Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations

We have developed a fluorescence-based single strand conformation polymorphism (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer for unique color detection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell lines with reported mutations in p53 exons 5-8 were analyzed by SSCP using various gel conditions. The sensitivity for mutation detection was 95% for all cell lines studied, and no false positives were seen in 10 normal DNA samples for all four exons. Experiments mixing known amounts of tumor and normal DNA showed that mutations were detected even when tumor DNA was mixed with 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a useful tool in cancer research, where screening large numbers of samples for p53 mutations is desired.     

Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome.

Germ-line mutations of the tumor-suppressor gene p53 have been observed in some families with the Li-Fraumeni syndrome (LFS), a familial cancer syndrome in which affected relatives develop a diverse set of early-onset malignancies including breast carcinoma, sarcomas, and brain tumors. The analysis of the p53 gene in LFS families has been limited, in most studies to date, to the region between exon 5 and exon 9. In order to determine the frequency and distribution of germ-line p53 mutations in LFS, we sequenced the 10 coding exons of the p53 gene in lymphocytes and fibroblast cell lines derived from 15 families with the syndrome. Germ-line mutations were observed in eight families. Six mutations were missense mutations located between exons 5 and 8. One mutation was a nonsense mutation in exon 6, and one mutation was a splicing mutation in intron 4, generating aberrant shorter p53 RNA(s). In three families, a mutation of the p53 gene was observed in the fibroblast cell line derived from the proband. However, the mutation was not found in affected relatives in two families and in the blood from the one individual, indicating that the mutation probably occurred during cell culture in vitro. In four families, no mutation was observed. This study indicates that germ-line p53 mutations in LFS are mostly located between exons 5 and 8 and that approximately 50% of patients with LFS have no germ-line mutations in the coding region of the p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in tumour suppressor gene p53 in human gliomas from Indian patients

Alterations in the tumour suppressorp53 gene are among the most common defects seen in a variety of human cancers. In order to study the significance of thep53 gene in the genesis and development of human glioma from Indian patients, we checked 44 untreated primary gliomas for mutations in exons 5–9 of thep53 gene by PCR-SSCP and DNA sequencing. Sequencing analysis revealed six missense mutations. The incidence of p53 mutations was 13⋅6% (6 of 44). All the six mutations were found to be located in the central core domain of p53, which carries the sequence-specific DNA-binding domain. These results suggest a rather low incidence but a definite involvement of p53 mutations in the gliomas of Indian patients.     

Detection of clinically relevant point mutations by a novel piezoelectric biosensor

Piezoelectric sensing is here applied to point mutation detection in human DNA. The mutation investigated is in the TP53 gene, which results inactivated in most cancer types. TP53 gene maps on chromosome 17 (17p13.1). It contains 11 exons and codifies for the relative protein, involved in cell proliferation. The TP53 gene has a wide mutation spectrum that is related to different tumours. In particular, those occurring in the structurally important L2 and L3 zinc-binding domains, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. For this reason, the identification of these mutations represents an important clinical target and biosensors could represent good candidate for fast mutation screening. In this paper, a DNA-based piezoelectric biosensor for the detection of the TP53 gene mutation at codon 248 is reported. A biotinylated probe was immobilised on the sensor surface via dextran-streptavidin modified surfaces. The sensor was optimised using synthetic oligonucleotides. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples of DNA extracted from two cell lines, one normal (wild-type) and one mutated, carrying the mutation at codon 248 of the TP53 gene. The results obtained demonstrate that the DNA-based piezoelectric biosensor is able to detect the point mutations in PCR-amplified samples showing the potentialities of this approach for routine analysis.     

TP53 mutations in human meningiomas

Overexpression of p53 has been reported to play a role in the development of neoplasms of the central nervous system. Meningiomas are generally benign intracranial tumors originating from the meninges. Overexpression of the p53 protein in meningiomas and an association with histological type and recurrence has been reported. Mutation of the TP53 gene leads to a more stable p53 protein in quantities high enough for detection by immunohistochemistry. In the search for these mutations the core domain of the TP53 gene of meningiomas has been analyzed. Only a very low incidence of mutations was reported. The apparent discordance between overexpression of p53 protein and TP53 gene mutations may be explained by mutations located outside the core domain. This issue was addressed in the present study. All 11 exons of 17 meningiomas were analyzed for DNA alterations by PCR single-strand conformation polymorphism (PCR-SSCP) analysis with subsequent sequencing. PCR-SSCP analysis showed a various number of band shifts and nucleotide alterations, caused either by alterations in the flanking introns or common polymorphisms (codon 36 and 72). The allele frequencies of the polymorphisms found in this small population of tumors resemble the frequencies reported in the literature. In addition, three nucleotide changes located in introns 2, 3 and 7 were found in 11, 3 and 4, respectively, of 17 specimens. Based on this study and on reports by others we conclude that it is not very likely that TP53 mutations are involved in the etiology of meningiomas.     

胃粘膜损伤中EB病毒感染与P53抑癌基因突变的研究

为了探讨EB病毒（EBV）感染与胃癌发生的关系,应用PCR技术对166例胃粘膜损伤标本的EBVDNA进行检测．其中66例应用银染PCR－SSCP分析技术检测了p53exon5～8突变情况,10例应用直接法原位PCR技术检测了EBV在组织中的感染情况。结果166例标本中EBV感染率为30．1％;EBV在细胞中感染大体是弥散型,主要存在于细胞核。66例标本中p53基因突变率为54．5％。对比分析,EBV阳性标本中p53基因突变率为75％（21／28）,EBV阴性标本中p53基因突变率为39．5％（15／38）。结果表明,EBV对胃粘膜组织细胞具有易感性,p53基因突变在胃粘膜病变中是一个常发事件,EBV感染与p53基因突变之间存在着高度相关性．对癌症的发生具有重要作用。     

Splicing mutations in TP53 in human squamous cell carcinoma lines influence immunohistochemical detection.

The mutational status of the tumor suppressor gene TP53 is often examined by immunohistochemistry. We compared the incidence of TP53 mutations in 12 permanent squamous cell carcinoma lines of the head and neck with the immunohistochemical staining obtained with two different antibodies. The mutational status of the TP53 gene was assessed by sequencing the complete coding frame of the TP53 mRNA. All 12 tumor cell lines had TP53 mutations. Six of them showed missense mutations and five had premature stop codons caused either by splicing mutations or nonsense mutations or by exon skipping. One tumor cell line was heterozygous, with a truncating splicing mutation and an additional missense mutation located on different alleles. In one case, an in-frame insertion of 23 extra codons was found. All missense mutations were positive in immunhistochemistry and Western blotting. The truncated p53 was not immunohistochemically detected in three cases with the DO-7 antibody and in five cases with the G59-12 antibody, giving false-negative results in 25% or 40%, respectively, of all tumor cell lines examined. We conclude that splicing mutations are common in squamous cell carcinoma lines and that the incidence of p53 inactiviation by erroneous splicing is higher than yet reported. Sequencing of only the exons of TP53 may miss intronic mutations leading to missplicing and may therefore systematically underestimate the TP53 mutation frequency.     

Multiple mutations of the p53 gene in human mammary carcinoma

Alteration of the p53 tumor suppressor gene is the most common genetic abnormality in human cancer. In breast cancer, depending on the stage of disease and method of detection, mutation rates of 25-60% have been observed. Multiple mutations of p53 gene in the same tumor however, are rarely reported. In this study we explored the frequency of multiple mutations of p53 gene in mammary carcinoma in a cohort of south Florida patients. Three hundred eighty-four cases of primary breast cancer diagnosed between 1984 and 1986 at the University of Miami, Jackson Medical Center were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on cloned PCR-amplified DNA of formalin-fixed, paraffin-embedded tumors. Two hundred thirty-four of 384 breast cancers (61%) had p53 mutation. Of those, 36 tumors showed more than one mutation; 31 tumors had two mutations, three showed three, one tumor had five mutations, and one case carried six mutations. The majority of mutations were missense (43) followed by silent (35); and most occurred within a single exon. Our study suggests that multiple mutations of p53 suppressor gene in breast cancer are more common than currently believed.     

Infrequent but significant p21/WAF1/CIP1 gene alteration in synchronous or metachronous transitional cell carcinoma

p53 inducible cyclin dependent kinase inhibitor, p21/WAF1/CIP1(p21), played a pivotal role for G1 arrest when cells received genotoxic stimuli. p21 could be a putative tumor suppressor gene, since its dysfunction may lead to accumulation of genomic alteration. We investigated the p21 and p53 status using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, in eight patients who had synchronous or metachronous urothelial tumors. Loss of heterozygosity (LOH) of p21 gene was detected in one coincidental tumor in one case. p21 positive cells were detected by immuno-histochemical staining in all tumors in one case, and in one coincidental tumor in two cases. Among p21 positive cells in these three cases, no p53 mutations were detected, whereas no p21 positive cells were detected in other cases with a p53 mutation. These findings suggested that in transitional cell carcinoma (TCC) p21 gene mutation is infrequent like the p53 gene mutation, but that LOH might be important in the inactivation of p21.     

Only Missense Mutations Affecting the DNA Binding Domain of P53 Influence Outcomes in Patients with Breast Carcinoma

The presence of a TP53 gene mutation can influence tumour response to some treatments, especially in breast cancer. In this study, we analysed p53 mRNA expression, LOH at 17p13 and TP53 mutations from exons 2 to 11 in 206 patients with breast carcinoma and correlated the results with disease-free and overall survival. The observed mutations were classified according to their type and location in the three protein domains (transactivation domain, DNA binding domain, oligomerization domain) and correlated with disease-free and overall survival. In our population, neither p53 mRNA expression nor LOH correlated with outcome. Concerning TP53 mutations, 27% of tumours were mutated (53/197) and the presence of a mutation in the TP53 gene was associated with worse overall survival (p = 0.0026) but not with disease-free survival (p = 0.0697), with median survival of 80 months and 78 months, respectively. When alterations were segregated into mutation categories and locations, and related to survival, tumours harbouring mutations other than missense mutations in the DNA binding domain of P53 had the same survival profiles as wild-type tumours. Concerning missense mutations in the DNA binding domain, median disease-free and overall survival was 23 months and 35 months, respectively (p = 0.0021 and p<0.0001, respectively), compared with 78 and 80 months in mutated tumours overall. This work shows that disease-free and overall survival in patients with a frameshift mutation of TP53 or missense mutation in the oligomerization domain are the same as those in wild-type TP53 patients.     

For novel gene mutations in five Japanese male patients with neonatal or late onset OTC deficiency: application of PCR-single-strand conformation polymorphisms for all exons and adjacent introns

Ornithine transcarbamylase deficiency (OTC), the most common inborn error of the urea cycle, shows an X-linked inheritance with frequent new mutations. Southern blots reveal only a small percent of the mutation, but amplification of cDNA or genomic DNA using the polymerase chain reaction (PCR) followed by DNA sequencing, has contributed greatly to overcoming this difficulty. Problems remaining are the limited availability of fresh liver samples for preparation of intact mRNA in the former case, and there are primer sequences for PCR for only some exons in the latter case. Here, we report the structures of intron sequences which are long enough to analyze all exons and adjacent introns of the OTC gene using PCR and PCR single-strand conformation polymorphisms (PCR-SSCP). We carried out a DNA analysis of findings in five Japanese male patients with neonatal or late onset form. Five patients had mutations in the protein coding region. C to G (S192R), A to T (D196V), A to G (T264A), T to C (M268T), and C to T (R277W) substitutions. The first four of these were novel missense mutations and the presence of the mutation was confirmed in the corresponding families.     

TP53 in gastric cancer: mutations in the l3 loop and LSH motif DNA-binding domains of TP53 predict poor outcome

The aim of this study was to clarify whether specific p53 mutations may have biological relevance in terms of disease relapse or death in gastric carcinomas (GC). Resected specimens from a consecutive series of 62 patients with GC undergoing potentially curative surgery were prospectively studied. The mutational status of exons 5-8 of the p53 gene was investigated in 62 cases using the PCR-SSCP and sequencing. Presence of microsatellite instability (MSI) was evaluated in 56 cases by analyzing loci highly sensitive of MSI. Twenty mutations of p53 were detected in 17 of the 62 cases analyzed (27%). Ten mutations (50%) occurred in highly conserved domains. According to the p53 specific functional domains: 4/20 mutations (20%) were in the L3 loop and 3/20 (15%) in LSH motif. Eight of the 56 GC resulted MSI-H, 5 (9%) MSI-L, and 43 (77%) MSI stable (MSS). None of the 8 (14%) MSI-H GC showed p53 mutations. p53 mutations were associated with intestinal histotype. Moreover, specific mutations in functional domain (L3 and LSH), together with advanced TNM stage, node involvement, depth of invasion, diffuse histotype, proved to be significantly related to quicker relapse and to shorter overall survival. Specific mutations in p53 functional domains, rather than any mutations in this gene, may be biologically more significant in terms of patients outcome, indicating that these mutations might have biological relevance to identify subgroups of patients at higher risk of relapse or death who might benefit from a more aggressive therapeutic approach.     

人类单纯性先天性心脏病中TBX5基因的突变及表达研究

本文首次较为完整地报道了藏汉通婚子代群体的14项肤纹参数（其中藏父汉思及汉父藏母各100 例）,并将这些肤纹参数分别与其藏汉父母样本的有关肤纹参数进行比较,再与1000例藏族及1040例 汉族两个大样本的有关肤纹参数进行比较。结果表明：藏汉后代的肤纹特征介于藏族和汉族之间,提示 肤纹参数的多因子遗传本质。     

Detection of K-ras and p53 mutations in sputum samples of lung cancer patients using laser capture microdissection microscope and mutation analysis

Mutations in the p53 tumor suppressor gene and the K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and other patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cytocentrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) for p53 mutations and by PCR and denaturing gradient gel electrophoresis (DGGE) for K-ras mutations. This method was used to analyze sputum of 15 Chinese women with lung cancer from Xuan Wei County, China and detected mutations in sputum of 7 (46.7%) patients, including 5 patients with p53 mutations, 1 patient with a K-ras mutation, and 1 patient with K-ras and p53 mutations. For comparison, only two of the mutations were detected by conventional methods. Therefore, the laser capture/mutation analysis method is sensitive and facilitates the detection of low-fraction mutations occurring throughout the p53 and K-ras genes in sputum of lung cancer patients. This method may be applicable to the analysis of epithelial cells from clinically normal sputum or BAL samples from individuals with a high risk for developing lung cancer.