title: MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults.
abstract: OBJECTIVE
  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 χ(2) 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).
  
  
  CONCLUSIONS
  In summary, MGMT and MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection.
authors.affiliation: Unidade Integrada de Farmacologia e Gastroenterologia, Universidade São Francisco, Bragança Paulista 12916-900, SP, Brazil.
authors.forename: Marisa C
authors.initials: MC
authors.lastname: Alvarez
authors.forename: Juliana C
authors.initials: JC
authors.lastname: Santos
authors.forename: Nathália
authors.initials: N
authors.lastname: Maniezzo
authors.forename: Marcelo S
authors.initials: MS
authors.lastname: Ladeira
authors.forename: Artur L C
authors.initials: AL
authors.lastname: da Silva
authors.forename: Isabel C A
authors.initials: IC
authors.lastname: Scaletsky
authors.forename: José
authors.initials: J
authors.lastname: Pedrazzoli
authors.forename: Marcelo L
authors.initials: ML
authors.lastname: Ribeiro
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000368:Aged; D001706:Biopsy; D016009:Chi-Square Distribution; D002648:Child; D002675:Child, Preschool; D002908:Chronic Disease; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D005260:Female; D005756:Gastritis / Q000175:diagnosis / Q000235:genetics* / Q000382:microbiology; D016481:Helicobacter Infections / Q000175:diagnosis / Q000235:genetics* / Q000382:microbiology; D016480:Helicobacter pylori / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D053842:Microsatellite Instability; D008875:Middle Aged; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012307:Risk Factors; D013274:Stomach Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000382:microbiology; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D055815:Young Adult
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes
keywords: Gastric cancer; Helicobacter pylori; MGMT; MLH1; Microsatellite instability; Promoter methylation