Brief Article
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World J Gastroenterol. May 28, 2013; 19(20): 3043-3051
Published online May 28, 2013. doi: 10.3748/wjg.v19.i20.3043
MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults
Marisa C Alvarez, Juliana C Santos, Nathália Maniezzo, Marcelo S Ladeira, Artur LC da Silva, Isabel CA Scaletsky, José Pedrazzoli Jr, Marcelo L Ribeiro
Marisa C Alvarez, Juliana C Santos, Nathália Maniezzo, José Pedrazzoli Jr, Marcelo L Ribeiro, Unidade Integrada de Farmacologia e Gastroenterologia, Universidade São Francisco, Bragança Paulista 12916-900, SP, Brazil
Marisa C Alvarez, Marcelo L Ribeiro, Programa de Pós Graduação em Genética e Biologia Molecular, State University of Campinas, Campinas 13083-970, SP, Brazil
Marcelo S Ladeira, Departamento de Clínica Médica, UNESP, Botucatu 18618-970, SP, Brazil
Artur LC da Silva, Departamento de Genética, Universidade Federal do Pará, Belém 68400-000, PA, Brazil
Isabel CA Scaletsky, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo 04021-001, SP, Brazil
Author contributions: Ribeiro ML contributed to research design; Alvarez MC performed the research; Santos JC and Maniezzo N contributed to contributions of new reagents/analytical tools; Alvarez MC and Ribeiro ML contributed to data analysis; Alvarez MC wrote the manuscript; da Silva ALC performed the MSI analysis and sequencing; Ladeira MS, Scaletsky ICA and Pedrazzoli Jr J provided specimens.
Supported by The Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, No. 2009/01813-0 and 2008/02678-6; the Conselho Nacional de Desenvolvimento Científico e Tecnológico, No. 471088/2007-2
Correspondence to: Marcelo L Ribeiro, PhD, Unidade Integrada de Farmacologia e Gastroenterologia, Universidade São Francisco, Av. São Francisco de Assis, 218. Jd. São José, Bragança Paulista 12916-900, SP, Brazil. marcelo.ribeiro@usf.edu.br
Telephone: +55-11-40348135 Fax: +55-11-40341825
Received: November 21, 2012
Revised: March 26, 2013
Accepted: April 9, 2013
Published online: May 28, 2013
Abstract

AIM: To evaluate the association between Helicobacter pylori (H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI).

METHODS: The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSP-PCR) in gastric biopsy samples from uninfected or H. pylori-infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ2 test with Yates continuity correction or Fisher’s exact test, and statistical significance for expression analysis was assessed using an unpaired Student’s t-test.

RESULTS: Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P < 0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori-infected chronic gastritis patients was statistically significant (P < 0.05). MLH1 methylation frequencies among H. pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori-infected adult chronic gastritis patients (P < 0.001 and P < 0.01, respectively). The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H. pylori-positive chronic gastritis samples (P < 0.05). The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P < 0.01).

CONCLUSION: In summary, MGMT and MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection.

Keywords: Helicobacter pylori, Microsatellite instability, Promoter methylation, MLH1, MGMT, Gastric cancer

Core tip: Gastric carcinogenesis is a multistep process that is triggered by Helicobacter pylori (H. pylori) infection and characterised by multiple genetic and epigenetic alterations, including the DNA repair genes. To date, few advances have been made to determine the time duration required for to H. pylori infection to induce such epigenetics alteration and thus potentially induce gastric carcinogenesis. The results presented in this study indicate that the methylation of the MGMT and MLH1 promoter regions might depend on the duration of infection because these methylation events were not observed in children.