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PubMed1996

Optimization of restriction fragment length polymorphism and single strand conformation polymorphism analysis for the detection of point mutations of ras oncogenes in paraffin-embedded tissues.

69 samples of formalin-fixed and paraffin-embedded tumor tissues (colon carcinomas n = 15, colon adenomas n = 15, transitional cell carcinomas of the urinary bladder n = 17, transitional cell papillomas of the urinary bladder n = 22) were investigated for point mutations of ras oncogenes using polymerase chain reaction (PCR) and nested-PCR followed by restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP), respectively. Results were proved by sequence analysis. Nested-PCR combined with SSCP analysis revealed five mutations of the Ki-ras oncogenes in five colon tumors. RFLP analysis revealed one point mutation in one sample of invasive transitional cell carcinomas. Using optimized PCR protocols, the methods described are applicable to paraffin- embedded tissues and offer a tool for mutation analysis in retrospective and prospective studies.

Base Sequence↗
PubMed1999

Catalytic activation of the membrane distal domain of protein tyrosine phosphatase epsilon, but not CD45, by two point mutations.

Most, if not all, of the catalytic activity of the tandem catalytic domain-containing receptor-like protein tyrosine phosphatases (PTPs) resides in the membrane proximal domains (D1), with little to no activity associated with the membrane distal domains (D2). Two point mutations in the D2 domain of PTPalpha, which restore invariant amino acids found in the KNRY motif and WPD loop of all active D1 domains, synergistically confer D1-equivalent kinetic properties towards the phosphotyrosine analogue pNPP, and activate PTPalpha-D2 catalysed phosphopeptide hydrolysis (Lim et al., J. Biol. Chem. 273 (1998) 28986-28993; Buist et al., Biochemistry 38 (1999) 914-922). As all D2 domains lack these two D1-invariant amino acids, we have investigated whether other D2 domains are activated by such point mutations. Mutant PTPepsilon-D2, closely related to PTPalpha-D2 and belonging to a subgroup of D2 domains with minimal and conservative substitutions of D1-invariant amino acids, exhibits synergistic activation towards pNPP but not towards a phosphopeptide substrate. CD45-D2, belonging to another subgroup of D2 domains with considerable substitutions in D1-invariant amino acids, is not activated by these mutations, even in the context of a third mutation which restores the minimal essential active site sequence C(X(5))R, indicating that additional defects are sufficient to preclude catalysis. The ability of the KNRY and WPD replacements to activate PTPepsilon-D2 and PTPalpha-D2, but not CD45-D2, in conjunction with the extent and nature of their wild-type amino acid substitutions, suggests that these D2 domains are representative of two functionally distinct groups of D2 domain.

Amino Acid Sequence↗
PubMed1999

Rapid screening of the LDL receptor point mutation FH-Genoa/Palermo. Mutation in brief no. 238. Online.

The LDL-receptor gene point mutation FH-Genoa/Palermo is the most frequent mutation responsible for Familial Hypercholesterolemia in Sicily. The mutation does not introduce or abolish any useful restriction site. We establish a GeneComb-based strategy to identify this mutation in a population of Sicilian unrelated clinically diagnosed FH probands. The method was very sensitive and specific; 12 out of 90 (13.3%) unrelated FH probands were found to carry the FH-Genoa/Palermo mutation. According to these results, the FH-Genoa/Palermo is the more frequent LDL-receptor gene mutation among the Sicilian FH patients. Moreover FH-Genoa/Palermo is the mutation cluster to date more represented in Southern Italy.

Genetic Testing↗
PubMed1995

Three unrelated Gaucher's disease patients with three novel point mutations in the glucocerebrosidase gene (P266R, D315H and A318D).

Three novel point mutations were detected in the glucocerebrosidase gene of three unrelated Gaucher's disease patients by direct sequencing of PCR products. The first is a C to G change at position 4263 in the genomic sequence (exon 7) which results in a proline to arginine change at position 266 in the mature enzyme (P266R). The second is a G to C change at position 5276 in the genomic sequence (exon 8) which results in an aspartic acid to histidine change at position 315 (D315H). The third is a C to A change at position 5286 in the genomic sequence (exon 8) which results in an alanine to aspartic acid change at position 318 (A318D). The first mutation destroys an AvaII restriction endonuclease site, the second creates a BspMI site and the third creates a BamH I site.

Adult↗
PubMed1993

Detection of point mutations in DNA using capillary electrophoresis in a polymer network.

The use of capillary electrophoresis (CE) in a polymer network for single-strand conformation polymorphism (SSCP) is investigated. SSCP is a method to detect DNA point mutations, essential in the diagnosis of several diseases. The PCR (polymerase chain reaction) amplified p53 gene, a tumour suppressor gene known to be frequently mutated in malignant cells, was subjected to CE analysis. Two single-strand DNA fragments of 372 bp in length differing in only one nucleotide could be separated. We conclude that SSCP using CE in a polymer network is a powerful method for the detection of point mutations in DNA sequences.

Base Sequence↗
PubMed1999

Two point mutations in the U3 region of the long terminal repeat convert a subgroup A transformation-defective rous sarcoma virus to a cytopathic virus.

To elucidate the mechanism of cytopathicity of the transformation-defective avian retrovirus tdPH2010, we examined the function of two point mutations we had previously found in the long terminal repeat U3 region of this virus. Our previous studies showed that the U3 region was responsible for the cytopathic effects. These mutations were a G-to-T mutation at position -126 from the transcription start site and a G-to-A mutation at -23. Site-directed mutagenesis was performed on a noncytopathic, wild-type virus BSU to alter the nucleotides at these two positions, one at a time, to those of tdPH2010. Cell growth assay using the altered viruses revealed that host cell growth was retarded only when both of these mutations were present. The two additional mutations previously found in the direct repeat 1-polypurine tract (DR1-PPT) region of tdPH2010 were present also in the noncytopathic strain tdPH2013. Site-directed mutagenesis confirmed that these two mutations had indeed no role in the cytopathic effect of tdPH2010. None of these mutations influenced virus production from the infected cells. We conclude that the cytopathic effects by tdPH2010 are ascribed to the two point mutations in the U3 region.

Animals↗
PubMed1995

[An autopsy-verified case of Creutzfeldt-Jakob disease with codon 129 polymorphism and codon 180 point mutation].

An autopsied case of Creutzfeldt-Jakob disease is reported. A 79-year-old Japanese female showed extrapyramidal sign (resting tremor, and rigidity) and dementia. She developed myoclonus and became akinetic within one year from the onset, and then died of pneumonia at age of 81. None of the members of her family had neuromuscular disorders. CT and MRI studies revealed progressive brain atrophy. Consecutive study of EEG did not reveal periodic synchronous discharges (PSD). Codon 129 polymorphism (Met/Val) and codon 180 point mutation (Val/Ile) were detected. The autopsy revealed spongiform change of cerebral cortex and negative Kuru plaques, confirming the diagnosis of Creutzfeldt-Jakob disease. Immunohistochemical study revealed weak synaptic prion staining. Western blot analysis showed positive Proteinase K resistant prion protein. Gene analysis of autopsied brain showed the same prion DNA polymorphism and mutation. The combination of codon 129 polymorphism and 180 point mutation might associate with an atypical clinical form of CJD, which shows the extrapyramidal signs at the onset, and negative PSD in EEG.

Aged↗
PubMed2000

Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants.

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.

Animals↗
PubMed2000

Absence of point mutations at codon 17 of the mdm2 gene (serine 17) in human primary tumors.

Mdm2 is a phosphoprotein that interacts with protein p53, inhibiting its activity. A serine located in position 17 of Mdm2, has been implicated in its phosphorylation process. We hypothesize that point mutations at serine 17 could block its phosphorylation and thereby increase the p53-Mdm2 interaction. This mechanism could increase the p53 degradation and cause a loss of the protective effect of p53 against tumorigenesis. This hypothesis was based on recent studies in vitro, demonstrating that when serine 17 is mutated, the DNA-dependent protein kinase, activated by genomic damage, is unable to phosphorylate it. Thus, we investigated whether structural point mutations at exon 3 of the Mdm2 gene, affecting codon 17, were present in 162 human primary tumors, 70 breast carcinomas, 14 bladder tumors, 18 colon adenocarcinomas and 60 testicular tumors. Direct sequencing of a fragment (204 bp) of exon 3 of the Mdm2 gene that contains the codon 17 showed no mutations at this position, independently of the presence or absence of p53 gene mutations in the same tumors. These results do not support the hypothesis that mutations in the Mdm2 gene at this level are involved in the tumorigenic process of human cancers.

Codon↗
PubMed2002

Loss of heterozygosity in parathyroid glands of familial hypercalcemia with hypercalciuria and point mutation in calcium receptor.

Development of sporadic parathyroid tumors is accompanied by loss of heterozygosity (LOH) on several chromosomes like 1p, 1q, 6q, 11q, and 15q. Here, we investigate a unique variant of familial hypercalcemia, unrelated to multiple endocrine neoplasia and hyperparathyroidism-jaw tumor syndromes, with hypercalcemia due to a point mutation in the intracellular part of the calcium receptor (CaR) gene. The hypercalcemia and hypercalciuria of the family is accompanied by age-related growth of the parathyroid glands and transition from diffuse to nodular parathyroid hyperplasia. Genome-wide screening for allelic loss was performed on nine enlarged parathyroid glands (weighing 40-680 mg) from eight parathyroidectomized members of the family (aged 22-66 yr). Using 139 fluorescent- or (32)P-labeled microsatellite markers, informative results were obtained on all examined chromosome arms and 1p, 1q, 6q, 11q, and 15q were investigated more closely. All parathyroid glands displayed allelic loss on at least one chromosomal arm (range 1-7). Most of the common loci for allelic loss corresponded to findings in sporadic parathyroid tumors, but the unique variant of familial hypercalcemia also exhibited frequent LOH on 12q (67%) and 7q (44%). LOH could not be detected at 3q, where the CaR gene is located, and additional somatic mutations in exons 2-7 of the CaR gene was not found by sequencing. The point mutation resulting in alteration of the intracellular portion of CaR seems to cause sensitivity to secondary genetic hits, with increased frequency of allelic loss (P < 0.01, r(2) = 0.66) and weight of parathyroid tumors with age in this family.

Adult↗
PubMed2000

Deletion mutagenesis downstream of the 5' long terminal repeat of human immunodeficiency virus type 1 is compensated for by point mutations in both the U5 region and gag gene.

We have studied the role of an RNA region at nucleotides (nt) +200 to +233, just downstream of the 5' long terminal repeat, in encapsidation of human immunodeficiency virus type 1 genomic RNA. Three deletion mutations, namely, BH-D0, BH-D1, and BH-D2, were generated to eliminate sequences at positions nt +200 to +219, +200 to +226, and +200 to +233. The result in each case was decreased levels of packaging of viral RNA into the mutated viruses, with the BH-D2 virus being the most severely affected. Consistently, all three deletions resulted in impaired viral infectiousness and the BH-D2 mutation showed the most dramatic impact in this regard. Further analysis revealed additional defects in Gag precursor processing and in the extension efficiency of the tRNA(3)(Lys) primer in reverse transcription reactions performed with these mutated viruses. To shed further light on the function of these deleted sequences in viral replication, the mutated viruses were cultured in MT-2 cells over prolonged periods to enable them to reacquire wild-type replication kinetics. Sequencing of the reverted viruses revealed point mutations in both the noncoding region and the gag gene. In the case of the BH-D0 revertant, two mutations were observed at positions G112A in the U5 region, termed M1, and T24I in the nucleocapsid protein, termed MNC, respectively. Either of these two mutations was able to confer wild-type replication capacity on BH-D0. In the case of BH-D1, each of the M1 mutations, a mutation termed M2, i.e., C227T, just downstream of the primer binding site, a mutation termed MP2 (T12I) in the p2 protein, and the MNC mutation were observed. A combination of either M1 and M2 or MP2 and MNC was able to rescue BH-D1. In the case of the BH-D2 deletion-containing viruses, three point mutations, i.e., M1, MP2, and MNC, were observed and the presence of all three was required to restore viral replication to wild-type levels.

Animals↗
PubMed1992

[Oncogene expression and point mutation in human pancreatic adenocarcinomas].

By nucleic acid hybridization and PCR techniques, oncogene expression in normal human pancreatic tissue as well as 4 human pancreatic adenocarcinoma cell lines were studied. Additionally, c-Ki-ras gene point mutation were also detected in the paraffin embedded sections collected from 34 cases of surgical specimens resected due to pancreatic adenocarcinoma. The results showed that none of the 5 oncogenes described was expressed in the normal human pancreatic tissue, whereas, all the human pancreatic adenocarcinoma cell lines expressed c-myc and c-Ki-ras oncogenes. 28 out of 34 cases (82.4%) of human pancreatic adenocarcinomas had c-Ki-ras gene codon 12 point mutation.

Adenocarcinoma↗
PubMed1998

Impairment of tRNA processing by point mutations in mitochondrial tRNA(Leu)(UUR) associated with mitochondrial diseases.

Several point mutations in mitochondrial tRNA genes have been linked to distinct clinical subgroups of mitochondrial diseases. A particularly large number of different mutations is found in the tRNA(Leu)(UUR) gene. We show that base substitutions at nucleotide position 3256, 3260, and 3271 of the mitochondrial genome, located in the D and anticodon stem of this tRNA, and mutation 3243 changing a base involved in a tertiary interaction, significantly impair the processing of the tRNA precursor in vitro. In correlation with other studies, our results suggest that inefficient processing of certain mutant variants of mitochondrial tRNA(Leu)(UUR) is a primary molecular impairment leading to mitochondrial dysfunction and consequently to disease.

Anticodon↗
PubMedDate not supplied

Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor.

Peptide nucleic acids (PNAs) containing an insert of three chiral monomers based on D-lysine ('chiral box') were synthesized and used as probes in Biospecific Interaction Analysis (BIA) for the recognition of DNA containing the W1282X point mutation of the cystic fibrosis gene. Hybridization experiments carried out in solution showed enhanced mismatch recognition when compared with the analogous achiral PNAs and oligonucleotides. The signal intensity was lower, but the selectivity of the Biacore response was found to be much higher than that observed with achiral PNAs. The newly designed chiral PNA probes were also found to hybridize with a 1:1 mixture of normal (N-W1282X) and mutated (M-W1282X) DNA oligomers immobilized on the biosensor, thus allowing discrimination not only between a normal and a mutated sequence (healthy/homozygous), but also between homo- and heterozygous individuals. These results suggest that 'chiral box' PNAs are potential powerful tools for the analysis of single point mutations of biological/biomedical relevance.

Cystic Fibrosis↗
PubMed1992

Trapped-oligonucleotide nucleotide incorporation (TONI) assay, a simple method for screening point mutations.

We present a simple screening method for detecting a known point mutation, using only one 5'-biotinylated oligonucleotide primer, with its 3' end adjacent to the mutation site. In parallel reactions, an amplified DNA template encompassing the biotinylated oligonucleotide and mutation site undergoes 40 step-cycles of single nucleotide incorporation using Taq thermostable DNA polymerase and only one radioactive [alpha-32P]dNTP, specified by either the normal or mutant sequence. The oligonucleotides, now radioactively labelled at the 3' end according to the template sequence, are then trapped by streptavidin-coated magnetic beads, and the percent of radiolabel incorporated is determined directly by the Cerenkov method in a scintillation counter. The trapped-oligonucleotide nucleotide incorporation (TONI) assay has been used for the screening of a mitochondrial polymorphism, and has also been shown to distinguish the genotypes of hemoglobin A/C, A/A, A/S, and S/S. It is reproducible over at least a 100-fold range of radioisotope and a 10-fold range of oligonucleotide primer. This method is particularly useful for diagnosing mutations which do not produce alterations detectable by restriction enzyme analysis, since optimization of conditions is rarely necessary. In addition, it requires only a single oligonucleotide, and no electrophoretic separation of the allele-specific products. It thus represents an improved and simplified modification of the existing allele-specific primer extension methods (Kuppuswamy et al., Proc Natl Acad Sci USA 88:1143-1147, 1991; Sokolov, Nucl Acids Res 18:3671, 1989; Syvanen et al., Genomics 8:684-692, 1990).

Base Sequence↗
PubMed2005

Specific point mutations may not accumulate with aging in the mouse mitochondrial DNA control region.

Increasing evidence suggests that mitochondrial function declines during aging in various tissues and in a wide range of organisms. This correlates with an age-dependent large accumulation of specific point mutations in the mtDNA control region that was reported recently in human fibroblast and skeletal muscle. However, evaluations of aging-related mtDNA mutations in other model animal systems. In this study, we analyzed mtDNA control regions of brain, skeletal muscle, heart, and other tissues from aged mice, in search of specific point mutations. A 948-bp fragment covering the entire mtDNA control region from various tissues of mice at the age of 25-26 months was sequenced. The sequence analysis was accomplished with a newly developed program Mutation Quantifier, which was able to accurately detect mutations with frequencies as low as 3%. Probably due to the relative shorter life-span, unlike what has been reported in human mtDNA, our results indicated there might be no significant accumulation of specific mutations in mouse mtDNA control region during aging.

Aging↗
PubMed2005

Comparison of amplification refractory mutation system and polymerase chain reaction-restriction fragment length polymorphism techniques used for the investigation of MEFV gene exon 10 point mutations in familial Mediterranean fever patients living in Cukurova region (Turkey).

Familial Mediterranean fever (FMF) is an autosomal recessive inherited disease characterized by recurrent fever, serositis and arthritis. The disease is highly prevalent in Mediterranean basin populations. Recently, the gene responsible for FMF (MEFV) was cloned and at least 40 MEFV gene mutations have been identified. The most frequently observed mutations in the MEFV gene are M694V, M694I, M680I, and V726A. These occur within exon 10 of the gene, and account for 85% of the known MEFV alleles. In this study, the reliability and economical aspects of amplification refractory mutation system (ARMS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques were compared for analyzing the frequencies of the major point mutations of 90 unrelated patients with FMF from the Cukurova region in Turkey. Both techniques yielded similar results: The ratio of independent alleles of 90 patients carrying one of the tested mutations was 81.1%; patients consisted of 12 different genotypes. In 64 of 90 patients (71.1%) mutations were observed in both alleles. Thirty-six patients (40%) were homozygous for the same mutation, 28 (31.1%) were heterozygous for different mutations. Eighteen patients (20%) were heterozygous for one allele with one of the four mutations but the other allele was unknown. In 8 patients (8.8%) no mutation could be detected. The most frequently observed mutation was M694V (51.66%), followed by M680I (17.22%), V726A (10.55%), and M694I (1.66%). In conclusion ARMS and PCR-RFLP techniques were equally reliable to detect the mutations in Turkish FMF patients. However, the ARMS technique was found to be more rapid and economical than the PCR-RFLP techniques.

Exons↗