DNA methyltransferase-1(DNMT1) is an important enzyme in determining genomic methylation patterns in mammalian cells. We investigated the associations between SNPs in the DNMT1 gene and risks of developing H. pylori seropositivity, gastric atrophy and gastric cancer in the Chinese population.
The study consisted of 447 patients with gastric cancer; 111 patients with gastric atrophy; and 961 healthy controls. Five SNPs, rs10420321, rs16999593, rs8101866, rs8111085 and rs2288349 of the DNMT1 gene were genotyped. Anti-H.pylori IgG was detected by ELISA. Gastric atrophy was screened by the level of serum pepsinogen Ιand II and then confirmed by endoscopy and histopatholgical examinations.
The age- and sex-adjusted OR of H. pylori seropositivity was 0.67 (95%CI: 0.51–0.87) for rs8111085 TC/CC genotypes, significantly lower than the TT genotype in healthy controls. The adjusted OR of H.pylori seropositivity was 0.68 (95%CI: 0.52–0.89) for rs10420321 AG/GG genotypes. In addition, patients carrying rs2228349 AA genotype have a significantly increased risk for H.pylori seropositivity (OR = 1.67; 95%CI: 1.02–2.75). Further haplotype analyses also showed that the ATTTG and ATCTA are significantly associated with increased risks in H.pylori infection compared to the GTCCG haplotype (OR = 1.38, 95%CI: 1.08–1.77; OR = 1.40, 95% CI: 1.09–1.80). The adjusted ORs of gastric atrophy were 1.66 (95%CI: 1.06–2.61) for rs10420321 GG genotype, and 1.67 (95%CI 1.06–2.63, P = 0.03) for rs8111085 CC genotype, but no association was found between SNPs in the DNMT1 gene and risk of developing gastric cancer.
Individuals with rs10420321 GG and rs8111085 CC genotype of the DNMT1 gene were associated with reduced risks for H.pylori infection. On the other hand, higher risks of gastric atrophy were found in the carriers with these two genotypes compared to other genotypes. Our results suggested that SNPs of DNMT1 could be used as genotypic markers for predicting genetic susceptibilities to H.pylori infection and risks in gastric atrophy.
Citation: Jiang J, Jia Z, Cao D, Jin M-S, Kong F, et al. (2012) Polymorphisms of the DNA Methyltransferase 1 Associated with Reduced Risks of Helicobacter pylori Infection and Increased Risks of Gastric Atrophy. PLoS ONE 7(9): e46058. doi:10.1371/journal.pone.0046058
Editor: Masaru Katoh, National Cancer Center, Japan
Received: June 26, 2012; Accepted: August 28, 2012; Published: September 25, 2012
Copyright: © Jiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study has been supported by National Natural Science Foundation of China (30940060 and 81072369).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Gastric cancer is the most common malignancy of gastrointestinal tract in East Asians, and the third most common cause of cancer-related deaths in China , . Helicobacter pylori (H.pylori) infection has been found to be a major risk factor for gastric cancer, through induction of gastric atrophy and progression to precancerous lesions , . Although H.pylori is estimated to be found in at least half of the world’s population, few develop to gastric adenocarcinoma. The extent of gastric damages induced by H.pylori infection seems to vary between one person to another, suggesting that the interaction between the host genetic traits and the bacterial virulence plays an important role in long-term outcomes of H.pylori infection , , . Single nucleotide polymorphisms (SNPs) are most common forms of genetic traits which may contribute genetic susceptibilities to gastric carcinogenesis.
DNA methylation is important in transcription regulation and chromatin remodeling in mammalian cells . Aberrant DNA methylation of CpG islands is a common epigenetic change found in gastric cancers and H.pylori infection has been shown to induce alterations of DNA methylation in gastric mucosae , . Increasing evidence suggests that aberrant methylation in gastric mucosae creates a field for cancerization happening in the early and precancerous stages. However, those studies focus on the impacts of environment factors (H.pylori), not host factors, such as methyltransferase (DNMT), which may affect epigenetic regulation.
DNA methyltransferase-1 (DNMT1) catalyzes the DNA post-replication methylation and maintains DNA methylation patterns during cell divisions . Several studies have found that DNMT1 is over-expressed in human cancers including gastric cancer, suggesting that it may be involved in tumorgenesis and tumor progression , . The DNMT1 gene is located on chromosome 19p13.2 with a total size of 62 kb and is constituted of 40 exons. Mutations in coding regions of the DNMT1 have been reported in colorectal cancer, such as a single base deletion in exon 23 resulting in deletion of the whole catalytic domain; a point mutation in exon 35 resulting in an amino acid substitution in the catalytic domain . The inactivation of DNMT1 by mutations can cause a genome-wide alteration of the DNA methylation status.
Two SNPs rs2241531 and rs4804490 of the DNMT1 gene have been identified to be associated with clearance of HBV infection in the Korean population . Polymorphism rs75616428, a nonsynonymous SNP in exon 4 of the DNMT1 was demonstrated to be weakly associated with an increased production of anti-SSB (La) antibody in systemic lupus erythematosus patients . Furthermore, an association was reported between the haplotypes of DNMT1 and sensitivities to exposure of bensopyrene diol epoxide, supporting involvements of these SNPs in protecting the cell from DNA damage and reducing the intrinsic susceptibility to cancer .
These results suggested that the genetic variants of the DNMT1 gene may modulate susceptibilities to virus infection and cancer development. Therefore, the aim of this investigation is to assess associations between polymorphisms in the DNMT1 gene with H.pylori infection and clinical outcomes of H.pylori infection within the Chinese population.
Four hundred and forty seven gastric cancer cases were selected from the Department of Gastric and Colorectal Surgery, First Hospital of Jilin University, between 2008 and 2010. All patients underwent tumor resections with histological confirmed gastric adenocarcinoma. Individuals with gastric atrophy and healthy controls were recruited from examinees in the health check-up centre of the same hospital from 2009 to 2010. In brief, a total of 1111 individuals without cancer history (654 males and 457 females, ages of 35 to 80 years old) participated in the study. The examinees were Han descent from the area of Changchun. 150 subjects were found to have gastric atrophy by serum PG examination and 111 of them were confirmed by biopsy and histopathological examinations; 39 subjects were excluded from the study as they either had been diagnosed pseudopositive for gastric atrophy (17 cases) or had rejected endoscopic examination (22 cases). The remaining examinees (961) were included in the control group. Informed consent was obtained from all patients and the study protocol was approved by the ethics committee of the first affiliated hospital, Jilin University.
Tests for H.pylori Infection and Diagnosis of Gastric Atrophy
Serum immunoglobulin (Ig) G antibodies to H. pylori were detected using a kit for H. pylori -Ig G enzyme-linked immunosorbent assay (ELISA) (Biohit, Helsink, Finland). The antibody titers were quantified by optical density (OD) readings according to the manufacturer’s protocol and titres higher than the threshold value of 30EIU were considered as positive for H. pylori infection. Levels of Pepsinogen Ι and II (PG Ι and PG II) in serum were measured using ELISA kits (Biohit, Helsink, Finland). For gastric atrophy screening, cut-off points used in this study were <82.3 ng/ml for PG Ι and <6.05 for PGΙ/PG II ratio, as they had previously been validated against histological confirmatory studies for gastric atrophy . The quality control samples in kits showed coefficients of variation of 4.5%, 4.3% and 4.7% for H.pylori, PG Ι and PG II, respectively. All gastric atrophy suspected cases, based on the serum screening, were confirmed by endoscopy, biopsy and histological examinations for final diagnosis.
Selection of Tagging SNPs
The principal hypothesis underlying this study is that one or more common SNPs in the DNMT1 gene are associated with risks for H.pylori infection, gastric atrophy and gastric cancer. Thus, the aim of SNP tagging is to identify a set of SNPs that efficiently tag all known SNPs. We postulate that such SNPs are also likely to tag any hitherto unidentified SNPs in the DNMT1 gene. Haplotype tagging SNPs (htSNPs) were selected from Han Chinese data in the HapMap Project (06-02-2009 HapMap) using the SNPbrowser™ Software v4.0 to capture SNPs with a minimum minor allele frequency (MAF) of 0.05 with a pair-wise r square of 0.8 or greater . There are 27 SNPs at minor allele frequencies >0.05 in the DNMT1 gene in Chinese on HapMap, Three SNPs lie in coding regions, including non-synonymous SNPs rs16999593 (His97Arg) in exon 4, rs8111085 (Ile327Val) in exon 12 and a synonymous SNP rs2228611 in exon 17. 24 SNPs reside in introns of the DNMT1 gene and 21 of them are shown in a complete linkage disequilibrium (LD) with Tag SNP (D′ = 1 and r2>0.8). The synonymous SNP rs2228611 was excluded from genotyping due to being in an absolute LD (D′ = 1 and r2 = 1) with rs8101866. Finally, five SNPs rs10420321, rs16999593, rs8101866, rs2288349 and rs8111085 were selected for a larger-scale genotyping.
Genomic DNA from whole blood sample was extracted with an AxyPrep blood Genomic DNA extraction Kit (AP-MN-BL-GDNA-250, Axygen Biosciences, Union City, USA). Polymerase chain reactions (PCR) were carried out in a 5 ul per reaction on the genomic DNA (10 ng) using a TaqMan universal PCR master mix (Applied Biosystems). Forward, reverse primers, FAM and VIC labeled probes were designed by Applied Biosystems (ABI Assay-by-Designs). Sequences of primers and probes are available on request. Amplification conditions on BIO-RAD S1000TM thermal cyclers (Bio-Rad Laboratories, Hercules, California) were as follows: 1 cycle of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The PCR products were genotyped on an ABI PRISM 7900 HT Sequence Detector in end-point mode using the Allelic Discrimination Sequence Detector Software V2.3 (Applied Biosystems). For the software to recognize genotypes, two non-template controls were included in each of 384-well plates. All samples were arrayed together in four 384-well plates, and the fifth plate contained eight duplicate samples from each of four plates to ensure the quality of genotyping (the concordance was >99% for all SNPs).
Deviations of genotype frequencies in controls from those expected under the Hardy-Weinberg equilibrium were assessed by a goodness-of-fit x2-test. Linkage disequilibrium (LD) between pairs of biallelic loci was determined using two measures, D´ and r2. Either Chi-square test or Fisher’s exact test was performed by comparing distributions of genotype frequencies between patients and controls. Risks associated with rare genotypes were estimated as odds ratios (ORs). Corresponding 95% confidence intervals (CIs) by unconditional logistic regression were adjusted by age (scale variable), sex (nominal variable) and H. pylori antibody (nominal variable). For haplotypes with frequencies >1%, risks were compared to those of the reference haplotype(major haplotype in the control group)using an unconditional logistic regression model with the HAPSTAT version 3.0 software (Tammy Bailey, Danyu Lin and the University of North Carolina, NC, USA) , . All statistical tests were two-tailed and P-values less than 0.05 were considered to be statistically significant. All analyses were performed using statistical software for windows, SAS version 9.2 (SAS Institute, Cray, NC, USA). The power of the statistical tests was calculated using the QUANTO Version 1.2.3 software program (Jim Gauderman and John Morrison, University of Southern California, CA, USA).
Allele Frequencies of the htSNPs
A total of 447 patients with gastric cancer (322 males and 125 females, aged between 35 to 80 years old), 111 subjects with gastric atrophy and 961 subjects who passed health checks were recruited in this study. The characteristics of subjects are summarized in table 1. The mean age was older in gastric cancer patients compared to the control group (61.6 vs. 50.6 years; P<0.001). There were more females in the control group (P<0.001). Prevalence rates of H.pylori seropositivity were significantly higher in the gastric cancer and gastric atrophy groups compared to the control group (69.1%, 75.7% vs. 49.7%, P<0.001). Genotype distributions for five SNPs in the control group were consistent with the Hardy-Weinberg equilibrium (P values were 0.32, 0.91, 0.31, 0.14 and 0.55 respectively). Rs10420321 and rs8111085 genotype distributions in the gastric atrophy group were found to be statistically significantly different to the control group and gastric cancer (P values were 0.04 and 0.05), however no statistically significant difference were found between groups for the remaining three SNPs.
Table 1. Characteristics of the subjects and the DNMT1 polymorphisms.doi:10.1371/journal.pone.0046058.t001
Associations of SNPs with H.pylori Seropositivity, Gastric Atrophy and Gastric Cancer
Rates of H.pylori seropositivity were statistically significantly reduced in subjects bearing with rs8111085 TC and CC genotypes (P value = 0.01). The age-adjusted and sex-adjusted ORs relative to the TT genotype were 0.70 (95%CI: 0.52–0.93) and 0.60 (95%CI: 0.42–0.86), respectively, showing a dominant effect of the rare allele (OR = 0.67, 95%CI: 0.51–0.87). A similar association was found between rs10420321 and risk of H.pylori seropositivity, a dominant effect of the rare allele G. ORs were 0.62 (95%CI: 0.43–0.90) for the AG, 0.70 (95%CI: 0.53–0.94) for the GG genotype, and 0.68 (95%CI: 0.52–0.89) for the combined AG/GG. The heterogeneity test of association was not statistically significant for rs2228349 (P = 0.68), but evidence suggested a recessive mode of the rare allele (OR = 1.67; 95%CI: 1.02–2.75). Haplotypes with frequencies ≥1% are shown in table 2. Five major haplotypes accounted for over 98% of the distribution. Furthermore, the haplotype analysis revealed that the ATTTG and ATCTA were statistically significantly associated with increased risks for H.pylori seropositivity versus the GTCCG haplotype (OR = 1.38, 95%CI: 1.08–1.77; OR = 1.40, 95%CI: 1.09–1.80).
Table 2. DNMT1 polymorphisms for H.pylori seropositivity in control groups.doi:10.1371/journal.pone.0046058.t002
The age-, sex- and H.pylori antibody-adjusted ORs for gastric atrophy were 1.67 (95%CI 1.06–2.63) for the CC compared to the TT genotype at rs8111085 and 1.66 (95%CI: 1.06–2.61) for the GG versus the AA genotype at rs10420321. However, the haplotype analysis did not show association between the haplotype and the risk in gastric atrophy. Associations between the SNPs in the DNMT1 gene and gastric cancer were also examined, but no significant correlation was found between the SNPs and gastric cancer risk (Table 3). In subgroup analysis, no correlation was found between the SNPs and Lauren’s classification, tumor differentiation and TNM staging (data not shown).
Previous studies have shown that H.pylori infection could induce aberrant DNA methylation at the CpG islands, resulting in inactivations of tumor suppressor genes in gastric mucosa, and creating predisposed fields for cancerization. In the present study, we investigated impacts of host factors on H. pylori infection and carcinogenesis. The association between the SNPs of the DNMT1 gene with H.pylori infection, gastric atrophy and gastric cancer in the Chinese Han population were studied. The results showed that the subjects bearing rs2228349 AA genotype has a significantly increased risk for H.pylori infection. In addition, our results revealed that C allele at rs8111085 and G allele at rs10420321 in the DNMT1 gene protect the carrier against H.pylori infection, while, surprisingly, they are associated with increased risks in developing gastric atrophy.
Colonization of H.pylori at the human gastric mucosa is associated with complex local and systemic immune responses. In response to H.pylori infection, uncommitted naïve CD4+ T-cell precursors differentiate into several T helper (Th) cell subsets (Th1, Th2, Th17 and T regulatory cells) and secrete distinct cytokines. IFN-γ producing Th1 cells are important for the clearance of intracellular pathogens and IL-4 producing Th2 cells are crucial for humoral immunity and the clearance of extracellular pathogens –. Significant inverse correlations were found between bacterial colonization and gastric expressions of IFN-γ, TNF-α, IL-12 and IL-17 . Increasing evidences showed that Th1 response, induced by H.pylori infection, contributes to the development of gastric atrophy . In contrast, the Th2 cytokine IL-4 limits H.pylori associated gastric inflammation and favours H.pylori clearance . Epigenetics are involved in the regulation of these developmental programs through activation or silencing of specifying genes during T-cell differentiation. IL-2, IL-3, IL-5, IL-10, IL-13, especially IL-4 and IFN-γ expressions were all up-regulated in CD4 and CD8 T cells with reduced DNA methylation, due to the lack of DNMT1 maintenance –. Therefore, we propose that the polymorphisms of DNMT1 may impair the function of the DNMT1 protein, and fail to maintain the silenced state of Th1 signature genes in Th2 cells, skewing T-cell response towards Th1 responses, resulting in clearance of H.pylori infection and causing relative severe gastric atrophy at the same time.
So far, the functions of DNMT1 polymorphisms are still unknown. The polymorphism rs8111085 at exon 12 leads to an amino acid change from Ile to Val, which may affect the corresponding DNMT1 protein structure or function. The protein may be impaired in its ability to stimulate de novo DNA methylation and to maintain the methylation status . Rs10420321 and rs2288349 are both located within introns of DNMT1. Using bioinformatic tool (http://compbio.cs.queensu.ca/F-SNP/), rs10420321 on intron 1 is predicted to affect the transcriptional regulation of the DNMT1 mRNA, while rs2288349 does not appear to reside in any transcription factor binding sites or splicing sites . Further LD analysis showed a complete LD (D′ = 0.996 and r2 = 0.985) between rs10420321 and the functional polymorphism rs8111085, but rs2288349 only has a much weaker LD with rs8111085 (r2 = 0.266). Therefore, it is plausible that the association with H.pylori infection is due to rs2288349 in a complete or perfect LD with unidentified causative SNPs. Further studies of DNMT1 sequence variants and their biologic functions may shed light on associations of the DNMT1 polymorphism and the risk for H.pylori infection and for gastric atrophy.
Over-expression of DNMT1 mRNA and protein is detected in gastric cancer suggesting that DNMT1 may contribute to tumorgenesis , . Moreover, our study showed DNMT1 polymorphisms are not associated with gastric cancer, consistent with previous findings in which no correlation was found between the polymorphisms of the DNMT1 gene and the risk for gastric cancer or colorectal cancer in the Iranian population , . Conflicting results were reported in several association studies on polymorphisms of the DNMT1 gene in relation to risks in breast cancer and ovarian cancer. While rs16999593 in exon 4 has been reported to be associated with an increased risk of sporadic infiltrating ductal breast carcinoma in Han Chinese female patients in one study , others found no apparent association between common DNMT1 polymorphisms or haplotypes and breast cancer risk among Chinese and European women –. The minor allele of rs9305012 at intron 23 of the DNMT1 gene was found to be associated with a decreased risk for ovarian cancer among Caucasian multivitamin supplement users . The difference in results from various studies may be due to different ethnic groups, unknown environmental factors or the interplay between environmental factors and genetic predisposition. Further studies are required to understand interactions of host and environmental factors and how and when these epigenetic interactions occur.
The limitation of our study is the small sample size for the H.pylori (+) cohort due to a low incidence in patients with gastric atrophy in China. A large-scale study recruiting more patients with gastric atrophy is required to confirm our findings in the future.
In conclusion, this study provides the first evidence of strong associations between polymorphisms and common haplotypes of the DNMT1 gene with decreased risks for H.pylori infection in the Chinese population. In addition, polymorphisms of the DNMT1 gene are also significantly associated with increased risks of developing gastric atrophy, suggesting that they could be used as biomarkers for predicting genetic susceptibilities to H.pylori infection and risk assessments in gastric atrophy. Further investigations are required to fully understand the biological importance of these polymorphisms.
The authors would like to thank everybody participating in this study, particularly to Mr Chang-Song Guo and Ms Ying Song for their technical support.
Conceived and designed the experiments: JJ XC. Performed the experiments: JJ ZJ XC DC MSJ FK. Analyzed the data: JJ ZJ. Contributed reagents/materials/analysis tools: MSJ DC FK JS. Wrote the paper: JJ XC.
- 1. Hamiliton SA (2000) WHO classification of tumors. Lyon: IARC Press.
- 2. (2011) Yearbook of health in the people’s republic of China 2010: China Yearbook Press.
- 3. Lochhead P, El-Omar EM (2007) Helicobacter pylori infection and gastric cancer. Best Pract Res Clin Gastroenterol 21: 281–297. doi: 10.1016/j.bpg.2007.02.002
- 4. Kabir S (2009) Effect of Helicobacter pylori eradication on incidence of gastric cancer in human and animal models: underlying biochemical and molecular events. Helicobacter 14: 159–171. doi: 10.1111/j.1523-5378.2009.00677.x
- 5. Amieva MR, El-Omar EM (2008) Host-bacterial interactions in Helicobacter pylori infection. Gastroenterology 134: 306–323. doi: 10.1053/j.gastro.2007.11.009
- 6. Snaith A, El-Omar EM (2008) Helicobacter pylori: host genetics and disease outcomes. Expert Rev Gastroenterol Hepatol 2: 577–585. doi: 10.1586/17474188.8.131.527
- 7. Hishida A, Matsuo K, Goto Y, Hamajima N (2010) Genetic predisposition to Helicobacter pylori-induced gastric precancerous conditions. World J Gastrointest Oncol 2: 369–379. doi: 10.4251/wjgo.v2.i10.369
- 8. Cedar H, Bergman Y (2012) Programming of DNA methylation patterns. Annu Rev Biochem 81: 97–117. doi: 10.1146/annurev-biochem-052610-091920
- 9. Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, et al. (2006) High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res 12: 989–995. doi: 10.1158/1078-0432.ccr-05-2096
- 10. Nakajima T, Yamashita S, Maekita T, Niwa T, Nakazawa K, et al. (2009) The presence of a methylation fingerprint of Helicobacter pylori infection in human gastric mucosae. Int J Cancer 124: 905–910. doi: 10.1002/ijc.24018
- 11. Okano M, Xie S, Li E (1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet 19: 219–220.
- 12. Kanai Y, Ushijima S, Kondo Y, Nakanishi Y, Hirohashi S (2001) DNA methyltransferase expression and DNA methylation of CPG islands and peri-centromeric satellite regions in human colorectal and stomach cancers. Int J Cancer 91: 205–212. doi: 10.1002/1097-0215(200002)9999:9999<::aid-ijc1040>3.0.co;2-2
- 13. Saito Y, Kanai Y, Sakamoto M, Saito H, Ishii H, et al. (2001) Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis. Hepatology 33: 561–568. doi: 10.1053/jhep.2001.22507
- 14. Kanai Y, Ushijima S, Nakanishi Y, Sakamoto M, Hirohashi S (2003) Mutation of the DNA methyltransferase (DNMT) 1 gene in human colorectal cancers. Cancer Lett 192: 75–82. doi: 10.1016/s0304-3835(02)00689-4
- 15. Chun JY, Bae JS, Park TJ, Kim JY, Park BL, et al. (2009) Putative association of DNA methyltransferase 1 (DNMT1) polymorphisms with clearance of HBV infection. BMB Rep 42: 834–839. doi: 10.5483/bmbrep.2009.42.12.834
- 16. Park BL, Kim LH, Shin HD, Park YW, Uhm WS, et al. (2004) Association analyses of DNA methyltransferase-1 (DNMT1) polymorphisms with systemic lupus erythematosus. J Hum Genet 49: 642–646. doi: 10.1007/s10038-004-0192-x
- 17. Leng S, Stidley CA, Bernauer AM, Picchi MA, Sheng X, et al. (2008) Haplotypes of DNMT1 and DNMT3B are associated with mutagen sensitivity induced by benzo[a]pyrene diol epoxide among smokers. Carcinogenesis 29: 1380–1385. doi: 10.1093/carcin/bgn121
- 18. Cao Q, Ran ZH, Xiao SD (2007) Screening of atrophic gastritis and gastric cancer by serum pepsinogen, gastrin-17 and Helicobacter pylori immunoglobulin G antibodies. J Dig Dis 8: 15–22. doi: 10.1111/j.1443-9573.2007.00271.x
- 19. De La Vega FM (2007) Selecting single-nucleotide polymorphisms for association studies with SNPbrowser software. Methods Mol Biol 376: 177–193. doi: 10.1007/978-1-59745-389-9_13
- 20. Lin DY, Zeng D, Millikan R (2005) Maximum likelihood estimation of haplotype effects and haplotype-environment interactions in association studies. Genet Epidemiol 29: 299–312. doi: 10.1002/gepi.20098
- 21. Zeng D, Lin DY, Avery CL, North KE, Bray MS (2006) Efficient semiparametric estimation of haplotype-disease associations in case-cohort and nested case-control studies. Biostatistics 7: 486–502. doi: 10.1093/biostatistics/kxj021
- 22. Bodger K, Crabtree JE (1998) Helicobacter pylori and gastric inflammation. Br Med Bull 54: 139–150. doi: 10.1093/oxfordjournals.bmb.a011664
- 23. D’Elios MM, Amedei A, Del Prete G (2003) Helicobacter pylori antigen-specific T-cell responses at gastric level in chronic gastritis, peptic ulcer, gastric cancer and low-grade mucosa-associated lymphoid tissue (MALT) lymphoma. Microbes Infect 5: 723–730. doi: 10.1016/s1286-4579(03)00114-x
- 24. Haeberle HA, Kubin M, Bamford KB, Garofalo R, Graham DY, et al. (1997) Differential stimulation of interleukin-12 (IL-12) and IL-10 by live and killed Helicobacter pylori in vitro and association of IL-12 production with gamma interferon-producing T cells in the human gastric mucosa. Infect Immun 65: 4229–4235.
- 25. Bergman M, Del Prete G, van Kooyk Y, Appelmelk B (2006) Helicobacter pylori phase variation, immune modulation and gastric autoimmunity. Nat Rev Microbiol 4: 151–159. doi: 10.1038/nrmicro1344
- 26. Flach CF, Ostberg AK, Nilsson AT, Malefyt Rde W, Raghavan S (2011) Proinflammatory cytokine gene expression in the stomach correlates with vaccine-induced protection against Helicobacter pylori infection in mice: an important role for interleukin-17 during the effector phase. Infect Immun 79: 879–886. doi: 10.1128/iai.00756-10
- 27. Smythies LE, Waites KB, Lindsey JR, Harris PR, Ghiara P, et al. (2000) Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice. J Immunol 165: 1022–1029.
- 28. Makar KW, Perez-Melgosa M, Shnyreva M, Weaver WM, Fitzpatrick DR, et al. (2003) Active recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes and T cells. Nat Immunol 4: 1183–1190. doi: 10.1038/ni1004
- 29. Penix LA, Sweetser MT, Weaver WM, Hoeffler JP, Kerppola TK, et al. (1996) The proximal regulatory element of the interferon-gamma promoter mediates selective expression in T cells. J Biol Chem 271: 31964–31972. doi: 10.1074/jbc.271.50.31964
- 30. Bruniquel D, Schwartz RH (2003) Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat Immunol 4: 235–240. doi: 10.1038/ni887
- 31. Fitzpatrick DR, Shirley KM, McDonald LE, Bielefeldt-Ohmann H, Kay GF, et al. (1998) Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells. J Exp Med 188: 103–117. doi: 10.1084/jem.188.1.103
- 32. Makar KW, Wilson CB (2004) DNA methylation is a nonredundant repressor of the Th2 effector program. J Immunol 173: 4402–4406.
- 33. Svedruzic ZM (2011) Dnmt1 structure and function. Prog Mol Biol Transl Sci 101: 221–254.
- 34. Lee PH, Shatkay H (2009) An integrative scoring system for ranking SNPs by their potential deleterious effects. Bioinformatics 25: 1048–1055. doi: 10.1093/bioinformatics/btp103
- 35. Etoh T, Kanai Y, Ushijima S, Nakagawa T, Nakanishi Y, et al. (2004) Increased DNA methyltransferase 1 (DNMT1) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpG islands in gastric cancers. Am J Pathol 164: 689–699. doi: 10.1016/s0002-9440(10)63156-2
- 36. Khatami F, Noorinayer B, Ghiasi S, Mohebi R, Hashemi M, et al. (2009) Lack of effects of single nucleotide polymorphisms of the DNA methyltransferase 1 gene on gastric cancer in Iranian patients: a case control study. Asian Pac J Cancer Prev 10: 1177–1182.
- 37. Khatami F, Noorinayer B, Mohebi SR, Ghiasi S, Mohebi R, et al. (2009) Effects of amino acid substitution polymorphisms of two DNA methyltransferases on susceptibility to sporadic colorectal cancer. Asian Pac J Cancer Prev 10: 1183–1188.
- 38. Xiang G, Zhenkun F, Shuang C, Jie Z, Hua Z, et al. (2010) Association of DNMT1 gene polymorphisms in exons with sporadic infiltrating ductal breast carcinoma among Chinese Han women in the Heilongjiang Province. Clin Breast Cancer 10: 373–377. doi: 10.3816/cbc.2010.n.049
- 39. Ye C, Beeghly-Fadiel A, Lu W, Long J, Shu XO, et al. (2010) Two-stage case-control study of DNMT-1 and DNMT-3B gene variants and breast cancer risk. Breast Cancer Res Treat 121: 765–769. doi: 10.1007/s10549-009-0569-9
- 40. Cebrian A, Pharoah PD, Ahmed S, Ropero S, Fraga MF, et al. (2006) Genetic variants in epigenetic genes and breast cancer risk. Carcinogenesis 27: 1661–1669. doi: 10.1093/carcin/bgi375
- 41. Harlid S, Ivarsson MI, Butt S, Hussain S, Grzybowska E, et al. (2011) A candidate CpG SNP approach identifies a breast cancer associated ESR1-SNP. Int J Cancer 129: 1689–1698. doi: 10.1002/ijc.25786
- 42. Kelemen LE, Sellers TA, Schildkraut JM, Cunningham JM, Vierkant RA, et al. (2008) Genetic variation in the one-carbon transfer pathway and ovarian cancer risk. Cancer Res 68: 2498–2506. doi: 10.1158/0008-5472.can-07-5165