A new method for FMR1 gene methylation screening by multiplex methylation-specific real-time polymerase chain reaction.

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation in men. It is caused by abnormalities in the FMR1 gene that are associated with CGG repeat expansion and the hypermethylation status of its promoter. Methylated alleles lead to transcriptional inhibition and consequent loss of Fragile X Mental Retardation Protein. Chemical modification of cytosine to uracil by bisulfite treatment has proved to be an attractive method for DNA methylation studies that precludes labor-intensive Southern blot analysis, which is the gold standard test for FXS. In this report, bisulfite-treated DNA samples were amplified using real-time multiplex methylation-specific polymerase chain reaction followed by melting curve analysis. Our results show that all control samples with known CGG repeat numbers and methylation statuses were correctly diagnosed. The samples from 43 male patients were also successfully diagnosed, which were in complete agreement with the results of Southern blotting. By such means, 39 patients were found to have an unmethylated allele; 3, a fully mutated allele; and 1, both methylated and unmethylated alleles, thus implying a diagnosis of mosaicism. In conclusion, we find our method to be convenient for screening a large number of male patients with FXS, because it is rapid and easy to perform, especially when there is a low quantity of DNA that must be sensitively and accurately assayed.

Specific intronic p53 mutation in esophageal squamous cell carcinoma in Southern Thailand.

AIM: To investigate p53 mutations in esophageal cancer in a high-risk population, and correlate them with smoking, alcohol consumption and betel chewing. METHODS: One hundred and sixty-five tumor samples of esophageal squamous cell carcinoma (ESCC) obtained from a university hospital in Songkhla province, Southern Thailand were investigated for p53 mutations in exons 5-8, using polymerase chain reaction-single strand conformation polymorphism analysis, followed by direct sequencing. A polymerase chain reaction-restriction fragment length polymorphism (RFLP) assay was additionally used to confirm possible germline mutation in intron 6. A history of risk habits was obtained by interviews. The association between risk habits and mutation frequency was evaluated using the χ(2) test. RESULTS: The studied specimens were from 139 male and 26 female patients with ESCC, treated at Songklanagarind Hospital. Most of the patients were smokers (86.7%) and alcohol consumers (72.73%), and 38.3% were betel chewers. Forty-three mutations of the p53 gene were detected in 25.5% (42/165) of tumor samples. Mutations were most commonly found in exon 5 (25.6%) and exon 8 (25.6%). Mutations in the hot-spot codon 248 were found in four cases (9.3% of all mutations). G:C→C:G (30.23%), G:C→A:T (27.90%) and G:C→T:A (16.28%) were the prevalent spectra of mutations. Unexpectedly, among 10 intronic mutations, eight cases harbored a similar mutation: G→C substitution in intron 6 (nucleotide 12759, GenBank NC_000017). These were additionally confirmed by the RFLP technique. Similar mutations were also detected in their matched blood samples using RFLP and direct sequencing, which suggested germline mutations. There was no significant correlation between risk habits and p53 mutation frequency. CONCLUSION: A proportion of Thai ESCC patients harbored specific intronic p53 mutations, which might be germline mutations. Further studies are needed to explore this novel finding.

p53 mutations in betel-associated oral cancer from Thailand.

The p53 mutations in betel-related oral cancer have been studied but the results may be confounded by tobacco and alcohol due to tobacco components in the quid or concomitant habits of smoking and drinking. In this study, p53 mutations in tumors from Thai betel chewers who neither smoke nor drink were studied in comparison to tumors from smokers/drinkers. PCR-SSCP analysis and direct sequencing on exon 5-8 were performed. Mutations were detected in 11.8% (8/68) of betel-related tumors and 7 of 8 mutations were G:C to A:T transitions. By contrast, mutations were found in 22.4% (13/58) of smoking/drinking-related tumors with various base substitutions. The results demonstrated different mutation profiles between the two exposed groups. The type of mutation detected in betel chewers suggested a possible role of areca-specific nitrosamine as a causative carcinogen.