RESUMO
OBJECTIVE: Establishing a new method on the basis of multiplex PCR-high performance liquid chromatography (HPLC) for screening a large deletion in mismatch repair genes. METHODS: Thirty-five pairs of primers were used to amplify all 16 exons of MSH2 and all 19 exons of MLH1 gene in 8 multiplex PCR. The products of multiplex PCR were analysed for the large deletion with Double Strand DNA Analysis System of HPLC. Firstly, validation of the method was tested on positive and negative controls in blind analysis. Secondly, 14 blood cell DNA samples from hereditary nonpolyposis colorectal cancer (HNPCC) patients and 13 colorectal cancer (CRC) tissues DNA samples from sporadic CRC patients were checked with the new developed method. RESULTS: (1) the genomic deletions in all 4 of positive controls were identically uncovered with the new method; (2) a novel germline and a novel somatic large deletions were unveiled in 1/14 HNPCC patients and in 1/13 CRC tissues. CONCLUSION: The method developed on multiplex PCR-HPLC is reliable for uncovering large genomic deletion in mismatch repair genes, and can be taken as a valuable addition to mutation screening system.
Assuntos
Pareamento Incorreto de Bases/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Proteínas de Transporte , Cromatografia Líquida de Alta Pressão , Humanos , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas NuclearesRESUMO
OBJECTIVE: To set up a sensitive and stable technique which has high throughout to detect the instability of microsatellite DNA. METHODS: Genomic DNA extracted from the cancer tissues and their normal tissues were subjected to microsatellite instability(MSI) analysis on five of DNA markers in 115 sporadic colorectal cancers by means of PCR and ion-pair reversed-phase high performance liquid chromatography. Genomic DNA extracted from lymphocytes in blood of 20 normal persons were analysed and used as the standard control. RESULTS: Seventeen (14.8%) MSI-H and 23(20.0%) MSI-L were found in 115 sporadic colorectal cancers. The rates of MSI in the young patients and old patients were much higher than that in the middle-age patients (P<0.05). And the rate of MSI in low differentiation group was also much higher than that in high or middle differentiation groups (P<0.05). CONCLUSION: The method the authors developed is a sensitive and accurate technique to detect MSI and has a high throughput.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , DNA de Neoplasias/genética , Repetições de Microssatélites/genética , Adulto , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , DNA de Neoplasias/análise , Humanos , Perda de Heterozigosidade , Pessoa de Meia-Idade , Neoplasias Retais/genética , Neoplasias Retais/patologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To investigate the etiological role of function and construction alteration in mismatch repair genes MSH2 and MLH1 in the patients onset of colorectal cancers (CRC) at early ages. METHODS: The genomic DNA was extracted from the tumor tissues and normal colon tissues during operation and subjected to analysis of microsatellite instability (MSI) in 42 Chinese patients aged less than 50 with CRC. Mutation screenings were performed with denaturing high-performance liquid chromatography (DHPLC) followed by DNA sequencing of DNA samples with variant peaks, and genomic deletion detection with quantitative multiplex PCR (Q-M-PCR) in the patients uncovered with MSI(+). RESULTS: 22 out of the 42 (52.4%) patients investigated were microsatellite instability positive (MSI(+)), 10/42 MSI(+)-H and 12/42 MSI(+)-L. 8 kinds of DNA germline alterations, 5 polymorphisms and 3 novel point mutations, were found in 9 patients with MSI(+). A large DNA (exon 1-6) deletion in MSH2 gene and a missense mutation Met242Ile in MLH1 gene were unveiled in CRC tissues of two patients with MSI(+)-H. CONCLUSION: Mutations of mismatch repair genes are frequent in Chinese patients of CRC with onset at early ages.