| IntroductionGastric carcinoma is the most vicious malignant tumor in our country both in terms of incidence rate and mortality rate. It is especially important and urgent to explore the molecular mechanism of gastric carcinoma and to make guidance on prevention and treatment. The occurrence of gastric carcinoma is a complex molecular biology process which multi - genes, multi - stages and multi - factors are involved in and multiple tumor suppressor genes are correlated with. Two hit hypothesis (Knudson, 1971) is a scientific prophecy about the mechanism of tumorigenesis which is generally accepted by cancer geneticists , and has been the theoretical basis of tumorigenesis mechanism. In 1998, Guilford et al found three families of gastric carcinoma with germ line E - cadherin mutation in New Zealand, which brought about the key evidence of CDH1 gene as a tumor suppressor gene and a hereditary predisposition gene. In addition, amplification and over - expression of cyclin E frequently occurred in gastric carcinoma, loss of heterozigosity is also observed on chromosome 4q, which revealed in-activation of Ago gene. Based on these data, we screened mutations of CDHl and Ago genes by applying combination of PCR ( polymerase chain reaction) and DHPLC( denatured high performance liquid chro-matography).Materials and Methods1. Samples83 cases of matched normal and tumorous tissues from patients who suffered from progressive stage gastric carcinomas were confirmed by pathologist, including 34 cases of moderate differentiated gastric carcinomas and 49 cases of poor differentiated gastric carcinomas. For germ line mutation screening, 14 cases of peripheral blood of young gastric carcinoma were collected, as well. All the samples were obtained from oncology department of the No. 1 affiliated hospital of China Medical University during surgery.2. Genomic DNA extractionFresh cancerous tissues, noncancerous tissues and peripheral blood were obtained at surgery and stored in liquid nitrogen. Genomic DNA was isolated using standard methods.3. Mutation Screening for CDH1 and Ago GeneIndividual exons of the CDH1 gene were first amplified by PCR and then subjected to DHPLC and applied to sequencing to confirm mutations.3.1 PCR Amplification; All the intron - complementary primers which enabled us to amplify all exons and the corresponding intron-exon junctions were devised to fully cover the coden sequence and the splice site of CDH1 and Ago gene.3.2 Denatured temperature of individual amplicons were determined according to the Tm score and melting curve. On the basis of these data, mutation screening were performed using DHPLC.3.3 Sequencing Analysis; for cases with abnormal chromatogra-phy compared with matched normal ones, PCR were reperformed and products were purified for sequencing.Results1. Polymorphisms of CDH1 geneA total of two novel polymorphic sequence variants have been i-dentified in 14 patients. Among them, one is in the coding region. Sequencing showed a C to T transition in the third position of codon 2254 in exon14, leading to no Asn amino acid changes. The other is in the noncoding region with polymorphism (the 688 +33G-A variant) . The former variant showed a relatively frequent occurrence (137 97) and the latter one existed in only one case (1/97).2. Mutation screening result of Ago geneThere were no Ago gene mutation in 22 cases of progressive gastric carcinoma(mostly are poor differentiated type).Conclusion1. Two previously unreported single nucleotide polymorphisms (SNPs) of CDH1 gene were identified in intron5 and exon14, respectively. Polymorphism in exon14 occurred frequently in gastric carcinoma (13/97).2. Mutations in the Ago gene might not be related to the process of gastric carcinogenesis.3. Combined application of PCR - DHPLC is a powerful tool in mutation screening.
|
PDF Full Text Request