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PubMed1996

Auditory findings in patients with maternally inherited diabetes and deafness harboring a point mutation in the mitochondrial transfer RNA(Leu) (UUR) gene.

Five patients with sensorineural hearing loss, who harbored a point mutation in the mitochondrial transfer RNA (tRNA) gene tRNA(Leu) (UUR), from five unrelated family pedigrees were examined. In these families diabetes and deafness were maternally inherited. Bilateral hearing was more severely impaired at higher frequencies. Audiometric test results revealed that hearing loss involved the cochlea. Hearing gradually deteriorated; the progression rate ranged from 1.5 to 7.9 dB per year. Proportion of mutant mitochondrial DNAs (mtDNAs) in the leukocytes was not related to the rate or degree of hearing loss, although hearing loss appeared at a younger age in patients with higher heteroplasmy. We speculate that after the proportion of damaged mtDNAs, mostly as a result of mutation, exceeds the expression threshold for deficiencies in mitochondrial protein synthesis and oxygen consumption, a drop in adenosine triphosphate level could lead to an imbalance of ion concentration, resulting in cell death in the cochlea.

Adult↗
PubMed1989

Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase. Southern hybridization analysis of the alpha-galactosidase gene in affected hemizygous males from 130 unrelated families with Fabry disease revealed six with different gene rearrangements and one with an exonic point mutation resulting in the obliteration of an Msp I restriction site. Five partial gene deletions were detected ranging in size from 0.4 to greater than 5.5 kb. Four of these deletions had breakpoints in intron 2, a region in the gene containing multiple Alu repeat sequences. A sixth genomic rearrangement was identified in which a region of about 8 kb, containing exons 2 through 6, was duplicated by a homologous, but unequal crossover event. The Msp I site obliteration, which mapped to exon 7, was detected in an affected hemizygote who had residual enzyme activity. Genomic amplification by the polymerase chain reaction and sequencing revealed that the obliteration resulted from a C to T transition at nucleotide 1066 in the coding sequence. This point mutation, the first identified in Fabry disease, resulted in an arginine356 to tryptophan356 substitution which altered the enzyme's kinetic and stability properties. The detection of these abnormalities provided for the precise identification of Fabry heterozygotes, thereby permitting molecular pedigree analysis in these families which revealed paternity exclusions and the first documented new mutations in this disease.

Blotting, Southern↗
PubMed1990

Tissue-specific transformation by epidermal growth factor receptor: a single point mutation within the ATP-binding pocket of the erbB product increases its intrinsic kinase activity and activates its sarcomagenic potential.

Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal, ligand-binding domain by retroviral insertion. The insertionally activated transcripts encode protein products that have constitutive tyrosine kinase activity and that can induce erythro-leukemia but not sarcomas. We have found that a single point mutation within the ATP-binding pocket of the tyrosine kinase domain in this truncated molecule can increase the ability of this oncogene to induce anchorage-independent growth of fibroblasts in vitro and fibrosarcoma formation in vivo. Associated with this increased transforming potential is a corresponding increase in the kinase activity of the mutant erbB protein product. The mutation, which converts a valine to isoleucine at position 157 of the insertionally activated c-erbB product, is at a residue that is highly conserved within the protein kinase family. To our knowledge, this is the first demonstration of a point mutation in the ATP-binding pocket that activates a tyrosine kinase.

Adenosine Triphosphate↗
PubMed2000

[Point mutations of N-ras oncogene and abnormal expression of rasp21, p53 proteins in orbital rhabdomyosarcoma].

OBJECTIVE: The point mutations of N-ras oncogene and the abnormal expression of rasp21 and p53 proteins in orbital rhabdomyosarcoma (RMS) were analyzed in order to explore the role of oncogene mutations in the pathogenesis of orbital RMS and its relation to patients' prognosis. METHOD: 21 human orbital RMS tissues were analyzed by dot-blot hybridization and immunohistochemistry using synthetic specific oligonucleotide probes and mutant rasp21, p53 monoclonal antibodies. RESULTS: The second base mutation of codon 12 of the N-ras oncogene was found in 4 patients. The third base mutation of codon 61 was found in 5 patients. The mutant rate was 33.3%. By immunohistochemistry analysis, the overexpression of rasp21 and p53 protein level was found in 42.9% and 76.2% respectively. The prognosis of patients with overexpression of ras p21, p53 was worse than that of normal expression (P < 0.01). CONCLUSION: The mutants of ras and p53 oncogene play important roles in the pathogenesis of orbital RMS, and the abnormal expression of their protein products is related to patients' prognosis.

Adolescent↗
PubMed1993

Biliary papillomatosis with the point mutation of K-ras gene arising in congenital choledochal cyst.

Biliary papillomatosis is a rare entity. A case of biliary papillomatosis associated with congenital choledochal cyst and intrahepatic gallstones is reported here. Percutaneous transhepatic cholangioscopy revealed multiple papillary lesions of the right intrahepatic duct and the common bile duct. Microscopically, the papillary mucosal lesion showed papillary proliferations of bile duct epithelial cells with mild atypia. Furthermore, a point mutation at codon 12 of the K-ras oncogene was found in the papillary lesion. To the best of our knowledge, this is the first case of biliary papillomatosis arising in congenital choledochal cyst. Although the pathogenesis of biliary papillomatosis in our case was unclear, biliary irritation associated with choledochal cyst may be related to biliary papillomatosis with point mutation at codon 12 of K-ras gene.

Aged↗
PubMed1991

Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies.

To characterize the region on human IgG1 responsible for its high-affinity interaction with the human Fc gamma receptor class I (Fc gamma RI), we have analyzed the binding properties of a series of genetically engineered chimeric antidinitrophenyl antibodies with identical murine antibody combining sites and hybrid IgG1/IgG2 human constant (C) regions. In addition, we have investigated a panel of reciprocally point-mutated IgG1 and IgG2 chimeric antibodies to identify the amino acid residues that confer cytophilic properties to human IgG1. Our data unambiguously indicate that cytophilic activity of IgG1 is an intrinsic property of its heavy-chain C region 2 (CH2) domain. We report that the entire sequence spanning residues 234-237 (LLGG) is required to restore full binding activity to IgG2 and IgG4 and that individual amino acid substitutions failed to render IgG2 active. Nevertheless, the reciprocal single point mutations in IgG1 either significantly lowered its activity or abolished it completely. Finally, we observed that an IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was more active than wild-type IgG1. This finding suggests that in addition to the primary contact site identified in the N terminus of the gamma 1 CH2 domain, secondary sites involving residues from the C-terminal half of the domain may also contribute to the IgG1-Fc gamma RI interaction.

Amino Acid Sequence↗
PubMed2002

[The transcriptional regulation study on human delta globin gene with CAAT box C-->T point mutation in its promoter].

OBJECTIVE: To study the transcriptional regulation of human delta globin gene with C-->T point mutation at -64 in its promoter. METHODS: Human delta globin genes including wild CAAT box and mutant CAAT box (-64C-->T) were separately cloned into eukaryotic expression vector pcDNA3.1 (-)/Myc-His A which was cut out the strong promoter CMV, transfected MEL cells, and induced by DMSO to express. The transcriptional regulation of human delta globin gene was analysed using semi-quantitative RT-PCR. RESULTS: The expression level of human delta globin gene with mutant CAAT box was 2.2-fold as high as that with wild CAAT box. CONCLUSION: The defective CAAT box of human delta globin gene promoter region may be one of the major reasons for its low expression level.

CCAAT-Enhancer-Binding Proteins↗
PubMed1992

Characterization of linker insertion and point mutations in the NS-1 gene of minute virus of mice: effects on DNA replication and transcriptional activation functions of NS-1.

The NS-1 gene of minute virus of mice encodes a multifunctional protein required for replication of the viral genome and for transcriptional regulation of the two MVM promoters. To study the localization of activities required for DNA replication and transactivation of the capsid gene promoter, insertion and point mutations were introduced into the NS-1 gene. The mutant NS-1 genes were expressed in COS-7 cells by using an SV 40 promoter driven NS-1 expression vector. The ability of the mutant proteins to complement a replication defective NS-1 mutant of the infectious MVM plasmid pMM984 and to activate transcription from the capsid gene promoter in chloramphenicol acetyl transferase expression assays was determined. Two point mutations Ser-249 to Ala and Lys-250 to Gln and a one amino acid insertion between Asp-606 and Leu-607 had no effect on viral DNA replication and transactivation activities. Six independent insertions of between 2 and 12 amino acids inhibited the DNA replication activity of NS-1 between 20- and at least 100-fold. There was no apparent correlation between the extent of inhibition of parvoviral DNA replication and the location of the mutations. The transcriptional activation function of NS-1 was inhibited between 1.5- and at least 20-fold and was therefore overall relatively less sensitive to mutagenesis than was its DNA replication function. An exception to this was a 5 amino acid insertion between Tyr-543 and Gln-544 that abolished transactivation as well as the ability of NS-1 to complement viral DNA replication.

Amino Acid Sequence↗
PubMed1995

Robust nonradioactive oligonucleotide ligation assay to detect a common point mutation in the CYP2D6 gene causing abnormal drug metabolism.

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simultaneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.

Adenosine Triphosphate↗
PubMed1992

Application of a PCR-mismatch technique to the BCL-2 gene: detection of point mutations in BCL-2 genes of malignancies with A t(14,18).

The ability to conveniently detect single-base mutations in the DNA of clinical material without prior knowledge of the mutant sequence remains a diagnostic challenge. Most techniques suffer from a lack of general applicability to all DNA sequences, poor sensitivity, requirement for RNA samples rather than DNA, or necessity for performing DNA sequencing to uncover unknown point mutations. Recently, Montandon, et al. (8) described a novel method whereby segments of DNA amplified by the Polymerase Chain Reaction (PCR) can be rapidly screened for mutations through their formation of heteroduplexes with an end-labeled reference DNA followed by site-specific chemical cleavage at mispaired bases. Here we have applied this PCR-mismatch technique to a portion of the BCL-2 gene, using DNA samples derived from the biopsy specimens of patients with lymphomas or lymphocytic leukemias. The BCL-2 gene becomes activated through a t(14;18) chromosomal translocation in the majority of non-Hodgkin's lymphomas. Somatic point mutations were detected in the BCL-2 genes of 3 of 5 patient samples that contained a t(14;18). No mutations were observed for lymphomas lacking a t(14;18), nor in the DNA of over 20 normal persons. Further analysis excluded the possibility that the detected mutations represented hereditary polymorphisms or PCR-artifacts. Based on comparisons with direct DNA sequencing, the PCR-mismatch technique appeared to be both highly specific and sensitive. The possible mechanisms responsible for these somatic mutations in translocated BCL-2 genes and their functional significance are discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Sensitivity of anticancer drugs in Saos-2 cells transfected with mutant p53 varied with mutation point.

A human osteosarcoma cell line, Saos-2, which is devoid of endogenous p53 gene, and clones of Saos-2 cells, which were transfected with wild type p53 or mutant p53 genes (123A, 143A, 175H and 273H), were observed for their surviving fraction after treatment with the commonly used anticancer drugs, cisplatin (CDDP), nimustine (ACNU), adriamicin (ADR) and bleomicin (BLM). The transfectants of the mutant 143A, 175H and 273H were significantly more resistant to CDDP than the transfectant of pOPI3 (expression plasmid only). The transfectants of the wild type p53 and mutant 123A were significantly more sensitive to ACNU than the transfectant of pOPI3. The transfectant of mutant 123A was more sensitive to ADR than the transfectant of pOPI3. There was no significant difference in sensitivity to BLM between Saos-2 and all kinds of transfectants. Thus the sensitivity of the cells to anticancer drugs varied with the mutation point of the p53 gene.

Bleomycin↗
PubMed2001

A point mutation in a domain of gamma interferon receptor 1 provokes severe immunodeficiency.

Gamma interferon (IFN-gamma) and the cellular responses induced by it are essential for controlling mycobacterial infections. Most patients bearing an IFN-gamma receptor ligand-binding chain (IFN-gammaR1) deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome. In this report a new mutation that affects the IFN-gammaR1 ligand-binding domain in a Spanish patient with mycobacterial disseminated infection and multifocal osteomyelitis is characterized. The mutation generates an amino acid change that does not abrogate protein expression on the cellular surface but that severely impairs responses after the binding of IFN-gamma (CD64 and HLA class II induction and tumor necrosis factor alpha and interleukin-12 production). A patient's younger brother, who was also probably homozygous for the mutation, died from meningitis due to Mycobacterium bovis. These findings suggest that a point mutation may be fatal when it affects functionally important domains of the receptor and that the severity is not directly related to a lack of IFN-gamma receptor expression. Future research on these nontruncating mutations will make it possible to develop new therapeutical alternatives in this group of patients.

Base Sequence↗
PubMed2001

Restoration of function of carboxy-terminally truncated pseudorabies virus glycoprotein B by point mutations in the ectodomain.

Glycoprotein B (gB) of pseudorabies virus (PrV) is essential for virus entry into target cells and direct viral cell-to-cell spread. Recently, we described a carboxy-terminally truncated derivative of PrV gB, gB-007, which was inefficiently incorporated into virions, was unable to complement infectivity, but was fully capable of restoring direct viral cell-to-cell spread of gB-negative PrV (R. Nixdorf, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 74:7137-7145, 2000). Since recombinant PrV-007, which expresses gB-007 instead of wild-type gB, was able to spread directly from cell to cell, we attempted to obtain compensatory mutations leading to restoration of the entry defect by performing serial passages in cell culture. This procedure has previously been used to successfully restore entry defects in gD- or gL-deficient PrV mutants. From an initial titer of 100 PFU per ml in the supernatant, titers increased, reaching wild-type levels of up to 10(7) PFU after ca. 20 passages. One single-plaque isolate of the passaged mutant, designated PrV-007Pass, was further characterized. PrV-007Pass gB was efficiently incorporated into the viral envelope and restored infectivity to a gB-negative PrV mutant, PrV-gB(-). Interestingly, localization of PrV-007Pass gB in the plasma membrane was similar to that of PrV-007. In contrast, wild-type gB is mainly found in intracellular vesicles. Marker rescue experiments and trans-complementation assays demonstrated the presence of compensatory mutations within the gB gene of PrV-007Pass. DNA sequencing revealed two point mutations in the gB open reading frame of PrV-007Pass, resulting in amino acid substitutions at positions 305 and 744 of gB, both of which are required for compensation of the defect in PrV-007. Our data again demonstrate the power of reversion analysis of herpesviruses and suggest that cytosolic and ectodomains play a role in incorporation of gB into virions.

Amino Acid Sequence↗
PubMed2004

Characterization of a novel large deletion and single point mutations in the BRCA1 gene in a Greek cohort of families with suspected hereditary breast cancer.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 predispose to breast and ovarian cancer. A multitude of mutations have been described and are found to be scattered throughout these two large genes. We describe analysis of BRCA1 in 25 individuals from 18 families from a Greek cohort. METHODS: The approach used is based on dHPLC mutation screening of the BRCA1 gene, followed by sequencing of fragments suspected to carry a mutation including intron--exon boundaries. In patients with a strong family history but for whom no mutations were detected, analysis was extended to exons 10 and 11 of the BRCA2 gene, followed by MLPA analysis for screening for large genomic rearrangements. RESULTS: A pathogenic mutation in BRCA1 was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. Single base putative pathogenic mutations were identified by dHPLC and confirmed by sequence analysis in 4 families: 5382insC (in two families), G1738R, and 5586G > A (in one family each). In addition, 18 unclassified variants and silent polymorphisms were detected including a novel silent polymorphism in exon 11 of the BRCA1 gene. Finally, MLPA revealed deletion of exon 20 of the BRCA1 gene in one family, a deletion that encompasses 3.2 kb of the gene starting 21 bases into exon 20 and extending 3.2 kb into intron 20 and leads to skipping of the entire exon 20. The 3' breakpoint lies within an AluSp repeat but there are no recognizable repeat motifs at the 5' breakpoint implicating a mechanism different to Alu-mediated recombination, responsible for the majority of rearrangements in the BRCA1 gene. CONCLUSIONS: We conclude that a combination of techniques capable of detecting both single base mutations and small insertions/deletions and large genomic rearrangements is necessary in order to accurately analyze the BRCA1 gene in patients at high risk of carrying a germline mutation as determined by their family history. Furthermore, our results suggest that in those families with strong evidence of linkage to the BRCA1 locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements.

Age of Onset↗
PubMed1979

Isoelectric focusing and electrophoresis combined as a method for defining new point mutations in the mouse.

Isoelectric focusing followed by electrophoresis is a method that allows the survey of the expression of an enormous number of genes in one organism when this method is used for separation of complex protein solutions from the tissue of the organism (protein-mapping method). This makes it possible to use this method for developing a test system aiming at the detection of newly induced or spontaneous point mutations in mammals. A large number of loci could be tested on a relatively small number of individuals. The protein-mapping method was used for the separation of mouse tissue extracts containing soluble proteins or total cell proteins. Several modifications of the method are described. The applicability of this method for defining genetic differences of proteins was demonstrated by an investigation of different strains of mice. Thereafter, a mutagenicity test was initiated with the protein-mapping method. Mice were treated with methyl-nitroso-urea as a potential mutagenic substance and the fetuses of the F1 generation were investigated. Preliminary results suggest that new point mutations were detected with this method.

Animals↗
PubMed1992

A point mutation of c-Ki-ras gene was found in human esophageal carcinoma cell lines but not in primary esophageal carcinomas.

Activation of the ras oncogene in primary human esophageal carcinomas and cell lines established from such carcinomas was analyzed using the polymerase chain reaction (PCR)-direct sequencing method. This analysis revealed a GC----AT transition at the second base in the 12th codon of the c-Ki-ras gene in TE1 and TE2 esophageal carcinoma cell lines. In contrast, no point mutation was detected in the 12th, 13th, and 61st codon of the c-Ki-ras and c-Ha-ras gene in 31 primary esophageal carcinomas including those from which TE1 and TE2 cell lines were established. These results demonstrate that while activation of the c-Ki-ras gene by point mutation occurred in a subset of esophageal carcinoma cell lines during establishment of the cell lines, the activation events are not important in the transformation of human esophageal epithelial cells.

Adenocarcinoma↗
PubMed1989

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.

Adenosine Deaminase↗
PubMed2001

Point mutations in homology domain II modify the sensitivity of rat Nav1.8 sodium channels to the pyrethroid insecticide cismethrin.

Two point mutations in homology domain II of the housefly Vsscl voltage-sensitive sodium channel subunit, M918T and L1014F are associated with resistance to pyrethroid insecticides and reduce the pyrethroid sensitivity of Vsscl sodium channels expressed in Xenopus laevis oocytes. To assess the impact of these residues as determinants of pyrethroid sensitivity in another sequence context, we mutated the corresponding positions of the rat pyrethroid-sensitive, TTX-resistant peripheral nerve sodium channel (rNav1.8; also called SNS or PN3) and determined the sensitivity of native and mutated channels expressed in Xenopus oocytes to the pyrethroid insecticide cismethrin. The rNav1.8 channel, like other vertebrate sodium channel isoforms, contains a conserved isoleucine residue at sequence position 780 that aligns with the conserved methionine at position 918 of Vsscl and other insect sodium channels. Channels mutated to contain methionine at position 780 (1780M) exhibited enhanced sensitivity to cismethrin and larger decay constants for pyrethroid-modified channel states. In contrast, the mutation corresponding to M918Tin the Vssc1 channel (1780T) profoundly decreased the cismethrin sensitivity of expressed channels. Insertion of the mutation corresponding to L1014F (L879F in rNav1.8) reduced the cismethrin sensitivity of channels having either isoleucine or methionine at position 780, whereas channels containing the 1780T/L879F double mutation were insensitive to this insecticide. Mutations at Ile780 and Leu879 also modified the voltage dependence of rNav1.8 channels, but these effects were not related to changes in pyrethroid sensitivity. These results confirm the importance of residues in homology domain II as fundamental determinants of the pyrethroid sensitivity of sodium channels.

Animals↗