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Results for “mutation analysis”

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At least 379 records · Page 21Linked to original sources
PubMed1999

Mutation analysis of transforming growth factor beta type II receptor, Smad2, and Smad4 in hepatocellular carcinoma.

Mutations in the transforming growth factor beta type II receptor (TGFbetaRII), Smad2, and Smad4 genes have been detected in several human cancers. However, there are no reports of mutation analysis of the entire coding regions in these genes in hepatocellular carcinoma, and the roles of these genes in hepatocarcinogenesis remain unknown. We screened 30 hepatocellular carcinomas for mutations of these genes using polymerase chain reaction single-strand conformation polymorphism. We detected no mutations, but did find 3 cases of loss of heterozygosity of chromosome 17p13.1. These results suggest that mutations of the TGFbetaRII, Smad2, and Smad4 genes are rare, and that genetic instability is uncommon in human hepatocellular carcinoma.

Carcinoma, Hepatocellular↗
PubMed1988

Mutational analysis of the tobacco mosaic virus 5'-leader for altered ability to enhance translation.

Mutational analysis of the 5'-untranslated leader sequence (omega) of tobacco mosaic virus (TMV) was carried out to determine those sequences necessary for the translational enhancement associated with omega. Five deletion mutants, a single base substitution, and a 25 base replacement mutant were tested for alterations in omega's ability to enhance expression of beta-glucuronidase (GUS) mRNA in tobacco mesophyll protoplasts and Escherichia coli or chloramphenicol acetyltransferase (CAT) mRNA in Xenopus laevis oocytes. Alteration of an eight base subsequence required for the binding of a second ribosome resulted in the loss of translational enhancement in X. laevis oocytes but not in protoplasts. Substantial increases in enhancement were observed for several of the mutants in E. coli.

Animals↗
PubMed2000

Mutation analysis in glycogen storage disease type 1 non-a.

We report molecular and clinical findings in 13 patients with rare types of glycogen storage disease 1 (GSD1 non-a). Analysis of G6PT encoding a microsomal transporter protein has revealed mutations on both chromosomes in each case, four of which are novel. Diagnosis has been confirmed in three patients suspected of having GSD1 non-a without enzymatic studies involving liver biopsy, thus emphasising the advantage of G6PT mutation analysis for all GSD1 non-a patients.

Adolescent↗
PubMed1999

An NMR and mutational analysis of an RNA pseudoknot of Escherichia coli tmRNA involved in trans-translation.

Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed trans -translation. According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350-400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes. A mutational analysis using a number of Escherichia coli tmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in trans -translation. Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudoknot structure in the presence of Mg(2+). Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems. Structural instability of the pseudoknot structure, even in the presence of Mg(2+), was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activities indicate that trans -translation requires the pseudoknot structure stabilized by Mg(2+)and specific residues G61 and G62 in loop 3.

Base Pairing↗
PubMed2006

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase.

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.

3' Flanking Region↗
PubMed1999

Peutz-Jeghers syndrome: four novel inactivating germline mutations in the STK11 gene. Mutations in brief no. 227. Online.

The diagnosis of Peutz-Jeghers syndrome is based on the occurrence of hamartomatous gastrointestinal polyps and perioral pigment spots. In view of the development of hamartomatous polyps in several syndromes and the variability of pigment spots in Peutz-Jeghers patients, identification of affected individuals is difficult. Recently, germline mutations in the STK11 gene have been reported as a molecular cause of Peutz-Jeghers syndrome. We present four novel inactivating mutations identified by direct sequencing of all 9 exons of the STK11 gene in 4 patients suggestive of Peutz-Jeghers syndrome: three frameshift mutations (125-137del; 474-480del; 516-517insT) and one nonsense mutation (Q220X). Our data obtained in these patients and in those reported previously emphasize the diagnostic value of histological discrimination between different types of hamartomatous polyps and of molecular analysis, particularly in cases with no family history of the disease.

AMP-Activated Protein Kinase Kinases↗
PubMed2005

Linkage and mutational analysis of familial thyroid dysgenesis demonstrate genetic heterogeneity implicating novel genes.

The pathophysiology of thyroid dysgenesis (TD) is not elucidated yet in the majority of cases. The unexpected familial clustering of congenital hypothyroidism due to TD suggests a genetically determined disorder. Four genes have been hitherto involved in thyroid development, including migration and growth. Three of these encode transcription factors (the thyroid transcription factors 1 and 2 (TTF1 or NKX2.1 and TTF2 or FOXE1) and PAX8) while the other encodes the thyrotropin hormone receptor (TSHR). Some mutations have been reported in patients affected by thyroid defects, which supports the relevance of these four genes in TD. However, their involvement in the general TD population remains questionable. Therefore, to document their involvement, we performed a linkage analysis followed by mutational analysis in 19 multiplex TD families. The LOD score results failed to prove linkage between any of the four genes and the TD phenotype, whatever the postulated mode of inheritance. Manual extended haplotypes showed allele sharing among affected individuals of at least one of these four genes in the majority of families. Nevertheless, mutational analysis did not identify mutations in these cases, arguing in favor of identity by descent and not identity by state. Furthermore, as a main result of the present study, extended haplotypes confirmed by mutational analysis showed that the four genes were excluded in five out of the 19 investigated families, demonstrating the relevance of other genes. In conclusion, the present study demonstrates genetic heterogeneity in the TD disorder and suggests the involvement of novel genes.

DNA Mutational Analysis↗
PubMed2001

Mutational analysis within the 3' region of the PKD1 gene in Japanese families.

Autosomal dominant polycystic kidney disease (ADPKD) is a widespread genetic disease that causes renal failure. One of the genes that is responsible for this disease, PKD1, has been identified and characterized. Many mutations of the PKD1 gene have been identified in the Caucasian population. We investigated the occurrence of mutations in this gene in the Japanese population. We analyzed each exon in the 3' single copy region of the gene between exons 35 and 46 in genomic DNA obtained from 69 patients, using a PCR-based direct sequencing method. Four missense mutations (T3509M, G3559R, R3718Q, R3752W), one deletion mutation (11307del61bp) and one polymorphism (L3753L) were identified, and their presence confirmed by allele-specific oligonucleotide (ASO) hybridization. These were novel mutations, except for R3752W, and three of them were identified in more than two families. Mutation analysis of the PKD1 gene in the Japanese population is being reported for the first time.

Alleles↗
PubMed1995

Mutational analysis of multiple endocrine neoplasia type 2A associated with Hirschsprung's disease.

BACKGROUND: The clinical association of multiple endocrine neoplasia type 2A (MEN 2A) and Hirschsprung's disease (HD), although rare, has been previously observed. Recently, germline mutations in the RET proto-oncogene, a transmembrane receptor with tyrosine kinase activity, have been detected in patients with familial HD. RET is also the predisposition gene for the inherited cancer syndrome MEN 2A. METHODS: We describe a DNA sequence variation within the coding region of RET in two large unrelated kindreds with MEN 2A (with 83 and 42 persons affected) in which HD cosegregated with MEN 2A in seven patients. Mutational analysis was performed with a highly sensitive polymerase chain reaction-based denaturing gradient gel electrophoresis technique followed by direct sequencing of mutants. RESULTS: Genetic analysis by denaturing gradient gel electrophoresis detected mutant bands in RET exon 10 in patients with MEN 2A from both kindreds. Direct DNA sequencing of mutants revealed a thymine-to-adenine base change in codon 618, resulting in a cysteine-to-serine substitution. The identical mutation was present in all seven children with HD. Of these children five underwent thyroidectomy for C-cell abnormalities; one 3-year-old child is awaiting thyroid surgery, and the remaining patient died at age of 12 weeks. CONCLUSIONS: The RET codon 618 Ser mutation could predispose patients with MEN 2A to HD. RET may assume a critical role in embryologic enteric nerve migration and tumorigenesis of cells from neural crest lineage.

Amino Acid Sequence↗
PubMed2005

Mutational analysis of the pH signal transduction component PalC of Aspergillus nidulans supports distant similarity to BRO1 domain family members.

The alkaline ambient pH signal transduction pathway component PalC has no assigned molecular role. Therefore we attempted a gene-specific mutational analysis and obtained 55 new palC loss-of-function alleles including 24 single residue substitutions. Refined similarity searches reveal conserved PalC regions including one with convincing similarity to the BRO1 domain, denoted PCBROH, where clustering of mutational changes, including PCBROH key residue substitutions, supports its structural and/or functional importance. Since the BRO1 domain occurs in the multivesicular body (MVB) pathway protein Bro1/Vps31 and also the pH signal transduction protein PalA (Rim20), both of which interact with MVB component (ESCRT-III protein) Vps32/Snf7, this might reflect a further link between the pH response and endocytosis.

Amino Acid Sequence↗
PubMed2005

Mutational analysis of the SH2-kinase linker region of Bruton's tyrosine kinase defines alternative modes of regulation for cytoplasmic tyrosine kinase families.

Bruton's tyrosine kinase (Btk) plays critical roles in B cell development and activation. Mutations of Btk cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. An Src homology domain 2-kinase linker region exists in all Src, Abl, ZAP70/Syk and Btk/Tec non-receptor tyrosine kinase families. Missense mutations in the Btk linker region can cause XLA, supporting an essential role for this protein segment. We investigated the regulatory role of the linker region in Btk function by mutational analysis. XLA-causing mutations L369F and R372G abolished Btk-mediated calcium response without affecting Btk protein stability and kinase activity significantly. Although mutation of a well-conserved tryptophan (W260A) in the linker region of the Src family kinase Hck has been shown to cause a hyperactive kinase, an analogous mutation in Btk (W395A) dramatically decreased Btk kinase activity. Tyrosine phosphorylation in the linker region was previously shown to regulate the function of Abl and ZAP70/Syk kinases. Even though tyrosine phosphorylation was detected on tyrosine 375 in the Btk linker region, no significant alteration was observed in Btk-signaling activity and biological function when this tyrosine was mutated in DT-40 cells or in Y375F knock-in mice. Our data and previous studies suggest that each cytoplasmic tyrosine kinase family has evolved a unique strategy to utilize the linker region to regulate the function of the enzyme.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Mutation analysis of the Fanconi anaemia A gene in breast tumours with loss of heterozygosity at 16q24.3.

The recently identified Fanconi anaemia A (FAA) gene is located on chromosomal band 16q24.3 within a region that has been frequently reported to show loss of heterozygosity (LOH) in breast cancer. FAA mutation analysis of 19 breast tumours with specific LOH at 16q24.3 was performed. Single-stranded conformational polymorphism (SSCP) analysis on cDNA and genomic DNA, and Southern blotting failed to identify any tumour-specific mutations. Five polymorphisms were identified, but frequencies of occurrence did not deviate from those in a normal control population. Therefore, the FAA gene is not the gene targeted by LOH at 16q24.3 in breast cancer. Another tumour suppressor gene in this chromosomal region remains to be identified.

Breast Neoplasms↗
PubMed2005

A novel missense mutation of doublecortin: mutation analysis of Korean patients with subcortical band heterotopia.

The neuronal migration disorders, X-linked lissencephaly syndrome (XLIS) and subcortical band heterotopia (SBH), also called "double cortex", have been linked to missense, nonsense, aberrant splicing, deletion, and insertion mutations in doublecortin (DCX) in families and sporadic cases. Most DCX mutations identified to date are located in two evolutionarily conserved domains. We performed mutation analysis of DCX in two Korean patients with SBH. The SBH patients had mild to moderate developmental delays, drug-resistant generalized seizures, and diffuse thick SBH upon brain MRI. Sequence analysis of the DCX coding region in Patient 1 revealed a c.386 C>T change in exon 3. The sequence variation results in a serine to leucine amino acid change at position 129 (S129L), which has not been found in other family members of Patient 1 or in a large panel of 120 control X-chromosomes. We report here a novel c.386 C>T mutation of DCX that is responsible for SBH.

Adolescent↗
PubMed2003

Mutation analysis of the BRG1 gene in prostate cancer clinical samples.

Previous studies in hereditary and sporadic prostate cancer have indicated the existence of a tumor suppressor gene in chromosomal region 19p13. The BRG1 gene in this region is one of the possible candidates, based on both the frequency of inactivating mutations in human cancer cell lines, including the prostate cancer cell line DU145, and its functional properties. To our knowledge, no studies have been done to evaluate possible involvement of the BRG1 gene in clinical prostate cancer. To accomplish this, we carried out a complete mutation analysis of all 35 BRG1 exons in tumor and constitutional DNA samples from 21 prostate cancer patients. We report the absence of somatic mutations in the panel of samples employed, but the existence of five germline single nucleotide polymorphisms (SNPs) in CpG islands of the BRG1 gene, among them, three novel ones. In conclusion, the study excludes the presence of common BRG1 mutations in prostate cancer.

Age of Onset↗
PubMed2000

Mutation analysis and description of sixteen RSH/Smith-Lemli-Opitz syndrome patients: polymerase chain reaction-based assays to simplify genotyping.

We report the clinical and molecular data of 16 patients with RSH/Smith-Lemli-Opitz syndrome (RSH/SLOS) with varying phenotypic severity, for which we have identified mutations in both alleles. RSH/SLOS is an autosomal recessive malformation syndrome caused by mutations in the gene encoding the sterol Delta(7)-reductase. This protein catalyzes the reduction of 7-dehydrocholesterol to cholesterol in the last step of cholesterol biosynthesis via the Kandutsch-Russell pathway. In addition to previously reported mutations (T93M, L109P, G147D, W151X, T154M, R242C, A247V, T289I, IVS8-1G-->C, Y408H, and E448K), we have identified six previously undescribed mutations (321G-->C, W177R, R242H, Y318N, L341P, and C444Y). We also report rapid polymerase chain reaction (PCR)-based assays developed to detect four of the recurring mutations (T93M, W151X, V326L, and R404C) and six other RSH/SLOS mutations (321G-->C, L109P, T154M, T289I, Y318N, and L341P). The purpose of this article is to correlate detailed clinical information with molecular data in order to improve our understanding of the genotype-phenotype correlation of RSH/SLOS and to report the development of PCR-based assays that will allow more rapid mutation analysis.

Alleles↗
PubMed2000

Mutational analysis of the mitochondrial copper metallochaperone Cox17.

The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochrome c oxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome c oxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys --> Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17 was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys --> Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.

Amino Acid Sequence↗