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At least 577 records · Page 32Linked to original sources
PubMed2006

Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF).

OBJECTIVES: YhiV (MdtF) is an resistance nodulation division (RND) type efflux pump in Escherichia coli with significant homology to AcrB but usually expressed at a low level in clinical isolates. When overexpressed the pump confers decreased susceptibility to a variety of substances including erythromycin and ethidium bromide (EtBr). We characterized two mutants of E. coli E12 (DeltaacrB DeltaacrF) overexpressing yhiV that showed surprising differences in their spectrum of multidrug resistance (MDR). METHODS: The two mutants obtained after repeated exposure of E. coli E12 to levofloxacin were tested for antimicrobial susceptibility to a variety of agents and for intracellular accumulation of selected pump substrates. Gene expression was studied by quantitative RT-PCR, and yhiV was sequenced. Gene inactivation and replacement were done by phage lambda-based homologous recombination. RESULTS: Mutant DKO20/1 overexpressed yhiV, showed a wild-type yhiV sequence and had >2-fold increased MICs of fluoroquinolones, novobiocin, macrolides/ketolides, EtBr, oxacillin and Phe-Arg-beta-naphthylamide (PAbetaN, a putative efflux pump inhibitor) compared with the E12 parent. A second mutant, strain DKO1/17 that had the Val-610-->Phe point mutation in YhiV differed from DKO20/1 by faster growth, >2-fold increased MICs of linezolid and tetracycline, but >2-fold decreased MICs of PAbetaN, azithromycin and telithromycin. Inactivation of yhiV in DKO1/17 and reintroduction of the wild-type and mutant yhiV sequence confirmed that the differing MICs of most of the drugs were associated with the observed single point mutation. Intracellular drug accumulation studies with linezolid and PAbetaN were consistent with the MIC results. CONCLUSIONS: The region around amino acid Val-610 in YhiV appears to be involved in determining recognition and efficiency of export of a number of MDR efflux pump substrates. This single point mutation in the periplasmic loop of the pump can increase resistance to a given drug such as a fluoroquinolone while decreasing resistance to another one.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Malignant fibrous histiocytomas and H-ras-1 oncogene point mutations.

AIMS: To investigate the types and the frequencies of H-ras-1 gene mutations in malignant fibrous histiocytomas. METHODS: Thirty five samples of malignant fibrous histiocytoma tissue were searched for point mutations within "hot spot" codons 12 and 13 of the H-ras-1 oncogene by the specific "nested" polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) and a direct cycle sequencing procedure. RESULTS: In contrast to previous reports, none of the tumours contained a point mutation or any other changes within or around the hot spot gene sequences. CONCLUSIONS: These data indicate that H-ras-1 oncogenic activation is not required in the molecular pathway of malignant fibrous histiocytoma formation and cannot be used as a discriminating factor for diagnostic sarcoma typing.

Exons↗
PubMed2002

Detection of K-ras point mutation at codon 12 in pancreatic diseases: a study in a Brazilian casuistic.

OBJECTIVE: To clarify the sensitivity and the validity of K-ras point mutational analysis at codon 12 in Brazilian patients with pancreatic diseases, and the possible correlation between the presence of the mutation and the histopathological findings. PATIENTS: Ninety-seven Brazilian patients with pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumors and chronic pancreatitis were enrolled in this study. Forty-five patients (46%) were female and 52 patients (54%) were male, having an average age of 60.2+/-9.2 years for adenocarcinoma (n=52), 45.1+/-19.4 years for pancreatic neuroendocrine tumors (n=20), and 46.4+/-11.2 years for chronic pancreatitis (n=25). DNA extracted from 11 normal human peripheric lymphocytes was utilized as a control. RESULTS: The sensitivity of K-ras mutational analysis was 83.3% (25/30) in paraffin-embedded samples and 72.7% (16/22) in surgically resected specimens of the malignancy. On the other hand, no mutations were found in pancreatic neuroendocrine tumors or in chronic pancreatitis. Regarding the histopathological grading, the higher positivity rate was found in poorly-differentiated adenocarcinoma (100%), and progressively decreased in moderately-differentiated adenocarcinoma (72.2%), and well-differentiated adenocarcinoma (66.6%). The positivity rate in non-classified adenocarcinoma was 81.8%. CONCLUSION: K-ras point mutation, in our study, is notably prevalent in malignancies and is absent in chronic pancreatitis and pancreatic neuroendocrine tumors. These results encourage us to consider the possibility of treatment strategies for this oncogene in the future.

Adenocarcinoma↗
PubMed2004

Identification of a new point mutation in hypoxanthine phosphoribosyl transferase responsible for hyperuricemia in a female patient.

A 29-year-old woman was referred to our department because of gout. Routine laboratory data showed hyperuricemia, a high level of plasma oxypurines, increased urinary uric acid excretion, and increased urinary oxypurine excretion, with decreased hypoxanthine phosphoribosyl transferase (HPRT) activity in the erythrocytes. From these findings, the patient was diagnosed with a partial deficiency of HPRT. To determine its properties, a cDNA sequence encoding HPRT and the androgen receptor AR XIST minimal promoter gene, as well as methylation of the AR gene were investigated. The HPRT cDNA sequence revealed a point mutation of G to A in nucleotide 40, which changed codon 14 from GAA (Glu) to AAA (Lys) in the mutant gene. In addition, the HPRT genomic DNA sequence, including the mutation site, revealed the same point mutation, indicating that the patient was heterozygote. Further analysis of the AR gene on the X chromosome suggested nonrandom X-chromosome inactivation, whereas the AR XIST minimal promoter gene was normal. Such results have not been previously reported in a female with partial HPRT deficiency.

Adenosine↗
PubMed1992

Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR.

About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.

Base Sequence↗
PubMed2002

PoPMuSiC, rationally designing point mutations in protein structures.

PoPMuSiC is an efficient tool for rational computer-aided design of single-site mutations in proteins and peptides. Two types of queries can be submitted. The first option allows to estimate the changes in folding free energy for specific point mutations given by the user. In the second option, all possible point mutations in a given protein or protein region are performed and the most stabilizing or destabilizing mutations, or the neutral mutations with respect to thermodynamic stability, are selected. For each sequence position or secondary structure the deviation from the most stable sequence is moreover evaluated, which helps to identify the most suitable sites for the introduction of mutations.

Computer-Aided Design↗
PubMed1998

The first pathogenic mitochondrial methionine tRNA point mutation is discovered in splenic lymphoma.

We describe a novel point mutation in the mitochondrial DNA transfer RNA methionine gene, a G-to-A transition at position 4450, in a patient with a splenic lymphoma with villous lymphocytes. The patient's lymphocytes were remarkable by the presence of large cytoplasmic inclusions demonstrated as abnormal mitochondria by electron microscopy and led to the discovery of the mutation using denaturing gradient gel electrophoresis as a screening procedure. The pathogenic potential of the mutation was clearly established by the following criteria. It was absent in a control population. It involves a nucleotide that is highly conserved along the phylogenetic tree. The mutation was heteroplasmic and, when present in a high proportion, was associated with morphological alterations of the mitochondria, with defects of respiratory chain complexes activities and with a decrease in the mitochondrially encoded cytochrome c oxidase subunit II. Transfer of the mutation in Rho0 cells allowed to demonstrate its association with a severe respiratory chain dysfunction. However, although the pathogenicity of the mutation was clearly demonstrated, its link with the patient disease remained disputable.

Aged↗
PubMed2002

Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene.

We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT-->GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10(7) normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10(10) normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.

Animals↗
PubMed1998

Neuroradiological features of six kindreds with MELAS tRNA(Leu) A2343G point mutation: implications for pathogenesis.

OBJECTIVE: To determine the neuroradiological abnormalities associated with subjects carrying the mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) tRNA(Leu)(UUR) A3243G point mutation METHODS: Mitochondrial genetic analysis was performed on 24 subjects from six kindreds with the MELAS tRNA(Leu)(UUR) A3243G point mutation. Cerebral CT and MRI were performed on 24 patients and 15 patients respectively. Previous neuroradiological investigations including cerebral CT from four deceased members of the families were also reviewed. Histological examination of postmortem specimens of two patients within the kindreds was performed. RESULTS: The commonest radiological finding was basal ganglia calcification. Other abnormalities included focal lesions and cerebellar and cerebral atrophy. Basal ganglia calcification was progressive, symmetric, and asymptomatic. Histologically, basal ganglia calcification in one patient was found to be in the pericapillary regions of the globus pallidus, with no neuronal involvement. Focal lesions most commonly involved the grey matter of the parietal and occipital lobes and cerebellum. Histopathological examination suggested that these were due to cellular rather than vascular dysfunction. Enlargement of the fourth ventricle was the first sign of cerebellar atrophy. Cerebral and cerebellar atrophy were only present with severe disease. CONCLUSIONS: These radiological findings, when considered in the context of the clinical and pathological findings, seem to reflect two major disease processes: an intermittent abrupt loss of function associated with cell injury from which there is at least partial recovery and a slowly progressive degenerative process causing basal ganglia calcification, and cerebral and cerebellar atrophy. The clinical and radiological features resulting from these processes are distinctive and provide insight into the consequences of mitochondrial dysfunction on the brain.

Adolescent↗
PubMed2003

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.

Amino Acid Sequence↗
PubMed2024

The Genetic Determinants and Genomic Consequences of Non-Leukemogenic Somatic Point Mutations.

Clonal hematopoiesis (CH) is defined by the expansion of a lineage of genetically identical cells in blood. Genetic lesions that confer a fitness advantage, such as point mutations or mosaic chromosomal alterations (mCAs) in genes associated with hematologic malignancy, are frequent mediators of CH. However, recent analyses of both single cell-derived colonies of hematopoietic cells and population sequencing cohorts have revealed CH frequently occurs in the absence of known driver genetic lesions. To characterize CH without known driver genetic lesions, we used 51,399 deeply sequenced whole genomes from the NHLBI TOPMed sequencing initiative to perform simultaneous germline and somatic mutation analyses among individuals without leukemogenic point mutations (LPM), which we term CH-LPMneg. We quantified CH by estimating the total mutation burden. Because estimating somatic mutation burden without a paired-tissue sample is challenging, we developed a novel statistical method, the Genomic and Epigenomic informed Mutation (GEM) rate, that uses external genomic and epigenomic data sources to distinguish artifactual signals from true somatic mutations. We performed a genome-wide association study of GEM to discover the germline determinants of CH-LPMneg. After fine-mapping and variant-to-gene analyses, we identified seven genes associated with CH-LPMneg (TCL1A, TERT, SMC4, NRIP1, PRDM16, MSRA, SCARB1), and one locus associated with a sex-associated mutation pathway (SRGAP2C). We performed a secondary analysis excluding individuals with mCAs, finding that the genetic architecture was largely unaffected by their inclusion. Functional analyses of SMC4 and NRIP1 implicated altered HSC self-renewal and proliferation as the primary mediator of mutation burden in blood. We then performed comprehensive multi-tissue transcriptomic analyses, finding that the expression levels of 404 genes are associated with GEM. Finally, we performed phenotypic association meta-analyses across four cohorts, finding that GEM is associated with increased white blood cell count and increased risk for incident peripheral artery disease, but is not significantly associated with incident stroke or coronary disease events. Overall, we develop GEM for quantifying mutation burden from WGS without a paired-tissue sample and use GEM to discover the genetic, genomic, and phenotypic correlates of CH-LPMneg.

Journal Article↗
PubMed1995

Sequence-based analysis of properdin deficiency: identification of point mutations in two phenotypic forms of an X-linked immunodeficiency.

Properdin deficiency is an inherited X-linked disorder causing increased susceptibility to meningococcal disease. Here, underlying genetic defects in the properdin gene were identified for the first time. Samples from individuals with type I deficiency, defined as complete absence of properdin in serum, and individuals with type II deficiency, characterized by low concentrations of properdin in serum, were analyzed by direct chromosome sequencing of overlapping PCR products. The complete gene, including 10 exons and 9 introns, covering 6460 bases of the region Xp11, was investigated by direct solid-phase sequencing. In the related individuals with type I deficiency a C to T mutation in exon 5 was identified, which gives rise to a stop codon TGA and thus a truncated gene product. In addition, point mutations were found in 4 introns and a silent mutation in exon 10. In the properdin gene from related individuals with type II deficiency two point mutations were found, one in intron 3 and one in exon 4. The latter mutation yields a substitution of arginine to tryptophan, which may affect folding, secretion, and/or turnover of the protein. The genetic and biochemical implications of these mutations are discussed.

Base Sequence↗
PubMed1989

Detection of ras point mutations by polymerase chain reaction using mutation-specific, inosine-containing oligonucleotide primers.

The use of DNA primers with 3'-ends complementary to specific genetic point mutations allowed for the rapid detection of such mutations in genomic DNA by polymerase chain reaction. The sensitivity of this approach was such that mutations could be detected in DNA samples mixed with a 10(7)-fold excess of normal non-mutated DNA. To increase the practicality of this approach for the detection of point mutations affecting all 3 of the known ras oncogenes we synthesized mutation-specific primers complementary to all 3 genes by substituting inosine residues at positions corresponding to ambiguous bases on the genes.

Base Sequence↗
PubMed1991

Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.

Previously we demonstrated that transgenic mice expressing mutant basal epidermal keratin genes exhibited a phenotype resembling a group of autosomal dominant human skin disorders known as epidermolysis bullosa simplex (EBS). EBS diseases affect approximately 1: 50,000 and are of unknown etiology, although all subtypes exhibit blistering arising from basal cell cytolysis. We now demonstrate that two patients with spontaneous cases of Dowling-Meara EBS have point mutations in a critical region in one (K14) of two basal keratin genes. To demonstrate function, we engineered one of these point mutations in a cloned human K14 cDNA, and showed that a K14 with an Arg-125----Cys mutation disrupted keratin network formation in transfected keratinocytes and perturbed filament assembly in vitro. Since we had previously shown that keratin network perturbation is an essential component of EBS diseases, these data suggest that the basis for the phenotype in this patient resides in this point mutation.

Amino Acid Sequence↗
PubMed2004

BRAF point mutations in primary melanoma show different prevalences by subtype.

To elucidate the biological significance of activating mutations of BRAF in human malignant tumors, we performed a mutation analysis using 43 cell lines established from tumors that had developed in several kinds of human organs. Because the same V599E point mutation was observed in three of six melanoma cell lines and no such mutations were observed in other types of cancers, we focused further on melanoma, performed mutation analyses of NRAS, KRAS, CTNNB1, and p16/p14(ARF) in these cell lines, and found one NRAS mutation and three p16/p14(ARF) mutations. We further searched for mutations of BRAF and NRAS in 35 primary sporadic melanomas from 35 Japanese patients and detected the V599E BRAF point mutation in only nine (26%) of them. Significant differences in mutation frequency were observed among four histological subtypes; four (50%) of eight superficially spreading melanoma and five (33%) of 15 acral lentiginous melanoma had the mutation, whereas none of 12 other types (six nodular melanoma, five lentigo melanoma, and one mucosal melanoma) had it. The BRAF mutation was observed frequently even in small lesions, indicating that activation of this gene may be one of the early events in the pathogenesis of some melanomas.

Base Sequence↗
PubMed1989

Increased expression of ras genes in non-Hodgkin's lymphomas is not associated with oncogenic activation of those genes by point mutation.

Twenty-three cases of non-Hodgkin's lymphoma (NHL) were analyzed for expression of ras genes by in situ hybridization utilizing biotinylated DNA probes. Increased expression of Ki-ras, Ha-ras and N-ras genes was observed in 12 cases, 6 cases and 1 case of NHL, respectively. Genomic DNA extracted from these 23 cases of NHL was region-specifically amplified by means of polymerase chain reaction to examine the presence of point mutations at the 12th, 13th and 61st codons of Ki-, Ha- and N-ras genes. Dot hybridization assays with appropriate oligonucleotide probes showed no evidence of point mutation in any case of NHL examined. These results indicate that increased expression of ras genes in NHL is not associated with ras gene activation by point mutation.

Base Sequence↗
PubMed1998

Two missense point mutations in different alleles in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene produce 3-hydroxy-3-methylglutaric aciduria in a French patient.

Two novel point mutations in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene were found in a French patient with double heterozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by reverse transcriptase-polymerase chain reaction of the mRNA using five different pairs of oligonucleotides produced no differences in the fragments amplified with respect to the control. Single-strand conformation polymorphism analysis showed that only one amplified fragment was different in the patient vs. control. Sequencing of the amplified fragments showed two missense point mutations, A698G and T788C, each of them mixed with the wild-type sequence. These mutations produced the changes H233R and L263P, leading to changes in the enzyme activity, which was largely abolished. The father and one brother of the proband were heterozygous for the L263P mutation and the mother and one daughter were heterozygous for the H233R mutation, which confirms the double-heterozygous character of the patient. Another sibling was free of the mutations. An enzymatic restriction analysis has been proposed to screen the occurrence of these two mutations in future patients.

Alleles↗
PubMed1996

Fast capillary electrophoresis-laser induced fluorescence analysis of ligase chain reaction products: human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy.

High speed capillary electrophoresis-laser-induced fluorescence (CE-LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA-ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lacI gene fragment inserted in a Bluescript II phagemid. LCR-CE-LIF was then applied to detect point mutations in human mitochondrial DNA, resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatable.

Base Sequence↗