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At least 505 records · Page 28Linked to original sources
PubMed1997

Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3alpha and beta isoforms in patients with NIDDM.

Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active dephosphorylated state. In a search for genetic defects responsible for the decreased insulin stimulated glycogen synthesis seen in patients with non-insulin-dependent diabetes mellitus (NIDDM) and their glucose-tolerant first-degree relatives we have performed mutational analysis of the coding region of the 2 isoforms of GSK-3alpha and GSK-3beta in 72 NIDDM patients and 12 control subjects. No structural changes were detected apart from a few silent mutations. Mapping of the GSK-3alpha to chromosome 19q13.1-13.2 and the GSK-3beta to chromosome 3q13.3-q21 outside known genetic loci linked to NIDDM further makes it unlikely that these genes are involved in the pathogenesis of common forms of NIDDM.

Alleles↗
PubMed2006

Immunohistochemical and mutational analysis of apoptosis-inducing factor (AIF) in colorectal carcinomas.

Apoptosis-inducing factor (AIF) has a principal role in caspase-independent apoptosis. In addition, AIF has a vital oxidoreductase activity that is required for cell survival. It may be important to identify the alterations of the AIF gene and AIF protein expression to see the function of AIF in human cancers. In this study we analyzed the expression of AIF protein in 103 colorectal carcinomas by immunohistochemistry. We also analyzed the mutation in exons 10-15 of AIF encoding the region that possesses the cell death function of AIF by single-strand conformation polymorphism (SSCP) in 48 colorectal, 48 gastric, 48 breast and 48 hepatocellular carcinomas, and 48 leukemias. By immunohistochemistry, AIF protein expression was detected in both cancer cells and normal mucosal epithelial cells in all of the 103 colorectal carcinoma tissues. However, the cancer cells showed higher intensities of AIF immunostaining than the normal cells in 83 cases (80.5%). AIF immunoreactivities were observed in the cancers irrespective of their location or the depth of invasion. Mutational analysis detected one AIF mutation in the colorectal carcinomas, but none in the other cancers. The AIF mutation detected was a two-base deletion mutation in intron 15. The increased expression of AIF in the malignant colorectal epithelial cells compared to the normal mucosal epithelial cells suggests that AIF expression may play a role in colorectal tumorigenesis. The data also suggest that somatic mutation of AIF is a rare event in common human cancers.

Adenocarcinoma↗
PubMed2004

Mutational analysis of the N-terminal DNA-binding domain of sleeping beauty transposase: critical residues for DNA binding and hyperactivity in mammalian cells.

The N-terminal domain of the Sleeping Beauty (SB) transposase mediates transposon DNA binding, subunit multimerization, and nuclear translocation in vertebrate cells. For this report, we studied the relative contributions of 95 different residues within this multifunctional domain by large-scale mutational analysis. We found that each of four amino acids (leucine 25, arginine 36, isoleucine 42, and glycine 59) contributes to DNA binding in the context of the N-terminal 123 amino acids of SB transposase, as indicated by electrophoretic mobility shift analysis, and to functional activity of the full-length transposase, as determined by a quantitative HeLa cell-based transposition assay. Moreover, we show that amino acid substitutions within either the putative oligomerization domain (L11A, L18A, L25A, and L32A) or the nuclear localization signal (K104A and R105A) severely impair its ability to mediate DNA transposition in mammalian cells. In contrast, each of 10 single amino acid changes within the bipartite DNA-binding domain is shown to greatly enhance SB's transpositional activity in mammalian cells. These hyperactive mutations functioned synergistically when combined and are shown to significantly improve transposase affinity for transposon end sequences. Finally, we show that enhanced DNA-binding activity results in improved cleavage kinetics, increased SB element mobilization from host cell chromosomes, and dramatically improved gene transfer capabilities of SB in vivo in mice. These studies provide important insights into vertebrate transposon biology and indicate that Sleeping Beauty can be readily improved for enhanced genetic research applications in mammals.

Amino Acid Sequence↗
PubMed1992

Mutational analysis of the adeno-associated virus rep gene.

The replication (rep) gene of the human parvovirus adeno-associated virus (AAV) is a pleiotropic effector of numerous viral functions and experts profound effects on cellular transformation. Of the four Rep proteins, the primarily nuclear Rep78 and Rep68 direct AAV DNA replication, trans activation of the capsid (cap) gene promoter, and inhibition of cellular proliferation mediated by various oncogenes. In an initial attempt to define functional domains in Rep78, we have constructed a comprehensive set of XhoI linker insertion and deletion mutations in the rep gene. Each of the mutant genes has been expressed in cell culture and assayed for the following functions: (i) nuclear localization, (ii) AAV DNA replication, (iii) trans activation of the AAV capsid gene transcription promoter, and (iv) suppression of cellular transformation mediated by the adenovirus E1a and an activated ras oncogene pair. Modest disruptions in the normal conformation of Rep78 inactivated its AAV DNA replication function and trans activation of the cap gene promoter. Linker insertion mutations in the amino-terminal one-third of the protein inactivated Rep78's ability to suppress oncogene-mediated cellular transformation. The transformation suppression domains are not limited to the amino-terminal regions, however, since deletions throughout the protein altered its suppression capabilities. A putative nuclear localization signal that is essential for each of the above functions was found in the Rep proteins. These results provide a preliminary screening of the functional domains in the AAV Rep proteins and pave the way for more subtle mutational analysis.

3T3 Cells↗
PubMed2006

Ligand recognition by vitamin D receptor: total alanine scanning mutational analysis of the residues lining the ligand binding pocket of vitamin D receptor.

We performed total alanine scanning mutational analysis (ASMA) of the residues lining the ligand binding pocket (LBP) of the human vitamin D receptor (hVDR) to investigate allosteric effects of ligands in the function of nuclear receptors (NRs). This was accomplished for the first time in the NR superfamily. The effects of ligand structure were also examined in this system (termed 2D-ASMA) using 8 representative VDR ligands. The results clearly revealed the role and importance of all amino acid residues lining the LBP and the relationships between ligand binding and transcriptional potency. 2D-ASMA indicated ligand-specific ligand-protein interactions, which are essential in determining the transactivation potency of the ligand. Taking the results as a whole, we suggest a ligand-mediated allosteric network, which allows transmission of information from ligands to the interfaces of the VDR in association with protein cofactors and was shown to be linked to a part of the network identified by statistical coupling analysis (SCA).

Alanine↗
PubMed1996

Characterization of transmembrane domains 6, 7, and 8 of MalF by mutational analysis.

Oligonucleotide mutagenesis was used to isolate mutations in membrane-spanning segments 6, 7, and 8 of MalF. MalF is a cytoplasmic membrane component of the binding protein-dependent maltose transport system in Escherichia coli. The current structural model predicts eight transmembrane domains for MalF. Membrane-spanning segments 6, 7, and 8 of MalF flank or are part of the EAA-X3-G-X9-I-X-LP consensus region present in the cytoplasmic membrane subunits of the bacterial ABC transporter superfamily members. Mutations with two novel phenotypes with respect to substrate specificity of the maltose transport system were isolated. One mutant grew on minimal maltose media but not on media containing either maltoheptaose or maltoheptaose plus maltose and was thus termed dextrin dominant negative. The other class of mutations led to a maltose minus but maltoheptaose plus phenotype. Nine of the isolated mutations leading to changes in substrate specificity were tightly clustered on one face of the postulated transmembrane helix 6. A similar clustering of mutations was detected in transmembrane domain 7. The majority of mutations in membrane-spanning segment 7 led to a protease-sensitive or a conditional phenotype with respect to MalF function or both. Mutations in transmembrane domain 8 appeared to be more randomly distributed. The majority of mutations in membrane-spanning segment 8 caused a Mal+ Dex- phenotype. Six Mal+ suppressor mutations isolated to two mutations in transmembrane domain 7 changed amino acid residues in membrane-spanning segment 6 or 8.

ATP-Binding Cassette Transporters↗
PubMed2003

Mutational analysis of p53 in human tumors: immunocytochemistry.

Mutations of the p53 gene are the most common genetic changes in human malignancies; therefore their detection is of practical importance. In contrast to wild-type p53 in resting normal cells, mutant p53 proteins are easily detectable by immunocytochemical methods due to their abnormally extended half-life. Several methods of immunocytochemistry can be used to analyze the presence and localization of p53 protein in cells or tissues. The most important is immunocytochemical p53 staining of sections from paraffin embedded tissues. This method is used as a relatively reliable surrogate marker for p53 mutations and has the advantage of being easy, fast, and suitable for mass screening of large archival tissue banks. Frozen sections can also be used to detect and localize the p53 proteins. p53 can also be detected in tissue culture cells. p53 can be detected in situ through a secondary antibody coupled to a fluorescent dye or an enzymatic activity that reacts with certain chromogens.

DNA Mutational Analysis↗
PubMed1998

Mutation analysis of a putative sialyltransferase gene, the SFRS2 splicing factor gene and the c-myb ET-locus in two families with hereditary neuralgic amyotrophy (HNA).

HNA is an autosomal dominant recurrent focal neuropathy involving the brachial plexus. The etiology of HNA is unknown but the genetic defect most likely affects a non-neuronal tissue. We previously described linkage to chromosome 17q24-q25 in two HNA-families. Here we report the mutation analysis of two candidate genes: a cDNA encoding a putative sialyltransferase and the SFRS2 splicing factor including the c-myb ET-locus which is encoded on the opposite strand of the SFRS2 gene. The complete protein coding regions of both genes were studied by direct DNA sequencing. We did not find a disease associated mutation indicating that these genes are most likely not involved in the pathogenesis of HNA. However, we identified and characterized a rare AvaII polymorphism in the SFRS2 gene and detected a sequencing error, leading to an amino acid change (Val11Leu) in the published sequence of the putative sialyltransferase.

Brachial Plexus Neuritis↗
PubMedDate not supplied

A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC.

The most sensitive screening technique for genes that predispose patients for particular cancers is direct sequencing. However, sequencing of complex genes is technically demanding, costly and time-consuming. We have tested alternate screening techniques to find a fast sensitive method for detecting alterations of DNA in the large BRCA1 gene prior to sequencing. Sequencing of this gene is particularly arduous because it lacks clearly defined mutation sites. The single-strand conformation polymorphism (SSCP) technique is one of the most frequently used pre-screening methods but its sensitivity and efficiency is not completely satisfying. We have compared the SSCP assay with a newly developed technique called denaturing high performance liquid chromatography (DHPLC) to screen the BRCAl gene. We studied 23 patients at high risk for early onset breast and ovarian cancer and four controls. In these patients, a total of 113 fragments with sequence variations in the BRCA1 gene could be identified. The DHPLC technique resolved 100% of the DNA alterations that were observed in cycle sequencing. In contrast, mutation analysis by SSCP accounted for 94% of the detected variations. In addition, DHPLC screening allowed us to discriminate between different alterations in a single fragment, because of the characteristic elution profiles of the DNA molecules. Polymorphisms that were present in our samples could be predicted by means of DHPLC testing independently of sequence analysis. We conclude that DHPLC is a highly potent screening method for genetic analyses. It is highly sensitive, efficient and economical and can be automated.

Breast Neoplasms↗
PubMed2000

Pancreatic function and extended mutation analysis in DeltaF508 heterozygous infants with an elevated immunoreactive trypsinogen but normal sweat electrolyte levels.

BACKGROUND: Newborn screening for cystic fibrosis (CF) with immunoreactive trypsinogen (IRT) and DeltaF508 analysis followed by sweat testing misses some infants with CF and detects more DeltaF508 carriers than expected. Some of the apparent DeltaF508 carriers may be DeltaF508 compound heterozygotes with normal sweat electrolyte levels. METHODS: Infants identified by newborn screening with an elevated IRT level, one DeltaF508 allele, and a sweat chloride level <60 mmol/L underwent CF mutation analysis, pancreatic stimulation testing, and repeat IRT analysis followed by clinical review and repeat sweat test at 12 months. RESULTS: Over a 24-month period we identified 122 DeltaF508 heterozygotes and recruited 57; 4 had borderline sweat chloride levels (40 to 60 mmol/L), 5 (8.8%, 95% CI 1.4, 16.2) had a second CF mutation (R117H), and 11 (20%, 95% CI 10, 30) had the intron 8 5T allele. Three had clinical CF at 12 months (initial sweat chloride levels: 53, 51, and 32 mmol/L). Pancreatic electrolyte secretion in the subjects with a borderline sweat chloride level was similar to that in patients with known CF. CONCLUSION: The excess of DeltaF508 heterozygotes detected by IRT/DNA screening is associated with the presence of a second mutation or the 5T allele in some infants. Screened infants with borderline sweat chloride levels almost certainly have CF, but long-term follow-up of the infants with the genotype DeltaF508/R117H and DeltaF508/5T is required to determine their outcome. In the meantime, newborn screening should be confined to severe mutations associated with classic CF.

Cystic Fibrosis↗
PubMed1996

Mutational analysis of the mitogenic and transforming activities of the insulin-like growth factor I receptor.

THe type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in mitogenesis and transformation. It has been previously shown that mitogenic signaling and transforming activity of the IGF-IR can be dissociated: a receptor truncated at residue 1229 (C-terminus) is fully mitogenic, in terms of its response to IGF-I, but cannot transform 3T3-like cells that are devoid of endogenous IGF-IRs (R- cells). We have extended our mutational analysis of the C-terminus of the human IGF-IR, by stably transfecting several mutant receptors into R- cells, and testing the resulting cell lines for IGF-I-mediated mitogenic response and formation of colonies in soft agar. The results indicate that the transforming domain of the IGF-IR can be localized between residues 1245 and 1310, these sequences being not required for mitogenic signaling. Within these residues, there are at least two areas that contribute to the transforming activity of the receptor.

3T3 Cells↗
PubMed2000

Mutational analysis of BRCA1 and BRCA2 genes in Chinese ovarian cancer identifies 6 novel germline mutations.

Germline mutations in the BRCA1 and BRCA2 genes predispose women to breast and ovarian cancer. An incidence of 5% and 3.3% respectively has been reported of BRCA1 and BRCA2 mutations in women with ovarian cancer unselected for family history. The contribution of BRCA1 and BRCA2 mutations to ovarian cancer in Chinese women is unknown. A total of 60 samples of ovarian cancer diagnosed in Chinese unselected for age or family history were analyzed for BRCA mutations using the protein truncation test. The entire coding exon of BRCA1 of 53 cases and that of exon 11 of BRCA2 of 43 cases were successfully screened. Six germline (11.3%) mutations (633C>T, 1080delT, 1129delA, 2371-2372delTG, 3976-3979delGTGA, and IVS 22+7 A>G) were detected in BRCA1. One germline mutation (3337C>T) (2.1%) was detected in BRCA2. None of these seven cases were associated with strong family history of breast and/or ovarian cancer. Five out of our six BRCA1 mutations and the one BRCA2 mutation identified are novel. Our 11.3% incidence of BRCA1 mutations in ovarian cancer found amongst Chinese with insignificant family history is apparently higher than that previously reported in other populations. It suggests that BRCA1 mutation may play a significant role in the development of sporadic ovarian cancer in Chinese women.

Adult↗
PubMed2000

Mutational analysis of 30 Slovenian cystic fibrosis patients compared to known Slovenian and European CF mutation spectra.

More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity makes the search for molecular defects in cystic fibrosis difficult. Out of 100 Slovenian CF families, we have screened 30, using DGGE and SSCP as mutation detection techniques, while the remaining 70 have been studied previously. Together our and the previous studies have been able to indentify 18 CF mutations which cover 77.6% of the CF alleles in those families. The relative frequency of deltaF508 is 62.7% which is significantly higher than the average reported for the Mediterranean South European region (51.6%). At the same time, significant differences in mutation frequencies were found for the G542X, R1162X, W1282X, N1303K and 3905insT mutations. Several, otherwise rare mutations have been detected, such as: I148T, Q552X, 457TAT-->G, R1006H, 2907delTT, 3667ins4, A559T and G576A. An interesting fact is that A559T was so far found mostly in CF patients of African-American origin. These results imply that a high heterogeneity of CF mutations occurs within the small population of Slovenia, consisting only of 2 million inhabitants. In view of the spectrum and frequencies of detected mutations, Slovenian population expresses characteristics of Mediterranean and central European countries, and at the same time shows also distinctive differences and unique region specific CF mutations (Q685X, D192G, S4X).

Alleles↗
PubMed1999

EXT-mutation analysis and loss of heterozygosity in sporadic and hereditary osteochondromas and secondary chondrosarcomas.

Osteochondromas occur as sporadic solitary lesions or as multiple lesions, characterizing the hereditary multiple exostoses syndrome (EXT). Approximately 15% of all chondrosarcomas arise within the cartilaginous cap of an osteochondroma. EXT is genetically heterogeneous, and two genes, EXT1 and EXT2, located on 8q24 and 11p11-p12, respectively, have been cloned. It is still unclear whether osteochondroma is a developmental disorder or a true neoplasm. Furthermore, it is unclear whether inactivation of both alleles of an EXT gene, according to the tumor-suppressor model, is required for osteochondroma development, or whether a single EXT germline mutation acts in a dominant negative way. We therefore studied loss of heterozygosity and DNA ploidy in eight sporadic and six hereditary osteochondromas. EXT1- and EXT2-mutation analysis was performed in a total of 34 sporadic and hereditary osteochondromas and secondary peripheral chondrosarcomas. We demonstrated osteochondroma to be a true neoplasm, since aneuploidy was found in 4 of 10 osteochondromas. Furthermore, LOH was almost exclusively found at the EXT1 locus in 5 of 14 osteochondromas. Four novel constitutional cDNA alterations were detected in exon 1 of EXT1. Two patients with multiple osteochondromas demonstrated a germline mutation combined with loss of the remaining wild-type allele in three osteochondromas, indicating that, in cartilaginous cells of the growth plate, inactivation of both copies of the EXT1 gene is required for osteochondroma formation in hereditary cases. In contrast, no somatic EXT1 cDNA alterations were found in sporadic osteochondromas. No mutations were found in the EXT2 gene.

Adolescent↗
PubMed1994

In vivo expression and mutational analysis of the barley yellow dwarf virus readthrough gene.

The barley yellow dwarf virus (BYDV) coat protein gene is separated from an adjacent downstream open reading frame (ORF) by a single termination codon. Immunological analysis of this downstream "readthrough" region reveals multiple coat protein-readthrough products. A full-length 72-kDa (P72) coat protein-readthrough fusion product is detected in total lysates from infected cells. However, purified aphid transmissible virions contain only a 50-kDa (P50) coat protein-readthrough product. Virion-associated P50 lacks the C-terminal domain predicted by its ORF sequence. A separate 33-kDa polypeptide (P33) corresponding to the readthrough C-terminus domain is detected in the crude cellular membrane fraction. Site-directed and deletion mutational analysis demonstrate that the readthrough ORF is dispensable for BYDV replication and virion accumulation in protoplasts. In contrast, a mutant which results in a continuous fusion product of coat and readthrough sequences is not viable. Point mutations were used to map regions required for P50 and P72 synthesis. A model explaining the relationships between the three forms of the readthrough polypeptides is proposed.

Base Sequence↗
PubMed2000

Unilateral surgery supported by germline RET oncogene mutation analysis in patients with sporadic medullary thyroid carcinoma.

Compared to hereditary medullary thyroid carcinoma (MTC), sporadic MTC tends to be unicentric and confined to one lobe. Patients with sporadic MTC usually undergo total thyroidectomy because of a possible hereditary or bilateral process. We evaluated the usefulness of germline RET oncogene mutation analysis in surgery for apparently sporadic MTC and performed unilateral surgery on patients without detectable mutation. In 36 patients with a preoperative diagnosis of apparently sporadic MTC, we performed germline RET oncogene mutation analyses: before surgery in 8 recent patients and after surgery in 28 who had been treated before 1996. Of the latter, 5 had bilateral MTC. DNA samples were extracted from their peripheral blood, and the polymerase chain reaction products of the RET proto-oncogene were analyzed using single-strand conformation polymorphism analysis and the direct sequencing methods. Before 1996 we often performed total thyroidectomy but changed to hemithyroidectomy thereafter, except in one patient with associated Graves' ophthalmopathy. Our minimal standard practice included systematic central and ipsilateral neck dissection. The outcome was assessed in terms of gastrin- and calcium-stimulated plasma calcitonin levels. Germline RET mutations were found in six patients. Five of these patients had bilateral MTC, whereas all 30 patients without mutation had unilateral disease. Hemithyroidectomy in seven of our recent patients resulted in normalization of plasma calcitonin levels in all, although four were found to have microscopic lymph node involvement. In conclusion, hemithyroidectomy with systematic central and ipsilateral neck dissection is an appropriate procedure for patients with sporadic MTC without detectable germline RET mutations.

Adult↗
PubMed2004

Mutational analysis of the human SLC26A8 gene: exclusion as a candidate for male infertility due to primary spermatogenic failure.

SLC26A8 is an anion transporter that is solely expressed in the testes. It interacts with MgcRacGAP that shows strong structural similarity with the Drosophila protein RotundRacGAP, which is established to have an essential role for male fertility in the fruit fly. To explore whether the SLC26A8 gene has a role in human male infertility, we performed mutational analysis in the coding region of the SLC26A8 gene in 83 male infertility patients and two groups of controls using single-strand conformational polymorphism and direct sequencing methods. We found six novel coding sequence variations, of which five lead to amino acid substitutions. All variants were found with similar frequencies in both patients and controls, thus suggesting that none of them may be causally associated with infertility. We conclude that the SLC26A8 mutations are not a common cause of male infertility.

Adult↗
PubMed2004

Mutation analysis of suppressor of cytokine signalling 3, a candidate gene in Type 1 diabetes and insulin sensitivity.

AIMS/HYPOTHESIS: Beta cell loss in Type 1 and Type 2 diabetes mellitus may result from apoptosis and necrosis induced by inflammatory mediators. The suppressor of cytokine signalling (SOCS)-3 is a natural inhibitor of cytokine signalling and also influences insulin signalling. SOCS3 could therefore be a candidate gene in the development of Type 1 and Type 2 diabetes mellitus. METHODS: Mutation analysis of the SOCS3 gene was performed in 21 patients with Type 1 diabetes mellitus and in seven healthy subjects. An identified promoter variant was examined in (i) 250 families with Type 1 diabetic family members (1097 individuals); (ii) 212 glucose-tolerant first-degree relatives of Type 2 diabetic patients; and (iii) 370 population-based young, healthy subjects who were unrelated. RESULTS: Three mutations were identified in the promoter region, but none in the coding region or the 3'UTR. Two of the three mutations had allele frequencies below 1% whereas the C -920-->A substitution had a minor allele frequency of 8%. In the group of young healthy subjects the insulin sensitivity index was higher among homozygous carriers of the A-allele than among heterozygous and wild-type subjects ( p=0.027, uncorrected). The same trend was found in the group of first-degree relatives of Type 2 diabetic patients. No association or linkage was found to Type 1 diabetes mellitus. CONCLUSIONS/INTERPRETATION: Homozygosity for the A-allele of the C -920-->A promoter polymorphism of the SOCS3 gene may be associated with increased whole-body insulin sensitivity, but deserves further investigation.

Adolescent↗