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PubMed1994

[Detection of point mutation of p53 gene by non-isotopic PCR-SSCP in paraffin-embedded breast cancer tissue].

Non-isotopic PCR-SSCP method was used to detect point mutation of p53 gene in paraffin-embedded breast cancers. The abnormal shifting of the single-stranded DNA (ssDNA) was identified in 14 out of 60 cases. The point mutations of four cases were found in exons 5-6, three in exon 7 and seven in exon 8. The mutant p53 protein was detected by immunohistochemistry. The positive nuclear staining was observed in 13 cases, among which 11 cases showed abnormal shifting of ssDNA in SSCP analysis. The point mutations of the other two cases might occur in the regions which were not examined in this study. The point mutations of p53 were found in 5 cases in the absence of detectable protein. This probably resulted from the reduction or loss of antigenicity of p53 in some of the routinely processed tissues. Our results indicate that detection of point mutations of p53 gene by PCR-SSCP method is more accurate and reliable than that by immunohistochemistry and non-isotopic PCR-SSCP is especially useful in the screening of point mutations during handling a large number of samples.

Breast Neoplasms↗
PubMed1992

[Detection of point mutations of C-Ki-ras oncogene in colon cancer by PCR using specific oligonucleotide probes].

Activation of C-Ki-ras oncogene by point mutation within codon 12,13 was determined by Polymerase Chain Reaction (PCR) using specific oligonucleotide probes. In 9 of 42 colon cancer specimens point mutations in codon 12 of C-Ki-ras oncogene were found. The point mutations identified were GGT(Gly)----GAT(Asp) (4 cases), GGT (Gly)----TGT (Cys) (3 cases) and GGT(Gly)----GTT(Val) (2 cases), respectively. Two types of point mutations (GGT----GAT, GGT----TGT) were found simultaneously in one specimen. The results showed that there was no relationship between the point mutation of C-Ki-ras oncogene and patient's sex, age, state of metastasis or prognosis.

Adult↗
PubMed1997

Screening of H-ras gene point mutations in 50 cases of bladder carcinoma.

BACKGROUND: Mutation converts the H-ras gene into an activated oncogene in about 10% of human bladder cancers. Codons 12 and 61 are the major "hot spots" for activation. A simple and accurate method to detect point mutations in these codons may be clinically useful for early diagnosis of bladder cancer. METHODS: Bladder cancer samples from 50 patients, plus 10 samples of normal bladder mucosa, were analyzed for possible point mutation of the H-ras gene at either codon 12 or codon 61. The H-ras gene DNA segments that include these 2 codons were amplified by PCR methods, then the possible presence of a point mutation was evaluated at each codon by susceptibility of the respective DNA segments to digestion with the restriction enzyme and by dot blot hybridization assay. A bladder cancer patient who had an H-ras gene mutation was examined to see whether the mutation was also detectable in the cells released in the urine. RESULTS: Definite or possible point mutations were found in 6 (12%) out of 50 bladder cancer patients, while no mutation was detected in normal mucosa. A point mutation could also be detected in cells isolated from the patient's urine sample. CONCLUSION: The prevalence of point mutations at codon 12 or codon 61 of the H-ras gene found in this study was similar to that previously estimated for human bladder cancer by DNA transfection assay. The method we have used for detecting point mutations of the H-ras gene provides a simple and highly accurate way to detect mutated cancer cells even in the urine. It may be clinically usable for early diagnosis of bladder cancer.

Adult↗
PubMed2004

Shedding of sabin poliovirus Type 3 containing the nucleotide 472 uracil-to-cytosine point mutation after administration of oral poliovirus vaccine.

A uracil-to-cytosine point mutation at nucleotide (nt) 472 of Sabin oral poliovirus vaccine (OPV) type 3 is found in conjunction with vaccine-associated paralytic poliomyelitis (VAPP). Direct RNA extraction and mutant analysis by polymerase chain reaction and restriction enzyme cleavage were used to identify this point mutation in clinical samples. A total of 238 stool samples were obtained from 28 healthy infants for 6 weeks after OPV vaccination. More than 25% of infants shed OPV3 in the week after vaccination, with a decrease on day 6. A second wave of OPV3 shedding occurred beginning the second week after vaccination and was maintained through the end of the study period. During the first week after vaccination, the proportion of nt 472 mutants in the shed OPV3 increased from undetectable to almost 100%. During the second shedding period, the proportion of nt 472 mutants remained close to 100%. These results suggest that selective mutation drives the VAPP-associated nt 472 point mutation for OPV3 in the human gastrointestinal tract.

Base Sequence↗
PubMed2001

P16INK4a point mutations and promoter hypermetylation in bronchial preneoplastic lesions.

The aim of this study was to determine p16INK4a point mutations and promoter hypermethylation in tumour cells and bronchial preneoplastic lesions in 32 surgically resected lungs due to primary squamous cell carcinoma. P16 point mutations were detected in 1 (3%) and promoter hypermethylation in 12 (31%) of 32 squamous cell carcinomas. The status of p16 was further characterized in 38 premalignant lesions including squamous metaplasias without dysplasia, squamous metaplasias with mild, moderate and severe dysplasias and 4 carcinomas in situ. No p16 point mutations have been found in premalignant or CIS lesions. Methylation of p16 was detected in 1 of 8 (12.5%) cases of squamous metaplasias without dysplasia, in 1 of 10 (10%) cases of squamous metaplasias with mild dysplasia, in 1 of 9 (11%) cases of squamous metaplasia with moderate dysplasia and in 2 of 7 (28.5%) cases of severe dysplasias, as well as in 1 of 4 (25%) carcinomas in situ. This investigation indicates that P16INK4a supressor gene point mutations are rather late event and inactivation of this gene by promoter hypermethylation is early and likely critical in bronchial cancerogenesis.

Bronchial Neoplasms↗
PubMed2003

Transcription increases multiple spontaneous point mutations in Salmonella enterica.

The spontaneous rate of G.C-->A.T mutations and a hotspot T.A-->G.C transversion are known to increase with the frequency of transcription-increases that have been ascribed primarily to processes that affect only these specific mutations. To investigate how transcription induces other spontaneous point mutations, we tested for its effects in repair-proficient Salmonella enterica using reversion assays of chromosomally inserted alleles. Our results indicate that transcription increases rates of all tested point mutations in the induced gene: induction significantly increased the individual rates of an A.T-->T.A transversion, an A.T-->G.C transition and the pooled rates of the three other point mutations assayed. Although the S.enterica genome is thought to have a mutational bias towards G.C base pairs, transitions creating A.T pairs were approximately 10 times more frequent than the reverse mutation, resulting in an overall mutation pressure to lower G+C contents. Transitions occurred at roughly twice the rate of transversions, similar to results from sequence comparisons; however, several individual transversions are more frequent than the least common transition.

Drug Resistance, Bacterial↗
PubMed1998

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection.

Solution hybridization is an essential step in sequencing and some point mutation detection methods. In practice, this hybridization is hampered resulting in the need of additional purification of the amplification products. The use of T7 gene 6 exonuclease may lead to efficient production of single-stranded DNA. In this study, the effect of pretreatment with exonuclease on direct cycle sequencing and point mutation detection was analyzed. Exonuclease-treated products were directly cycle sequenced without further purification. This resulted in highly efficient quality improvement for sequencing allowing detection of heterozygotes. Point mutation detection by Point-EXACCT (exonuclease-amplification coupled capture technique) demonstrated detection of one cell containing a mutation in an excess of 75000 wild type cells. Exonuclease-enhanced detection methods offer simple, rapid detection strategies that are easily adaptable for widespread clinical laboratory use. With the use of exonuclease, the detection of heterozygosity using fluorescent cycle sequencing is becoming more reliable. The high sensitivity of Point-EXACCT due to the use of exonuclease makes it a highly promising method for large-scale screening of (pre)malignant changes in patients with a high risk for developing cancer.

DNA Mutational Analysis↗
PubMed2000

Clarithromycin-resistance and point mutations in the 23S rRNA gene in Helicobacter pylori isolates from Japan.

BACKGROUND: Resistance of Helicobacter pylori to clarithromycin is mostly due to the point mutations in the 23S rRNA. In Japan, however, the frequency of these mutations has not been fully investigated. Furthermore, no study has used gastric biopsy specimens to detect these point mutations. METHODS: The frequency of primary clarithromycin-resistant H. pylori was examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Eighty-two strains (42 isolated from patients with gastric cancer and 40 isolated from patients with chronic gastritis) were examined. Two biopsy specimens obtained from patients in whom eradication therapy including clarithromycin had failed were also studied. RESULTS: Either A2143G or A2144G point mutation was detected in 90% of clarithromycin-resistant H. pylori strains. Eight out of 82 strains (9.8%) had either A2143G or A2144G point mutation. Only one out of 42 strains in patients with gastric cancer had A2143G mutation, whereas five strains had A2144G and two had A2143G mutations in 40 strains isolated from control subjects. The proportion was significantly lower in patients with early gastric cancer (P < 0.05). This PCR-RFLP was also applicable for DNA samples extracted from biopsy specimens and infection of clarithromycin-resistant H. pylori was observed. CONCLUSION: The results suggest that the point mutation in the 23S rRNA gene is commonly seen in clarithromycin-resistant H. pylori and it contributes to the treatment failure in Japan. The PCR-RFLP system is a sensitive method by which to diagnose H. pylori infection as well as a simple method for detecting clarithromycin resistance without bacterial culture.

Anti-Bacterial Agents↗
PubMed1997

Detection of K-ras point mutation by enriched PCR-colorimetric plate assay.

Point mutations in the K-ras gene are frequently observed in a variety of human malignancies, including colorectal and pancreatic cancers. In this paper, we describe a sensitive procedure for the detection of point mutations of codon 12 of the K-ras gene. The assay employs a single-tube enriched PCR procedure, coupled to colorimetric detection. In the enriched PCR procedure, the first round of amplification introduces a restriction enzyme site in the wild type, but not in mutant K-ras PCR product. The wild type products are then digested and the second round of PCR enriches for the mutant sequences by amplifying the resistant products. The second round of amplification allows the incorporation of biotin and a substrate binding tag at opposite ends of the mutant product, thus allowing detection of the product by a simple colorimetric assay. The assay has been validated using DNA from a variety of cell lines known to contain either mutant or wild type K-ras. Under these conditions, the assay has proved both reproducible and sensitive, with the ability to detect one mutant molecule in a background of 1000 wild type molecules. The assay allowed discrimination of mutant from wild type K-ras in samples from colonic adenocarcinomas and normal colonic mucosa. The use of a colorimetric detection system reduces observer bias and facilitates analysis of large numbers of samples. As such, the assay may have specific application in the sensitive detection of K-ras mutations in a variety of clinical samples.

Adenocarcinoma↗
PubMed2004

[Single nucleotide polymorphisms and point mutations of ataxia telangiectasia mutated gene in Chinese of Han ethnicity].

OBJECTIVE: To explore the single nucleotide polymorphisms (SNPs) and point mutations of ataxia telangiectasia mutated (ATM) gene in Chinese of Han ethnicity. METHODS: The target fragments of the exon 39, exon 61 and exon 63 of ATM gene were first amplified using PCR technique, then screened for the SNPs and point mutations using single strand conformation polymorphism (SSCP) technique, finally the representative bands were sequenced for the verification of the new single nucleotide polymorphisms and point mutations using the automatic DNA sequencing technique. RESULTS: Six new SNPs were found in the exon 39, intron 61 and intron 63 of ATM gene. They are respectively the A/T polymorphisms at nucleotide 5689 and 5691 in exon 39, the T/G polymorphisms at nucleotide +69 and +99, the A/G polymorphism at nucleotide +94 in intron 61, the G/C polymorphism at nucleotide +17 in intron 63. Five new point mutations were found in the exon 61, intron 62 and exon 63 of ATM gene. They are respectively the T/G transversion at nucleotide 8618 in exon 61, the T/G transversion at nucleotide -13 in intron 62, the T/G transversion at nucleotide 8793, the G/A transitions at nucleotide 8816 and 8848 in exon 63. The known three SNPs, the G/A polymorphism at nucleotide 5557 in exon 39, the T/C polymorphism at nucleotide +104 in intron 61 and the T/C polymorphism at nucleotide -55 in intron 62, were verified in Chinese of Han ethnicity. CONCLUSION: There are great differences in the SNPs of ATM gene between Chinese of Han ethnicity and Caucasian.

Asian People↗
PubMed2000

The frequency of point mutations in mitochondrial DNA is elevated in the Alzheimer's brain.

Using a PCR-based strategy, we found that point mutation frequencies in mitochondrial DNA (mtDNA) were 2- to 3-fold higher in the parietal gyrus, hippocampus, and cerebellum from subjects with Alzheimer's disease (AD) compared to normal controls. In contrast, levels of a commonly studied deletion mutation, mtDNA(4977), were not elevated in AD. The frequency of point mutations did not vary significantly among the three brain areas, whereas the frequency of mtDNA(4977) was 15- to 25-fold lower in the cerebellum in comparison to the cortex; this regional variation was seen in both the normal and Alzheimer's brain. In blood mtDNA, point mutation frequencies were not elevated in AD patients. The elevated frequency of point mutations in all three brain regions is consistent with the idea that increased oxidant stress is associated with AD.

Adult↗
PubMed1997

Colourimetric point mutation assay: for detection of precore mutants of hepatitis B.

A colourimetric assay for the analysis of point mutations in PCR amplified DNA fragments from hepatitis B virus (HBV) is described. The method was applied for analysis of the single point mutation in codon 28 of the precore gene of HBV, which inhibits expression of HBe antigen. The assay, which uses a microtitre plate formate, incorporates fluorescein-labelled dideoxynucleotides as opposed to radioactively-labelled deoxynucleotides used in methods described previously. Synthetic control wild type and mutant oligonucleotides were tested to optimise the reaction conditions. The assay was thus shown to yield both qualitative and quantitative data on the relative proportions of wild type and mutant sequences within a given sample. Amplicons from clinical specimens of known sequence were analysed to validate the assay. Sixteen chronic carriers of HBV were tested using the codon 28 point mutation assay, and the results were confirmed by direct sequencing. The method described is suitable for applications where point mutations are of interest.

Colorimetry↗
PubMed1998

K-ras gene point mutations in human endometrial carcinomas: correlation with clinicopathological features and patients' outcome.

In order to evaluate the role of K-ras gene point mutations in the progression of endometrial carcinoma, we applied the polymerase chain reaction/restriction-fragment-length polymorphism technique to 57 tumours surgically removed from women of Polish origin. We assessed the relationship between K-ras gene activation and clinicopathological features as well as patients' outcome. Mutational activation in codon 12 of the K-ras gene was detected in 8 out of 57 (14%) endometrial carcinomas, while in codon 13 of the K-ras gene no point mutations were noted. A correlation between the histological type of the tumour and codon 12 K-ras gene mutation was noted (P < 0.05; Fisher exact test). K-ras gene mutation was not related to the patients' age, surgical stage, histological grade or to the depth of myometrial invasion. A trend towards a poorer prognosis was noted during the follow-up of patients whose tumours had shown K-ras codon 12 point mutations, but the difference was not significant (P = 0.06; log-rank test). Our data indicate that point mutations in codon 12 of the K-ras gene are a rare event in human endometrial carcinomas. The lack of correlation between K-ras point mutations and clinicopathological features (except histological type) supports the hypothesis of a random activation of the K-ras gene in human neoplastic endometrium.

Aged↗
PubMed1997

Point mutations in mitochondrial DNA of patients with dilated cardiomyopathy.

Mitochondrial (mt) DNA mutations are hypothesized to be involved in the pathogenesis of dilated cardiomyopathy, because the mtDNA encodes 13 polypeptides that are essential for oxidative phosphorylation, upon which the heart relies for energy. To test this hypothesis, we amplified the mitochondrial genome by long PCR and then used restriction analysis and direct sequencing to examine 58 unrelated patients with dilated cardiomyopathy and 49 controls for the detection of point mutations. The results demonstrated that point mutations were significantly more frequent in the mtDNA of patients than in that of controls (173 in 58 patients v 54 in 49 controls, 2=16.51, P<0.001). In addition to normal variants and mutations common to both patients and controls, 43 mutations were identified only in patients. All but four of these mutations were homoplasmic. Mutations involving the evolutionarily conserved residues of cytochrome c oxidase subunit I, NADH dehydrogenase 5, tRNAAla and tRNAArg were identified. As many as 13 point mutations were found in an 8-month-old patient. In conclusion, there exist significantly more point mutations in mtDNA of patients than in controls, suggesting that multiple mutations may exert a cumulative effect on heart function. Thus, by altering the function of respiratory enzyme subunits or tRNAs, mtDNA point mutations could be relevant for the pathogenesis of dilated cardiomyopathy.

Adult↗
PubMed2004

A point mutation in the lariat branch point of intron 6 of NPC1 as the cause of abnormal pre-mRNA splicing in Niemann-Pick type C disease.

The lariat branch point sequence (BPS) is crucial for splicing pre-mRNA even if BPS mutations have infrequently been reported in human disease. In two siblings with Niemann-Pick type C (NPC) disease we identified two mutations of the NPC1 gene: i) one in exon 20 (c.2932C>T) (p.R978C) previously reported in NPC patients; ii) the other (c.882-28A>G) unreported, in the highly conserved adenosine of a putative lariat BPS of intron 6. Using RT-PCR we found that, besides the normally spliced mRNA, patients' fibroblasts contained minute amounts of an mRNA devoid of exon 7. The exon 6--exon 8 junction in this mRNA causes a frameshift and a premature stop codon, predicted to result in a truncated protein. To assess the effect of c.882-28A>G mutation we constructed two minigenes (wild type and mutant), spanning from intron 5 to intron 8, which were inserted into a pTarget vector and transfected in COS1 cells. The wild type minigene generated an mRNA of the expected size and sequence; the mutant minigene generated only an mRNA devoid of exon 7. This is the first example of a splicing defect due to a mutation in the lariat BPS in an intron of NPC1 found in NPC patients.

Adult↗
PubMed1993

Charcot-Marie-Tooth disease type 1A. Association with a spontaneous point mutation in the PMP22 gene.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. CMT type 1A is associated with a 1.5-megabase DNA duplication in region p11.2-p12 of chromosome 17 in most patients. An increased dosage of a gene within the duplicated segment appears to cause the disease. The PMP22 gene, which encodes a myelin protein, has been mapped within the duplication and proposed as a candidate gene for CMT type 1A. METHODS: We analyzed DNA samples from a cohort of 32 unrelated patients with CMT type 1 who did not have the 1.5-Mb tandem duplication in 17p11.2-p12 for mutations within the PMP22 coding region. Molecular techniques included the polymerase chain reaction (PCR), heteroduplex analysis to detect point mutations, and direct nucleotide-sequence determination of amplified PCR products. RESULTS: A 10-year-old boy was identified with a point mutation in PMP22, which resulted in the substitution of cysteine for serine in a putative transmembrane domain of PMP22. Analysis of family members revealed that the PMP22 point mutation arose spontaneously and segregated with the CMT type 1 phenotype in an autosomal dominant pattern. The patients with the PMP22 point mutation had clinical and electrophysiologic phenotypes that were similar to those of patients with the 1.5-Mb duplication. CONCLUSIONS: The PMP22 gene has a causative role in CMT type 1. Either a point mutation in PMP22 or a duplication of the region including the PMP22 gene can result in the disease phenotype.

Amino Acid Sequence↗
PubMed2004

Screening for point mutations in the LDL receptor gene in Bulgarian patients with severe hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common, autosomal dominant disorder of lipid metabolism, caused by defects in the receptor-mediated uptake of LDL (low-density lipoproteins) due to mutations in the LDL receptor gene ( LDLR). Mutations underlying FH in Bulgaria are largely unknown. The aim of the present study was to provide information about the spectrum of point mutations in LDLR in a sample of 45 Bulgarian patients with severe hypercholesterolemia. Exons 3, 4, 6, 8, 9, and 14, previously shown to be mutational hot spots in LDLR, were screened using PCR-single-strand conformation polymorphism (SSCP). Samples with abnormal SSCP patterns were sequenced. Three different, hitherto undescribed point mutations (367T>A, 377T>A, 917C>A) and two previously described mutations (858C>A and 1301C>T) in eight unrelated patients were identified; four of the detected point mutations being missense mutations and one, a nonsense mutation. One of the newly described point mutations (917C>A) is a base substitution at a nucleotide position, at which two other different base substitutions have already been reported. Thus, all three possible base substitutions at this nucleotide position have been detected, making it a hot spot for point mutations causing FH. This is the first such mutational hot spot described in exon 6 of LDLR.

Bulgaria↗
PubMed2000

Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum beta-lactamases.

The aim of this study was to detect point mutations in extended-spectrum beta-lactamase (ESBL) genes in a background of wild-type (non-ESBL-producing) bacteria using a highly sensitive and specific method developed for this purpose. The ligase detection reaction-polymerase chain reaction (LDR-PCR) method was used to test different ESBL-producing strains and clinical isolates for a specific point mutation in the bla SHV-ESBL gene (glycine to serine mutation at position 238) and was compared with the commercially available E test ESBL (AB Biodisk, Sweden). Nine of the 40 clinical isolates tested were positive for the bla SHV-ESBL point mutation when tested by the LDR-PCR method but negative when tested by the E test. In contrast to the E test or other molecular genetic tests, the LDR-PCR method is able to identify a single bacterium with a point mutation in a background of 100,000 wild-type (non-ESBL-producing) bacteria.

Amino Acid Substitution↗