[Detection of point mutation of p53 gene in head and neck squamous cell carcinoma by non-isotopic PCR-SSCP].
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Non-isotopic PCR-SSCP technique was used to detect point mutation of p53 gene in fresh tissue taken from head and neck squamous cell carcinoma. The abnormal band of the single-stranded DNA (ssDNA) was identified in 7 of 13 cases. The results showed that point mutation occurred in the related DNA fragments. The point mutation of exon 5 was found in one case and that of exon 6, 7, 8 in two respectively. These results indicated that the p53 gene mutation was closely correlated with the development of head and neck squamous cell carcinoma. Compared with the traditional isotopic PCR-SSCP technique, the non-isotopic PCR-SSCP one is a kind of simpler, safer, quicker and more effective way to detect the mutation of p53 tumor suppressor gene. It is especially useful in the screening of point mutation when handling a large number of samples.Keywords:
Single-strand conformation polymorphism
Epidermoid carcinoma
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Single-strand conformation polymorphism
Epidermoid carcinoma
Silent mutation
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To study the phenotypes and genotypes of a protein C (PC) deficiency pedigree.Immunoassay (ELISA) was used for PC antigen and activated PC (APC) detection, PCR for amplification of the fragment of protein C gene exon II to exon IX, single-strand conformation polymorphism (SSCP) for difference of denatured cDNA and DNA sequencing for gene mutation.Four members in the pedigree were found to be PC antigen levels between 34.3% - 67.8% and PC activity between 22% - 49% which are lower in comparison with normal references (80% - 120% and 70% - 130%, respectively). A G-to-A mutation in exon VII of the protein C gene at 6 219 position was identified in 9 members. This mutation resulted in the substitution of Arg for Gln at 169 amino acid.The proband is of heterozygosity. The G6219 A mutation in exon VII of the protein C gene leads to the substitution of Arg 169 Gln. This mutation is reported for the first time in China.
Single-strand conformation polymorphism
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Oropharyngeal squamous cell carcinoma (SCC) is probably the most serious cancer to be encountered in the clinic.Exons 1-3 of the p16 and exon 2 of the p21 genes were examined for mutations by polymerase chain reaction (PCR)/direct DNA sequencing methods in 21 fresh-frozen tissue specimens of oropharyngeal SCCs previously studied for expression and mutation (exons 5-9) of the p53 gene. Mutations in exons 2 and 3 of the S100A4 gene were studied in 10 cases. Findings were examined for correlations with patients' clinicopathological parameters and data on survival.No p16 gene mutations were found in any of the cases examined. Only one tumour, which previously had shown expression and mutation in the p53, had a point mutation at codon 117 of exon 2 of the p21 gene with a resulting Cys-->Tyr amino acid substitution. Exons 2 and 3 of the S100A4 gene were not found mutated in the cases studied. Analysis of survival data showed that, among the 60% (12 out of 20) of patients who died, 50% (6 out of 12) had mutations in p53 while the remaining 50% had no mutations. Of the 50% with mutated p53, 17% (1 out of 6) also had mutation in p21. For the 40% (8 out of 20) who are alive, 63% (5 out of 8) had mutations in p53 and 37% (3 out of 8) had no mutations.These findings demonstrate that: (i) mutations of the cell cycle regulatory genes p16 and p21 and the S100A4 gene are infrequent and might be unnecessary in development and/or progression of oropharyngeal SCCs, (ii) the results were not found to relate to previous results of p53, (iii) loss of p21 and/or p53 might not predict for prognosis in these cancers, (iv) the absence of mutations of the three genes examined in the cases previously found mutated for p53 might suggest that these mutations are complementary to p53 mutations in the development of these cancers, but the lack of these mutations in the cases with no mutations in p53 might suggest that the pathogenesis of these cancers may follow other independent pathways. Since p53 and pRb (p16/pRb/cyclin-D1) pathways represent the two main mechanisms for cell cycle control at the G1-S checkpoint, further studies are necessary to examine the possible alterations in the pRb (p16/pRb/cyclin-D1) and the p53 pathways in oropharyngeal SCCs.
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Introduction. p53 gene is the most common tumor suppressor gene involved in pathogenesis oral squamous cell carcinoma (OSCC). Protein product of p53 gene contributes to cell cycle control and apoptosis. p53 gene mutations may lead to uncontrolled cell growth. The aim of this study was to determine the incidence of mutation in DNA-binding domain of p53 gene. Materials and Methods. In the 60 specimens, the presence of point mutation in exons 5, 6, 7 and 8 was detected using PCR-SSCP method. To confirm the presence of p53 mutation found by SSCP method, five samples were analyzed by sequencing of exon 5. Results. Point mutation affecting exons 5, 6, 7 and 8 were found in 60% of analyzed samples. A higher incidence of mutation was detected in exon 7 and 8 (60%), than in exon 5 and 6. Sequencing of exon 5, confirmed the presence of mutations revealed by SSCP method. Study of associations showed an increase of p53 mutations in poor differentiated and carcinoma of higher clinical stages. Conclusion. p53 gene is one of major factor in control of cell cycle and has important role in pathogenesis of oral squamous cell carcinoma.
Pathogenesis
Single-strand conformation polymorphism
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Multiple tumor suppressor gene 1 (MTS1) has been found mutated or deleted in a variety of human cancers. Our purpose was to identify and characterize MTS1 gene mutations in primary oral squamous cell carcinomas (SCCs) in each of the three exons of the MTS1 gene. Seventeen archival samples of oral SCC were evaluated for the presence of MTS1 mutations using single strand conformation polymorphism (SSCP) and DNA sequencing. Three of 17 tumors exhibited MTS1 gene mutations; one tumor exhibited a mutation in exon 2 and two tumors exhibited mutations at the splice site junction of intron 2 and exon 3. Three tumors also exhibited a common base change in the 3’untranslated region of exon 3, which is interpreted as a likely polymorphic variant. An examination of the three tumors exhibiting MTS1 point mutations revealed no unique characteristics relative to p53 immunohistochemical activity, mitotic frequency, or degree of histologic differentiation. This study indicates that MTS1 gene mutations may be involved in at least a minor proportion of oral SCCs.
Single-strand conformation polymorphism
Epidermoid carcinoma
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We have examined 63 methylcholanthrene (MCA)-induced mouse sarcomas for possible correlations of mutations involving the c-myc, ras and p53 genes. The c-myc gene was found to be amplified in 18 of these sarcomas (29%). Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and subsequent direct sequencing identified 18 cases carrying K-ras mutation at codons 12, 13 and 61 (29%). No mutation was detected in the H-ras and N-ras genes. Mutations of the p53 gene in exons 5 to 8 were found in 45 cases (71%). Comparison of these mutations revealed that out of 18 cases with c-myc gene amplifications, 10 carried K-ras mutations (56%) and 14 carried p53 mutations (78%). In contrast, among 45 cases of sarcomas without c-myc gene amplification, 8 were found to have K-ras mutations (18%). The same 45 cases were found to have 31 p53 mutations (69%). The present study suggests a strong correlation between c-myc gene amplification and K-ras gene mutation (P < 0.01). p53 gene mutation was frequently found among MCA-induced mouse sarcomas, indicating the importance of this mutation in the etiology of these tumors. However, p53 mutations were present in sarcomas regardless of the state of c-myc amplification and K-ras mutation. Therefore, a defect in the p53 gene is independent of amplification of the c-myc gene or point mutation of the K-ras gene.
Single-strand conformation polymorphism
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Single-strand conformation polymorphism
Coding region
Mutation frequency
Epidermoid carcinoma
Silent mutation
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Single-strand conformation polymorphism
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Non-isotopic PCR-SSCP technique was used to detect point mutation of p53 gene in fresh tissue taken from head and neck squamous cell carcinoma. The abnormal band of the single-stranded DNA (ssDNA) was identified in 7 of 13 cases. The results showed that point mutation occurred in the related DNA fragments. The point mutation of exon 5 was found in one case and that of exon 6, 7, 8 in two respectively. These results indicated that the p53 gene mutation was closely correlated with the development of head and neck squamous cell carcinoma. Compared with the traditional isotopic PCR-SSCP technique, the non-isotopic PCR-SSCP one is a kind of simpler, safer, quicker and more effective way to detect the mutation of p53 tumor suppressor gene. It is especially useful in the screening of point mutation when handling a large number of samples.
Single-strand conformation polymorphism
Epidermoid carcinoma
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Citations (1)
Polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis of the p53 tumor suppressor gene (from exon 2 to 9) was performed on samples from 47 adult patients with primary myelodysplastic syndrome (MDS). Point mutation was found in 5 (11%) patients: exon 7 in 3, exon 4 in 1 and intron 5 in 1. The frequency of p53 mutation was significantly higher at advanced stages (p = 0.048) and higher in patients with abnormal karyotypes (p = 0.023). Although all of the p53 mutations were detected at advanced stages, four of them were detected at initial diagnosis with very short survival. Sequential SSCP analysis in 20 transformed MDS patients revealed only one new p53 mutation during progression from early MDS phases. The results suggest that p53 mutation might occur as an early genetic event and is probably associated with rapid progression and poor survival in some MDS patients.
Single-strand conformation polymorphism
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