Detection of CDH1 gene promoter hypermethylation in gastric cancer and chronic gastritis.

Aim: The current study aimed to assess the frequency of CDH1 promoter gene hypermethylation in gastric cancer and chronic gastritis and its correlation with clinicopathological aspects. Methods: Methylation-specific PCR was used to detect CDH1 promoter gene hypermethylation in 53 chronic gastritis patients and 40 gastric cancer patients along with normal adjacent tissues. Results: The chronic gastritis group comprised 29 males and 24 females with a mean age of 51.8 ± 12.96 years, and 49.1 % of them were positive for H. pylori infection. The frequency of CDH1 hypermethylation in gastritis lesions was 18.8 %. CDH1 hypermethylation showed a significant correlation with H. pylori infection (p = 0.039), but no significant association was observed with other clinical features. The gastric cancer group consisted of individuals with a mean age of 65.4 ± 10.6, among them, 77.5 % were male and 22.5 % were female, 62.5 % had PT3 tumors, 40 % had PN1 lymph node involvement, and the majority (47.5 %) of samples were obtained from body segment. CDH1 hypermethylation was significantly associated with depth of invasion (p = 0.017) and nodal invasion (p = 0.041) in this group. In both groups, normal adjacent specimens lacked CDH1 hypermethylation, and there was no statistically significant correlation between CDH1 hypermethylation and age at which the tumor was diagnosed, gender, activity level, or tumor location. Conclusion: This study demonstrates that E-cadherin methylation is associated with some characteristics of chronic gastritis and gastric cancer. These findings support previous research indicating that CDH1 hypermethylation may play a significant role in the development of gastric cancer.

Profile and frequency of p53 gene alterations in gastritis lesions from Iran.

BACKGROUND: It has been frequently shown that p53 alterations have an important role in the development of gastric cancers but there is no data on p53 alteration in gastric cancer and its precancerous lesions from Iran although this country experiences one of the highest gastric cancer incidence and mortality rates in the world. The purpose of this study was to do a comprehensive assessment of p53 alterations in the Iranian population of gastritis patients and to evaluate the association between p53 alterations, microsatellite status and clinicopathological aspects. METHODS: After DNA extraction, PCR sequencing was done for exons 2-7. Also microsatellite status was evaluated using five microsatellite markers: NR-27, NR-21, NR-24, BAT-25 and BAT-26. RESULTS: The highest rate of alteration was seen in codons 72 (85.6%, SNP) and 248 (30.9%, mutation). Also, we found 2 new mutations in codons 9 and 146. In contrast with previous work, transition at the CpG codons was relatively rare. Nucleotide alterations were more prevalent in the Helicobacter pylori-positive group but not significantly. Neither nuclear staining for p53 protein nor microsatellite instability was seen in gastritis lesions. CONCLUSION: p53 alterations might contribute to the pathogenesis of gastritis and perhaps gastric cancer in Iran. However, the different spectrum seen here implies other mechanism(s) in gastritis and gastric cancer development in the Iranian population.

SMAD4 mutations identified in Iranian patients with colorectal cancer and polyp.

AIM: Search for SMAD4 mutations in Colorectal cancer (CRC) or polyp in Iran. BACKGROUND: Colorectal cancer is one of the five prevalent cancers among the Iranian population; however, its molecular mechanisms are not fully understood. The vast majority of CRCs arise from neoplastic polyp. METHODS: Colorectal cancer and polyp lesions with matched normal tissues from patients who had undergone colonoscopy in Taleghani Hospital (January 2009 - November 2010) were included in the study. DNA extraction and PCR-sequencing for exons 5-11 of the SMAD-4 gene were carried out on 39 and 30 specimens of polyp and adenocarcinoma, respectively. RESULTS: Of cancer and polyp specimens, 33.3% and 28.2%, respectively, were mutated in the Smad-4 gene. The majority of SMAD4 mutations, especially in the MH2 domain were missense mutations (63.6% and 68.75, respectively). In cancer, codon 435 and in polyp, codons 435 and 399 were the most common alterations. Unlike cancer specimens, transversion was found frequently in the polyp (56.25% vs. 35.7%). CG>TA transition was about 18.75% and 14.3% in cancer and polyp samples, respectively. Mutations of codon 264 and C.483-4 were seen both in cancer and neoplastic polyps. CONCLUSION: As frequent alterations, missense mutations are presumably selected during tumorigenesis and polyposis due to their structural impacts on SMAD4 functions and TGF-ß signaling pathway. The lower frequency of CG>TA can be attributed to global genome hypomethylation. Presumably, SMAD4 mutations had occurred in the primary polyps, and some of these mutated cells then developed into carcinoma. On the other hand, polyp-specific mutations may lower the risk of CRC.

Evaluation of global genome methylation in gastritis lesion and its correlation with clinicopatological findings.

Global genome hypomethylation as an epigenetic phenomenon may induce (pre)neoplastic transformation through inducing chromosomal and genomic instability and activating oncogenes. Global genome hypomethylation has a fundamental role in early stages of tumorigenesis but little is known about this epigenetic event in gastric precancerous lesions such as gastritis. Therefore, we decided to evaluate this issue in gastritis lesion for obtaining new insight toward molecular biology of gastric cancer. Here we used a technique composed of restriction enzyme digestion and pyrosequencing known as luminometric methylation assay to evaluate this issue. DNA obtained from normal and gastritis lesions was digested with HpaII (sensitive to methylation in its cut site) and MspI (insensitive). Overhangs resulting from these enzymes then fill in by polymerase extension assay using pyrosequencing instrument. Nucleotide incorporation during polymerase extension generates light, which expresses as pick in the pyrogram. By comparing the height of picks obtained form both enzymes it can be possible to evaluate and compare global genome methylation level of gastritis and normal tissues. If the target site is fully methylated, the HpaII/MspI (their pick height) will approach zero. If not, this ratio will be around 1. In the other conditions this ratio varies between 0 and 1. Comparing the ratio of normal and gastritis sample, it can be inferred whether or not gastritis is hypomethylated. This study was performed on 83 gastritis and normal adjacent tissues. The patients included 34 male and 49 female and were 15 to 83 years old. According to our study, gastritis tissue was hypomethylated more than the normal tissue (p = 0.028). Global genome methylation has no significant correlation with MSI, pathological findings, age, and gender. We conclude that global genome hypomethylation occurs in the gastritis level. This reduction probably continues in the next steps toward gastric cancer and may induce other epigenetic and/or genetic changes (such as MSI) that promote carcinogenesis.

Correlation between global genome methylation and mutation at CpG codons of p53 gene.

OBJECTIVE: Hypomethylation within the body of the p53 gene, which is normally methylated, has been found in neoplasms. Also, the CG → TA transition was not detected in the CpG codons of the p53 gene in gastritis lesions in Iranian patients. Therefore, an evaluation of the probable correlation between global genome methylation and alteration at CpG codons of p53 gene was needed. METHODS: For defining the genotypes of CpG codons, DNA sequencing was performed on 90 paired samples of gastritis and normal tissues. To measure global genome methylation status, the extracted DNA was digested with HpaII (methylation sensitive) and MspI (insensitive). Then, enzymatic digestion was quantitated using Pyrosequencing as peak height. By calculating the HpaII/ MspI peak ratio it is possible to evaluate the methylation level of normal and gastritis tissues. RESULTS: Codons 9, 245 and 248 underwent a CG → AT transversion but not a CG → TA transition. In addition, the mean methylation level was significantly different between the patients with GG and GT genotypes at codon 245 (P = 0.019). CONCLUSIONS: As CG → AT transversion at codon 245 is associated with global genome methylation, GG hypomethylation may induce different pattern of mutations, for example, C → A instead of C → T at the CpG codons of the p53 gene during gastritis development in Iranian patients.

Association of intron and exon polymorphisms of p53 gene in Iranian patients with gastritis.

AIM: The propose of this study was to evaluate the probable correlation between exon and intron polymorphisms of p53 gene and their association with clinicopathological aspects of gastritis. BACKGROUND: Regarding to the decisive role of p53 in the development of a variety of human cancers, a comprehensive study concerning probable correlation between polymorphisms in the p53 intron and exon in gastritis lesions, may open new insight toward gastric cancer development and prevention. PATIENTS AND METHODS: PCR-Sequencing was done for exons and introns 2-7 on the 97 gastritis and normal samples, age range of 15-83 years. Also, microsatellite status was evaluated using five mono nucleotide repeat markers. Variation at codon 72 was associated with IVS2 + 38, p53INS3 and IVS3-29. In addition, IVS2 + 38 had association with polymorphism at codon 36 & 245. Gastritis samples had stable microsatellite except nine patients showing polymorphism for NR-21 and one for Bat-25. RESULTS: Most of patients with stable microsatellites (83.9%) had allele G at codon72 without p53INS3. In addition, all patients with GA and CG at codon 36 / IVS2 + 38 had stable microsatellites. Severity and activity of gastritis were in association with genotypes combined of codon 36/IVS2 + 38 and 245/IVS2 + 38 respectively. In addition, the profiles of combined variation at codon 72/IVS3-29 and codon 72/IVS6 + 31 were different between patients with ages less and greater than 45 years. CONCLUSION: As, some exon variations of p53 gene specially codon 72, were in association with alterations at introns and their combined genotypes were correlated with microsatellite status, pathological findings and age, therefore, it could be inferred that the these combinations of p53 gene polymorphisms work as a whole, not as single.

Detection of p53 common intron polymorphisms in patients with gastritis lesions from Iran.

BACKGROUND: p53 alterations have been implicated in the development of many cancers, such as gastric cancer, but there is no evidence of p53 intron alterations in gastritis lesions. The aim of this study was to investigate the p53 intron alterations in gastritis along with p53 and mismatch repair protein expression and microsatellite status. MATERIALS AND METHODS: PCR-sequencing was conducted for introns 2-7 on DNA extracted from 97 paired samples of gastritis lesions and normal adjacent tissue. Abnormal accumulation of p53 and mismatch repair proteins was investigated using immunohistochemistry. In addition, microsatellite status was evaluated with reference to five mononucleotide markers. RESULTS: Gastritis cases included 41 males and 56 females in the age range of 15-83 years, 87.6% being H.pylori positive. IVS2+38, IVS3ins16 and IVS7+72 were the most polymorphic sites. Their minor allele frequency values were as follows: 0.38, 0.21 and 0.06, respectively. Samples with GG genotype at IVS2+38 and CT at IVS7+72 had no insertion. Moreover, most of the stable samples (91.9 %) had a G allele at IVS2+38. All of the samples were IHC negative for p53 protein, microsatellite stable and expressed mismatch repair proteins. p53 alterations were prominent in the HP+ group, but without statistical significance. CONCLUSIONS: According to our results, some p53 polymorphisms such as IVS2+38, IVS3ins16 and IVS7+72, because of their correlations together or with microsatellite status may contribute to gastritis development. However, so far effects on p53 expression and function remain unclear. Therefore, a comprehensive survey is needed to delineate their biological significance.

Association between the 1793G> A MTHFR polymorphism and sporadic colorectal cancer in Iran.

BACKGROUND: It has been proposed that folate and polymorphisms of the enzyme methylenetetrahydrofolate reductase (MTHFR), which regulates influx of folate for methylation reactions for DNA synthesis and repair, are involved in colorectal cancer. This study was designed to determine the influence of a genetic variant (MTHFR G1793A) and folate on colon cancer in Iran. MATERIALS AND METHODS: We analyzed 227 cases and 239 normal unmatched controls using pyrosequencing. Odds ratios and 95% confidence intervals (95% CI) were calculated to evaluate associations of the MTHFR gene polymorphism with colorectal cancer risk. RESULTS: A significantly reduced risk of recurrence was observed in patients heterozygous for the MTHFR G1793A polymorphism (OR: 0.17; 95% CI, 0.05-0.52). The frequency of GG, GA and AA genotypes of MTHFR among the colorectal cancer patients were 98%, 2% and 0% respectively, while the frequencies among controls were 90%, 10% and 0%, respectively. Furthermore, a significant reduction in recurrence risk was seen in MTHFR G1793A heterozygotes limited to those who received folate supplements. CONCLUSION: Our study is compatible with previous findings concerning a reverse association between the MTHFR 1793G> A genotype with cancers in different populations.