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PubMed1992

A point mutation (Ala229 to Thr) in the hinge domain of the c-erbA beta thyroid hormone receptor gene in a family with generalized thyroid hormone resistance.

Various point mutations in the c-erbA thyroid hormone receptor (TR) beta gene of unrelated kindreds have been reported to be responsible for different phenotypes of generalized thyroid hormone resistance. We now report a new point mutation, Td, in one of two TR beta alleles of three affected members of one family, designated family T. In contrast to the previously described point mutations, all located in the T3-binding domain of the TR beta gene, mutation Td was identified in the carboxy-terminal part of the hinge domain. Direct sequencing of the polymerase chain reaction-amplified whole coding region of the patients' fibroblast TR beta genes displayed a single guanine to adenine transition at cDNA nucleotide position 985. This altered alanine (GCC) to threonine (ACC) in codon 229. Garnier prediction of the consequence of the mutation indicated an altered secondary structure. The G----A nucleotide substitution was not present in 80 random TR beta alleles, suggesting that this point mutation is responsible for generalized thyroid hormone resistance in family T. The in vitro expressed mutant TR beta was shown to bind with high affinity to various thyroid hormone response elements. However, the affinity of the TR beta to bind to T3 was reduced 3-fold, indicating that the hinge domain of the TR beta is important for full ligand-binding activity. Moreover, it seems that multiple subdomains of the TR beta interact cooperatively to achieve optimal T3 activity.

Alleles↗
PubMed1992

A comparison of single nucleotide primer extension with mispairing PCR-RFLP in detecting a point mutation.

A recent report by Petruzzella et al. (BBRC 186, 491-497, 1992) raised a question as to whether a point mutation in the mitochondrial ND2 gene (BBRC 182, 238-246, 1992) is relevant to Alzheimer's disease. The argument was based on their inability to detect the point mutation at position 5460 in codon 331 in the DNAs extracted from 15 patients with Alzheimer's disease using mispairing PCR-RFLP. To clarify the discrepancy, we tested the DNAs reported by Petruzzella et al. for the mutation by single-nucleotide primer extension. The present work confirms our previous report and extends our finding of the point mutation in 8 of the 15 AD DNAs.

Aged↗
PubMed1994

Extremely high levels of mutant mtDNAs co-localize with cytochrome c oxidase-negative ragged-red fibers in patients harboring a point mutation at nt 3243.

A single mtDNA point mutation at nt 3243 has been associated with two different clinical phenotypes: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes ('MELAS3243') and progressive external ophthalmoplegia ('PEO3243'). It has been shown that there is a much higher proportion of ragged-red fibers (RRF) with cytochrome c oxidase (COX) deficiency in PEO3243 than in MELAS3243. Using PCR/RFLP analysis of isolated individual skeletal muscle fibers from patients with both syndromes, we found a direct correlation between the localized concentration of the nt 3243 mutation and impairment of COX function at the single muscle fiber level: we found relatively low levels of mutant mtDNAs (56 +/- 21%) in 'normal' fibers; high levels (90 +/- 6%) in COX-positive RRF; and an almost complete segregation of mutant mtDNAs (95 +/- 3%) in COX-negative RRF. Thus, the differential distribution of fibers with extremely high concentrations of mutant mtDNAs characterizes, and probably distinguishes, the skeletal muscle of PEO and MELAS patients harboring the same nt-3243 mutation.

Analysis of Variance↗
PubMed2005

Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations.

We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time 19.1 min) and could screen multiple mutations simultaneously.

Base Sequence↗
PubMed2005

Contribution of de novo point mutations to the overall mutational burden in mitochondrial DNA of adult rats.

This study analyzed the incidence of point mutations in mitochondrial DNA of brain and muscle tissues from young (6-month) and old (24-month) male F344 rats. Coding sequence mutations in subunit 5 of the NADH dehydrogenase gene were detected after high-fidelity PCR amplification and cloning by denaturing gradient gel electrophoresis (DGGE) assay followed by sequencing of detected mutants. In total, almost a thousand individual clones were analyzed both in brain and muscle samples. On average, mtDNA from brain tissue showed a 66% increase with age in mutation frequencies (2.3+/-1.9 vs. 3.8+/-4.5 x 10(-4) mutations/bp, mean+/-SD), which failed to reach statistical significance (p=0.45). Muscle tissues yielded substantially fewer mutants with average mutant frequencies for both young and old rats almost 10 times lower than the corresponding values in the brain tissue (0.3+/-0.4 and 0.5+/-0.6 x 10(-4), respectively). The difference in mutation accumulation between muscle and brain was highly significant in both the younger group (Chi-squared=9.7, p < or = 0.01) and in older animals (Chi-squared=10.9, p < or = 0.001). Molecular analysis of the mutated sequences revealed that almost half were identical sequences recurring in different samples and tissues. Our findings indicate that the process of mutation accumulation in mitochondria begins in the germ-line and/or during earlier stages of life, contributing up to half of the total mutational burden, whereas de novo spontaneous formation of point mutations in adulthood is far less than was anticipated.

Aging↗
PubMed1992

Detection of point mutations and a gross deletion in six Hunter syndrome patients.

We have used screening with the polymerase chain reaction and chemical mismatch detection of amplified cDNA to detect and characterize deletions and point mutations in six Hunter Syndrome patients. A high degree of mutational heterogeneity was observed. The first patient is completely deleted for the gene coding for alpha-L-iduronate sulfate sulfatase, while the second has a point mutation that creates a stop codon. The third patient shows a point mutation that creates a novel splice site that is preferentially utilized and results in partial loss of one exon in the RNA. Patients 4, 5, and 6 have point mutations resulting in single amino acid substitutions. Four of the six single-base changes observed in this study were examples of transitions of the highly mutable dinucleotide CpG to TpG. This study has demonstrated a procedure capable of detecting all types of mutation that affect the function of the IDS protein and should enable direct carrier and prenatal diagnosis for Hunter syndrome families.

Amino Acid Sequence↗
PubMed2003

Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome.

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10,000 and 5/100,000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 x 10(-4). Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10(-6) using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (approximately 100 bp) juxtaposed with a 'clamp', a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent approximately 9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 x 10(-5) in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 x 10(4) turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10(5) persons.

Cell Line↗
PubMed1999

Spontaneous and chemically induced point mutations in HPRT cDNA of the metabolically competent human lymphoblastoid cell line, MCL-5.

Thioguanine-resistant clones of the human lymphoblastoid cell, MCL-5, which carries two recombinant plasmids expressing xenobiotic metabolizing enzymes, were obtained spontaneously and after treatment with 0.1 microgram/ml benzo[a]pyrene (BaP), 1.0 microgram/ml 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), and 10 micrograms/ml cigarette smoke condensate (CSC). Treatment with the chemicals reduced the cloning efficiency (CE) of MCL-5 cells from about 30% in untreated cultures to about 10% after treatment with NNK and to about 1% or to less than 1% after treatment with CSC or BaP, respectively. At the same time, the mutant frequencies were increased about sevenfold above those of untreated cultures. Among a total of 138 independent mutant clones that had resulted from 55 separate cultures, 60 point mutations were identified within the hypoxanthine guanine phosphoribosyltransferase (HPRT) reading frame by sequencing full-size reverse transcription polymerase chain reaction (RT-PCR) products from thioguanine-resistant clones. The identified 53 coding errors were distributed among 33 locations and types. Among the 30 types of single basepair substitutions leading to coding errors, 12 had not been described before. In the present set of point mutations, the distribution of base substitution types as well as of mutated sites appeared to be influenced by the treatment with the chemicals. Thus, the ratio of G to T transversions increased from 3 among 19 spontaneous point mutations in the HPRT coding region to 9 among 21 BaP-induced point mutations. The G119T and G208T transversions were found three times each, exclusively after treatment with BaP, while the accumulation of two to eight incidences of the G97T, CG142/3TA, C508T, T583A and G599A mutations was split among different treatments. All eight identified point mutations identified after NNK treatment were at G or T residues on either strand that were followed by additional G or T residues.

Benzo(a)pyrene↗
PubMed2004

Flow-stress-induced discrimination of a K-ras point mutation by sandwiched polymer microsphere-enhanced surface plasmon resonance.

The highly sensitive detection of a K-ras point mutation with the aid of DNA-carrying microspheres as a flow-stress receptor is proposed at the surface of a surface plasmon resonance (SPR) biosensor. Single-stranded DNAs were immobilized onto epoxy-group-derivatized gold surfaces and the hybridization of DNA targets was monitored. The subsequent interaction with DNA-carrying micospheres enhanced the SPR response. The increase of flow rate during the event of dissociation changed the amount of detachment of the DNA-carrying microspheres for the mismatched pair. In addition, the viscosity was changed by addition of glycerol to the buffer. The increase of shear stress from the flow resulted in detachment of DNA-carrying microspheres hybridized with the mismatched sequence and increased the ability to discriminate a point mutation. This is a new method which not only increases the lower detection limit of evanescent wave-based biosensors, but also the ability to discriminate a point mutation which is a critical factor for ultrasensitive DNA detection in flow devices.

DNA↗
PubMed1998

Mitochondrial DNA point mutation at nucleotide pair 3316 in a Japanese family with heterogeneous phenotypes of diabetes.

A mitochondrial DNA (mtDNA) point mutation at nucleotide pair (np) 3316 has been reported in relation to diabetes. We recently encountered a non-obese family with this type of mutation. The proband in the affected family, a 49-year-old woman who had been previously diagnosed as having an insulin-requiring non-insulin-dependent diabetes mellitus (NIDDM), was referred to our hospital for treatment of diabetic gangrene in her left foot. Her insulin secretory capacity was markedly reduced, but the insulin sensitivity evaluated by the euglycemic hyperinsulinemic clamp technique was normal. In addition, her serum lactate level was markedly increased after a 5 min ambulation, although her serum pyruvate and ketones remained within the normal range. Twenty-year-old twin sons had been treated with insulin since the age of 7, when both were diagnosed with insulin-dependent diabetes mellitus (IDDM). The proband's mother, a 68-year-old, was nondiabetic at this time. MtDNA analysis revealed a point mutation at np 3316 in all family members, which was homoplasmic for the mutation on a photograph of agarose gel electrophoresis containing ethidium bromide under ultraviolet light. This mutation seemed to be maternally transmitted in the family, and the onset of diabetes was occurring earlier and the insulin secretory capacity was declining from generation to generation, so that these findings suggest that the point mutation at np 3316 is associated with various phenotypes of diabetes.

DNA, Mitochondrial↗
PubMedDate not supplied

Significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas.

The significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas is still unclear. The aim of this study was to evaluate the significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas. All of the 78 reports written from 1988 to 1996 on K-ras point mutation of carcinoma, mucin-producing tumors, and hyperplastic epithelia of the pancreas in both surgical or autopsy specimens and pancreatic juice are reviewed. As results, in surgical or autopsy specimens, K-ras mutation was found in 81% of ordinary duct cell carcinoma and in 53% of mucin-producing tumor of the pancreas; this mutation was also found in hyperplastic epithelia in chronic pancreatitis (7%) and in autopsy cases without pancreatic diseases. In pancreatic juice, K-ras mutation was found in 72% of ordinary pancreatic carcinoma and in 53% of mucin-producing tumor, respectively. In conclusion, most previous reports have indicated that K-ras mutation in pancreatic juice is useful for a diagnosis of pancreatic carcinoma. However, since K-ras gene mutation was also detected in non-tumorous lesions, the diagnosis of pancreatic carcinomas is not necessarily correct if it is based solely on the detection of K-ras mutation in pancreatic juice. Future studies should focus on analyzing the amino acid sequence of K-ras mutation or the combination of this mutation with other parameters such as tumor markers in pancreatic juice, to enhance its specificity and accuracy.

Carcinoma↗
PubMed1994

The rat N-ras gene; interference of pseudogenes with the detection of activating point mutations.

The PCR technique in combination with selective hybridization to mutation specific oligonucleotides, is a widely used methodology for the detection of activating point mutations in ras oncogenes. In the present paper we demonstrate for the N-ras gene of the rat that processed pseudogenes do interfere with this method. A first indication for this interference came from the sequence analysis of cloned PCR fragments of exon 1, amplified with primers derived from previously reported exon sequences of the mouse N-ras gene. Between different clones originating from one PCR reaction, a marked sequence heterogeneity is observed and this is shown to be the result of the presence of at least two different processed pseudogenes of the rat N-ras gene. These two pseudogenes, together with the wildtype N-ras gene and a small 3' part of the unr gene, were eventually cloned and their genomic organization and nucleotide sequences determined. Furthermore, representative examples of the confounding effects of these pseudogenes on the screening for activating point mutations are presented. Taken together, our results demonstrate that intron-specific amplification is a prerequisite for the unambiguous detection of activating point mutations in the N-ras gene of the rat.

Amino Acid Sequence↗
PubMed2003

Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5'-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations.

Base Sequence↗
PubMed1999

The p53 tumor suppressor protein reduces point mutation frequency of a shuttle vector modified by the chemical mutagens (+/-)7, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, aflatoxin B1 and meta-chloroperoxybenzoic acid.

p53 has been postulated to be the guardian of the genome. However, results supporting the prediction that point mutation frequencies are elevated in p53-deficient cells either have not been forthcoming or have been equivocal. To analyse the effect of p53 on point mutation frequency, we used the supF gene of the pYZ289 shuttle vector as a mutagenic target. pYZ289 was treated in vitro by ultraviolet irradiation, aflatoxin B1, (+/-)7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and meta-chloroperoxybenzoic acid and then transfected into p53-deficient cells with or without a p53 expression vector. p53 reduced the mutant frequency up to fivefold when pYZ289 was treated with aflatoxin B1, (+/-)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene or meta-chloroperoxybenzoic acid but not when it was ultraviolet-irradiated. The p53-dependent mutation frequency reduction was higher at a higher level of premutational lesions for aflatoxin B1 and (+/-)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and at a lower level of lesions for meta-chloroperoxybenzoic acid. This suggests that the chemical mutagens produce, in a dose-dependent fashion, two kinds of DNA damage, one subject to p53-dependent mutation frequency reduction and the other not. These results indicate that p53 can reduce the point mutation frequency in a shuttle vector treated by chemical mutagens and suggest that p53 can act as guardian of the genome for at least some kinds of point mutations.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

[Immune response to p53 protein in patients with squamous epithelial carcinoma of the upper aerodigestive tract is independent of p53 point mutation].

OBJECTIVE: Mutations of the p53 gene are the most prevalent genomic changes in human malignancies. The detection of these mutations is usually carried out by gene sequencing or with immunohistochemical methods. A new approach is to search for p53 antibodies in serum of cancer patients. The cause for the production of antibodies against p53 is unclear. The aim of this study was to show the correlation between p53 pointmutations and p53 antibodies in patients with squamous cell carcinoma of the head and neck (SCCHN). METHODS: A semiquantitative ELISA was employed to screen the serum of 117 patients with SCCHN for p53 antibodies. Sixty-one tumor samples were evaluated with SSCP-analysis for point mutations in exons 4 to 9. RESULTS: Twenty-three (19.7%) of the cancer patients had antibodies to p53. There was no significant difference in the frequency of p53 point mutations in exon 4 to 9 between the whole cancer patient group and the p53 antibody positive group. CONCLUSIONS: The accumulation of p53 in the tumor cell is one hypothesis as to how p53 becomes immunogenic. This leads to a decrease in tolerance after a certain amount of time. In contrast to this it is assumed that after a p53 point mutation, important protein determinants are changed and p53 becomes antigenic. The results of this study indicate that p53 point mutations in SCCHN seem to have no influence on the development of p53 antibodies. Therefore this study supports the theory that the loss of tolerance and subsequently the antibody production is induced by an accumulation of p53. The type of p53 point mutations on exon 4-9 seems to have no influence that p53 gains immunogenic potential in SCCHN:

Antibodies, Neoplasm↗
PubMed2002

Phosphorylation-dependent down-regulation of c-Myb DNA binding is abrogated by a point mutation in the v-myb oncogene.

The viral Myb (v-Myb) oncoprotein of the avian myeloblastosis virus (AMV) is an activated form of the cellular transcription factor c-Myb causing acute monoblastic leukemia in chicken. Oncogenic v-Myb alterations include N- and C-terminal deletions as well as point mutations. Whereas truncations in Myb cause loss of various protein modifications, none of the point mutations in v-Myb has been directly linked to protein modifications. Here we show that the DNA-binding domain of c-Myb can be phosphorylated on serine 116 by the catalytic subunit of protein kinase A. Phosphorylation of Ser(116) differentially destabilizes a subtype of c-Myb-DNA complexes. The V117D mutation of the AMV v-Myb oncoprotein abolishes phosphorylation of the adjacent Ser(116) residue. Modification of Ser(116) was also detected in live cells in c-Myb, but not in AMV v-Myb. Phosphorylation-mimicking mutants of c-Myb failed to activate the resident mim-1 gene. Our data imply that protein kinase A or a kinase with similar specificity negatively regulates c-Myb function, including collaboration with C/EBP, and that the leukemogenic AMV v-Myb version evades inactivation by a point mutation that abolishes a phosphoacceptor consensus site. This suggests a novel link between Myb, a signal transduction pathway, cooperativity with C/EBP, and a point mutation in the myb oncogene.

Amino Acid Sequence↗
PubMed2002

Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein.

CREB-binding protein (CBP) is a multifunctional cofactor implicated in many intracellular signal transduction pathways. We aimed to investigate the involvement of CBP in the cAMP response element-binding protein (CREB)-mediated pathway. The point mutation Tyr658Ala in the CREB-binding domain (CBD) was shown to abolish the binding activity of CBP to phospho-CREB, the activated form of CREB. By using a mutant Cre/loxP recombination system, this point mutation was aimed to be generated in the mouse genome in a tissue- and time-specific manner. A targeting construct in which CBD exon 5 and inverted exon 5* containing the point mutation flanked by two mutant loxP sites (lox66 and lox71) oriented in a head-to-head position was generated. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase, the favorable reaction leads to a product where the mutated exon 5* is placed into the position to be correctly transcribed and spliced. Inversion was observed to be complete in both bacteria and mouse embryonic stem cells. Our results indicate that this Cre- mediated inversion method is a valuable tool to introduce point mutations in the mouse genome in a regulatable manner.

Animals↗
PubMed2000

[p16 gene deletion and point mutation in highly purified fresh breast neoplasm].

OBJECTIVE: To study the form and frequency of p16 gene deletion and point mutation in human breast neoplasm and the effective detection. METHODS: Cell culture was made using repeat-adhering method to purify human breast neoplasm cells and PCR-SSCP analysis in 14 highly purified fresh breast neoplasm, 14 fresh none-purified specimens, 27 paraffin-embedded specimens and corresponding normal tissues beside neoplasms. RESULTS: The rate of p16 gene deletion and point mutation was 42.9% (6/14) in the highly purified group, in which exon 1st - 3rd, showed homozygous deletion (5 cases) and exon 2nd showed mutation (1 case). The rate of p16 gene mutation in the other three groups was 7.1%, 7.4% and 4.9%. The detection rate of mutation was significantly higher in the highly purified group than in the other three groups (P < 0.01), but it was not significantly different in the other three groups (P > 0.05). CONCLUSIONS: p16 gene deletion and point mutation exist in primary breast cancer. Homozygous deletion and small fragment deletion may be an important mechanism of inactivation in human breast neoplasm. Purified cancer cells will enhance the detection rate.

Adult↗