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Mutation Sites (mutation + site)
Selected AbstractsMDM2 SNP309 T>G alone or in combination with the TP53 R72P polymorphism does not appear to influence disease expression and age of diagnosis of colorectal cancer in HNPCC patientsINTERNATIONAL JOURNAL OF CANCER, Issue 3 2007Bente A. Talseth Abstract Disease expression in hereditary nonpolyposis colorectal cancer (HNPCC) cannot be readily explained by mutation site in the respective DNA mismatch repair genes associated with this disorder. One explanation is the role of modifying genes that can either promote or prevent disease development on a background of increased risk. Two single nucleotide polymorphisms in MDM2 and TP53 have been shown to be associated with younger ages of disease onset in HNPCC (TP53) and Li-Fraumeni syndrome (MDM2). In this study 220 HNPCC patients were examined, from Australia and Poland, all characterized at the molecular level to determine the frequency of the MDM2 SNP309 T>G and to assess its influence on disease expression. The results were then pooled with the results of a previous study to assess the combined influence of the MDM2 SNP309 T>G and TP53 SNP R72P. A significant difference was observed between CRC patients and unaffected MMR gene mutation carriers over the age of 45 years (p = 0.01). The unaffected MMR gene mutation carriers over the age of 45 years who carry the G allele have a reduced risk of developing CRC. The results indicate that the MDM2 SNP309, alone or in combination with TP53 R72P, does not influence age of diagnosis of CRC in individuals with HNPCC. In conclusion, the data indicates the G allele of MDM2 SNP309 might have a protective effect on disease development in HNPCC patients and that age of diagnosis of CRC is not associated with MDM2 SNP309 or TP53 R72P either as single SNPs or combined. © 2006 Wiley-Liss, Inc. [source] Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-,2(I) and Severe Osteogenesis ImperfectaJOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2001Bonnie G. Campbell Abstract RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-,2(I) suggested comigration with the similarly sized pro-,2(I) derived from the mutant allele. Furthermore, ,-chains were overhydroxylated and the ratio of ,1(I):,2(I) was 3.2:1, consistent with the presence of ,1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-,2(I) C-propeptide and confirmed a diagnosis of OI. [source] The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide-oligonucleotide adducts determined using nanoLC/MS/MSJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2009James Glick Abstract Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, ( ± )-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [( ± )- anti -benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5,-ACCCG5CG7TCCG11CG13C-3,/5,-GCGCGGGCGCGGGT-3,) derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational ,hot spots' and have also been reported as chemical ,hot spots' for the formation of BPDE-DNA adducts. In evaluating the effect of cytosine methylation on BPDE-DNA adduct binding, it was found that codon 156, containing the nucleobase G5 instead of the mutational hot spot codons 157 (G7) and 158 (G11), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G5, G11 > G13 > G7. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G11 CpG site (codon 158) approximately two-fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G5, G7 > G11 > G13. The three- to four-fold increase in adduction at the mutational hot spot codon 157 (G7) relative to the unmethylated or single-stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run. Copyright © 2009 John Wiley & Sons, Ltd. [source] Codon 101 of PRKCG, a preferential mutation site in SCA14MOVEMENT DISORDERS, Issue 12 2007Dagmar Nolte [source] Identification of the Rdl mutation in laboratory and field strains of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae)PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 12 2004Chris Bass Abstract In many insect species, resistance to cyclodiene insecticides is caused by amino acid substitutions at a single residue (A302) within the M2 transmembrane region of the ,-aminobutyric acid (GABA) receptor sub-unit termed Rdl (resistance to dieldrin). These mutations (A302S and A302G) have also been shown to confer varying levels of cross-resistance to fipronil, a phenylpyrazole insecticide with a similar mode of action to cyclodienes. To investigate the possible occurrence of these mutations in the cat flea, Ctenocephalides felis (Bouché), a 176-bp fragment of the cat flea Rdl gene, encompassing the mutation site, was PCR amplified and sequenced from nine laboratory flea strains. The A302S mutation was found in eight of the nine strains analysed, although the relative frequency of the mutant allele varied between strains. Only one strain (R6) was found to be homozygous for the S302 allele in all the individuals tested, and this correlated with previous reports of low-level fipronil resistance in this strain. A PCR-based diagnostic assay, capable of screening individual fleas for this mutation, was developed and used to survey a range of fleas collected at random from veterinary clinics in the UK and USA. The A302S mutation was present at a high frequency in these domestic pet populations. Copyright © 2004 Society of Chemical Industry [source] Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X-ray crystallographic analysesFEBS JOURNAL, Issue 2 2009Muhammad S. Rohman The Arg97 , Gly and Asp136 , His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7 °C, respectively, in Tm, and 3.5 and 6.1 kJ·mol,1, respectively, in ,G(H2O). These mutations also stabilized the So-RNase HI derivative (4×-RNase HI) with quadruple thermostabilizing mutations in an additive manner. As a result, the resultant sextuple mutant protein (6×-RNase HI) was more stable than the wild-type protein by 28.8 °C in Tm and 27.0 kJ·mol,1 in ,G(H2O). To analyse the effects of the mutations on the protein structure, the crystal structure of the 6×-RNase HI protein was determined at 2.5 Å resolution. The main chain fold and interactions of the side-chains of the 6×-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites. These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive. The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6×-RNase HI protein. We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability. [source] Crystal structure of a staphylokinase variantFEBS JOURNAL, Issue 2 2002A model for reduced antigenicity Staphylokinase (SAK) is a 15.5-kDa protein from Staphylococcus aureus that activates plasminogen by forming a 1 : 1 complex with plasmin. Recombinant SAK has been shown in clinical trials to induce fibrin-specific clot lysis in patients with acute myocardial infarction. However, SAK elicits high titers of neutralizing antibodies. Biochemical and protein engineering studies have demonstrated the feasibility of generating SAK variants with reduced antigenicity yet intact thrombolytic potency. Here, we present X-ray crystallographic evidence that the SAK(S41G) mutant may assume a dimeric structure. This dimer model, at 2.3-Å resolution, could explain a major antigenic epitope (residues A72,F76 and residues K135-K136) located in the vicinity of the dimer interface as identified by phage-display. These results suggest that SAK antigenicity may be reduced by eliminating dimer formation. We propose several potential mutation sites at the dimer interface that may further reduce the antigenicity of SAK. [source] Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss,HUMAN MUTATION, Issue 4 2002Alessandro Ferraris Abstract Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. Hum Mutat 20:312,320, 2002. © 2002 Wiley-Liss, Inc. [source] HATODAS II , heavy-atom database system with potentiality scoringJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3 2009Michihiro Sugahara HATODAS II is the second version of HATODAS (the Heavy-Atom Database System), which suggests potential heavy-atom reagents for the derivatization of protein crystals. The present expanded database contains 3103 heavy-atom binding sites, which is four times more than the previous version. HATODAS II has three new criteria to evaluate the feasibility of the search results: (1) potentiality scoring for the predicted heavy-atom reagents, (2) exclusion of the disordered amino acid residues based on the secondary structure prediction and (3) consideration of the solvent accessibility of amino acid residues from a homology model. In the point mutation option, HATODAS II suggests possible mutation sites into reactive amino acid residues such as Met, Cys and His, on the basis of multiple sequence alignments of homologous proteins. These new features allow the user to make a well informed decision as to the possible heavy-atom derivatization experiments of protein crystals. [source] Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infectionJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 5 2000Yu-Fen Fan Abstract Background and Aims: The expression of hepatitis B viral (HBV) antigens in liver tissue reflects the replicative status of chronic HBV infection. We have previously recognized a novel marginal pattern of hepatitis B surface antigen (HBsAg) in hepatocytes, which usually clusters in groups and emerges at the late non-replicative phase. This study was designed to investigate whether the marginal-type HBsAg represented the gene product of a specific HBV-surface mutant. Methods: Microdissection of cirrhotic nodules homogeneously expressing marginal HBsAg was performed on two of 12 resected livers from HBsAg-seropositive patients with hepatocellular carcinoma. The gene presumably encoding marginal HBsAg was polymerase chain reaction (PCR)-cloned, sequenced and analysed. In vitro transfection and expression of the cloned surface mutant plasmids were performed on the Huh7 cell line to illustrate intrahepatic HBsAg expression. Results: Immunohistochemical staining revealed that the marginal HBsAg was positive for pre-S1 and thus contained large surface proteins. The PCR cloning and sequencing of the genes presumably encoding marginal-type HBsAg in both cases revealed the same deletion at the 5, terminus (nt 2,55) of pre-S2. A point mutation on the small-surface (S) antigen was also found in one case. The pre-S2 deletion sequence and the mutation sites of the S gene coincide with human lymphocyte antigen-restricted T- and/or B-cell epitopes. In vitro transfection of the mutant plasmid revealed a blot-like retention or accumulation of HBsAg in the cytoplasm or at the periphery of hepatocytes, accompanied by a decreased secretion of HBsAg in the culture supernatant, mimicking intrahepatic expression. Conclusion: A natural pre-S2 deletion mutant was identified in hepatocytes expressing a novel marginal pattern of HBsAg, which probably contains mutant, large, surface proteins. The biological significance of the pre-S2 deletion mutant should be interesting in view of the clustering proliferation of hepatocytes expressing marginal HBsAg. [source] Oculocutaneous albinism type 4: six novel mutations in the membrane-associated transporter protein gene and their phenotypesPIGMENT CELL & MELANOMA RESEARCH, Issue 5 2006Katsuhiko Inagaki Summary Oculocutaneous albinism type 4 (OCA4) is an autosomal recessive hypopigmentary disorder caused by mutations in the Membrane-Associated Transporter Protein gene (SLC45A2). The SLC45A2 protein is a 530-amino-acid polypeptide that contains 12 putative transmembrane domains, and appears to be a transporter that mediates melanin synthesis. Eighteen pathological mutations have been reported so far. In this study, six novel mutations, p.Y49C (c.146A > G), p.G89R (c.265G > A), p.C229Y (c.686G > A), p.T437A (c.1309A > G), p.T440A (c.1318A > G) and p.G473D (c.1418G > A) were found in eight Japanese patients with various clinical phenotypes. The phenotypes of OCA4 were as various as the other types of OCA and probably depended on the mutation sites in the SLC45A2 gene. [source] Structure of human erythrocyte NADH-cytochrome b5 reductaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Sachiko Bando Erythrocyte NADH-cytochrome b5 reductase reduces methaemoglobin to functional haemoglobin. In order to examine the function of the enzyme, the structure of NADH-cytochrome b5 reductase from human erythrocytes has been determined and refined by X-ray crystallography. At 1.75,Å resolution, the root-mean-square deviations (r.m.s.d.) from standard bond lengths and angles are 0.006,Å and 1.03°, respectively. The molecular structure was compared with those of rat NADH-cytochrome b5 reductase and corn nitrate reductase. The human reductase resembles the rat reductase in overall structure as well as in many side chains. Nevertheless, there is a large main-chain shift from the human reductase to the rat reductase or the corn reductase caused by a single-residue replacement from proline to threonine. A model of the complex between cytochrome b5 and the human reductase has been built and compared with that of the haem-containing domain of the nitrate reductase molecule. The interaction between cytochrome b5 and the human reductase differs from that of the nitrate reductase because of differences in the amino-acid sequences. The structures around 15 mutation sites of the human reductase have been examined for the influence of residue substitutions using the program ROTAMER. Five mutations in the FAD-binding domain seem to be related to cytochrome b5. [source] Structures of Cytochrome b5 Mutated at the Charged Surface-Residues and Their Interactions with Cytochrome c,CHINESE JOURNAL OF CHEMISTRY, Issue 11 2002Jlan Wu Abstract Glu44, Glu48, Ghi56 and Asp60 are the negatively charged residues located at the molecular surface of cytochrome b5. Two mutants of cytochrome b5 were prepared, in which two or all of these four residues were mutated to alanines. The mutations give rise to slightly positive shifts of the redox potentials of cytochrome b5 and obvious decrease of the cytochrome b5 -cytochrome c binding constants and electron transfer rates. The crystal structures of the two mutants were determined at 0.18 nm resolution, showing no alteration in overall structures and exhibiting slight changes in the local conformations around the mutation sites as compared with the wild-type protein. Based on the crystal structure of the quadruple-site mutant, a model for the binding of this mutant with cytochrome c is proposed, which involves the salt bridges from Glu37, Glu38 and heme propionate of cytochrome b5 to three lysines of cytochrome c and can well account for the properties and behaviors of this mutant. [source] Deletion hotspot in the argininosuccinate lyase gene: association with topoisomerase II and DNA polymerase , sites ,HUMAN MUTATION, Issue 11 2006John Christodoulou Abstract Molecular analysis of argininosuccinate lyase (ASAL) deficiency has led to the identification of a deletion hotspot in the ASL gene. Six individuals with ASAL deficiency had alleles that led to a complete absence of exon 13 from the ASL mRNA; each had a partial deletion of exon 13 in the genomic DNA. In all six patients, the deletions begin 18 bp upstream of the 3, end of exon 13. In four cases, the deletions were 13 bp in length, and ended within exon 13, whereas in two other patients the deletions were 25 bp and extended into intron 13. The sequence at which these deletions begin overlaps both a putative topoisomerase II recognition site and a DNA polymerase , mutation/frameshift site. Moreover, the topoisomerase II cut site is situated precisely at the beginning of the deletions, which are flanked by small (2- and 3-bp) direct repeats. We note that a similar concurrence of these two putative enzyme sites can be found in a number of other deletion sites in the human genome, most notably the ,F508 deletion in the CFTR gene. These findings suggest that the joint presence of these two enzyme sites represents a DNA sequence context that may favor the occurrence of small deletions. Hum Mutat 27(11), 1065,1071, 2006. © 2006 Wiley-Liss, Inc. [source] | ||||||||||||||||