Point mutations and allelic loss in the TP53 locus of cutaneous malignant melanomas.
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Alleles↗
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EVIDENCE OF AN ESSENTIAL DIFFERENCE BETWEEN POINT MUTATIONS AND CHROMOSOME BREAKS INDUCED BY TRIETHYLENE MELAMINE IN DROSOPHILA SPERMATOZOA.
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Animals↗
Structural and functional defects caused by point mutations in the alpha-crystallin domain of a bacterial alpha-heat shock protein.
The diverse family of alpha-crystallin-type small heat shock proteins (alpha-Hsps or sHsps) is characterised by a central, moderately conserved alpha-crystallin domain. Oligomerisation followed by dissociation of subparticles is thought to be a prerequisite for chaperone function. We demonstrate that HspH, a bacterial alpha-Hsp from the soybean-symbiont Bradyrhizobium japonicum, assembles into dynamic complexes freely exchanging subunits with homologous and heterologous complexes. The importance of the alpha-crystallin domain for oligomerisation and chaperone activity was tested by site-directed mutagenesis of 12 different residues. In contrast to mammalian alpha-Hsps, the majority of these mutations elicited severe structural and functional defects in HspH. The individual exchange of five amino acid residues throughout the alpha-crystallin domain was found to compromise oligomerisation to various degrees. Assembly defects resulting in complexes of reduced size correlated with greatly decreased or abolished chaperone activity, reinforcing that complete oligomerisation is required for functionality. Mutation of a highly conserved glycine (G114) at the C-terminal end of the alpha-crystallin domain specifically impaired chaperone activity without interfering with oligomerisation properties, indicating that this residue is critical for substrate interaction. The structural and functional importance of this and other residues is discussed in the context of a modeled three-dimensional structure of HspH.
Amino Acid Sequence↗
Complementation of one RecA protein point mutation by another. Evidence for trans catalysis of ATP hydrolysis.
The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament.
Adenosine Diphosphate↗
Haemophilia B: database of point mutations and short additions and deletions, fifth edition, 1994.
The fifth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of < 30bp) identified in haemophilia B patients. The 1,142 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.
Databases, Factual↗
Haemophilia B: database of point mutations and short additions and deletions--eighth edition.
The eighth edition of the haemophilia B database (http://www.umds.ac. uk/molgen/haemBdatabase.htm ) lists in an easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1713 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.
Computer Communication Networks↗
Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors.
A challenge in strain construction is that unmarked deletion and nucleotide substitution alleles generally do not confer selectable phenotypes. We describe here a rapid and efficient strategy for transferring such alleles via generalized transduction. The desired allele is first constructed and introduced into the chromosome by conventional allelic-exchange methods. The suicide vector containing the same allele is then integrated into the mutant chromosome, generating a tandem duplication homozygous for that allele. The resulting strain is used as a donor for transductional crosses, and selection is made for a marker carried by the integrated suicide vector. Segregation of the tandem duplication results in haploid individuals, each of which carries the desired allele. To demonstrate this mutagenesis strategy, we used bacteriophage P22HTint for generalized transduction-mediated introduction of unmarked mutations to Salmonella enterica serovar Typhimurium. This method is applicable to any species for which generalized transduction is established.
Bacteriophage P22↗
Detection of codon 61 point mutations of the K-ras gene in lung and colorectal cancers by enriched PCR.
Mutation of the Kirsten ras (K-ras) gene is one of most common alterations in solid tumors including lung and colorectal cancers. We developed new enriched PCR-RFLP assay to detect mutations of K-ras codon 61 at the 1st and 2nd letters and non-enriched PCR-RFLP assay to detect the 3rd letter mutation. One mutant allele among 10(3) wild-type alleles was detected by enriched PCR-RFLP assay, while one mutant in 10 wild-type alleles was detected by non-enriched PCR-RFLP assay for codon 61 3rd letter. We then examined K-ras codon 12, 13 and 61 mutations in lung and colorectal cancers using these assays. K-ras codon 12 mutation was detected in 10 of 109 (9%) lung cancer and 19 of 83 (23%) colorectal cancer cases. K-ras codon 13 mutation was detected in 2 of 83 (2%) colorectal and 0 of 109 NSCLC cases, respectively. There was no K-ras codon 61 mutation in either type of cancer. Our results demonstrate that enriched PCR-RFLP is a sensitive assay to detect K-ras codon 61 mutation, however, it was extremely rare in lung and colorectal cancers, suggesting organ-specific pathways in mutagenesis of the ras gene family.
Aged↗
[Incidence of point mutations in Ki-ras codon 12 and 13 in squamous epithelial carcinomas of the head-neck region].
BACKGROUND: Point mutations of ras genes are one of the most frequent genetic alterations in human malignancies with up to over 95% in certain tumors. Some studies have presumed a low incidence of point mutations in Ki-ras gene codons 12 and 13 in squamous cell carcinoma of the head and neck (SCCHN). The objective of this study was to gain more information about the significance of point mutations in Ki-ras codons 12 and 13 by investigation of a high number of cases. METHODS: By employing the polymerase chain reaction with specific primers, 63 fresh tumor biopsies were investigated by the nonradioisotopic single-strand conformation polymorphism (SSCP) analysis. To cooperate the efficiency and to characterize the nature of the point mutations, direct fluorescence-labeled DNA sequencing was performed in two cases. RESULTS: Five out of the 63 (7.9%) SCCHN samples exhibited point mutations in codons 12 and 13 by SSCP analysis. In two out of these mutated cases, a G to T transversion in codon 12.2 of the Ki-ras gene was detected. The analysis of the patient's clinical parameters as well as the histopathological grading and tumor staging revealed no correlation with the Ki-ras point mutation. CONCLUSIONS: Ki-ras point mutations in codons 12 and 13 are a rare event in SCCHN. These data give additional evidence that these genetic alterations do not play a major role in the development of malignancies of the upper aerodigestive tract.
Aged↗
Antisense inhibition of ras p21 expression that is sensitive to a point mutation.
Many genetic disorders result from a single point mutation, and many tumor oncogenes have been found to be altered by a point mutation. The ability to inhibit selectively the expression of the mutated form of a protein without affecting its normal counterpart is central to many therapeutic strategies, since the normal protein may serve indispensable functions. Antisense oligonucleoside methylphosphonates and their psoralen derivatives directed at either normal human Ha-ras p21 or ras p21 that is mutated at a single base in codon 61 have been examined for their efficacy and specificity as inhibitors of p21 expression. Mixed cultures of cells expressing both forms of p21 were treated with the antisense oligomer complementary to the normal p21 or with the antisense oligomer complementary to the point-mutated p21. Each of the antisense oligomers specifically inhibited expression of only the form of ras p21 to which it was completely complementary and left the other form of p21 virtually unaffected.
Base Sequence↗
Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia.
Recently, point mutations in the gene of the granulocyte colony-stimulating factor (G-CSF) receptor have been reported in two patients with severe congenital neutropenia who developed acute myeloid leukemia (AML). We investigated the frequency of these specific G-CSF receptor mutations in patients with congenital neutropenia undergoing treatment with r-metHuG-CSF (Filgrastim) and the clinical relevance of these mutations. Nucleotides 2306 to 2561 including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene were amplified by reverse transcriptase-polymerase chain reaction. Detection of point mutations was performed with specific restriction enzyme analysis, as well as sequencing of PCR products. Both genomic DNA and cDNA from neutrophils and mononuclear cells were analyzed from 28 patients with severe congenital neutropenia. Four of 28 patients with congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. The point mutations replace a glutamine codon by a stop codon of the G-CSF receptor gene. Among these four congenital neutropenia patients with a mutated G-CSF receptor, two developed AML. All four patients were investigated regularly and no correlation between occurrence of G-CSF receptor mutation and time or dose of r-metHuG-CSF treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 24 congenital neutropenia patients. Furthermore, we tested six family members of the two patients with AML including mothers and fathers, one sister, and one brother who suffers from congenital neutropenia, as well. All family members displayed a normal G-CSF receptor gene. After the acquisition of the G-CSF receptor mutations, the congenital neutropenia patients continued to respond to G-CSF therapy with an increase in absolute neutrophils in the peripheral blood. We conclude that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur spontaneously and are not inherited. From our data, we suggest that the described G-CSF receptor point mutations do not alter the response to treatment with r-metHuG-CSF and are not the cause of severe congenital neutropenia.
Acute Disease↗
Joint effect of G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism on the risk of premature coronary artery disease.
The study sought an association between the G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism and premature coronary artery disease (CAD), and the interactive effect on CAD risk between the G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism as well as between tested polymorphisms and traditional risk factors. 167 patients with CAD younger than 55 years were compared with 132 healthy subjects. The frequency of factor V point mutation was 7.8 % among Slovene patients with premature CAD, and 4.5 % among controls. No association was found between either the factor V point mutation (AG genotype) or M1M1 genotype of factor VII Arg/Gln(353) gene polymorphism and the risk of CAD in Slovenia using univariate analysis (factor V point mutation: OR = 1.8, 95% CI = 0.7-4.9; p = 0.25; factor VII Arg/Gln(353) gene polymorphism: OR = 1, 95 % CI = 0.6-1.7; p = 0.9). However, a joint effect on the risk of CAD was found between factor V point mutation (AG genotype) and M1M1 genotype (OR = 3.6, 95 % CI = 1-12.9; p = 0.03). Additionally, an interactive effect on CAD risk was found between AG genotype and metabolic risk factors (OR = 3.8, 95% CI = 1.1-13.6; p = 0.03). In conclusion, we provide evidence for a joint effect on CAD risk between G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism as well as between factor V point mutation and metabolic risk factors.
Age of Onset↗
A single point mutation is the cause of the Greek form of hereditary persistence of fetal haemoglobin.
In normal humans the fetal stage-specific gamma-globin genes are silenced after birth and not expressed in the adult. Exceptions are seen in cases of hereditary persistence of fetal haemoglobin (HPFH). These are clinically important because the elevated levels of gamma-globin can alleviate beta-thalassaemia and sickle cell anaemia. One class of mutations is associated with point mutations in the promoter of the gamma-globin genes (non-deletion HPFH), whereas others seem to be caused by large deletions 3' to the gamma-globin genes. To test whether the point mutation found in the Greek non-deletion HPFH (guanine to adenine at nucleotide position -117) is the cause of the raised gamma-globin levels in the adult stage and is not just a linked polymorphism, we engineered this mutation into a gamma-globin gene. When this gene was introduced into mice, the presence of the -117 mutation results in persistence of gamma-globin expression at a high level and a concomitant decrease in beta-globin expression in fetal and adult mice. We show that these changes correlate with the loss of binding of the transcription factor GATA1 to the gamma-globin promoter, suggesting that it may act as a negative regulator of the gamma-globin gene in adults.
Animals↗
Molecular analysis of the RET proto-oncogene in patients with sporadic medullary thyroid carcinoma: a novel point mutation in the extracellular cysteine-rich domain.
Germline point mutations in the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (2A and 2B) and familial medullary thyroid carcinoma. On the other hand, somatic point mutations of RET have been described in a subset of sporadic medullary thyroid carcinomas (MTCs). We examined tumor and blood DNA of thirteen apparently sporadic MTC patients for mutations in RET exons 10, 11, 13, 15 and 16 to determine whether they had true sporadic tumors or either de novo or occult germline mutations. Three different somatic missense mutations were documented in seven patients. In five patients a mutation in exon 16, codon 918, (ATG-->ACG) causing a Met-->Thr substitution was found. In the remaining two patients the mutation affected exon 11: codon 630 in one case and codon 634 in the other. In both cases a T-->C transversion was identified causing a Cys-->Arg substitution. In conclusion, absence of a germline mutation in RET exons 10, 11, 13 or 16 is evidence against an inherited form in all cases. In seven patients, identification of a somatic mutation supported the previous clinical diagnosis of sporadic medullary thyroid carcinoma; in one of them we identified a hitherto undescribed somatic point mutation at codon 630.
Adult↗
Concomitant point mutation of tumor suppressor gene p53 and oncogene c-N-ras in malignant neuroendocrine pancreatic tumor.
Activation by point mutation of ras family genes as well as point mutations of the p53 tumor suppressor gene are found in many tumors. Here we describe a rare case of malignant neuroendocrine pancreatic tumor with multiple metastases in different organs showing strong positivity for synaptophysin, glucagon-like peptide 1, pan-cytokeratin, moderate positivity for chromogranin, Phe-5 and calcitonin and weak positivity for vasointestinal peptide. We found a point mutation at codon 61 of the c-N-ras oncogene, and point mutations in the p53 tumor suppressor gene in the primary tumor as well as in its metastases in liver. The mutation in the c-N-ras gene was a cytosine to adenine transversion, resulting in the amino-acid lysine. Allele specific hybridization showed that the mutation involved one of two c-N-ras alleles as the oligonucleotide for the normal codon also hybridized to amplified tumor DNA. Concomitant mutation of the p53 tumor suppressor gene at codons 248 and 249 was found. The mutation in codon 248 was a cytosine to guanine transversion resulting in the amino-acid glycine. The mutation in codon 249 was a third base, G- > T, transversion leading to a change from arginine to serine. This is the first time that concomitant point mutations in c-N-ras and p53 have been found in a neuroendocrine pancreatic tumor. Based upon these and our previous results, we concluded that these genetic changes may play a role in the development of this particular pancreatic tumor.
Biomarkers, Tumor↗
Diagnosis of pancreatic cancer by K-ras point mutation and cytology of pancreatic juice.
OBJECTIVES: Recently, it was reported that detection of K-ras point mutation at codon 12 in pancreatic juice is an objective method for the diagnosis of pancreatic cancer, but a few reports have suggested that this might represent an early event in pancreatic oncogenesis. In the present study we examined, in various patients, the occurrence of K-ras codon 12 point mutation in pancreatic juice and compared it with pancreatic juice cytology, which is also a reliable diagnostic method. METHODS: Pancreatic juice was obtained endoscopically from patients with various pancreatic disorders and those without definite diseases, and was examined cytologically and for the occurrence of K-ras codon 12 point mutation. The K-ras gene was amplified by polymerase chain reaction (PCR) and the mutation at codon 12 (GGT-->GAT) was examined by slot blot hybridization analysis. RESULTS: K-ras point mutation at codon 12 was detected in seven of 14 (50%) pancreatic cancers, in four of 10 (40%) mucin-producing tumors, in four of 13 (31%) chronic pancreatitis, and in two of 10 (20%) pancreas without definite disorders. K-ras point mutation was detected in nine of 18 (50%) pancreatic juice samples containing cancer cells, in eight of 18 (44%) pancreatic juice samples containing atypical cells, but in none of such samples containing only normal cells. CONCLUSION: Cancer cells were detected from pancreatic cancer exclusively, but K-ras point mutation at codon 12 was detected in pancreatic juice, not only from pancreatic cancer, but also from other diseases.
Aged↗
Detection of high incidence of H-RAS oncogene point mutations in acute myelogenous leukemia.
We have been analyzing RAS p21 proteins and the DNA sequence of leukemic cells. We report here that these cells have high expression of H-RAS p21, which originates from point mutations of RAS oncogenes. The leukemic cells from six patients with acute myelogenous leukemia were separated from heparinized whole blood and bone marrow by a density gradient technique. The expression of RAS oncogenes was analyzed by a fluorescence-activated cell sorting with a panel of monoclonal antibodies. The high expression of DWP, which was reported to recognized activated RAS oncogene, was found in two patients and was associated with high levels of H-RAS expression. These facts prompted us to analyze the DNA sequence of RAS genes with an automated DNA sequencer. Unexpectedly, various kinds of H-RAS point mutations were found in all six cases, including two cases of hot-spot point mutation at codon 12, whereas K-RAS point mutation (no hot-spot point mutations) was found in six cases. The same H-RAS point mutations, at codons 10, 11, and 15, were found in all six cases. To our knowledge, there is no report on H-RAS point mutation in human leukemias. On the basis of these findings, we suggest that H-RAS point mutation together with p53 gene mutation may play an important role in leukemogenesis.
Base Sequence↗
The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non HCC prevalent area in China.
AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.
Base Sequence↗