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At least 397 records · Page 22Linked to original sources
PubMed1996

Detection of K-ras point mutations at codon 12 in pure pancreatic juice for the diagnosis of pancreatic cancer by PCR-RFLP analysis.

The present study was undertaken to detect K-ras point mutations at codon 12 in pure pancreatic juice (PPJ) for the diagnosis of pancreatic cancer (PC) using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. PPJ was collected through a cannula under a duodenal fiberscope from 26 patients with PC and 32 patients with chronic pancreatitis (CP). DNA was extracted from PPJ and was used as the template for PCR. Analysis of PPJ by PCR-RFLP with BstNI revealed that the incidence of K-ras point mutations at codon 12 was 81% (21/26) in patients with PC and 6% (2/32) in those with CP. With reference to the location of PC, the incidence of K-ras mutations was 79% (11/14) in the head, 86% (6/7) in the body, and 80% (4/5) in the tail of the pancreas. The incidence of K-ras mutants was 50% (1/2) in tumor size 1 (TS1; < or = 2.0 cm in size), 71% (5/7) in TS2 (2.1 to < or = 4.0 cm), 89% (8/9) in TS3 (4.1 to < or = 6.0 cm), and 88% (7/8) in TS4 (> 6.1 cm). These results suggested that analysis of K-ras point mutations at codon 12 in PPJ using the PCR-RFLP method is a promising new genetic test for the diagnosis of PC.

Adult↗
PubMed2001

A novel point mutation (C571T) in the tissue-non-specific alkaline phosphatase gene in a case of adult-type hypophosphatasia.

OBJECTIVE: Hypophosphatasia (HOPS) is an inheritable disorder characterized by defective skeletal mineralization, deficiency of tissue-non-specific alkaline phosphatase (TNSALP) activity and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In this study we analysed the genomic TNSALP gene from a patient with HOPS, her family, and unrelated normal controls. MATERIALS AND METHODS: The proband was a 52-year-old Japanese woman with adult onset HOPS. The patient showed deficiency in alkaline phosphatase (ALP) activity, increased urinary excretion of phosphoethanolamine and severe periodontal disease. Genomic DNA was extracted from the peripheral leukocytes of the subjects. Based on published sequence data in the TNSALP gene, 11 pairs of polymerase chain reaction (PCR) primers were used to amplify the coding exons. The PCR amplified samples were subjected to PCR-single strand conformation polymorphism (SSCP) analysis and PCR-allele specific oligonucleotide (ASO) analysis. RESULTS: By PCR-SSCP analysis of the patient's genomic DNA, fragments containing exon 5 revealed abnormal mobility. This abnormal mobility (exon 5) was also found in the genomic DNA in her mother's sister, but were not detected in her father, brothers or sisters, and unrelated normal controls. Sequencing analysis of the abnormal band extracted from the SSCP gel revealed a C to T transition at nucleotide position 571 (C571T) in exon 5. This mutation resulted in a substitution of Ala-115 with a Val in the mature TNSALP polypeptide. PCR-ASO analysis also confirmed this missense point mutation. The result of this study showed that the pro-band has inherited the C571T mutation in exon 5 from her mother alone and the disease in this family was inherited as an autosomal dominant trait from the pedigree. CONCLUSIONS: The C571T mutation is a new missense point mutation and appears to cause significant changes in the structure and function of TNSALP because Ala-115 is highly conserved in rat TNSALP and human tissue-non-specific, intestinal and placental ALPs.

Alanine↗
PubMed2003

Short communication: point mutations in the dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Colombia.

Point mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum can lead to an increasing resistance of P. falciparum to pyrimethamine/sulphadoxine. We examined the prevalence of these mutations in 36 samples from Colombia. Analysed by polymerase chain reaction (PCR) for infection with P. falciparum, 25 (69%) tested positive. These positive isolates were tested further for point mutations in the genes of DHFR (codons 16, 51, 59, 108 and 164) and DHPS (codons 436, 437, 540, 581 and 613) by nested PCR and following mutation-specific restriction enzyme digestion. Gene mutations occurred in both the DHFR and DHPS gene of the Colombian isolates, suggesting that resistance to antifolate drugs exists or may develop soon in Colombia.

Adult↗
PubMed1988

Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. We have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A we have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. In one of the commonest, G6PD Mediterranean, which is associated with favism among other clinical manifestations, a single amino acid replacement was found (serine----phenylalanine): it must be responsible for the decreased stability and the reduced catalytic efficiency of this enzyme. Single point mutations were also found in G6PD Metaponto (Southern Italy) and in G6PD Ilesha (Nigeria), which are asymptomatic, and in G6PD Chatham, which was observed in an Indian boy with neonatal jaundice. In G6PD "Matera," which is now known to be the same as G6PD A-, two separate point mutations were found, one of which is the same as in G6PD A. In G6PD Santiago, a de novo mutation (glycine----arginine) is associated with severe chronic hemolytic anemia. The mutations observed show a striking predominance of C----T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

Amino Acid Sequence↗
PubMed2003

Hydrogel-microsphere-enhanced surface plasmon resonance for the detection of a K-ras point mutation employing peptide nucleic acid.

Highly-sensitive detection of a K-ras point mutation in codon 12, frequently found in pancreatic cancer, based on DNA-carrying hydrogel microspheres as a response enhancer for surface plasmon resonance (SPR), is described. Acrylamide-based microspheres with carboxyl groups were conjugated with DNA probes. Use of the DNA-carrying microsphere in the sandwich method, that is, binding of the microspheres with target DNAs at the sensor surface, enhanced the SPR response as a combined result of increased dielectric constant by the DNA-carrying microspheres. Microspheres lead to response enhancement, as shown by a 100-fold increase in sensitivity compared to that of non-amplified DNA target hybridization. In addition, the advantage of peptide nucleic acid (PNA) in the detection of a K-ras point mutation at the sensor surface by increasing temperature and flow rate is discussed. Results illustrate that the sandwich method through DNA-carrying microspheres for a SPR sensor is a promising approach for ultrasensitive DNA detection.

DNA Probes↗
PubMed2006

Real-time quantitative polymerase chain reaction analysis of mitochondrial DNA point mutation.

Mitochondrial respiratory chain disorders are a group of clinically and genetically heterogeneous diseases. Several mitochondrial (mt)DNA point mutations are responsible for common mitochondrial diseases. These pathogenic mtDNA point mutations are usually heteroplasmic. Molecular diagnosis of the disease requires both qualitative detection of the mutation and quantitative analysis of the mutant heteroplasmy. In this report, two methods based on real-time quantitative polymerase chain reaction (PCR) analysis are used. The first method utilizes wild-type or mutant sequence-specific TaqMan probe, which is labeled with a fluorescent reporter molecule at the 5'-end of the oligonucleotide probe and a quencher at the 3'-end of the probe. The second method utilizes sequence-specific primers to amplify the wild-type or mutant sequence followed by SYBR green detection of PCR products. Both methods allow simultaneous detection and quantification of the mutant mtDNA. In this chapter, we describe the detailed procedures regarding the application of fluorescent probes, and real time quantitative PCR in the molecular diagnosis of mitochondrial DNA disorders.

Animals↗
PubMed1990

Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer.

The p53 gene initially was thought to be an oncogene, but recent evidence suggests that wild-type p53 can function as a tumor suppressor gene in lung, colon, and breast cancer as well as less common malignancies. This study reports the first identification of intronic point mutations as a mechanism for inactivation of the p53 tumor suppressor gene. Abnormally sized p53 mRNAs found in a small cell and a non-small cell lung cancer cell line were characterized by sequence analysis of cDNA/PCR products, the RNase protection assay and immunoprecipitation. These mRNAs were found to represent aberrant splicing leading to the production of abnormal or no p53 protein. Sequence analysis of genomic DNA revealed that a point mutation at the splice acceptor site in the third intron or the splice donor site in the seventh intron accounts for the abnormal mRNA splicing. In one patient the same intronic point mutation was found in the tumor cell line derived from a bone marrow metastasis and in multiple liver metastases but not in normal DNA, indicating that it occurred as a somatic event before the development of these metastases. These findings further support the role of inactivation of the p53 gene in the pathogenesis of lung cancer and indicate the role of intronic point mutation in this process.

Amino Acid Sequence↗
PubMed1995

Gene conversion vs point mutation in generating variability at the antigen recognition site of major histocompatibility complex loci.

In order to assess the roles of gene conversion followed by natural selection and balancing selection for point mutations in polymorphisms at major histocompatibility complex (MHC) loci, DNA sequences of several mammalian taxa were analyzed. Synonymous and nonsynonymous diversities were estimated separately for the antigen recognition site (ARS) and the remaining region of class I and class II genes. In some sequence pairs, the number of nonsynonymous substitutions exceeds that of synonymous substitutions at the ARS. This result indicates that some kind of balancing selection for point mutation is operating. In other sequence pairs (particularly of bovine and of rabbit), the number of synonymous substitutions at the ARS exceeds the same number at the remaining region. This result indicates that gene conversion involving a short region followed by natural selection is important. In general, a combination of gene conversion, point mutation, natural selection and random drift is thought to have contributed to polymorphisms.

Animals↗
PubMed2000

p53 intronic point mutation, aberrant splicing and telomeric associations in a case of B-chronic lymphocytic leukaemia.

We report a case of chronic lymphocytic leukaemia (CLL) with telomeric associations and a p53 intronic point mutation. Karyotypic analysis revealed clonal and non-clonal telomeric associations, accompanied by clonal cytogenetic abnormalities and also in isolation. The p53 mutation, which occurred at the invariant base pair -2 of the splice acceptor site in intron 7 resulted in the abolition of correct splicing of exon 7 to exon 8. Multiple aberrant splice products were characterized, all of which differed from wildtype in the DNA binding domain. Fluorescence in situ hybridization demonstrated that the clone retained two copies of the p53 gene and wild-type p53 transcript was detected on cloning of reverse transcriptase polymerase chain reaction (RT-PCR) product, indicating that one wild-type allele remained. However, a plasmid clone with correct splicing at the exon 7/8 boundary, but with a 21 bp deletion in exon 8, was also found at low frequency. This finding indicates clonal evolution, resulting in complete loss of wild-type p53. The intronic point mutation was not present in DNA extracted from cervical tissue indicating that it was a leukaemic phenomenon. This is the first case of an intronic point mutation to be reported in CLL. This mutation led to chaotic p53 expression and, interestingly, occurred in a case showing telomeric associations, a rare phenomenon in B-CLL.

Amino Acid Sequence↗
PubMed1994

A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense.

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.

Amino Acid Sequence↗
PubMed1993

No evidence for a point mutation at codon 713 and 717 of amyloid precursor protein gene in Japanese schizophrenics.

A point mutation at codon 717 of amyloid precursor protein (APP) gene has been demonstrated to play an important pathogenic role in some cases of familial Alzheimer's disease (FAD). Recently, a single case of chronic schizophrenia with a point mutation at codon 713 of APP gene which sits very close to the mutation in FAD was reported. We screened for these two kinds of mutations in 39 schizophrenic patients using polymerase chain reaction (PCR) and restriction enzyme technique. A mutation of codon 713 creates a MaeIII restriction site and that of codon 717 creates a BclI site. Enzyme digestion with amplified PCR product revealed no restriction site in all subjects. None of our subjects had either of these two kinds of mutations. Our findings support the hypothesis that the case of a mutation at codon 713 of APP gene is a natural non-pathogenic variant and, as well as a mutation at codon 717, has no relation with the genetic predisposition to schizophrenia.

Adult↗
PubMed1992

Absence of ras family point mutations at codons 12, 13 and 61 in N-ethyl-N-hydroxyethylnitrosamine- or N-nitrosomorpholine-induced renal cell tumors in rats.

The prevalence of Ki-ras, Ha-ras and N-ras point mutation within exons 1 and 2 was studied in 17 cases of renal cell tumors (8 carcinomas and 9 adenomas) induced by N-ethyl-N-hydroxyethyl-nitrosamine or N-nitrosomorpholine. DNA samples prepared from acetone-fixed, paraffin-embedded tissues were amplified by means of the polymerase chain reaction, and point mutations at codons 12, 13 and 61 were analyzed by direct sequence methods with oligonucleotide primers. No mutations were detected in any of the renal tumors. The results thus indicated that ras family point mutation is not necessary for kidney tumor development in rats, supporting the view that ras mutations may not be generally relevant to neoplastic development in various organs in different species.

Adenoma↗
PubMed2005

Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples.

MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 microg of Thermus thermophilus his6-MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations.

Cell Cycle Proteins↗
PubMed1997

Allele-specific PCR analysis for detection of the gld Fas-ligand point mutation.

The discovery of a naturally occurring missense point mutation in the gene encoding Fas-ligand (FasL/CD95L) in generalized lymphoproliferative disease (gld) mice has lead to the characterization of FasL as an important mediator of apoptosis. Further analysis of FasL function can be facilitated by crossing the gld mutation onto other mouse-strains, for example those carrying mutations affecting other molecules involved in apoptosis, or disease-prone genetic backgrounds. The success of this is dependent on a quick and reliable screening method. Here we report an allele-specific PCR for detection of the gld mutation. This approach permits the screening of back-crossed F1 progeny within one day, using whole blood samples as a source of genomic DNA. The technique is fast, robust, easily learnt, and unambiguous.

Alleles↗
PubMed1994

Association of deletion and homoplasmic point mutation of the mitochondrial DNA in an ocular myopathy.

The mitochondrial DNA of a 41 year old patient with ocular myopathy was explored. We found a deletion of 3540 base pair in about 50% of the mitochondrial genomes associated with a homoplasmic point mutation. The mutation at nucleotide pair 7444 converts stop codon AGA into lysine codon AAA (human mitochondrial genetic code). The synergistic effect between two point mutations has already been described in mitochondrial pathology but this is the first time that an association between a deletion and a point mutation is shown.

Adult↗
PubMed1988

Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion.

The rat neu gene, which encodes a receptor-like protein homologous to the epidermal growth factor receptor, is frequently activated by a point mutation altering a valine residue to a glutamic acid residue in its predicted transmembrane domain. Additional point mutations have been constructed in a normal neu cDNA at and around amino acid position 664, the site of the naturally arising mutation. A mutation which causes a substitution of a glutamine residue for the normal valine at residue 664 leads to full oncogenic activation of the neu gene, but five other substitutions do not. Substituted glutamic acid residues at amino acid positions 663 or 665 do not activate the neu gene. Thus only a few specific residues at amino acid residue 664 can activate the oncogenic potential of the neu gene. Deletion of sequences of the transforming neu gene demonstrates that no more than 420 amino acids of the 1260 encoded by the gene are required for full transforming function. Mutagenesis of the transforming clone demonstrates a correlation between transforming activity and tyrosine kinase activity. These data indicate that the activating point mutation induces transformation through (or together with) the activities of the tyrosine kinase.

Animals↗
PubMed1992

Point mutations in the upstream region of the alpha-galactosidase A gene exon 6 in an atypical variant of Fabry disease.

Single point mutations in the upstream region of exon 6 of the alpha-galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; 301Arg----Gln (902G----A) in a case that has already been published and 279Gln----Glu (835C----G) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; 328Gly----Arg (982G----A) in the downstream region of exon 6 in one case and two combined mutations, 66Glu----Gln (196G----C)/112Arg----Cys (334C----T), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the alpha-galactosidase A gene.

Adolescent↗
PubMed1994

A rapid and sensitive PCR screening method for point mutations associated with mitochondrial encephalomyopathies.

Alterations of the mitochondrial DNA, encoding important parts of the cellular energy-generating system (oxidative phosphorylation, OXPHOS), are often associated with the occurrence of degenerative neuromuscular diseases. Especially point mutations in the mitochondrial tRNA genes, which cannot be complemented by the nuclear encoded tRNAs, are candidates for severe defects of the OXPHOS system. An A to G transition at nt 8344 in the tRNA(Lys) gene has been associated with MERRF disease whereas an A to G substitution at nt 3243 in the tRNA(Leu) gene has been linked to the MELAS syndrome. These two mtDNA alterations as well as point mutations in protein-coding genes can be detected simultaneously by an allele-specific amplification of the altered mtDNA. This assay allows the reliable detection of heteroplasmic point-mutations, even if the mutated DNA appears to a small extent of less than 1%.

Alleles↗