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PubMed1999

A novel 8-bp insertion in codon 281 of p53 in a patient with acute lymphoblastic leukaemia and 2 separate leukaemic clones. Mutations in brief no. 219. Online.

We report a novel p53 insertion in a case of aggressive acute lymphoblastic leukaemia in a 16 year old male, in which 2 separate leukaemic clones were previously identified by T-cell receptor sigma and immunoglobulin heavy chain gene rearrangement studies. Initial p53 mutation screening of blast cells from 29 patients with acute leukaemia by PCR-denaturing gradient gel electrophoresis showed that 2 had a silent codon 213 polymorphism and only the index case had a somatic mutation identified to be an 8 bp insertion in codon 281 (5'CCGGGGGG-3'). This insertion was associated with the second, more aggressive clone which underwent clonal evolution and became resistant to cytotoxic chemotherapy. With an allele-specific primer in PCR, we were able to demonstrate the presence of this clone as a minority at disease presentation, and in 2 of 3 collections of peripheral blood progenitor cells.

Adolescent↗
PubMed2000

Mutational analysis of the highly conserved C-terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators.

The XylS protein of the TOL plasmid of Pseudomonas putida belongs to the so-called AraC/XylS family of regulators, that includes more than 100 different bacterial proteins. A conserved stretch of about 100 amino acids is present at the C-terminal end. This conserved region is believed to contain seven alpha-helices, including two helix-turn-helix (HTH) DNA binding motifs (alpha(2)-T-alpha(3) and alpha(5)-Talpha-(6)), connected by a linker alpha-helix (alpha(4)), and two flanking alpha-helices (alpha(1) and alpha(7)). The second HTH motif is the region with the highest homology in the proteins of the family, with certain residues showing almost 90% identity. We have constructed XylS single mutants in the most conserved residues and have analysed their ability to stimulate transcription from its cognate promoter, Pm, fused to 'lacZ. The analysis revealed that mutations in the alpha(5)-helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the alpha(6)-helix was important for the activity. The strongest effect of the mutations was observed in conserved residues located outside the DNA binding domain, namely, Gly-290 in the turn between the two helices, Pro-309 located downstream of alpha(6), and Leu-313, in the small last helix alpha(7), that seems to play an important role in the activation of RNA-polymerase. Our analysis shows that conservation of amino acids in the family reflects structural requirements rather than functionality in specific DNA interactions.

Amino Acid Sequence↗
PubMed1998

Mutational analysis of the domain structure of mouse protein phosphatase 2Cbeta.

The structures of five distinct isoforms of mammalian protein phosphatase 2Cbeta (PP2Cbeta-1, -2, -3, -4 and -5) have previously been found to differ only at their C-terminal regions. In the present study, we performed mutational analysis of recombinant mouse PP2Cbeta-1 to determine the functional domains of the molecule and elucidate the biochemical significance of the structural differences in the isoforms. Differences in affinity for [32P]phosphohistone but not for [32P]phosphocasein were observed among the five PP2Cbeta isoforms. Deletion of 12 amino acids from the C-terminal end, which form a unique sequence for PP2Cbeta-1, caused a 35% loss of activity against [32P]phosphohistone but no loss of activity against [32P]phosphocasein. Deletion of up to 78 amino acids from this end did not cause any further alteration in activity, whereas deletion of 100 amino acids totally eliminated the activity against both [32P]phosphohistone and [32P]phosphocasein. On the other hand, deletion of 11 amino acids from the N-terminal end caused a 97% loss of enzyme activity, and further deletions caused a total loss of activity. Substitution of any of the six specific amino acids among 16 tested in this study, which were located among the 250 N-terminal residues, caused 98-100% loss of enzyme activity. Among these amino acids, three (Glu-38, -60 and -243) have recently been reported to be essential for the binding of metal ions in the catalytic site of the PP2C molecule [Das, Helps, Cohen and Barford (1996) EMBO J. 15, 6798-6809]. These observations indicate that PP2Cbeta is composed of at least two distinct functional domains, an N-terminal catalytic domain of about 310 amino acids and the remaining C-terminal domain, which is involved in determination of substrate specificity.

Animals↗
PubMed2002

Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance.

We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the Bunyaviridae family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol. 75:1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5'- and 3'-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and 2 to 4; site B, positions 8 and 8, where underlined nucleotides refer to positions in the vRNA 3' end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5' and 3' ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5' and 3' ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.

Animals↗
PubMed1996

Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS.

The Escherichia coli H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. H-NS negatively regulates the transcription of a number of apparently unlinked genes on the chromosome, suggesting that it functions as a global transcriptional repressor. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and protein-protein interaction (dimerization and/or oligomerization), respectively. We first isolated a number of hns mutations which resulted in derepression of the proVWX operon. These included 20 independent missence mutations each resulting in a single amino acid substitution, and six nonsense mutations each giving a C-terminally truncated form of H-NS. The substituted amino acids were revealed to be located non-randomly in the primary sequence of H-NS. This set of hns mutants was examined extensively in terms of phenotypes and biochemical properties. Based on the in vivo and in vitro results, together with the locations of the altered amino acids, three distinct functional domains were identified in H-NS. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA. The N-terminal domain was suggested to be involved in the ability to repress transcription. Mutations in this region abolished its ability to repress the transcription of proV, in vivo and in vitro, without loss of its DNA-binding activity. None of the mutants examined was impaired in the formation of a dimer and/or oligomers, suggesting that the central region of H-NS is involved in oligomerization. These results are discussed with special reference to the molecular mechanism underlying the function of H-NS as a transcriptional repressor. In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS.

Amino Acid Sequence↗
PubMed2006

Immunohistochemical and mutational analysis of PDGF and PDGFR in desmoid tumours: is there a role for tyrosine kinase inhibitors in c-kit-negative desmoid tumours?

AIM: To determine the platelet-derived growth factor (PDGF) alpha and beta status of desmoid tumours. Desmoid tumours are rare monoclonal neoplasms that appear to have no metastatic potential. Surgical resection and radiotherapy in the event of a positive surgical margin is the first-line treatment. Recurrences are frequent. Treatment results using non-steroidal anti-inflammatory agents, anti-oestrogen compounds and other agents such as Imatinib mesylate have been published. Therapy with Imatinib has been proposed as a therapeutic option, although in most reports desmoid tumours are reported to be c-kit-. METHODS AND RESULTS: We performed immunohistochemical analysis on 124 archived samples (85 patients) of desmoid tumours using antibodies to PDGFalpha, PDGFbeta, PDGFRalpha and PDGFRbeta. All desmoid tumours showed immunoreactivity with antibodies to PDGFalpha and PDGFRalpha, whereas with antibodies to PDGFbeta and PDGFRbeta no specific reaction could be detected. Mutational analysis of PDGFRalpha (exons 11, 12, 17 and 18) and PDGFRbeta (exon 12) on frozen material from 14 patients was performed, but no mutations leading to amino acid changes in the mature protein were identified. CONCLUSION: The absence of an activating mutation in a protooncogene does not exclude the efficacy of tyrosine kinase inhibitors through other possible mechanisms, and these might be a therapeutic option for patients with desmoid tumours in whom established local and systemic approaches fail to control the disease.

Adult↗
PubMed2005

Mutational analysis of idiopathic renal hypouricemia in Korea.

Idiopathic renal hypouricemia is a hereditary disease characterized by abnormally high renal uric acid clearance. Most patients are clinically silent, but acute renal failure (ARF), urolithiasis, or hematuria may develop. A defect in the SLC22A12 gene, which encodes the renal uric acid transporter, URAT1, is the known major cause of this disorder. We performed a mutational analysis of the SLC22A12 gene in five Korean patients with idiopathic renal hypouricemia in this study. Two patients presented with microscopic hematuria, one with uric acid urolithiasis, and one with exercise-induced ARF. One patient was asymptomatic. Three different mutations, W258X, R90H and R477H, were detected in four of the patients. However, no mutation was found in the fifth ARF patient. This is the first study of SLC22A12 mutations in a country other than Japan. W258X was found to be the predominant SLC22A12 mutation in Korean renal hypouricemia patients, as has been reported in Japan.

Acute Kidney Injury↗
PubMedDate not supplied

Haplotype and mutation analysis in Greek patients with Wilson disease.

In this study, we report the results of haplotype and mutation analysis of the ATP7B gene in Wilson disease (WD) patients of Greek origin. We have analysed 25 WD families and two single patients and characterised 94% of the WD chromosomes investigated. We have found 12 different molecular defects (three frameshifts, two splice site, two nonsense, five missense mutations), four of which are novel. Five of the mutations are widely prevalent accounting for 74% of the WD chromosomes analysed. These results may enable preclinical diagnosis in the large majority of WD patients of Greek descent, thereby improving genetic counselling and disease management.

Adolescent↗
PubMed1988

Mutational analysis of the chicken beta-globin enhancer reveals two positive-acting domains.

We report a mutational analysis of the chicken beta-globin enhancer, using transient expression in primary embryonic erythrocytes. A series of 27 scanning mutants differing only by consecutive 4-base substitutions was tested. Previous experiments, using protection from DNase I digestion, demonstrated four regions in the enhancer that bind factors. Mutations in two of the four regions (denoted I and III) have no effect on enhancer activity. The other two regions (II and IV) mediate all of the positive-acting activity. Region II appears to consist of two subregions, mutation in either of which leads to the same loss in activity as mutation of both. Like region II, mutation of either half of region IV results in reduced enhancer activity. However, in contrast to region II, mutation of both halves of region IV results in more loss of enhancer activity than either single mutation. These data suggest that the half-sites in region II must interact in order to cause enhancement, whereas the half-sites in region IV act independently. Triplications of regions II or IV are able to enhance to a similar degree as the parental enhancer, but single copies of regions II or IV enhance only weakly. Thus, as measured by transient expression in embryonic erythrocytes, the beta-globin enhancer may involve the action of as few as three DNA-binding proteins acting at two sites.

Acetyltransferases↗
PubMed2005

3-Methylcrotonyl-CoA carboxylase deficiency: mutation analysis in 28 probands, 9 symptomatic and 19 detected by newborn screening.

Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder that appears to be the most frequent organic aciduria detected in tandem mass spectrometry (TMS)-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. We report mutation analysis in 28 MCC-deficient probands, 19 of whom were asymptomatic newborns detected by TMS newborn screening, and nine presented with clinical symptoms. Ten have mutations in MCCA, and 18 in MCCB. We identified 10 novel MCCA and 14 novel MCCB mutant alleles including missense, nonsense, frameshift and splice site mutations, and show that three of the missense mutations result in severely decreased MCC activity when expressed in MCC-deficient cell lines. Our data demonstrate no clear correlation between genotype and phenotype suggesting that factors other than the genotype at the MCC loci have a major influence on the phenotype of MCC deficiency.

Alleles↗
PubMed1995

Characterization of glucocerebrosidase in Greek Gaucher disease patients: mutation analysis and biochemical studies.

Gaucher disease is the most frequent lysosomal storage disease in Greece, accounting for 24% of all lysosomal disorders diagnosed during the last 13 years at the Institute of Child Health in Athens. The nature of the defects in glucocerebrosidase in Greek Gaucher patients with non-neuronopathic (type 1) and neuronopathic (types 2 and 3) phenotypes was investigated at the level of the glucocerebrosidase gene and enzyme activity. Mutation analysis performed in 10/23 Gaucher patients with different types of the disorder led to the identification of four mutations, N370S, L444P, R463C and D409H, comprising 75% of the investigated alleles. N370S was only found in association with type 1 disease. The genotype D409H/R463C was identified for the first time and was associated with the severe type 2 disorder. There was no correlation between residual in vitro enzyme activity and either phenotype or genotype. However, in cultured fibroblast of the neuronopathic cases, glucocerebrosidase protein concentration was reduced and the capacity to degrade exogenous C6NBD-glucosylceramide was more severely impaired.

Cells, Cultured↗
PubMed2005

Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth.

Sotos syndrome is characterized by pre- and post-natal overgrowth, typical craniofacial features, advanced bone age, and developmental delay. Some degree of phenotypic overlap exists with other overgrowth syndromes, in particular with Weaver syndrome. Sotos syndrome is caused by haploinsufficiency of the NSD1 (nuclear receptor SET domain containing gene 1) gene. Microdeletions involving the gene are the major cause of the syndrome in Japanese patients, whereas intragenic mutations are more frequent in non-Japanese patients. NSD1 aberrations have also been described in some patients diagnosed as Weaver syndrome. Some authors have suggested a certain degree of genotype-phenotype correlation, with a milder degree of overgrowth, a more severe mental retardation, and a higher frequency of congenital anomalies in microdeleted patients. Data on larger series are needed to confirm this suggestion. We report here on microdeletion and mutation analysis of NSD1 in 59 patients with congenital overgrowth. Fourteen novel mutations, two previously described and one microdeletion were identified. All patients with a NSD1 mutation had been clinically classified as "classical Sotos," although their phenotype analysis demonstrated that some major criteria, such as overgrowth and macrocephaly, could be absent. All patients with confirmed mutations shared the typical Sotos facial gestalt. A high frequency of congenital heart defects was present in patients with intragenic mutations, supporting the relevance of the NSD1 gene in the pathogenesis of this particular defect.

Adolescent↗
PubMed2003

Mutational analysis of major, sequential IgE-binding epitopes in alpha s1-casein, a major cow's milk allergen.

BACKGROUND: Allergy to cow's milk is common in early childhood, and no therapy other than avoidance exists. In murine models of peanut allergy, immunotherapy with mutated, engineered, proteins appears promising. OBJECTIVE: We sought to identify the critical amino acids (AAs) for immunoglobulin E (IgE) binding within the major B-cell epitopes of alpha(s1)-casein, a major cow's milk allergen. This will provide the necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE-binding capacity. METHODS: For mutational analysis of the IgE-binding epitopes, peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single or multiple AA substitutions. Membranes were immunolabeled with pooled sera from 15 cow's-milk-allergic patients and with 8 individual sera. RESULTS: With the pooled sera, substitution of a single AA led to complete abrogation of IgE binding to 2 of 8 peptides and diminished binding in the remainder. Substitution of multiple AAs led to an abrogation of binding in the remaining peptides. In 4 of the 8 peptides, the critical AA identified with pooled sera did not result in significant reduction of IgE binding with 1 or more individual patients. For these patients, other critical AAs were identified, indicating a more heterogeneous pattern in IgE recognition. CONCLUSION: This study indicates that single or multiple AA substitutions within IgE-binding epitopes result in reduced binding of milk-specific IgE antibodies by patients' sera. However, for future immunotherapeutic interventions with mutated peptides, critical AAs should be evaluated with individual patient sera to determine B-cell-epitope heterogeneity.

Adolescent↗
PubMed1996

Mutational analysis of TNF-alpha gene reveals a regulatory role for the 3'-untranslated region in the genetic predisposition to lupus-like autoimmune disease.

Lupus-prone mice show reduced production of TNF-alpha and, upon long-term treatment with recombinant TNF-alpha, significant protection from disease development. Mutational analysis of the 5'-untranslated region (UTR) and 3'-UTR of the mouse TNF-alpha gene reveals a marked degree of polymorphism. Transient expression experiments in the RAW 264.7 macrophage-like cell line using the luciferase reporter system suggest an important role for the mutations in the 3'-UTR in the biosynthesis of TNF-alpha, and provide a molecular explanation for the reduced TNF-alpha production in lupus-prone NZW mice.

Animals↗
PubMed2005

Mutational analysis of the "regulatory module" of hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) is a rate-limiting enzyme in lipolysis that displays broad substrate specificity. HSL function is regulated by reversible phosphorylation that occurs within a 150 aa "regulatory module" of the protein. The current studies used mutational analysis to dissect the contribution of the "regulatory module" in HSL activity and substrate specificity. Deletion of the entire "regulatory module" or replacement of the "regulatory module" with the "lid" of lipoprotein lipase resulted in enzymatically inactive proteins. Deletion of sequentially longer stretches of the "regulatory module" resulted in a stepwise reduction in hydrolytic activity. Analysis of 7-19 amino acid deletional mutants that spanned the "regulatory module" showed that the N-terminal partial deletion mutants retained normal hydrolytic activity and activation by PKA. In contrast, the C-terminal partial deletion mutants displayed reduced hydrolytic activities, with preferential loss of activity against lipid-, as opposed to water-soluble, substrates. Single amino acid mutations of F650C, P651A, and F654D reduced activity against lipid-, but not water-soluble, substrates. The current results suggest that the length of the "regulatory module" and specific sequences within the C-terminal portion of the "regulatory module" of HSL (amino acids 644-683) are crucial for activity and appear to be responsible for determining lipase activity.

Amino Acid Motifs↗
PubMed2001

Mutation analysis of the p51 gene and correlation between p53, p73, and p51 expressions in prostatic carcinoma.

BACKGROUND: p73 and p51 are genes possessing amino-acid similarities to p53. We previously found no mutation in p73 in prostatic carcinoma, but did find abnormal expression of the gene. Involvement of these genes in prostatic carcinogenesis is still poorly understood. METHODS: Mutation analysis of the p51 gene and allelotyping of 3q28, on which p51 lies, were performed. Expression of p53, p73, and p51 was examined using reverse transcription-polymerase chain reaction, and expression levels were compared. RESULTS: No mutation in p51 was found (0/55 cases). Loss of heterozygosity at 3q28 was detected in 6 of 28 cases (21.8%). By expression analysis we found that in p53, 4 of 38 cases (10.5%) showed downregulation. No cases showed upregulation of p53. In contrast, p73 and p51 were downregulated in 42.1 and 39.5% of cases, respectively, and upregulated in 31.5 and 34.2% of cases, respectively. Expression levels of p51 corresponded with those of p73 in 25 of 38 cases (65.8%). CONCLUSIONS: Somatic mutations in p73 and p51 are not important in prostatic carcinogenesis. These genes may be associated with tumors by expression levels and may have roles in addition to tumor suppression.

Aged↗
PubMed2003

Cloning and mutation analysis of ZFP276 as a candidate tumor suppressor in breast cancer.

Loss of heterozygosity (LOH) involving chromosome 16q23.4 occurs frequently in breast tumors, which suggests that this region may contain a tumor suppressor gene. Since ZFP276 is located in this region, we have therefore cloned and performed mutation analysis of its coding region in 70 breast tumors. One silent polymorphism and two alterations predicted to result in amino acid changes were detected. Absence of the wild-type allele in tumors carrying the E530D variant suggests a possible role for this change in tumorigenesis.

Alleles↗
PubMed1995

Mutation analysis of the BRCA1 gene in 76 Japanese ovarian cancer patients: four germline mutations, but no evidence of somatic mutation.

To investigate the putative role of BRCA1, a gene involved in hereditary breast and ovarian cancer, in sporadic ovarian tumors among Japanese women, we examined 76 unselected primary ovarian cancers for mutations in the coding region of BRCA1 using the single-strand conformation polymorphism technique. Although no somatic mutations were detected in any of the tumors, constitutional mutations were identified in four cases: two frameshifts, one nonsense mutation and one intronic base substitution 32 bp downstream of exon 22; RT-PCR experiments revealed that the single-base substitution in the intron seemed to increase the transcript lacking exon 22. All four cases were judged to involve truncation of the gene product. The evidence reported here supports a rather limited role of BRCA1 in ovarian carcinogenesis in the Japanese population.

BRCA1 Protein↗