714. [DOI] [PubMed] [Google Scholar]
- 10.Stern LL, Mason JB, Selhub J, Choi SW. Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the Methylenetetrahydrofolate reductase gene. Cancer Epidemiol Biomarkers Prev. 2000 Aug;9:849–53. [PubMed] [Google Scholar]
- 11.Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, et al. Folate deficiency causes uracil mis-incorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc Natl Acad Sci USA. 1997 Apr;94(7):3290–5. doi: 10.1073/pnas.94.7.3290. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Robien K, Ulrich CM. 5, 10-methylenetetrahydrofolate reductase polymorphisms and leukemia risk: a HuGE mini review. Am J Epidemiol. 2003 Apr;157(7):571–82. doi: 10.1093/aje/kwg024. [DOI] [PubMed] [Google Scholar]
- 13.Taioli E, Garza MA, Ahn YO, Bishop DT, Bost J, Budai B, et al. Meta- and pooled analyses of the Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and colorectal cancer: a HuGE-GSEC review. Am J Epidemiol. 2009 Nov;170:1207–21. doi: 10.1093/aje/kwp275. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Qi X, Ma X, Yang X, Fan L, Zhang Y, Zhang F, et al. Methylenetetrahydrofolate reductase polymorphisms and breast cancer risk: a meta-analysis from 41 studies with 16,480 cases and 22,388 controls. Breast Cancer Res Treat. 2010 Sep;123:499–506. doi: 10.1007/s10549-010-0773-7. [DOI] [PubMed] [Google Scholar]
- 15.Langevina SM, Lin D, Matsuo K, Gao CM, Takezaki T, Stolzenberg-Solomon RZ, et al. Review and pooled analysis of studies on MTHFR C677T polymorphism and esophageal cancer. Toxicol Lett. 2009 Jan;184(2):73–80. doi: 10.1016/j.toxlet.2008.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Dong X, Wu J, Liang P, Li J, Yuan L, Liu X. Methylenetetrahydrofolatereductase C677T and A1298C polymorphisms and gastric cancer: a meta-analysis. Arch Med Res. 2010 Feb;41(2):125–33. doi: 10.1016/j.arcmed.2010.01.001. [DOI] [PubMed] [Google Scholar]
- 17.Mohagheghi MA, Mosavi-Jarrahi A, Malekzadeh R, Parkin M. Cancer incidence in Tehran metropolis: the first report from the Tehran Population-based Cancer Registry, 1998-2001. Arch Iran Med. 2009 Jan;12(1):15–23. [PubMed] [Google Scholar]
- 18.Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histological classification. An attempt at a histological classification. 1965;64:31–49. doi: 10.1111/apm.1965.64.1.31. [DOI] [PubMed] [Google Scholar]
- 19.Mohammadi M, Talebkhan Y, Khalili G, Mahboudi F, Massarrat S, Zamaninia L, et al. Advantage of using a home-made ELISA kit for detection of H. Indian J Med Microbiol. 2008 Apr-Jun;26(2):127–31. doi: 10.4103/0255-0857.40525. [DOI] [PubMed] [Google Scholar]
- 20.Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb;16(3) doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Wang Y, Guo W, He Y, Chen Z, Wen D, Zhang X, et al. Association of MTHFR C677T and SHMT1 C1420T with susceptibility to ESCC and GCA in a high incident region of Northern China. Cancer Causes Control. 2007;18(2):143–52. doi: 10.1007/s10552-006-0097-4. [DOI] [PubMed] [Google Scholar]
- 22.De ReV, Cannizzaro R, Canzonieri V, Cecchin E, Caggiari L, De MattiaE, et al. MTHFR polymorphisms in gastric cancer and in first-degree relatives of patients with gastric cancer. Tumour Biol. 2010 Jan;31(1):23–32. doi: 10.1007/s13277-009-0004-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Rashid A, Issa JP. CpG island methylation in gastroenterologic neoplasia: a maturing field. Gastroenterology. 2004 Nov;127(5):1578–88. doi: 10.1053/j.gastro.2004.09.007. [DOI] [PubMed] [Google Scholar]
- 24.Tahara T, Shibata T, Nakamura M, Yamashita H, Yoshioka D, Okubo M, et al. MTHFR 677T carrier influences the methylation status of H. pylori-infected gastric mucosa in older subjects. Dig Dis Sci. 2009 Nov;54(11):2391–8. doi: 10.1007/s10620-008-0624-0. [DOI] [PubMed] [Google Scholar]
- 25.Hercberg S, Galan P. Nutritional anaemias. Baillieres Clin Haematol. 1992 Jan;5(1):143–68. doi: 10.1016/s0950-3536(11)80039-9. [DOI] [PubMed] [Google Scholar]
- 26.Wroblewski LE, Peek RMJr, Wilson KT. pylori and gastric cancer: factors that modulate disease risk. Clin Microbiol Rev. 2010;23(4):713–39. doi: 10.1128/CMR.00011-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002 Dec;420(6917):860–7. doi: 10.1038/nature01322. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Salgueiro J, Zubillaga M, Goldman C, Barrado A, Martinez SarrasagueM, Leonardi N, et al. Review article: is there a link between micronutrient malnutrition and Helicobacter pylori infection. Aliment Pharmacol Ther. 2004 Nov;20(10):1029–34. doi: 10.1111/j.1365-2036.2004.02265.x. [DOI] [PubMed] [Google Scholar]
- 29.Neves FilhoEH, Alves MK, Lima VP, Rabenhorst SH. polymorphism and differential methylation status in gastric cancer: an association with H. pylori infectio. pylori infection. 2010 Dec;457(6):627–33. doi: 10.1007/s00428-010-0996-3. [DOI] [PubMed] [Google Scholar]
- 30.Zhang FF, Terry MB, Hou L, Chen J, Lissowska J, Yeager M, et al. Genetic polymorphisms in folate metabolism and the risk of stomach cancer. Cancer Epidemiol Biomarkers Prev. 2007 Jan;16:115–21. doi: 10.1158/1055-9965.EPI-06-0513. [DOI] [PubMed] [Google Scholar]
- 31.Gotze T, Rocken C, Rohl FW, Wex T, Hoffmann J, Westphal S, et al. Gene polymorphisms of folate metabolizing enzymes and the risk of gastric cancer. Cancer Lett. 2007 Jun;251(2):228–36. doi: 10.1016/j.canlet.2006.11.021. [DOI] [PubMed] [Google Scholar]
- 32.Walmsley CM, Bates CJ, Prentice A, Cole TJ. Relationship between cigarette smoking and nutrient intakes and blood status indices of older people living in the UK: further analysis of data from the national diet and nutrition survey of people aged 65 years and over, 1994/95. Public Health Nutr. 1999 Jun;2(2):199–208. doi: 10.1017/s1368980099000257. [DOI] [PubMed] [Google Scholar]
- 33.Mannino DM, Mulinare J, Ford ES, Schwartz J. Tobacco smoke exposure and decreased serum and red blood cell folate levels: data from the third national health and nutrition examination survey. Nicotine Tob Res. 2003 Jun;5(3):357–62. doi: 10.1080/1462220031000094330. [DOI] [PubMed] [Google Scholar]
- 34.Ueland PM, Hustad S, Schneede J, Refsum H, Vollset SE. Biological and clinical implications of the MTHFR C677T polymorphism. Trends Pharmacol Sci. 2001 Apr;22(4):195–201. doi: 10.1016/s0165-6147(00)01675-8. [DOI] [PubMed] [Google Scholar]
- 35.Lacasana-Navarroa M, Galvan-Portilloa M, Chenb J, López-Cervantes M, López-Carrillo L. Methylenetetra-hydrofolate reductase 677C>T polymorphism and gastric cancer susceptibility in Mexico. Eur J Cancer. 2006 Mar;42(4):528–33. doi: 10.1016/j.ejca.2005.10.020. [DOI] [PubMed] [Google Scholar]
- 36.Hosseini M, Houshmand M, Ebrahimi A. MTHFR polymorphisms and breast cancer risk. Arch Med Sci. 2011 Feb;7(1):134–7. doi: 10.5114/aoms.2011.20618. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Fard-Esfahani P, Fard-Esfahani A, Saidi P, Fayaz S, Mohabati R, Majdi M. An increased risk of differentiated thyroid carcinoma in Iran with the 677C→T homozygous polymorphism in the MTHFR Gene. Cancer Epidemiol. 2011;35(1):56–8. doi: 10.1016/j.canep.2010.10.001. [DOI] [PubMed] [Google Scholar]
- 38.Safarinejad MR, Shafiei N, Safarinejad S. Relationship between three polymorphisms of Methylenetetrahydrofolate reductase (MTHFR C677T, A1298C, and G1793A) gene and risk of prostate cancer: a case-control study. Prostate. 2010 Nov;70(15):1645–57. doi: 10.1002/pros.21200. [DOI] [PubMed] [Google Scholar]